Correspondence should be addressed to Andreas E Zautner azautnegwdgde
Received 14 July 2014 Revised 22 August 2014 Accepted 28 August 2014 Published 17 September 2014
Academic Editor Gundlapally S Reddy
Copyright copy 2014 Hagen Frickmann et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Atypical and multidrug resistance especially ESBL and carbapenemase expressing Enterobacteriaceae is globally spreadingTherefore it becomes increasingly difficult to achieve therapeutic success by calculated antibiotic therapy Consequently rapidantibiotic resistance testing is essential Various molecular and mass spectrometry-based approaches have been introducedin diagnostic microbiology to speed up the providing of reliable resistance data PCR- and sequencing-based approaches arethe most expensive but the most frequently applied modes of testing suitable for the detection of resistance genes evenfrom primary material Next generation sequencing based either on assessment of allelic single nucleotide polymorphismsor on the detection of nonubiquitous resistance mechanisms might allow for sequence-based bacterial resistance testingcomparable to viral resistance testing on the long term Fluorescence in situ hybridization (FISH) based on specific bindingof fluorescence-labeled oligonucleotide probes provides a less expensive molecular bridging technique It is particularlyuseful for detection of resistance mechanisms based on mutations in ribosomal RNA Approaches based on MALDI-TOF-MS alone or in combination with molecular techniques like PCRelectrospray ionization MS or minisequencing providethe fastest resistance results from pure colonies or even primary samples with a growing number of protocols This reviewdetails the various approaches of rapid resistance testing their pros and cons and their potential use for the diagnosticlaboratory
1 Introduction
Generation of antimicrobial susceptibility patterns remainsone of the most important tasks of clinical microbiologylaboratories The effective calculated antimicrobial therapyof infectious disease patients is consistently challenged bythe rapidly rising prevalence of resistant and multidrug-or even pandrug-resistant pathogens worldwide In recentyears this trend was accompanied by a shift from Gram-positive to Gram-negative bacteria like multidrug-resistantEnterobacteriaceae strains (MRE resistant to three ormore classes of antibiotics) as well as multidrug-resistant
nonfermenters (Pseudomonas aeruginosa and Acinetobacterbaumannii) [1ndash4] In particular carbapenemase express-ing Enterobacteriaceae coresistant to non-beta-lactam anti-biotics like quinolones aminoglycosides colistin and fos-fomycin are a recent major public health concern [5ndash8] Col-onization byMRE is highly region and patient group specificFor example in the French capital Paris a tenfold increasein the intestinal colonization rate of healthy individualswith extended-spectrum beta-lactamase- (ESBL-) producingbacteria was observed during the last half decade [9] ESBLcolonization was with 46 particularly in French childrenaged 6ndash24 months significantly above average [10] Long
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 375681 19 pageshttpdxdoiorg1011552014375681
2 BioMed Research International
lasting persistence of MRE as demonstrated by a Swedishand a French study contributes to the increase in the MREprevalence sometimes even years after infection [11 12] Amedian MRE-colonization period of 125 months could bedetected in a cohort of newborn children in Norway [13] AnEnglish studywas able to verify persistence of resistance geneseven in the absence of antibiotic pressure [14] Furthermorethe colonization rate also differs between healthy subjects andpatients at risk It could be demonstrated in aKorean endemicarea that 203 of healthy individuals were colonized withESBL producers while high-risk patients were colonized in425 of cases [15] However the risk of faecal colonizationdepends mainly on the local prevalence For example anESBL prevalence of 657 has been demonstrated in healthyadults inThailand [16] while another study showed an ESBLprevalence of 113 in outpatients in England [17] Farmanimals are another reservoir for multidrug-resistant bacte-ria A survey in the Netherlands demonstrated that chickensare colonized with ESBL-producing Enterobacteriaceae tomore than 70 while swine and cattle are known reservoirsfor livestock-associated methicillin resistant Staphylococcusaureus (laMRSA) [18]
The multiresistances of the Gram-negative bacteria rep-resent a major challenge for the traditional culture-basedmicrobiology Furthermore the limited treatment optionsfor a calculated therapy and therewith the risk of an inap-propriate therapy are an intensifying factor of this problem[19] As a consequence morbidity and mortality of outpa-tient and nosocomial-acquired infections with multidrug-resistant Gram-negative bacteria are significantly increasedSimilarly Mycobacterium tuberculosis has posed a serioushealth threat as a result of multidrug resistance In its2013 global report on tuberculosis WHO estimates that36 (95 confidence interval 21ndash52) of new cases and202 (95 confidence interval 133ndash272) of previouslytreated cases had multidrug-resistant (MDR) tuberculosis(defined as tuberculosis caused by M tuberculosis isolatesthat are resistant to rifampicin and isoniazid) and 13 millionTB deaths [20] On the other hand CMV resistance hasbeen reported to be on the rise in transplant recipients[21 22]
Information on antimicrobial susceptibility aids a clini-cian in prescribing an appropriate antimicrobial drug for aparticular infection Due to the rapid rise in antimicrobialresistance worldwide [1] it is becoming increasingly impor-tant for a clinician to rapidly receive information on theantimicrobial susceptibility profile of the isolated pathogenfor appropriate treatment to be initiated Traditionally clin-ical microbiology laboratories have relied on phenotypicmethods to determine the antibiotic susceptibility profilesof pathogens [23] These methods remain useful and haveadvantages such as low costs as well as being easy to performand having established interpretation criteria But they lackthe ability to generate timely susceptibility results hencedelaying initiation of treatment [24] Furthermore currentlythere is a need to establish adequate and standardized screen-ing and isolation procedures for carbapenemase-producingbacteria especially in risk patients as well as in patients
in which MRE colonizationinfection has been previouslyshown These limitations have been found to have conse-quences in patient management for example delay in theinitiation of antibacterial treatment has led to increases inmortality [25] as well as in hospitalization time [26] andmake it challenging to implement the back-end approachof the antimicrobial stewardship program which has shownrewarding results in patientmanagement and the fight againstantimicrobial resistance [27]
In response to the limitations of phenotypic methodsand the desires to improve patient management and curbthe spread of antimicrobial resistance rapid antimicrobialsusceptibility testing methods are continuously developedThese methods have been found to identify a pathogenand its antimicrobial susceptibility profile within a shortperiod of time There are basically five different ways toaccelerate susceptibility testing in clinical diagnostics (I)bypassing conventional culture by direct detection of thepathogen or resistance mechanism in the primary sample(II) bypassing plate or broth culture dependent susceptibilitytesting (secondary culture) (III) avoiding time consumingwork stepsmethods (IV) increasing the sensitivity to thedetection of the infectious agent that means detecting theinfectious agent in earlier disease stages at lower viral ormicrobial loads and (V) earlier detection of an evolvingdrug resistance during treatment in spreading less susceptiblequasispecies
For example real-time quantitative PCR (qPCR) hasmade it possible to detect multidrug-resistant tuberculosis(MDR TB) in a sample within an hour hence immediatelyinitiating appropriate treatment and control measures [28]Also MALDI-TOF mass spectrometry (MS) has made itpossible to detect the most pathogens in a sample withinminutes with high sensitivity and specificity [29] In additionthese methods have made it possible to control the spreadof resistant strains reduce the length of patient stay inhospitals and enhance the implementation of antimicrobialstewardship programs
In this review we detail the rapid antimicrobial suscep-tibility testing methods that have been developed recentlyThey include classical agglutination assays molecular testingmethods for example qPCR DNA microarrays LuminexxMAP assays and next generation sequencing fluores-cence in situ hybridization (FISH) and mass spectrometry-based methods for example phyloproteomics assays usingstable isotope labeling of amino acids mass spectro-metric beta-lactamase assays PCRelectrospray ionization-mass spectrometry (PCRESI MS) minisequencing andmass spectrometry-based comparative sequence analysis(MSCSA) In addition we discuss the impact that these tech-niques are likely to bring for the patient management and thereduction of antimicrobial resistance
2 Agglutination Assays as RapidCulture-Associated Options
Agglutination assays are based on a suspension of micropar-ticles coated with specific antibodies leading to agglutination
BioMed Research International 3
in contact with their specific antigens Such proceduresare useful for a preliminary resistance screening from purebacterial colonies if the resistance mechanism of interest isassociated with a single antigen only which is expressedon the surface of the pathogen Accordingly agglutinationassays are unfeasible for the screening for complex resistancepatterns which may be associated with multiple structurallydifferent families of enzymes as in the case of extended-spectrum beta-lactamases (ESBL) or carbapenemases inGram-negative rod-shaped bacteria
Agglutination assays for the rapid identification of bacte-rial resistance patterns are widely restricted to the identifica-tion of the penicillin binding protein 2a (PBP-2a) the majorresistance determinant ofMethicillin resistant Staphylococcusaureus (MRSA) Different agglutination kits show specifici-ties of 913 to 100 if applied to MRSA colony material[30ndash32] The sensitivity is even more restricted rangingbetween 827 and 941 [30ndash32] If sufficient quantities ofcolony material are used agglutination testing allows for theidentification of small-colony variant MRSA strains as well[33]
The lack of sensitivity seems to be associated with certainstaphylococcal cassette chromosome (SCC-mecA) types withtype IV scoring particularly poor [31] Furthermore aggluti-nation kits are only positive if methicillin resistance is due tothe mecA gene If mecC a divergent mecA homologue is thecause of the resistance agglutination usually fails as observedfor 10 out of 10 mecC-positive live-stock associated MRSAstrains [34]
Of note agglutination based PBP-2a testing is possiblefrom liquid sample materials as well However the sensitivityis poor From blood culture pellets PBP-2a agglutinationshowed sensitivity of only 18 in a recent study In contrastspecificity was excellent with 100 [35]
The usage of genotypic methods in the rapid detectionof antimicrobial resistance genes is gradually shifting fromacademic research laboratories to diagnostic laboratories andpoint-of-care testing The attractiveness of these methods indetermination of antimicrobial resistance has been attributedto two factors firstly their capability to generate resultswithin a short time as compared to phenotypic methods sec-ondly their capability to detect antimicrobial genes directlyfrom the patient sample without necessarily waiting forculture results [36]These two attributes aid clinicians in pre-scribing appropriate treatment to patients at the opportunetime hence making a positive contribution to antimicrobialstewardship programs [27] However genotypic tools for thedetection of antimicrobial resistance may generate false neg-ative results due to (i) their inability to detect new resistancemechanisms or (ii) false-positive results because they maydetect inactive or incomplete resistance genes in a specimenwhich have not inferred resistance to the antimicrobial drugunder test [37]
Current genotypic methods that are used for the rapiddetection of antimicrobial resistance genes include (i) nucleicacid amplification methods particularly real-time quantita-tive PCR (qPCR) (ii) DNA hybridization based methodsparticularly DNAmicroarrays (iii) Luminex xMAP technol-ogy and (iv) next generation sequencingmethods Below is abrief description on the application of each of thesemolecularmethods for the rapid detection of antimicrobial resistance
31 Nucleic Acid Amplification Methods Recently one of thePCR techniques that has received a wide application in clin-ical microbiology is the quantitative real-time PCR (qPCR)technique [38] This has been attributed to its flexibility andcapability to rapidly and simultaneously identify multiplepathogens in a clinical specimen and the presence of antimi-crobial resistance genes in the identified pathogens [39] Asa result numerous qPCR assays for rapid identification ofpathogens in clinical specimens have been developed butmost of the available qPCR assays for detection of microbialresistance genes are limited to the detection of antibioticresistance In short most of the available commercial qPCRassays detect the presence of mecA and mecC which confermethicillin resistance in S aureus the vanA and vanB geneswhich confer glycopeptide resistance and genes that encodeextended-spectrum 120573-lactamases (for detailed review oneach assay see Maurin 2012 [39]) One outstanding featureof all these qPCR assays is their capability to simultaneouslyand accurately detect resistance genes within a remarkablyshorter time period of 4ndash6 hours Similarly qPCR assaysfor rapid detection of resistance against rifampin (RIF) andisoniazid (INH) have been introduced Ramirez and cowork-ers have recently combined qPCR and high-resolution melt(HRM) technology to develop an assay which rapidly andsimultaneously identifiesmultidrug-resistantM tuberculosismutations in the rpoB gene conferring resistance to RIF andmutations in the katG and inhA genes conferring resistanceto INH [28] This assay produces results within 6 hours ascompared to GenoType MTBDRplus assay (Hain LifescienceGmbH Germany) and culture susceptibility testing whichtake 8 hours and 56 days to generate results In the recenttime several in-house qPCR assays for rapid and simulta-neous detection of genes encoding Klebsiella pneumoniaecarbapenemase (blaKPC) andNewDelhi metallo-120573-lactamase(blaNDM) inGram-negative rod-shaped bacteria [40ndash43] havebeen introduced Similarly several in-house qPCR assays forrapid and simultaneous detection of blaOXA-48 blaVIM andblaIMP carbapenemase genes in Enterobacteriaceae have beenestablished [44ndash46]
PCR-based MRSA testing has found wide applicationsin microbiological routine laboratories Next to in-houseassays commercially available molecular MRSA testing plat-forms comprise for example BD GeneOhm MRSA (BectonDickinson Heidelberg Germany) GT MRSA DirectGQMRSA (Hain Lifescience Nehren Germany) Hyplex Staphy-loResist (Amplex Gieszligen Germany) LightCycler (RocheDiagnostics Ltd Rotkreuz Switzerland) kits like LC MRSAAdvanced CepheidXpertGeneExpert (Cepheid SunnyvaleCA USA) and TIB Molbiol LightMix MRSA (TIB Molbiol
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Berlin Germany) All test systems showed reliable results ina recent external laboratory control evaluation in Germany[47] Similarly commercial PCR assays for the detection ofESBL-associated blaCTX-M beta-lactamases and only partiallyESBL-associated blaTEM and blaSHV as well as OXA1-typecarbapenemases (the latter combined in a consensus run)were introduced (Amplex Gieszligen Germany) [48] Similarmultiplex PCR systems are available for the most frequentlydetected carbapenemases which are particularly useful forthe follow-up during hospital outbreak events (AmplexGieszligen Germany) even from primary sample materials [4950]The switch ofmolecular carbanemase detection to robustloop-mediated isothermal amplification (LAMP) [46] allowsfor commercial point-of-care testing (POCT) compatible testsolutions for bedside testing for example the eazyplex Super-BugCRE system (Amplex Gieszligen Germany) which providesresults within 10 minutes However the great number ofdifferent possible cephalosporin and carbapenem resistancemechanisms finally exceeds any multiplexing capacity ifcompleteness is aspired
Nevertheless in addition to rapid and simultaneous pro-viding of reliable results qPCR has been found to be afford-able sensitive specific user friendly not space demandingand deliverable [37ndash39 51] Due to these attributes qPCR hasfound various applications in point-of-care testing (POCT)For example the Xpert MTBRIF test (Cepheid SunnyvaleCA USA) is a qPCR-based assay that has been developedto rapidly and simultaneously detect M tuberculosis andrifampicin (USAN rifampin) resistance To evaluate itsusefulness in POCT a large multicentre study involving6069 cases from six unrelated sites was performed In thisstudy Xpert MTBRIF detected rifampicin resistance casesin 1 hour as compared to line-probe assay and phenotypicdrug susceptibility testing that detected the same cases in 20days and 106 days respectively [52] As mentioned above asimilarGenXpert-basedPOCT test forMRSA screening fromclinical sample materials is available as well
Multiplex PCR assays have also been developed to rapidlyand simultaneously identify multiple pathogens in clinicalspecimens as well as the presence of antimicrobial resistancegenes in the identified pathogens Strommenger and cowork-ers developed amultiplex PCR which simultaneously detects9 resistance genes in S aureus directly from clinical speci-men within 6 hours [53] These 9 resistance genes includemecA (methicillin resistance) aacA-aphD (aminoglycosideresistance) tetK tetM (tetracycline resistance) ermA ermC(macrolide-lincosamide-streptogramin B resistance) vatAvatB and vatC (streptogramin A resistance) [53]
Like qPCR multiplex PCR assays were used as POC teststo facilitate patient management One example is the mul-tiplex PCR-based Unyvero Pneumonia Application (UPA)assay (Curetis AG Holzgerlingen Germany) that has beendeveloped to rapidly and simultaneously detect 18 bacterialspeciesPneumocystis jirovecii and 22 resistancemarkers fromrespiratory specimens (httpwwwcuretiscom) In one ofthe studies showing its suitability for POC testing the UPAassay detected multiple antibiotic resistances within 1 hour(as compared to phenotypic methods that took 96 hours)in a group of 56 hospitalized patients with respiratory tract
infections whowere under treatmentThis finding influencedthe modification of treatment in fifteen patients with severepneumonia leading to their recovery [54] The UPA assayis of course not able to replace conventional testing due toits design because it is not able to detect further microbialspecies and resistance mechanisms besides the implementedones
The continuous development of PCR-based assays withthe capability to rapidly and simultaneously detect pathogensand presence of resistance genes in specimens coupledwith their application in POCT may further improve themanagement of patients as long as appropriate quality controlis ensured
32 DNAMicroarray Technology The biggest challenge asso-ciated with the unprecedented rise of antimicrobial drugresistance worldwide is the scarce availability of assays thatare able to rapidly and simultaneously identify a causativepathogen and generate its antimicrobial resistance profileRecent oligonucleotide-based DNA microarrays match thischallenge In a recent study Zhang and coworkers describedthat CapitalBio DNAmicroarray (CapitalBio Corp) could ina mean time of 58 hours simultaneously identify Mycobac-terium species and detect mutations that confer isoniazid(INH) and rifampicin (RMP) resistance in specimens col-lected from spinal tuberculosis patients as compared toconventional culture and drug susceptibility testing whichtook a mean time of 568 days [55] Briefly oligonucleotideprobes which had been designed to identify Mycobacteriumspecies based on 16S rRNA sequences andmutations of rpoBinhA and katG that confer INH and RMP resistance werecovalently linked to the surface of aldehyde-activated slidesDNAwas extracted from specimens PCRwas used to amplifythe resistance genes and amplicons hybridized on the slidesThe emitted fluorescent signals were analyzed Guo andcoworkers evaluated the ability of a biochip which is basedon the same principle to rapidly and simultaneously identifymultidrug-resistant M tuberculosis (MRTB) and mutationsof rpoB inhA and katG that confer INH and RMP resistancein clinical sputum specimens [56] This group found thatthe biochip could in a mean time of 6 hours simultaneouslyidentifyM tuberculosis and detectmutations that confer INHand RMP resistance
Recent reports have also reported the availability ofCheck-Pointrsquos ESBLKPC DNA microarray for the identi-fication and detection of extended-spectrum 120573-lactamases(ESBLs) and Klebsiella pneumoniae carbapenemases (KPCcarbapenemases) [57 58] This array uses a methodologyknown as multiplex ligation detection to identify ESBL-associated or at least partially ESBL-associated genes (blaTEMblaSHV and blaCTX-M) and blaKPC genes (for details see [57])In a study to evaluate the rapidness at which this arraycould identify and detect these genes Naas and coworkersfound that Check-Pointrsquos ESBLKPC DNA microarray couldidentify them in 7-8 hours as compared to conventionalsusceptibility testing that took a mean time of 54 hoursSimilar results were observed by Willemsen and coworkersin a study that was aimed at evaluating the rapidness at
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which this array could identify and detect these ESBLKPCgenes in hospitals in the Netherlands [58] In addition todetecting and identifying ESBLKPC resistance in gastroin-testinal tract infections caused by Enterobacteriaceae Check-Pointrsquos ESBLKPC DNA microarray has also been usedto detect and identify KPC resistance in hospital-acquiredpneumonia caused by Klebsiella pneumoniae [59] Based onthese experiences the Check-MDR CT 102 DNA microarrayfor the detection of the most prevalent carbapenemase genes(blaNDM blaVIM blaKPC blaOXA-48 and blaIMP) and extended-spectrum120573-lactamase- (ESBL-) related gene families (blaSHVblaTEM and blaCTX-M) has been developed The evaluation ofthe rapidness of the Check-MDR CT 102 DNA microarrayto detect these genes has shown that it yields results 5 hoursfaster than Check-Pointrsquos ESBLKPC DNAmicroarray [60]
At present the DNA microarray technology is mostlyused in the routine detection of antimicrobial resistance ofTB and HIV [61ndash66] The routine use of systems such asMVPlex (Genaco Biomedical Products Huntsville USA) andStaphPlex systems (Genaco Biomedical Products HuntsvilleUSA) which combine both qPCR and DNA microarraytechnology suggest that independent DNA microarray tech-nology might find further applications in the routine clinicalmicrobiology [67 68] The MVPlex system detects the nucmecA (SCCmec)-orfX vanA vanB ddl and tuf genes toscreen for MRSA in nasal swabs [69] and the relatedStaphPlex system performs simultaneous species-level iden-tification (nuc versus tuf ) and detection of mecA aacAermA ermC tetM and tetK as well as Panton-Valentineleukocidin (PVL) for the rapid detection and characterizationof staphylococci directly from positive blood culture bottles[70]
33 Luminex xMAP Technology The description of cooc-curring single nucleotide polymorphism (SNP) mutations inantimicrobial resistance associated genes allows for targetedresistance testing For example unequivocally genetic studieshave proven that there are 5 different mutations in quinoloneresistance-determining region (QRDR) of gyrA gyrB andparE within Salmonella typhi [71] Similar studies havealso shown distinct mutations in the quinolone resistance-determining region (QRDR) of gyrA within Campylobacterjejuni and Campylobacter coli [72]
Rapid simultaneous detection of cooccurring singlenucleotide polymorphism (SNP) mutations in antimicrobialresistance associated genes remains however challengingMost molecular assays such as qPCR and pyrosequencinglack the capability to simultaneously detect cooccurringsingle nucleotide polymorphism (SNP)mutations in differentgenes in a given specimen [73] However this challengehas been overcome by Luminex xMAP Technology a multi-plexing technology which allows for simultaneous detectionof multiple nucleic acid sequences in a single reaction[74] During operation microtiter plates are loaded withmicrospheres that is coated and color-coded beads Themicrospheres are mixed with purified nucleic acids of the testorganism and allowed to hybridize emitting monochromaticlight which the Luminex analyzer reads and interprets At
present this technology has been used to simultaneouslydetect 11 mutations in gyrA gyrB and parE of SalmonellaTyphi and Salmonella Paratyphi A [75] Further it has beenused to simultaneously detect mutations in gyrA of C jejuniandC coli [76] In comparison to sequencing andmicroarraytechnology Luminex xMAPTechnology has been found to beflexible rapid and cost effective [74ndash76]
34 Next Generation Sequencing (NGS) Near whole genomesequencing (WGS) or next generation sequencing (NGS)allows for the assessment of bacterial genomes within severalhours A variety of different technological solutions havebeen introduced including laser printer sized benchtopdevices like 454GS Junior (Roche Basel Switzerland)MiSeq(Illumina San Diego CA USA) and Ion Torrent PGM (LifeTechnologies Grand Island NYUSA) In a previous analysisthe MiSeq (Illumina) system scored best regarding boththroughput per run and error rates while both the 454 GSJunior (Roche) and the Ion Torrent PGM (Life Technologies)systems were prone to homopolymer-associated indel errors[77]
Result interpretation of whole bacterial genomes is basedon either allelic comparisons [78] or single nucleotide poly-morphism (SNP) analysis [79] Data assessment and inter-pretation can be facilitated by commercial software packageslike SeqSphere+ (Ridom BIOINFORMATICS Ltd MunsterGermany) or BioNumerics (Applied Maths Sint-Martens-Latem Belgium)
NGS allows for resistance identification by the presenceof the underlying mechanism rather than just in pharma-codynamic terms [80] so it may revolutionize microbialresistance testing on the long termThis comprises the identi-fication and characterization of resistance genes encoding forextended-spectrum 120573-lactamases (eg 119887119897119886CTX-M 119887119897119886TEM and119887119897119886SHV) plasmid-mediated AmpCs (eg 119887119897119886CMY) quinoloneresistance (eg mutations in gyrA parC or qnr elements)aminoglycoside resistance (eg aminoglycosides modifyingenzymes 16S rRNA methylases) or carbapenemases (eg119887119897119886KPC 119887119897119886NDM) [81]
NGS-based resistance testing is of particular interest forslowly growing infectious agents with atypical resistancepatterns like multidrug-resistant (MDR) or extensive-drugresistance (XDR) M tuberculosis for which rapid identi-fication or exclusion of resistance determinants is of highrelevance for the therapeutic approach Ion Torrent full-gene sequencing with consecutive complete genetic analysiswithin 5 days (Table 5) allowed for reliable resistance detec-tion in M tuberculosis isolates of Burmese Hmong andIndian immigrants in the USA [82] Similar WGS data weredescribed for drug-resistant strains from Russia harbouringalmost all known drug-resistance associated mutations [83]In a direct comparison of Ion Torrent sequencing withphenotypic Bactec MGIT 960 (Becton Dickinson FranklinLakes NJ USA) analysis and genotypicHain line-probe assay(LPA) (Hain Lifescience Ltd Nehren Germany) there wascomplete concordance of NGS to phenotypic resistance andgenotypic rpoB and katG results for the analyzed M tuber-culosis isolates Even more Ion Torrent sequencing detected
6 BioMed Research International
uncommon substitutions and previously uncharacterizedresistance mutations in rpoB rrs and pncA [84] FurtherNGS is able to discriminate mixed mycobacterial genotypesin patient isolates based on single nucleotide variations(SNVs) [85] So it might be suitable to identify resistancemutations in genotypes that occur inminor proportions only
HoweverNGS-based resistance testing is not restricted tomycobacteria Recently NGSwas used to identify transmissi-ble plasmids in multidrug-resistant E coli isolates expressingan ESBL phenotype and transferring their cefotaxime resis-tance marker at high frequency in laboratory conjugationexperiments [86] High-throughput sequencing successfullyproved to be a valuable tool for tracing resistance plasmidsin the course of outbreaks as well [87] However a commer-cial NGS assay (Hospital Acquired Infection BioDetectionSystem Pathogenica Boston MA USA) for investigationsof outbreaks with ESBL-positive Enterobacteriaceae showedgood sensitivity (98) but failed to discriminate betweenESBL and non-ESBL TEM and SHV beta-lactamases or tospecify CTX-M genes by group [88]
Current obstacles to a routine use of NGS technologiesin diagnostic microbiology and resistance testing comprisecosts and scarcely available user-friendly bioinformatics plat-forms [89] Nevertheless NGS technologies provide high-resolution genotyping in a short time frame of only two tofive days [89] Therefore NGSWGS in the microbiologicallaboratory will be the logical next step for the routinediagnosis of infection and the prediction of antimicrobialsusceptibility [90] potentially replacing traditional culturalapproaches on the intermediate or long term
4 Fluorescence In Situ Hybridization (FISH)for the Detection of Bacterial Resistance
FISH (fluorescence in situ hybridization) is a cheap andconvenient option for the identification and resistance testingof bacterial pathogens Traditional FISH is based on specifichybridization of short usually 18ndash25 bases long fluorescent-labelled single-stranded oligonucleotide probes to ribosomalRNA (rRNA) of the target organismwith subsequent analysisunder the fluorescence microscope usually allowing forthe identification of microbes at genus or species level Inprinciple each kind of intracellular RNA can be hybridizedwith FISH probes However rRNA is particularly well suitedas a FISH target because ribosomes are numerous in aprotein-synthesizing cell thus allowing for a boostering offluorescence intensity [91]
This traditional FISH method is both rapid and easy tostandardize so it can be applied for molecular rapid testingSmall modifications of the procedure comprise the use ofpatent-protected commercial peptide nucleic acid (PNA)probes or probes containing locked nucleic acids (LNA)instead of simple single-stranded DNA probes PNA-FISHtechnology reduces nonspecific probe attachment due tothe electrically neutral backbone of the oligonucleotides andis recommendable for routine diagnostics due to a higherdegree of standardization However patent-protected PNA
probes are expensive although they are well suited for thediagnostic routine setting [92]
FISH is particularly suitable for the detection of resistancedeterminants if two prerequisites are guaranteed Ribosoma-llymediated resistance for example affecting antibiotic drugslike macrolide or linezolid is well suited because riboso-mal RNA copies are numerous in living cells allowing forbright fluorescence signals Further FISH can be successfullyapplied if only one or few variable bases provide resistanceso there is no need for a large number of probes in the probepanel
These prerequisites are fulfilled in case of clarithromycinresistance testing in Helicobacter pylori Therefore FISH-based resistance testingwas early evaluated for this indication[93] Clarithromycin in H pylori is basically mediated bythree point mutations in the ribosomal 23S rRNA [94]which can be addressed by three described FISH probesClaR1 ClaR2 and ClaR3 [93] (Table 1) While ClaR1 isassociated with a minimum inhibitory concentration (MIC)of gt64mgL ClaR2 and ClaR3 are associated with varyingMICs between 8mgL and 64mgL [94]
The FISH probes for clarithromycin resistance testing inH pylori were successfully applied to bacteria both fromculture and in bioptic material and extensively assessed invarious studies [93 95ndash97] Reliable test results can even beachieved in formalin-fixed paraffin-embedded tissue afteradequate deparaffination [98] The combined use of probeslabelled with different fluorescence molecules allows for theidentification of coinfections with clarithromycin-sensitiveand -resistant H pylori strains by FISH [99]
Commercial test providers distributed the robust andeasy-to-apply procedure In one study with such a com-mercial test kit [100] a sensitivity of 90 and a specificityof 100 were achieved for the detection of clarithromycin-resistant H pylori within bioptic material In another studyoccasional false-positive H pylori detections were generated[101] although the results of FISH-based resistance test-ing of correctly identified H pylori proved to be reliableRecently a PNA probe-based approach for clarithromycinresistance testing in H pylori showed perfect matching withPCRsequencing in a retrospective studywith formalin-fixedparaffin-embedded tissues (Table 2) [102]
Similar to H pylori FISH-based clarithromycin resis-tance testing could be successfully demonstrated for ther-motolerant Campylobacter spp with a wild-type probe anda clarithromycin resistance probe targeting the A2059Gmutation in the 23S rRNA gene (Table 3) The observedsensitivity and specificity with culture material were 100[103]
Comparable to clarithromycin resistance linezolid resis-tance is ribosomally mediated In enterococci it is typicallycaused by a 2567GgtT base substitution in the 23S rRNA(Table 4) In a collection of 106 enterococcal isolates acorresponding linezolid resistance FISH assay succeeded inpredicting phenotypic resistance in 100 of cases [104]Even a single mutated allele was associated with strongfluorescence signals
First successful attempts of FISH-based resistance testingwere described for non-rRNA-based resistance mechanisms
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Table 1 DNA-FISH-probes detecting clarithromycin resistance in H pylori Russmann et al 2001a [93]
Target Probe Probe sequenceWild type ClaWT 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Table 2 PNA-FISH-probes detecting clarithromycin resistance in H pylori Cerqueira et al 2013 [102] shortened versions of the DNA-FISH-probes from Table 1
Target Probe Probe sequenceWild type HpWT 51015840-GGT-CTT-TCC-GTC-T-31015840
Table 3 DNA-FISH-probes detecting clarithromycin resistance in thermotolerantCampylobacter spp Haas et al 2008 [103] Of note probeC wt 23S is identical with probe ClaWT probe C res 23S 2059AgtG with probe ClaR2 (Table 1)
Target Probe Probe sequenceWild type C wt 23S 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Clarithromycin resistance mutation (A2059G) C res 23S 2059AgtG 51015840-CGG-GGT-CTC-TCC-GTC-TT-31015840
Table 4 DNA-FISH-probes detecting linezolid resistance in enterococci Locked nucleic acids (LNA) were used at the mismatch position(bold underlined print) within in probes
Target Probe Probe sequenceWild type LZD-WT 51015840-CCC-AGC-TCG-CGT-GC-31015840
Agglutination assays lt5 minutes mdash lt100C LowFluorescence in situ hybridization 1-2 hours lt1500000C 100ndash800C IntermediateReal-time PCR(including DNA preparation) 4ndash6 hours 3500000ndash6000000C 1500ndash2500C Strongly depending on
the test systemLoop-mediated isothermal amplification(LAMP) assays lt1 hour 200000ndash400000C 1500ndash2500C Intermediate
Next generation sequencing (NGS) 2ndash5 days 35000000ndash75000000C 7500ndash80000C Very highMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry(MALDI-TOF-MS)
lt5 minutes 7500000ndash30000000C lt100C High
as well FISH-based detection of blaSHV-238240 one of thegenes coding for extended-spectrum 120573-lactamases (ESBL) isan example of a non-rRNA-based FISHprotocol for detectinga particular resistance determinant using the probe 51015840-GAC-CGG-AGC-TAG-CAA-GCG-31015840 [105] However the ESBLphenotype can be associated with a variety of different allelesso this particular probe will be of use only in case of a specificsuspicion for example during an outbreakAccordingly such
a procedure will be reserved for very few if any indications inthe diagnostic routine
Further progression of FISH technology comprisessignal-amplified catalyzed reported deposition (CARD)FISH doubly labeled oligonucleotide probe- (DOPE-) basedFISH combinatorial labelling and spectral imaging (CLASI)FISH and the combination of FISH with other diagnosticapproaches aswell as FISHprocedures for gene identification
8 BioMed Research International
requiring in situ amplification of the respective gene as incase of the rolling circle amplification (RCA) FISH [106]RCA-FISH was successfully applied for the identification ofthe mecA gene in Methicillin resistant Staphylococcus aureus(MRSA) based on the mecA-probes MR-1 51015840-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-31015840 andMR-2 51015840-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-31015840 [107]
FISH-based detection of resistance determinants is apromising diagnostic approach due to its rapidity conve-nience and cost effectiveness The associated rapid detectionof antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associ-ated benefits for the patientrsquos health The main advantage ofFISH is its potential use for resistance testing directly fromprimarymaterial including tissuewith low effort So FISHcanalso be applied in resource-limited settings where expensivetechnologies are not available (Figure 1) In contrast to PCRFISH can also attribute a particular resistance mechanism toa microscopically observed bacterium
However so far FISH is restricted to very few indicationsfor which protocols have been described As a furtherdrawback standardization of FISH-based resistance testing iswidely missing If applied from primary samplematerials liketissue tissue autofluorescence has to be considered requiringconsiderable experience to interpret such diagnostic resultsTo reduce potential interpretation errors FISH from tissuefurther requires counterstaining with a pan-eubacterial FISHprobe and nonspecificDNA staining for example withDAPI(410158406-diamidino-2-phenylindole) to confirm the presence ofnucleic acids of the detected pathogens as recently demanded[108]
Given all these limitations FISH for resistance testingwillpresumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market
5 Direct Fluorescent Imaging ofResistance Determinants by FluorescenceResonance Energy Transfer (FRET)
Nonnucleotide probes labelled with reporter and quenchermolecules allowing for fluorescence energy transfer (FRET)can be used to detect enzymatic resistance mechanisms asdescribed for 120573-lactamases [109] After enzymatic hydrolyza-tion of probes to separate the quencher from the reporter thehydrolyzed probes attach the resistance enzymes as reactiveelectrophiles However this mechanism has so far been onlydescribed for 120573-lactamases in a proof-of-principle analysis[109] and broad evaluation studies are missing Its practicalrelevance for the microbiological routine diagnostics willrequire further evaluation
6 Mass Spectrometric Approaches
Matrix-assisted laser desorption ionization time-of-flightmass spectrometry- (MALDI-TOF MS-) based intact cellmass spectrometry (ICMS) has recently advanced to the stan-dard method for species identification for cultured bacteria
and fungi [24 110ndash114] Promising approaches have beenmade using ICMS spectra for subspecies identification [115]This technique bears a high potential for the fast identi-fication of susceptibility associated biomarker ions that islately only marginally realized in clinical routine diagnosticsThus phyloproteomic approaches help to identify indirectlymostly chromosomal encoded resistance genes by identifyingphylogenetic relatedness [116ndash121] MS can be used to detectchanges in the bacterial or fungal proteome induced byexposition to antimicrobials [24 122ndash124] Whole proteomechanges in consequence of exposition to antimicrobials canbe also detected using stable isotope labeled amino acids(SILAC) [125 126] One very promising approach is the so-calledmass spectrometric beta-lactamase (MSBL) assay [127ndash131] which is based on the mass spectrometric detection ofhydrolyzed beta-lactams Finally there is the combination ofgenotypic and mass spectrometric methods PCR ampliconscan be characterized by PCRelectrospray ionization-massspectrometry (PCRESI MS) [132] and minisequencing [133134] and mass spectrometry-based comparative sequenceanalysis [135 136] can be used to detect susceptibility changesassociated with point mutations
61 Prediction of Broad Spectrum Resistant Clonal Groupsby Phyloproteomics MALDI-TOFMS-based intact cell massspectrometry (ICMS) is potentially able to characterizestrains at the subspecies level and could act as useful toolfor taxonomy and epidemiology [137 138] For the discrim-ination of representative strains particular biomarker ionsthat were completely present or absent as well as shiftsin biomarker masses in a particular subset of strains wereconsidered Using different mathematical algorithms it wasfor example feasible to discriminate Salmonella enterica sspenterica serovar Typhi from other less virulent Salmonellaenterica ssp enterica serotypes [139] to distinguish Campy-lobacter jejuni MLST-ST22 and ST45 from other MLSTsequence types [140] or to perform phyloproteomic analysisof Rhodococcus erythropolis [141] Pseudomonas putida [142]or Neisseria menigitidis [143]
Thefirst approaches to associateMSfingerprintswith sus-ceptibility patterns were designed to differentiate methicillinsusceptible Staphylococcus aureus (MSSA) from methicillinresistant Staphylococcus aureus (MRSA) [144ndash148] Thesewere mostly not standardized and hardly reproducible Butrelatively good reproducibility was demonstrated for thediscrimination of the five major MRSA clonal complexesCC5 CC8 CC22 CC30 and CC45 corresponding to thefive major PFGE MRSA types regardless of their methicillinsensitivity [149 150] A study by Lu and coworkers identifieda set of biomarkers that were able to distinguish betweenmethicillin resistant and vancomycin-intermediate S aureus(VISA) strains and vancomycin-susceptible S aureus strainsas well as between SCCmec types IV and V isolates andSCCmec types IndashIII isolates [151] Further studies demon-strated that isogenic S aureus lacking or artificially harboringSCCmec could not be distinguished in a mass range from2000 to 15000119898119911 [152] whereas isogenic MRSA whichspontaneously reverted to MSSA could be discriminated byMALDI-TOF MS [153]
BioMed Research International 9
(a)
(b)
(c)
(d)
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
[1] WHO Antimicrobial Resistance Global Report on SurveillanceWHO 2014
[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
[20] WHO Global Tuberculosis Report 2013 World Health Organi-zation Geneva Switzerland 2013
[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
BioMed Research International 15
[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
lasting persistence of MRE as demonstrated by a Swedishand a French study contributes to the increase in the MREprevalence sometimes even years after infection [11 12] Amedian MRE-colonization period of 125 months could bedetected in a cohort of newborn children in Norway [13] AnEnglish studywas able to verify persistence of resistance geneseven in the absence of antibiotic pressure [14] Furthermorethe colonization rate also differs between healthy subjects andpatients at risk It could be demonstrated in aKorean endemicarea that 203 of healthy individuals were colonized withESBL producers while high-risk patients were colonized in425 of cases [15] However the risk of faecal colonizationdepends mainly on the local prevalence For example anESBL prevalence of 657 has been demonstrated in healthyadults inThailand [16] while another study showed an ESBLprevalence of 113 in outpatients in England [17] Farmanimals are another reservoir for multidrug-resistant bacte-ria A survey in the Netherlands demonstrated that chickensare colonized with ESBL-producing Enterobacteriaceae tomore than 70 while swine and cattle are known reservoirsfor livestock-associated methicillin resistant Staphylococcusaureus (laMRSA) [18]
The multiresistances of the Gram-negative bacteria rep-resent a major challenge for the traditional culture-basedmicrobiology Furthermore the limited treatment optionsfor a calculated therapy and therewith the risk of an inap-propriate therapy are an intensifying factor of this problem[19] As a consequence morbidity and mortality of outpa-tient and nosocomial-acquired infections with multidrug-resistant Gram-negative bacteria are significantly increasedSimilarly Mycobacterium tuberculosis has posed a serioushealth threat as a result of multidrug resistance In its2013 global report on tuberculosis WHO estimates that36 (95 confidence interval 21ndash52) of new cases and202 (95 confidence interval 133ndash272) of previouslytreated cases had multidrug-resistant (MDR) tuberculosis(defined as tuberculosis caused by M tuberculosis isolatesthat are resistant to rifampicin and isoniazid) and 13 millionTB deaths [20] On the other hand CMV resistance hasbeen reported to be on the rise in transplant recipients[21 22]
Information on antimicrobial susceptibility aids a clini-cian in prescribing an appropriate antimicrobial drug for aparticular infection Due to the rapid rise in antimicrobialresistance worldwide [1] it is becoming increasingly impor-tant for a clinician to rapidly receive information on theantimicrobial susceptibility profile of the isolated pathogenfor appropriate treatment to be initiated Traditionally clin-ical microbiology laboratories have relied on phenotypicmethods to determine the antibiotic susceptibility profilesof pathogens [23] These methods remain useful and haveadvantages such as low costs as well as being easy to performand having established interpretation criteria But they lackthe ability to generate timely susceptibility results hencedelaying initiation of treatment [24] Furthermore currentlythere is a need to establish adequate and standardized screen-ing and isolation procedures for carbapenemase-producingbacteria especially in risk patients as well as in patients
in which MRE colonizationinfection has been previouslyshown These limitations have been found to have conse-quences in patient management for example delay in theinitiation of antibacterial treatment has led to increases inmortality [25] as well as in hospitalization time [26] andmake it challenging to implement the back-end approachof the antimicrobial stewardship program which has shownrewarding results in patientmanagement and the fight againstantimicrobial resistance [27]
In response to the limitations of phenotypic methodsand the desires to improve patient management and curbthe spread of antimicrobial resistance rapid antimicrobialsusceptibility testing methods are continuously developedThese methods have been found to identify a pathogenand its antimicrobial susceptibility profile within a shortperiod of time There are basically five different ways toaccelerate susceptibility testing in clinical diagnostics (I)bypassing conventional culture by direct detection of thepathogen or resistance mechanism in the primary sample(II) bypassing plate or broth culture dependent susceptibilitytesting (secondary culture) (III) avoiding time consumingwork stepsmethods (IV) increasing the sensitivity to thedetection of the infectious agent that means detecting theinfectious agent in earlier disease stages at lower viral ormicrobial loads and (V) earlier detection of an evolvingdrug resistance during treatment in spreading less susceptiblequasispecies
For example real-time quantitative PCR (qPCR) hasmade it possible to detect multidrug-resistant tuberculosis(MDR TB) in a sample within an hour hence immediatelyinitiating appropriate treatment and control measures [28]Also MALDI-TOF mass spectrometry (MS) has made itpossible to detect the most pathogens in a sample withinminutes with high sensitivity and specificity [29] In additionthese methods have made it possible to control the spreadof resistant strains reduce the length of patient stay inhospitals and enhance the implementation of antimicrobialstewardship programs
In this review we detail the rapid antimicrobial suscep-tibility testing methods that have been developed recentlyThey include classical agglutination assays molecular testingmethods for example qPCR DNA microarrays LuminexxMAP assays and next generation sequencing fluores-cence in situ hybridization (FISH) and mass spectrometry-based methods for example phyloproteomics assays usingstable isotope labeling of amino acids mass spectro-metric beta-lactamase assays PCRelectrospray ionization-mass spectrometry (PCRESI MS) minisequencing andmass spectrometry-based comparative sequence analysis(MSCSA) In addition we discuss the impact that these tech-niques are likely to bring for the patient management and thereduction of antimicrobial resistance
2 Agglutination Assays as RapidCulture-Associated Options
Agglutination assays are based on a suspension of micropar-ticles coated with specific antibodies leading to agglutination
BioMed Research International 3
in contact with their specific antigens Such proceduresare useful for a preliminary resistance screening from purebacterial colonies if the resistance mechanism of interest isassociated with a single antigen only which is expressedon the surface of the pathogen Accordingly agglutinationassays are unfeasible for the screening for complex resistancepatterns which may be associated with multiple structurallydifferent families of enzymes as in the case of extended-spectrum beta-lactamases (ESBL) or carbapenemases inGram-negative rod-shaped bacteria
Agglutination assays for the rapid identification of bacte-rial resistance patterns are widely restricted to the identifica-tion of the penicillin binding protein 2a (PBP-2a) the majorresistance determinant ofMethicillin resistant Staphylococcusaureus (MRSA) Different agglutination kits show specifici-ties of 913 to 100 if applied to MRSA colony material[30ndash32] The sensitivity is even more restricted rangingbetween 827 and 941 [30ndash32] If sufficient quantities ofcolony material are used agglutination testing allows for theidentification of small-colony variant MRSA strains as well[33]
The lack of sensitivity seems to be associated with certainstaphylococcal cassette chromosome (SCC-mecA) types withtype IV scoring particularly poor [31] Furthermore aggluti-nation kits are only positive if methicillin resistance is due tothe mecA gene If mecC a divergent mecA homologue is thecause of the resistance agglutination usually fails as observedfor 10 out of 10 mecC-positive live-stock associated MRSAstrains [34]
Of note agglutination based PBP-2a testing is possiblefrom liquid sample materials as well However the sensitivityis poor From blood culture pellets PBP-2a agglutinationshowed sensitivity of only 18 in a recent study In contrastspecificity was excellent with 100 [35]
The usage of genotypic methods in the rapid detectionof antimicrobial resistance genes is gradually shifting fromacademic research laboratories to diagnostic laboratories andpoint-of-care testing The attractiveness of these methods indetermination of antimicrobial resistance has been attributedto two factors firstly their capability to generate resultswithin a short time as compared to phenotypic methods sec-ondly their capability to detect antimicrobial genes directlyfrom the patient sample without necessarily waiting forculture results [36]These two attributes aid clinicians in pre-scribing appropriate treatment to patients at the opportunetime hence making a positive contribution to antimicrobialstewardship programs [27] However genotypic tools for thedetection of antimicrobial resistance may generate false neg-ative results due to (i) their inability to detect new resistancemechanisms or (ii) false-positive results because they maydetect inactive or incomplete resistance genes in a specimenwhich have not inferred resistance to the antimicrobial drugunder test [37]
Current genotypic methods that are used for the rapiddetection of antimicrobial resistance genes include (i) nucleicacid amplification methods particularly real-time quantita-tive PCR (qPCR) (ii) DNA hybridization based methodsparticularly DNAmicroarrays (iii) Luminex xMAP technol-ogy and (iv) next generation sequencingmethods Below is abrief description on the application of each of thesemolecularmethods for the rapid detection of antimicrobial resistance
31 Nucleic Acid Amplification Methods Recently one of thePCR techniques that has received a wide application in clin-ical microbiology is the quantitative real-time PCR (qPCR)technique [38] This has been attributed to its flexibility andcapability to rapidly and simultaneously identify multiplepathogens in a clinical specimen and the presence of antimi-crobial resistance genes in the identified pathogens [39] Asa result numerous qPCR assays for rapid identification ofpathogens in clinical specimens have been developed butmost of the available qPCR assays for detection of microbialresistance genes are limited to the detection of antibioticresistance In short most of the available commercial qPCRassays detect the presence of mecA and mecC which confermethicillin resistance in S aureus the vanA and vanB geneswhich confer glycopeptide resistance and genes that encodeextended-spectrum 120573-lactamases (for detailed review oneach assay see Maurin 2012 [39]) One outstanding featureof all these qPCR assays is their capability to simultaneouslyand accurately detect resistance genes within a remarkablyshorter time period of 4ndash6 hours Similarly qPCR assaysfor rapid detection of resistance against rifampin (RIF) andisoniazid (INH) have been introduced Ramirez and cowork-ers have recently combined qPCR and high-resolution melt(HRM) technology to develop an assay which rapidly andsimultaneously identifiesmultidrug-resistantM tuberculosismutations in the rpoB gene conferring resistance to RIF andmutations in the katG and inhA genes conferring resistanceto INH [28] This assay produces results within 6 hours ascompared to GenoType MTBDRplus assay (Hain LifescienceGmbH Germany) and culture susceptibility testing whichtake 8 hours and 56 days to generate results In the recenttime several in-house qPCR assays for rapid and simulta-neous detection of genes encoding Klebsiella pneumoniaecarbapenemase (blaKPC) andNewDelhi metallo-120573-lactamase(blaNDM) inGram-negative rod-shaped bacteria [40ndash43] havebeen introduced Similarly several in-house qPCR assays forrapid and simultaneous detection of blaOXA-48 blaVIM andblaIMP carbapenemase genes in Enterobacteriaceae have beenestablished [44ndash46]
PCR-based MRSA testing has found wide applicationsin microbiological routine laboratories Next to in-houseassays commercially available molecular MRSA testing plat-forms comprise for example BD GeneOhm MRSA (BectonDickinson Heidelberg Germany) GT MRSA DirectGQMRSA (Hain Lifescience Nehren Germany) Hyplex Staphy-loResist (Amplex Gieszligen Germany) LightCycler (RocheDiagnostics Ltd Rotkreuz Switzerland) kits like LC MRSAAdvanced CepheidXpertGeneExpert (Cepheid SunnyvaleCA USA) and TIB Molbiol LightMix MRSA (TIB Molbiol
4 BioMed Research International
Berlin Germany) All test systems showed reliable results ina recent external laboratory control evaluation in Germany[47] Similarly commercial PCR assays for the detection ofESBL-associated blaCTX-M beta-lactamases and only partiallyESBL-associated blaTEM and blaSHV as well as OXA1-typecarbapenemases (the latter combined in a consensus run)were introduced (Amplex Gieszligen Germany) [48] Similarmultiplex PCR systems are available for the most frequentlydetected carbapenemases which are particularly useful forthe follow-up during hospital outbreak events (AmplexGieszligen Germany) even from primary sample materials [4950]The switch ofmolecular carbanemase detection to robustloop-mediated isothermal amplification (LAMP) [46] allowsfor commercial point-of-care testing (POCT) compatible testsolutions for bedside testing for example the eazyplex Super-BugCRE system (Amplex Gieszligen Germany) which providesresults within 10 minutes However the great number ofdifferent possible cephalosporin and carbapenem resistancemechanisms finally exceeds any multiplexing capacity ifcompleteness is aspired
Nevertheless in addition to rapid and simultaneous pro-viding of reliable results qPCR has been found to be afford-able sensitive specific user friendly not space demandingand deliverable [37ndash39 51] Due to these attributes qPCR hasfound various applications in point-of-care testing (POCT)For example the Xpert MTBRIF test (Cepheid SunnyvaleCA USA) is a qPCR-based assay that has been developedto rapidly and simultaneously detect M tuberculosis andrifampicin (USAN rifampin) resistance To evaluate itsusefulness in POCT a large multicentre study involving6069 cases from six unrelated sites was performed In thisstudy Xpert MTBRIF detected rifampicin resistance casesin 1 hour as compared to line-probe assay and phenotypicdrug susceptibility testing that detected the same cases in 20days and 106 days respectively [52] As mentioned above asimilarGenXpert-basedPOCT test forMRSA screening fromclinical sample materials is available as well
Multiplex PCR assays have also been developed to rapidlyand simultaneously identify multiple pathogens in clinicalspecimens as well as the presence of antimicrobial resistancegenes in the identified pathogens Strommenger and cowork-ers developed amultiplex PCR which simultaneously detects9 resistance genes in S aureus directly from clinical speci-men within 6 hours [53] These 9 resistance genes includemecA (methicillin resistance) aacA-aphD (aminoglycosideresistance) tetK tetM (tetracycline resistance) ermA ermC(macrolide-lincosamide-streptogramin B resistance) vatAvatB and vatC (streptogramin A resistance) [53]
Like qPCR multiplex PCR assays were used as POC teststo facilitate patient management One example is the mul-tiplex PCR-based Unyvero Pneumonia Application (UPA)assay (Curetis AG Holzgerlingen Germany) that has beendeveloped to rapidly and simultaneously detect 18 bacterialspeciesPneumocystis jirovecii and 22 resistancemarkers fromrespiratory specimens (httpwwwcuretiscom) In one ofthe studies showing its suitability for POC testing the UPAassay detected multiple antibiotic resistances within 1 hour(as compared to phenotypic methods that took 96 hours)in a group of 56 hospitalized patients with respiratory tract
infections whowere under treatmentThis finding influencedthe modification of treatment in fifteen patients with severepneumonia leading to their recovery [54] The UPA assayis of course not able to replace conventional testing due toits design because it is not able to detect further microbialspecies and resistance mechanisms besides the implementedones
The continuous development of PCR-based assays withthe capability to rapidly and simultaneously detect pathogensand presence of resistance genes in specimens coupledwith their application in POCT may further improve themanagement of patients as long as appropriate quality controlis ensured
32 DNAMicroarray Technology The biggest challenge asso-ciated with the unprecedented rise of antimicrobial drugresistance worldwide is the scarce availability of assays thatare able to rapidly and simultaneously identify a causativepathogen and generate its antimicrobial resistance profileRecent oligonucleotide-based DNA microarrays match thischallenge In a recent study Zhang and coworkers describedthat CapitalBio DNAmicroarray (CapitalBio Corp) could ina mean time of 58 hours simultaneously identify Mycobac-terium species and detect mutations that confer isoniazid(INH) and rifampicin (RMP) resistance in specimens col-lected from spinal tuberculosis patients as compared toconventional culture and drug susceptibility testing whichtook a mean time of 568 days [55] Briefly oligonucleotideprobes which had been designed to identify Mycobacteriumspecies based on 16S rRNA sequences andmutations of rpoBinhA and katG that confer INH and RMP resistance werecovalently linked to the surface of aldehyde-activated slidesDNAwas extracted from specimens PCRwas used to amplifythe resistance genes and amplicons hybridized on the slidesThe emitted fluorescent signals were analyzed Guo andcoworkers evaluated the ability of a biochip which is basedon the same principle to rapidly and simultaneously identifymultidrug-resistant M tuberculosis (MRTB) and mutationsof rpoB inhA and katG that confer INH and RMP resistancein clinical sputum specimens [56] This group found thatthe biochip could in a mean time of 6 hours simultaneouslyidentifyM tuberculosis and detectmutations that confer INHand RMP resistance
Recent reports have also reported the availability ofCheck-Pointrsquos ESBLKPC DNA microarray for the identi-fication and detection of extended-spectrum 120573-lactamases(ESBLs) and Klebsiella pneumoniae carbapenemases (KPCcarbapenemases) [57 58] This array uses a methodologyknown as multiplex ligation detection to identify ESBL-associated or at least partially ESBL-associated genes (blaTEMblaSHV and blaCTX-M) and blaKPC genes (for details see [57])In a study to evaluate the rapidness at which this arraycould identify and detect these genes Naas and coworkersfound that Check-Pointrsquos ESBLKPC DNA microarray couldidentify them in 7-8 hours as compared to conventionalsusceptibility testing that took a mean time of 54 hoursSimilar results were observed by Willemsen and coworkersin a study that was aimed at evaluating the rapidness at
BioMed Research International 5
which this array could identify and detect these ESBLKPCgenes in hospitals in the Netherlands [58] In addition todetecting and identifying ESBLKPC resistance in gastroin-testinal tract infections caused by Enterobacteriaceae Check-Pointrsquos ESBLKPC DNA microarray has also been usedto detect and identify KPC resistance in hospital-acquiredpneumonia caused by Klebsiella pneumoniae [59] Based onthese experiences the Check-MDR CT 102 DNA microarrayfor the detection of the most prevalent carbapenemase genes(blaNDM blaVIM blaKPC blaOXA-48 and blaIMP) and extended-spectrum120573-lactamase- (ESBL-) related gene families (blaSHVblaTEM and blaCTX-M) has been developed The evaluation ofthe rapidness of the Check-MDR CT 102 DNA microarrayto detect these genes has shown that it yields results 5 hoursfaster than Check-Pointrsquos ESBLKPC DNAmicroarray [60]
At present the DNA microarray technology is mostlyused in the routine detection of antimicrobial resistance ofTB and HIV [61ndash66] The routine use of systems such asMVPlex (Genaco Biomedical Products Huntsville USA) andStaphPlex systems (Genaco Biomedical Products HuntsvilleUSA) which combine both qPCR and DNA microarraytechnology suggest that independent DNA microarray tech-nology might find further applications in the routine clinicalmicrobiology [67 68] The MVPlex system detects the nucmecA (SCCmec)-orfX vanA vanB ddl and tuf genes toscreen for MRSA in nasal swabs [69] and the relatedStaphPlex system performs simultaneous species-level iden-tification (nuc versus tuf ) and detection of mecA aacAermA ermC tetM and tetK as well as Panton-Valentineleukocidin (PVL) for the rapid detection and characterizationof staphylococci directly from positive blood culture bottles[70]
33 Luminex xMAP Technology The description of cooc-curring single nucleotide polymorphism (SNP) mutations inantimicrobial resistance associated genes allows for targetedresistance testing For example unequivocally genetic studieshave proven that there are 5 different mutations in quinoloneresistance-determining region (QRDR) of gyrA gyrB andparE within Salmonella typhi [71] Similar studies havealso shown distinct mutations in the quinolone resistance-determining region (QRDR) of gyrA within Campylobacterjejuni and Campylobacter coli [72]
Rapid simultaneous detection of cooccurring singlenucleotide polymorphism (SNP) mutations in antimicrobialresistance associated genes remains however challengingMost molecular assays such as qPCR and pyrosequencinglack the capability to simultaneously detect cooccurringsingle nucleotide polymorphism (SNP)mutations in differentgenes in a given specimen [73] However this challengehas been overcome by Luminex xMAP Technology a multi-plexing technology which allows for simultaneous detectionof multiple nucleic acid sequences in a single reaction[74] During operation microtiter plates are loaded withmicrospheres that is coated and color-coded beads Themicrospheres are mixed with purified nucleic acids of the testorganism and allowed to hybridize emitting monochromaticlight which the Luminex analyzer reads and interprets At
present this technology has been used to simultaneouslydetect 11 mutations in gyrA gyrB and parE of SalmonellaTyphi and Salmonella Paratyphi A [75] Further it has beenused to simultaneously detect mutations in gyrA of C jejuniandC coli [76] In comparison to sequencing andmicroarraytechnology Luminex xMAPTechnology has been found to beflexible rapid and cost effective [74ndash76]
34 Next Generation Sequencing (NGS) Near whole genomesequencing (WGS) or next generation sequencing (NGS)allows for the assessment of bacterial genomes within severalhours A variety of different technological solutions havebeen introduced including laser printer sized benchtopdevices like 454GS Junior (Roche Basel Switzerland)MiSeq(Illumina San Diego CA USA) and Ion Torrent PGM (LifeTechnologies Grand Island NYUSA) In a previous analysisthe MiSeq (Illumina) system scored best regarding boththroughput per run and error rates while both the 454 GSJunior (Roche) and the Ion Torrent PGM (Life Technologies)systems were prone to homopolymer-associated indel errors[77]
Result interpretation of whole bacterial genomes is basedon either allelic comparisons [78] or single nucleotide poly-morphism (SNP) analysis [79] Data assessment and inter-pretation can be facilitated by commercial software packageslike SeqSphere+ (Ridom BIOINFORMATICS Ltd MunsterGermany) or BioNumerics (Applied Maths Sint-Martens-Latem Belgium)
NGS allows for resistance identification by the presenceof the underlying mechanism rather than just in pharma-codynamic terms [80] so it may revolutionize microbialresistance testing on the long termThis comprises the identi-fication and characterization of resistance genes encoding forextended-spectrum 120573-lactamases (eg 119887119897119886CTX-M 119887119897119886TEM and119887119897119886SHV) plasmid-mediated AmpCs (eg 119887119897119886CMY) quinoloneresistance (eg mutations in gyrA parC or qnr elements)aminoglycoside resistance (eg aminoglycosides modifyingenzymes 16S rRNA methylases) or carbapenemases (eg119887119897119886KPC 119887119897119886NDM) [81]
NGS-based resistance testing is of particular interest forslowly growing infectious agents with atypical resistancepatterns like multidrug-resistant (MDR) or extensive-drugresistance (XDR) M tuberculosis for which rapid identi-fication or exclusion of resistance determinants is of highrelevance for the therapeutic approach Ion Torrent full-gene sequencing with consecutive complete genetic analysiswithin 5 days (Table 5) allowed for reliable resistance detec-tion in M tuberculosis isolates of Burmese Hmong andIndian immigrants in the USA [82] Similar WGS data weredescribed for drug-resistant strains from Russia harbouringalmost all known drug-resistance associated mutations [83]In a direct comparison of Ion Torrent sequencing withphenotypic Bactec MGIT 960 (Becton Dickinson FranklinLakes NJ USA) analysis and genotypicHain line-probe assay(LPA) (Hain Lifescience Ltd Nehren Germany) there wascomplete concordance of NGS to phenotypic resistance andgenotypic rpoB and katG results for the analyzed M tuber-culosis isolates Even more Ion Torrent sequencing detected
6 BioMed Research International
uncommon substitutions and previously uncharacterizedresistance mutations in rpoB rrs and pncA [84] FurtherNGS is able to discriminate mixed mycobacterial genotypesin patient isolates based on single nucleotide variations(SNVs) [85] So it might be suitable to identify resistancemutations in genotypes that occur inminor proportions only
HoweverNGS-based resistance testing is not restricted tomycobacteria Recently NGSwas used to identify transmissi-ble plasmids in multidrug-resistant E coli isolates expressingan ESBL phenotype and transferring their cefotaxime resis-tance marker at high frequency in laboratory conjugationexperiments [86] High-throughput sequencing successfullyproved to be a valuable tool for tracing resistance plasmidsin the course of outbreaks as well [87] However a commer-cial NGS assay (Hospital Acquired Infection BioDetectionSystem Pathogenica Boston MA USA) for investigationsof outbreaks with ESBL-positive Enterobacteriaceae showedgood sensitivity (98) but failed to discriminate betweenESBL and non-ESBL TEM and SHV beta-lactamases or tospecify CTX-M genes by group [88]
Current obstacles to a routine use of NGS technologiesin diagnostic microbiology and resistance testing comprisecosts and scarcely available user-friendly bioinformatics plat-forms [89] Nevertheless NGS technologies provide high-resolution genotyping in a short time frame of only two tofive days [89] Therefore NGSWGS in the microbiologicallaboratory will be the logical next step for the routinediagnosis of infection and the prediction of antimicrobialsusceptibility [90] potentially replacing traditional culturalapproaches on the intermediate or long term
4 Fluorescence In Situ Hybridization (FISH)for the Detection of Bacterial Resistance
FISH (fluorescence in situ hybridization) is a cheap andconvenient option for the identification and resistance testingof bacterial pathogens Traditional FISH is based on specifichybridization of short usually 18ndash25 bases long fluorescent-labelled single-stranded oligonucleotide probes to ribosomalRNA (rRNA) of the target organismwith subsequent analysisunder the fluorescence microscope usually allowing forthe identification of microbes at genus or species level Inprinciple each kind of intracellular RNA can be hybridizedwith FISH probes However rRNA is particularly well suitedas a FISH target because ribosomes are numerous in aprotein-synthesizing cell thus allowing for a boostering offluorescence intensity [91]
This traditional FISH method is both rapid and easy tostandardize so it can be applied for molecular rapid testingSmall modifications of the procedure comprise the use ofpatent-protected commercial peptide nucleic acid (PNA)probes or probes containing locked nucleic acids (LNA)instead of simple single-stranded DNA probes PNA-FISHtechnology reduces nonspecific probe attachment due tothe electrically neutral backbone of the oligonucleotides andis recommendable for routine diagnostics due to a higherdegree of standardization However patent-protected PNA
probes are expensive although they are well suited for thediagnostic routine setting [92]
FISH is particularly suitable for the detection of resistancedeterminants if two prerequisites are guaranteed Ribosoma-llymediated resistance for example affecting antibiotic drugslike macrolide or linezolid is well suited because riboso-mal RNA copies are numerous in living cells allowing forbright fluorescence signals Further FISH can be successfullyapplied if only one or few variable bases provide resistanceso there is no need for a large number of probes in the probepanel
These prerequisites are fulfilled in case of clarithromycinresistance testing in Helicobacter pylori Therefore FISH-based resistance testingwas early evaluated for this indication[93] Clarithromycin in H pylori is basically mediated bythree point mutations in the ribosomal 23S rRNA [94]which can be addressed by three described FISH probesClaR1 ClaR2 and ClaR3 [93] (Table 1) While ClaR1 isassociated with a minimum inhibitory concentration (MIC)of gt64mgL ClaR2 and ClaR3 are associated with varyingMICs between 8mgL and 64mgL [94]
The FISH probes for clarithromycin resistance testing inH pylori were successfully applied to bacteria both fromculture and in bioptic material and extensively assessed invarious studies [93 95ndash97] Reliable test results can even beachieved in formalin-fixed paraffin-embedded tissue afteradequate deparaffination [98] The combined use of probeslabelled with different fluorescence molecules allows for theidentification of coinfections with clarithromycin-sensitiveand -resistant H pylori strains by FISH [99]
Commercial test providers distributed the robust andeasy-to-apply procedure In one study with such a com-mercial test kit [100] a sensitivity of 90 and a specificityof 100 were achieved for the detection of clarithromycin-resistant H pylori within bioptic material In another studyoccasional false-positive H pylori detections were generated[101] although the results of FISH-based resistance test-ing of correctly identified H pylori proved to be reliableRecently a PNA probe-based approach for clarithromycinresistance testing in H pylori showed perfect matching withPCRsequencing in a retrospective studywith formalin-fixedparaffin-embedded tissues (Table 2) [102]
Similar to H pylori FISH-based clarithromycin resis-tance testing could be successfully demonstrated for ther-motolerant Campylobacter spp with a wild-type probe anda clarithromycin resistance probe targeting the A2059Gmutation in the 23S rRNA gene (Table 3) The observedsensitivity and specificity with culture material were 100[103]
Comparable to clarithromycin resistance linezolid resis-tance is ribosomally mediated In enterococci it is typicallycaused by a 2567GgtT base substitution in the 23S rRNA(Table 4) In a collection of 106 enterococcal isolates acorresponding linezolid resistance FISH assay succeeded inpredicting phenotypic resistance in 100 of cases [104]Even a single mutated allele was associated with strongfluorescence signals
First successful attempts of FISH-based resistance testingwere described for non-rRNA-based resistance mechanisms
BioMed Research International 7
Table 1 DNA-FISH-probes detecting clarithromycin resistance in H pylori Russmann et al 2001a [93]
Target Probe Probe sequenceWild type ClaWT 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Table 2 PNA-FISH-probes detecting clarithromycin resistance in H pylori Cerqueira et al 2013 [102] shortened versions of the DNA-FISH-probes from Table 1
Target Probe Probe sequenceWild type HpWT 51015840-GGT-CTT-TCC-GTC-T-31015840
Table 3 DNA-FISH-probes detecting clarithromycin resistance in thermotolerantCampylobacter spp Haas et al 2008 [103] Of note probeC wt 23S is identical with probe ClaWT probe C res 23S 2059AgtG with probe ClaR2 (Table 1)
Target Probe Probe sequenceWild type C wt 23S 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Clarithromycin resistance mutation (A2059G) C res 23S 2059AgtG 51015840-CGG-GGT-CTC-TCC-GTC-TT-31015840
Table 4 DNA-FISH-probes detecting linezolid resistance in enterococci Locked nucleic acids (LNA) were used at the mismatch position(bold underlined print) within in probes
Target Probe Probe sequenceWild type LZD-WT 51015840-CCC-AGC-TCG-CGT-GC-31015840
Agglutination assays lt5 minutes mdash lt100C LowFluorescence in situ hybridization 1-2 hours lt1500000C 100ndash800C IntermediateReal-time PCR(including DNA preparation) 4ndash6 hours 3500000ndash6000000C 1500ndash2500C Strongly depending on
the test systemLoop-mediated isothermal amplification(LAMP) assays lt1 hour 200000ndash400000C 1500ndash2500C Intermediate
Next generation sequencing (NGS) 2ndash5 days 35000000ndash75000000C 7500ndash80000C Very highMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry(MALDI-TOF-MS)
lt5 minutes 7500000ndash30000000C lt100C High
as well FISH-based detection of blaSHV-238240 one of thegenes coding for extended-spectrum 120573-lactamases (ESBL) isan example of a non-rRNA-based FISHprotocol for detectinga particular resistance determinant using the probe 51015840-GAC-CGG-AGC-TAG-CAA-GCG-31015840 [105] However the ESBLphenotype can be associated with a variety of different allelesso this particular probe will be of use only in case of a specificsuspicion for example during an outbreakAccordingly such
a procedure will be reserved for very few if any indications inthe diagnostic routine
Further progression of FISH technology comprisessignal-amplified catalyzed reported deposition (CARD)FISH doubly labeled oligonucleotide probe- (DOPE-) basedFISH combinatorial labelling and spectral imaging (CLASI)FISH and the combination of FISH with other diagnosticapproaches aswell as FISHprocedures for gene identification
8 BioMed Research International
requiring in situ amplification of the respective gene as incase of the rolling circle amplification (RCA) FISH [106]RCA-FISH was successfully applied for the identification ofthe mecA gene in Methicillin resistant Staphylococcus aureus(MRSA) based on the mecA-probes MR-1 51015840-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-31015840 andMR-2 51015840-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-31015840 [107]
FISH-based detection of resistance determinants is apromising diagnostic approach due to its rapidity conve-nience and cost effectiveness The associated rapid detectionof antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associ-ated benefits for the patientrsquos health The main advantage ofFISH is its potential use for resistance testing directly fromprimarymaterial including tissuewith low effort So FISHcanalso be applied in resource-limited settings where expensivetechnologies are not available (Figure 1) In contrast to PCRFISH can also attribute a particular resistance mechanism toa microscopically observed bacterium
However so far FISH is restricted to very few indicationsfor which protocols have been described As a furtherdrawback standardization of FISH-based resistance testing iswidely missing If applied from primary samplematerials liketissue tissue autofluorescence has to be considered requiringconsiderable experience to interpret such diagnostic resultsTo reduce potential interpretation errors FISH from tissuefurther requires counterstaining with a pan-eubacterial FISHprobe and nonspecificDNA staining for example withDAPI(410158406-diamidino-2-phenylindole) to confirm the presence ofnucleic acids of the detected pathogens as recently demanded[108]
Given all these limitations FISH for resistance testingwillpresumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market
5 Direct Fluorescent Imaging ofResistance Determinants by FluorescenceResonance Energy Transfer (FRET)
Nonnucleotide probes labelled with reporter and quenchermolecules allowing for fluorescence energy transfer (FRET)can be used to detect enzymatic resistance mechanisms asdescribed for 120573-lactamases [109] After enzymatic hydrolyza-tion of probes to separate the quencher from the reporter thehydrolyzed probes attach the resistance enzymes as reactiveelectrophiles However this mechanism has so far been onlydescribed for 120573-lactamases in a proof-of-principle analysis[109] and broad evaluation studies are missing Its practicalrelevance for the microbiological routine diagnostics willrequire further evaluation
6 Mass Spectrometric Approaches
Matrix-assisted laser desorption ionization time-of-flightmass spectrometry- (MALDI-TOF MS-) based intact cellmass spectrometry (ICMS) has recently advanced to the stan-dard method for species identification for cultured bacteria
and fungi [24 110ndash114] Promising approaches have beenmade using ICMS spectra for subspecies identification [115]This technique bears a high potential for the fast identi-fication of susceptibility associated biomarker ions that islately only marginally realized in clinical routine diagnosticsThus phyloproteomic approaches help to identify indirectlymostly chromosomal encoded resistance genes by identifyingphylogenetic relatedness [116ndash121] MS can be used to detectchanges in the bacterial or fungal proteome induced byexposition to antimicrobials [24 122ndash124] Whole proteomechanges in consequence of exposition to antimicrobials canbe also detected using stable isotope labeled amino acids(SILAC) [125 126] One very promising approach is the so-calledmass spectrometric beta-lactamase (MSBL) assay [127ndash131] which is based on the mass spectrometric detection ofhydrolyzed beta-lactams Finally there is the combination ofgenotypic and mass spectrometric methods PCR ampliconscan be characterized by PCRelectrospray ionization-massspectrometry (PCRESI MS) [132] and minisequencing [133134] and mass spectrometry-based comparative sequenceanalysis [135 136] can be used to detect susceptibility changesassociated with point mutations
61 Prediction of Broad Spectrum Resistant Clonal Groupsby Phyloproteomics MALDI-TOFMS-based intact cell massspectrometry (ICMS) is potentially able to characterizestrains at the subspecies level and could act as useful toolfor taxonomy and epidemiology [137 138] For the discrim-ination of representative strains particular biomarker ionsthat were completely present or absent as well as shiftsin biomarker masses in a particular subset of strains wereconsidered Using different mathematical algorithms it wasfor example feasible to discriminate Salmonella enterica sspenterica serovar Typhi from other less virulent Salmonellaenterica ssp enterica serotypes [139] to distinguish Campy-lobacter jejuni MLST-ST22 and ST45 from other MLSTsequence types [140] or to perform phyloproteomic analysisof Rhodococcus erythropolis [141] Pseudomonas putida [142]or Neisseria menigitidis [143]
Thefirst approaches to associateMSfingerprintswith sus-ceptibility patterns were designed to differentiate methicillinsusceptible Staphylococcus aureus (MSSA) from methicillinresistant Staphylococcus aureus (MRSA) [144ndash148] Thesewere mostly not standardized and hardly reproducible Butrelatively good reproducibility was demonstrated for thediscrimination of the five major MRSA clonal complexesCC5 CC8 CC22 CC30 and CC45 corresponding to thefive major PFGE MRSA types regardless of their methicillinsensitivity [149 150] A study by Lu and coworkers identifieda set of biomarkers that were able to distinguish betweenmethicillin resistant and vancomycin-intermediate S aureus(VISA) strains and vancomycin-susceptible S aureus strainsas well as between SCCmec types IV and V isolates andSCCmec types IndashIII isolates [151] Further studies demon-strated that isogenic S aureus lacking or artificially harboringSCCmec could not be distinguished in a mass range from2000 to 15000119898119911 [152] whereas isogenic MRSA whichspontaneously reverted to MSSA could be discriminated byMALDI-TOF MS [153]
BioMed Research International 9
(a)
(b)
(c)
(d)
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
[1] WHO Antimicrobial Resistance Global Report on SurveillanceWHO 2014
[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
[20] WHO Global Tuberculosis Report 2013 World Health Organi-zation Geneva Switzerland 2013
[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
BioMed Research International 15
[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
in contact with their specific antigens Such proceduresare useful for a preliminary resistance screening from purebacterial colonies if the resistance mechanism of interest isassociated with a single antigen only which is expressedon the surface of the pathogen Accordingly agglutinationassays are unfeasible for the screening for complex resistancepatterns which may be associated with multiple structurallydifferent families of enzymes as in the case of extended-spectrum beta-lactamases (ESBL) or carbapenemases inGram-negative rod-shaped bacteria
Agglutination assays for the rapid identification of bacte-rial resistance patterns are widely restricted to the identifica-tion of the penicillin binding protein 2a (PBP-2a) the majorresistance determinant ofMethicillin resistant Staphylococcusaureus (MRSA) Different agglutination kits show specifici-ties of 913 to 100 if applied to MRSA colony material[30ndash32] The sensitivity is even more restricted rangingbetween 827 and 941 [30ndash32] If sufficient quantities ofcolony material are used agglutination testing allows for theidentification of small-colony variant MRSA strains as well[33]
The lack of sensitivity seems to be associated with certainstaphylococcal cassette chromosome (SCC-mecA) types withtype IV scoring particularly poor [31] Furthermore aggluti-nation kits are only positive if methicillin resistance is due tothe mecA gene If mecC a divergent mecA homologue is thecause of the resistance agglutination usually fails as observedfor 10 out of 10 mecC-positive live-stock associated MRSAstrains [34]
Of note agglutination based PBP-2a testing is possiblefrom liquid sample materials as well However the sensitivityis poor From blood culture pellets PBP-2a agglutinationshowed sensitivity of only 18 in a recent study In contrastspecificity was excellent with 100 [35]
The usage of genotypic methods in the rapid detectionof antimicrobial resistance genes is gradually shifting fromacademic research laboratories to diagnostic laboratories andpoint-of-care testing The attractiveness of these methods indetermination of antimicrobial resistance has been attributedto two factors firstly their capability to generate resultswithin a short time as compared to phenotypic methods sec-ondly their capability to detect antimicrobial genes directlyfrom the patient sample without necessarily waiting forculture results [36]These two attributes aid clinicians in pre-scribing appropriate treatment to patients at the opportunetime hence making a positive contribution to antimicrobialstewardship programs [27] However genotypic tools for thedetection of antimicrobial resistance may generate false neg-ative results due to (i) their inability to detect new resistancemechanisms or (ii) false-positive results because they maydetect inactive or incomplete resistance genes in a specimenwhich have not inferred resistance to the antimicrobial drugunder test [37]
Current genotypic methods that are used for the rapiddetection of antimicrobial resistance genes include (i) nucleicacid amplification methods particularly real-time quantita-tive PCR (qPCR) (ii) DNA hybridization based methodsparticularly DNAmicroarrays (iii) Luminex xMAP technol-ogy and (iv) next generation sequencingmethods Below is abrief description on the application of each of thesemolecularmethods for the rapid detection of antimicrobial resistance
31 Nucleic Acid Amplification Methods Recently one of thePCR techniques that has received a wide application in clin-ical microbiology is the quantitative real-time PCR (qPCR)technique [38] This has been attributed to its flexibility andcapability to rapidly and simultaneously identify multiplepathogens in a clinical specimen and the presence of antimi-crobial resistance genes in the identified pathogens [39] Asa result numerous qPCR assays for rapid identification ofpathogens in clinical specimens have been developed butmost of the available qPCR assays for detection of microbialresistance genes are limited to the detection of antibioticresistance In short most of the available commercial qPCRassays detect the presence of mecA and mecC which confermethicillin resistance in S aureus the vanA and vanB geneswhich confer glycopeptide resistance and genes that encodeextended-spectrum 120573-lactamases (for detailed review oneach assay see Maurin 2012 [39]) One outstanding featureof all these qPCR assays is their capability to simultaneouslyand accurately detect resistance genes within a remarkablyshorter time period of 4ndash6 hours Similarly qPCR assaysfor rapid detection of resistance against rifampin (RIF) andisoniazid (INH) have been introduced Ramirez and cowork-ers have recently combined qPCR and high-resolution melt(HRM) technology to develop an assay which rapidly andsimultaneously identifiesmultidrug-resistantM tuberculosismutations in the rpoB gene conferring resistance to RIF andmutations in the katG and inhA genes conferring resistanceto INH [28] This assay produces results within 6 hours ascompared to GenoType MTBDRplus assay (Hain LifescienceGmbH Germany) and culture susceptibility testing whichtake 8 hours and 56 days to generate results In the recenttime several in-house qPCR assays for rapid and simulta-neous detection of genes encoding Klebsiella pneumoniaecarbapenemase (blaKPC) andNewDelhi metallo-120573-lactamase(blaNDM) inGram-negative rod-shaped bacteria [40ndash43] havebeen introduced Similarly several in-house qPCR assays forrapid and simultaneous detection of blaOXA-48 blaVIM andblaIMP carbapenemase genes in Enterobacteriaceae have beenestablished [44ndash46]
PCR-based MRSA testing has found wide applicationsin microbiological routine laboratories Next to in-houseassays commercially available molecular MRSA testing plat-forms comprise for example BD GeneOhm MRSA (BectonDickinson Heidelberg Germany) GT MRSA DirectGQMRSA (Hain Lifescience Nehren Germany) Hyplex Staphy-loResist (Amplex Gieszligen Germany) LightCycler (RocheDiagnostics Ltd Rotkreuz Switzerland) kits like LC MRSAAdvanced CepheidXpertGeneExpert (Cepheid SunnyvaleCA USA) and TIB Molbiol LightMix MRSA (TIB Molbiol
4 BioMed Research International
Berlin Germany) All test systems showed reliable results ina recent external laboratory control evaluation in Germany[47] Similarly commercial PCR assays for the detection ofESBL-associated blaCTX-M beta-lactamases and only partiallyESBL-associated blaTEM and blaSHV as well as OXA1-typecarbapenemases (the latter combined in a consensus run)were introduced (Amplex Gieszligen Germany) [48] Similarmultiplex PCR systems are available for the most frequentlydetected carbapenemases which are particularly useful forthe follow-up during hospital outbreak events (AmplexGieszligen Germany) even from primary sample materials [4950]The switch ofmolecular carbanemase detection to robustloop-mediated isothermal amplification (LAMP) [46] allowsfor commercial point-of-care testing (POCT) compatible testsolutions for bedside testing for example the eazyplex Super-BugCRE system (Amplex Gieszligen Germany) which providesresults within 10 minutes However the great number ofdifferent possible cephalosporin and carbapenem resistancemechanisms finally exceeds any multiplexing capacity ifcompleteness is aspired
Nevertheless in addition to rapid and simultaneous pro-viding of reliable results qPCR has been found to be afford-able sensitive specific user friendly not space demandingand deliverable [37ndash39 51] Due to these attributes qPCR hasfound various applications in point-of-care testing (POCT)For example the Xpert MTBRIF test (Cepheid SunnyvaleCA USA) is a qPCR-based assay that has been developedto rapidly and simultaneously detect M tuberculosis andrifampicin (USAN rifampin) resistance To evaluate itsusefulness in POCT a large multicentre study involving6069 cases from six unrelated sites was performed In thisstudy Xpert MTBRIF detected rifampicin resistance casesin 1 hour as compared to line-probe assay and phenotypicdrug susceptibility testing that detected the same cases in 20days and 106 days respectively [52] As mentioned above asimilarGenXpert-basedPOCT test forMRSA screening fromclinical sample materials is available as well
Multiplex PCR assays have also been developed to rapidlyand simultaneously identify multiple pathogens in clinicalspecimens as well as the presence of antimicrobial resistancegenes in the identified pathogens Strommenger and cowork-ers developed amultiplex PCR which simultaneously detects9 resistance genes in S aureus directly from clinical speci-men within 6 hours [53] These 9 resistance genes includemecA (methicillin resistance) aacA-aphD (aminoglycosideresistance) tetK tetM (tetracycline resistance) ermA ermC(macrolide-lincosamide-streptogramin B resistance) vatAvatB and vatC (streptogramin A resistance) [53]
Like qPCR multiplex PCR assays were used as POC teststo facilitate patient management One example is the mul-tiplex PCR-based Unyvero Pneumonia Application (UPA)assay (Curetis AG Holzgerlingen Germany) that has beendeveloped to rapidly and simultaneously detect 18 bacterialspeciesPneumocystis jirovecii and 22 resistancemarkers fromrespiratory specimens (httpwwwcuretiscom) In one ofthe studies showing its suitability for POC testing the UPAassay detected multiple antibiotic resistances within 1 hour(as compared to phenotypic methods that took 96 hours)in a group of 56 hospitalized patients with respiratory tract
infections whowere under treatmentThis finding influencedthe modification of treatment in fifteen patients with severepneumonia leading to their recovery [54] The UPA assayis of course not able to replace conventional testing due toits design because it is not able to detect further microbialspecies and resistance mechanisms besides the implementedones
The continuous development of PCR-based assays withthe capability to rapidly and simultaneously detect pathogensand presence of resistance genes in specimens coupledwith their application in POCT may further improve themanagement of patients as long as appropriate quality controlis ensured
32 DNAMicroarray Technology The biggest challenge asso-ciated with the unprecedented rise of antimicrobial drugresistance worldwide is the scarce availability of assays thatare able to rapidly and simultaneously identify a causativepathogen and generate its antimicrobial resistance profileRecent oligonucleotide-based DNA microarrays match thischallenge In a recent study Zhang and coworkers describedthat CapitalBio DNAmicroarray (CapitalBio Corp) could ina mean time of 58 hours simultaneously identify Mycobac-terium species and detect mutations that confer isoniazid(INH) and rifampicin (RMP) resistance in specimens col-lected from spinal tuberculosis patients as compared toconventional culture and drug susceptibility testing whichtook a mean time of 568 days [55] Briefly oligonucleotideprobes which had been designed to identify Mycobacteriumspecies based on 16S rRNA sequences andmutations of rpoBinhA and katG that confer INH and RMP resistance werecovalently linked to the surface of aldehyde-activated slidesDNAwas extracted from specimens PCRwas used to amplifythe resistance genes and amplicons hybridized on the slidesThe emitted fluorescent signals were analyzed Guo andcoworkers evaluated the ability of a biochip which is basedon the same principle to rapidly and simultaneously identifymultidrug-resistant M tuberculosis (MRTB) and mutationsof rpoB inhA and katG that confer INH and RMP resistancein clinical sputum specimens [56] This group found thatthe biochip could in a mean time of 6 hours simultaneouslyidentifyM tuberculosis and detectmutations that confer INHand RMP resistance
Recent reports have also reported the availability ofCheck-Pointrsquos ESBLKPC DNA microarray for the identi-fication and detection of extended-spectrum 120573-lactamases(ESBLs) and Klebsiella pneumoniae carbapenemases (KPCcarbapenemases) [57 58] This array uses a methodologyknown as multiplex ligation detection to identify ESBL-associated or at least partially ESBL-associated genes (blaTEMblaSHV and blaCTX-M) and blaKPC genes (for details see [57])In a study to evaluate the rapidness at which this arraycould identify and detect these genes Naas and coworkersfound that Check-Pointrsquos ESBLKPC DNA microarray couldidentify them in 7-8 hours as compared to conventionalsusceptibility testing that took a mean time of 54 hoursSimilar results were observed by Willemsen and coworkersin a study that was aimed at evaluating the rapidness at
BioMed Research International 5
which this array could identify and detect these ESBLKPCgenes in hospitals in the Netherlands [58] In addition todetecting and identifying ESBLKPC resistance in gastroin-testinal tract infections caused by Enterobacteriaceae Check-Pointrsquos ESBLKPC DNA microarray has also been usedto detect and identify KPC resistance in hospital-acquiredpneumonia caused by Klebsiella pneumoniae [59] Based onthese experiences the Check-MDR CT 102 DNA microarrayfor the detection of the most prevalent carbapenemase genes(blaNDM blaVIM blaKPC blaOXA-48 and blaIMP) and extended-spectrum120573-lactamase- (ESBL-) related gene families (blaSHVblaTEM and blaCTX-M) has been developed The evaluation ofthe rapidness of the Check-MDR CT 102 DNA microarrayto detect these genes has shown that it yields results 5 hoursfaster than Check-Pointrsquos ESBLKPC DNAmicroarray [60]
At present the DNA microarray technology is mostlyused in the routine detection of antimicrobial resistance ofTB and HIV [61ndash66] The routine use of systems such asMVPlex (Genaco Biomedical Products Huntsville USA) andStaphPlex systems (Genaco Biomedical Products HuntsvilleUSA) which combine both qPCR and DNA microarraytechnology suggest that independent DNA microarray tech-nology might find further applications in the routine clinicalmicrobiology [67 68] The MVPlex system detects the nucmecA (SCCmec)-orfX vanA vanB ddl and tuf genes toscreen for MRSA in nasal swabs [69] and the relatedStaphPlex system performs simultaneous species-level iden-tification (nuc versus tuf ) and detection of mecA aacAermA ermC tetM and tetK as well as Panton-Valentineleukocidin (PVL) for the rapid detection and characterizationof staphylococci directly from positive blood culture bottles[70]
33 Luminex xMAP Technology The description of cooc-curring single nucleotide polymorphism (SNP) mutations inantimicrobial resistance associated genes allows for targetedresistance testing For example unequivocally genetic studieshave proven that there are 5 different mutations in quinoloneresistance-determining region (QRDR) of gyrA gyrB andparE within Salmonella typhi [71] Similar studies havealso shown distinct mutations in the quinolone resistance-determining region (QRDR) of gyrA within Campylobacterjejuni and Campylobacter coli [72]
Rapid simultaneous detection of cooccurring singlenucleotide polymorphism (SNP) mutations in antimicrobialresistance associated genes remains however challengingMost molecular assays such as qPCR and pyrosequencinglack the capability to simultaneously detect cooccurringsingle nucleotide polymorphism (SNP)mutations in differentgenes in a given specimen [73] However this challengehas been overcome by Luminex xMAP Technology a multi-plexing technology which allows for simultaneous detectionof multiple nucleic acid sequences in a single reaction[74] During operation microtiter plates are loaded withmicrospheres that is coated and color-coded beads Themicrospheres are mixed with purified nucleic acids of the testorganism and allowed to hybridize emitting monochromaticlight which the Luminex analyzer reads and interprets At
present this technology has been used to simultaneouslydetect 11 mutations in gyrA gyrB and parE of SalmonellaTyphi and Salmonella Paratyphi A [75] Further it has beenused to simultaneously detect mutations in gyrA of C jejuniandC coli [76] In comparison to sequencing andmicroarraytechnology Luminex xMAPTechnology has been found to beflexible rapid and cost effective [74ndash76]
34 Next Generation Sequencing (NGS) Near whole genomesequencing (WGS) or next generation sequencing (NGS)allows for the assessment of bacterial genomes within severalhours A variety of different technological solutions havebeen introduced including laser printer sized benchtopdevices like 454GS Junior (Roche Basel Switzerland)MiSeq(Illumina San Diego CA USA) and Ion Torrent PGM (LifeTechnologies Grand Island NYUSA) In a previous analysisthe MiSeq (Illumina) system scored best regarding boththroughput per run and error rates while both the 454 GSJunior (Roche) and the Ion Torrent PGM (Life Technologies)systems were prone to homopolymer-associated indel errors[77]
Result interpretation of whole bacterial genomes is basedon either allelic comparisons [78] or single nucleotide poly-morphism (SNP) analysis [79] Data assessment and inter-pretation can be facilitated by commercial software packageslike SeqSphere+ (Ridom BIOINFORMATICS Ltd MunsterGermany) or BioNumerics (Applied Maths Sint-Martens-Latem Belgium)
NGS allows for resistance identification by the presenceof the underlying mechanism rather than just in pharma-codynamic terms [80] so it may revolutionize microbialresistance testing on the long termThis comprises the identi-fication and characterization of resistance genes encoding forextended-spectrum 120573-lactamases (eg 119887119897119886CTX-M 119887119897119886TEM and119887119897119886SHV) plasmid-mediated AmpCs (eg 119887119897119886CMY) quinoloneresistance (eg mutations in gyrA parC or qnr elements)aminoglycoside resistance (eg aminoglycosides modifyingenzymes 16S rRNA methylases) or carbapenemases (eg119887119897119886KPC 119887119897119886NDM) [81]
NGS-based resistance testing is of particular interest forslowly growing infectious agents with atypical resistancepatterns like multidrug-resistant (MDR) or extensive-drugresistance (XDR) M tuberculosis for which rapid identi-fication or exclusion of resistance determinants is of highrelevance for the therapeutic approach Ion Torrent full-gene sequencing with consecutive complete genetic analysiswithin 5 days (Table 5) allowed for reliable resistance detec-tion in M tuberculosis isolates of Burmese Hmong andIndian immigrants in the USA [82] Similar WGS data weredescribed for drug-resistant strains from Russia harbouringalmost all known drug-resistance associated mutations [83]In a direct comparison of Ion Torrent sequencing withphenotypic Bactec MGIT 960 (Becton Dickinson FranklinLakes NJ USA) analysis and genotypicHain line-probe assay(LPA) (Hain Lifescience Ltd Nehren Germany) there wascomplete concordance of NGS to phenotypic resistance andgenotypic rpoB and katG results for the analyzed M tuber-culosis isolates Even more Ion Torrent sequencing detected
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uncommon substitutions and previously uncharacterizedresistance mutations in rpoB rrs and pncA [84] FurtherNGS is able to discriminate mixed mycobacterial genotypesin patient isolates based on single nucleotide variations(SNVs) [85] So it might be suitable to identify resistancemutations in genotypes that occur inminor proportions only
HoweverNGS-based resistance testing is not restricted tomycobacteria Recently NGSwas used to identify transmissi-ble plasmids in multidrug-resistant E coli isolates expressingan ESBL phenotype and transferring their cefotaxime resis-tance marker at high frequency in laboratory conjugationexperiments [86] High-throughput sequencing successfullyproved to be a valuable tool for tracing resistance plasmidsin the course of outbreaks as well [87] However a commer-cial NGS assay (Hospital Acquired Infection BioDetectionSystem Pathogenica Boston MA USA) for investigationsof outbreaks with ESBL-positive Enterobacteriaceae showedgood sensitivity (98) but failed to discriminate betweenESBL and non-ESBL TEM and SHV beta-lactamases or tospecify CTX-M genes by group [88]
Current obstacles to a routine use of NGS technologiesin diagnostic microbiology and resistance testing comprisecosts and scarcely available user-friendly bioinformatics plat-forms [89] Nevertheless NGS technologies provide high-resolution genotyping in a short time frame of only two tofive days [89] Therefore NGSWGS in the microbiologicallaboratory will be the logical next step for the routinediagnosis of infection and the prediction of antimicrobialsusceptibility [90] potentially replacing traditional culturalapproaches on the intermediate or long term
4 Fluorescence In Situ Hybridization (FISH)for the Detection of Bacterial Resistance
FISH (fluorescence in situ hybridization) is a cheap andconvenient option for the identification and resistance testingof bacterial pathogens Traditional FISH is based on specifichybridization of short usually 18ndash25 bases long fluorescent-labelled single-stranded oligonucleotide probes to ribosomalRNA (rRNA) of the target organismwith subsequent analysisunder the fluorescence microscope usually allowing forthe identification of microbes at genus or species level Inprinciple each kind of intracellular RNA can be hybridizedwith FISH probes However rRNA is particularly well suitedas a FISH target because ribosomes are numerous in aprotein-synthesizing cell thus allowing for a boostering offluorescence intensity [91]
This traditional FISH method is both rapid and easy tostandardize so it can be applied for molecular rapid testingSmall modifications of the procedure comprise the use ofpatent-protected commercial peptide nucleic acid (PNA)probes or probes containing locked nucleic acids (LNA)instead of simple single-stranded DNA probes PNA-FISHtechnology reduces nonspecific probe attachment due tothe electrically neutral backbone of the oligonucleotides andis recommendable for routine diagnostics due to a higherdegree of standardization However patent-protected PNA
probes are expensive although they are well suited for thediagnostic routine setting [92]
FISH is particularly suitable for the detection of resistancedeterminants if two prerequisites are guaranteed Ribosoma-llymediated resistance for example affecting antibiotic drugslike macrolide or linezolid is well suited because riboso-mal RNA copies are numerous in living cells allowing forbright fluorescence signals Further FISH can be successfullyapplied if only one or few variable bases provide resistanceso there is no need for a large number of probes in the probepanel
These prerequisites are fulfilled in case of clarithromycinresistance testing in Helicobacter pylori Therefore FISH-based resistance testingwas early evaluated for this indication[93] Clarithromycin in H pylori is basically mediated bythree point mutations in the ribosomal 23S rRNA [94]which can be addressed by three described FISH probesClaR1 ClaR2 and ClaR3 [93] (Table 1) While ClaR1 isassociated with a minimum inhibitory concentration (MIC)of gt64mgL ClaR2 and ClaR3 are associated with varyingMICs between 8mgL and 64mgL [94]
The FISH probes for clarithromycin resistance testing inH pylori were successfully applied to bacteria both fromculture and in bioptic material and extensively assessed invarious studies [93 95ndash97] Reliable test results can even beachieved in formalin-fixed paraffin-embedded tissue afteradequate deparaffination [98] The combined use of probeslabelled with different fluorescence molecules allows for theidentification of coinfections with clarithromycin-sensitiveand -resistant H pylori strains by FISH [99]
Commercial test providers distributed the robust andeasy-to-apply procedure In one study with such a com-mercial test kit [100] a sensitivity of 90 and a specificityof 100 were achieved for the detection of clarithromycin-resistant H pylori within bioptic material In another studyoccasional false-positive H pylori detections were generated[101] although the results of FISH-based resistance test-ing of correctly identified H pylori proved to be reliableRecently a PNA probe-based approach for clarithromycinresistance testing in H pylori showed perfect matching withPCRsequencing in a retrospective studywith formalin-fixedparaffin-embedded tissues (Table 2) [102]
Similar to H pylori FISH-based clarithromycin resis-tance testing could be successfully demonstrated for ther-motolerant Campylobacter spp with a wild-type probe anda clarithromycin resistance probe targeting the A2059Gmutation in the 23S rRNA gene (Table 3) The observedsensitivity and specificity with culture material were 100[103]
Comparable to clarithromycin resistance linezolid resis-tance is ribosomally mediated In enterococci it is typicallycaused by a 2567GgtT base substitution in the 23S rRNA(Table 4) In a collection of 106 enterococcal isolates acorresponding linezolid resistance FISH assay succeeded inpredicting phenotypic resistance in 100 of cases [104]Even a single mutated allele was associated with strongfluorescence signals
First successful attempts of FISH-based resistance testingwere described for non-rRNA-based resistance mechanisms
BioMed Research International 7
Table 1 DNA-FISH-probes detecting clarithromycin resistance in H pylori Russmann et al 2001a [93]
Target Probe Probe sequenceWild type ClaWT 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Table 2 PNA-FISH-probes detecting clarithromycin resistance in H pylori Cerqueira et al 2013 [102] shortened versions of the DNA-FISH-probes from Table 1
Target Probe Probe sequenceWild type HpWT 51015840-GGT-CTT-TCC-GTC-T-31015840
Table 3 DNA-FISH-probes detecting clarithromycin resistance in thermotolerantCampylobacter spp Haas et al 2008 [103] Of note probeC wt 23S is identical with probe ClaWT probe C res 23S 2059AgtG with probe ClaR2 (Table 1)
Target Probe Probe sequenceWild type C wt 23S 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Clarithromycin resistance mutation (A2059G) C res 23S 2059AgtG 51015840-CGG-GGT-CTC-TCC-GTC-TT-31015840
Table 4 DNA-FISH-probes detecting linezolid resistance in enterococci Locked nucleic acids (LNA) were used at the mismatch position(bold underlined print) within in probes
Target Probe Probe sequenceWild type LZD-WT 51015840-CCC-AGC-TCG-CGT-GC-31015840
Agglutination assays lt5 minutes mdash lt100C LowFluorescence in situ hybridization 1-2 hours lt1500000C 100ndash800C IntermediateReal-time PCR(including DNA preparation) 4ndash6 hours 3500000ndash6000000C 1500ndash2500C Strongly depending on
the test systemLoop-mediated isothermal amplification(LAMP) assays lt1 hour 200000ndash400000C 1500ndash2500C Intermediate
Next generation sequencing (NGS) 2ndash5 days 35000000ndash75000000C 7500ndash80000C Very highMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry(MALDI-TOF-MS)
lt5 minutes 7500000ndash30000000C lt100C High
as well FISH-based detection of blaSHV-238240 one of thegenes coding for extended-spectrum 120573-lactamases (ESBL) isan example of a non-rRNA-based FISHprotocol for detectinga particular resistance determinant using the probe 51015840-GAC-CGG-AGC-TAG-CAA-GCG-31015840 [105] However the ESBLphenotype can be associated with a variety of different allelesso this particular probe will be of use only in case of a specificsuspicion for example during an outbreakAccordingly such
a procedure will be reserved for very few if any indications inthe diagnostic routine
Further progression of FISH technology comprisessignal-amplified catalyzed reported deposition (CARD)FISH doubly labeled oligonucleotide probe- (DOPE-) basedFISH combinatorial labelling and spectral imaging (CLASI)FISH and the combination of FISH with other diagnosticapproaches aswell as FISHprocedures for gene identification
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requiring in situ amplification of the respective gene as incase of the rolling circle amplification (RCA) FISH [106]RCA-FISH was successfully applied for the identification ofthe mecA gene in Methicillin resistant Staphylococcus aureus(MRSA) based on the mecA-probes MR-1 51015840-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-31015840 andMR-2 51015840-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-31015840 [107]
FISH-based detection of resistance determinants is apromising diagnostic approach due to its rapidity conve-nience and cost effectiveness The associated rapid detectionof antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associ-ated benefits for the patientrsquos health The main advantage ofFISH is its potential use for resistance testing directly fromprimarymaterial including tissuewith low effort So FISHcanalso be applied in resource-limited settings where expensivetechnologies are not available (Figure 1) In contrast to PCRFISH can also attribute a particular resistance mechanism toa microscopically observed bacterium
However so far FISH is restricted to very few indicationsfor which protocols have been described As a furtherdrawback standardization of FISH-based resistance testing iswidely missing If applied from primary samplematerials liketissue tissue autofluorescence has to be considered requiringconsiderable experience to interpret such diagnostic resultsTo reduce potential interpretation errors FISH from tissuefurther requires counterstaining with a pan-eubacterial FISHprobe and nonspecificDNA staining for example withDAPI(410158406-diamidino-2-phenylindole) to confirm the presence ofnucleic acids of the detected pathogens as recently demanded[108]
Given all these limitations FISH for resistance testingwillpresumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market
5 Direct Fluorescent Imaging ofResistance Determinants by FluorescenceResonance Energy Transfer (FRET)
Nonnucleotide probes labelled with reporter and quenchermolecules allowing for fluorescence energy transfer (FRET)can be used to detect enzymatic resistance mechanisms asdescribed for 120573-lactamases [109] After enzymatic hydrolyza-tion of probes to separate the quencher from the reporter thehydrolyzed probes attach the resistance enzymes as reactiveelectrophiles However this mechanism has so far been onlydescribed for 120573-lactamases in a proof-of-principle analysis[109] and broad evaluation studies are missing Its practicalrelevance for the microbiological routine diagnostics willrequire further evaluation
6 Mass Spectrometric Approaches
Matrix-assisted laser desorption ionization time-of-flightmass spectrometry- (MALDI-TOF MS-) based intact cellmass spectrometry (ICMS) has recently advanced to the stan-dard method for species identification for cultured bacteria
and fungi [24 110ndash114] Promising approaches have beenmade using ICMS spectra for subspecies identification [115]This technique bears a high potential for the fast identi-fication of susceptibility associated biomarker ions that islately only marginally realized in clinical routine diagnosticsThus phyloproteomic approaches help to identify indirectlymostly chromosomal encoded resistance genes by identifyingphylogenetic relatedness [116ndash121] MS can be used to detectchanges in the bacterial or fungal proteome induced byexposition to antimicrobials [24 122ndash124] Whole proteomechanges in consequence of exposition to antimicrobials canbe also detected using stable isotope labeled amino acids(SILAC) [125 126] One very promising approach is the so-calledmass spectrometric beta-lactamase (MSBL) assay [127ndash131] which is based on the mass spectrometric detection ofhydrolyzed beta-lactams Finally there is the combination ofgenotypic and mass spectrometric methods PCR ampliconscan be characterized by PCRelectrospray ionization-massspectrometry (PCRESI MS) [132] and minisequencing [133134] and mass spectrometry-based comparative sequenceanalysis [135 136] can be used to detect susceptibility changesassociated with point mutations
61 Prediction of Broad Spectrum Resistant Clonal Groupsby Phyloproteomics MALDI-TOFMS-based intact cell massspectrometry (ICMS) is potentially able to characterizestrains at the subspecies level and could act as useful toolfor taxonomy and epidemiology [137 138] For the discrim-ination of representative strains particular biomarker ionsthat were completely present or absent as well as shiftsin biomarker masses in a particular subset of strains wereconsidered Using different mathematical algorithms it wasfor example feasible to discriminate Salmonella enterica sspenterica serovar Typhi from other less virulent Salmonellaenterica ssp enterica serotypes [139] to distinguish Campy-lobacter jejuni MLST-ST22 and ST45 from other MLSTsequence types [140] or to perform phyloproteomic analysisof Rhodococcus erythropolis [141] Pseudomonas putida [142]or Neisseria menigitidis [143]
Thefirst approaches to associateMSfingerprintswith sus-ceptibility patterns were designed to differentiate methicillinsusceptible Staphylococcus aureus (MSSA) from methicillinresistant Staphylococcus aureus (MRSA) [144ndash148] Thesewere mostly not standardized and hardly reproducible Butrelatively good reproducibility was demonstrated for thediscrimination of the five major MRSA clonal complexesCC5 CC8 CC22 CC30 and CC45 corresponding to thefive major PFGE MRSA types regardless of their methicillinsensitivity [149 150] A study by Lu and coworkers identifieda set of biomarkers that were able to distinguish betweenmethicillin resistant and vancomycin-intermediate S aureus(VISA) strains and vancomycin-susceptible S aureus strainsas well as between SCCmec types IV and V isolates andSCCmec types IndashIII isolates [151] Further studies demon-strated that isogenic S aureus lacking or artificially harboringSCCmec could not be distinguished in a mass range from2000 to 15000119898119911 [152] whereas isogenic MRSA whichspontaneously reverted to MSSA could be discriminated byMALDI-TOF MS [153]
BioMed Research International 9
(a)
(b)
(c)
(d)
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
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[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
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[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
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[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
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[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
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[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
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[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
Berlin Germany) All test systems showed reliable results ina recent external laboratory control evaluation in Germany[47] Similarly commercial PCR assays for the detection ofESBL-associated blaCTX-M beta-lactamases and only partiallyESBL-associated blaTEM and blaSHV as well as OXA1-typecarbapenemases (the latter combined in a consensus run)were introduced (Amplex Gieszligen Germany) [48] Similarmultiplex PCR systems are available for the most frequentlydetected carbapenemases which are particularly useful forthe follow-up during hospital outbreak events (AmplexGieszligen Germany) even from primary sample materials [4950]The switch ofmolecular carbanemase detection to robustloop-mediated isothermal amplification (LAMP) [46] allowsfor commercial point-of-care testing (POCT) compatible testsolutions for bedside testing for example the eazyplex Super-BugCRE system (Amplex Gieszligen Germany) which providesresults within 10 minutes However the great number ofdifferent possible cephalosporin and carbapenem resistancemechanisms finally exceeds any multiplexing capacity ifcompleteness is aspired
Nevertheless in addition to rapid and simultaneous pro-viding of reliable results qPCR has been found to be afford-able sensitive specific user friendly not space demandingand deliverable [37ndash39 51] Due to these attributes qPCR hasfound various applications in point-of-care testing (POCT)For example the Xpert MTBRIF test (Cepheid SunnyvaleCA USA) is a qPCR-based assay that has been developedto rapidly and simultaneously detect M tuberculosis andrifampicin (USAN rifampin) resistance To evaluate itsusefulness in POCT a large multicentre study involving6069 cases from six unrelated sites was performed In thisstudy Xpert MTBRIF detected rifampicin resistance casesin 1 hour as compared to line-probe assay and phenotypicdrug susceptibility testing that detected the same cases in 20days and 106 days respectively [52] As mentioned above asimilarGenXpert-basedPOCT test forMRSA screening fromclinical sample materials is available as well
Multiplex PCR assays have also been developed to rapidlyand simultaneously identify multiple pathogens in clinicalspecimens as well as the presence of antimicrobial resistancegenes in the identified pathogens Strommenger and cowork-ers developed amultiplex PCR which simultaneously detects9 resistance genes in S aureus directly from clinical speci-men within 6 hours [53] These 9 resistance genes includemecA (methicillin resistance) aacA-aphD (aminoglycosideresistance) tetK tetM (tetracycline resistance) ermA ermC(macrolide-lincosamide-streptogramin B resistance) vatAvatB and vatC (streptogramin A resistance) [53]
Like qPCR multiplex PCR assays were used as POC teststo facilitate patient management One example is the mul-tiplex PCR-based Unyvero Pneumonia Application (UPA)assay (Curetis AG Holzgerlingen Germany) that has beendeveloped to rapidly and simultaneously detect 18 bacterialspeciesPneumocystis jirovecii and 22 resistancemarkers fromrespiratory specimens (httpwwwcuretiscom) In one ofthe studies showing its suitability for POC testing the UPAassay detected multiple antibiotic resistances within 1 hour(as compared to phenotypic methods that took 96 hours)in a group of 56 hospitalized patients with respiratory tract
infections whowere under treatmentThis finding influencedthe modification of treatment in fifteen patients with severepneumonia leading to their recovery [54] The UPA assayis of course not able to replace conventional testing due toits design because it is not able to detect further microbialspecies and resistance mechanisms besides the implementedones
The continuous development of PCR-based assays withthe capability to rapidly and simultaneously detect pathogensand presence of resistance genes in specimens coupledwith their application in POCT may further improve themanagement of patients as long as appropriate quality controlis ensured
32 DNAMicroarray Technology The biggest challenge asso-ciated with the unprecedented rise of antimicrobial drugresistance worldwide is the scarce availability of assays thatare able to rapidly and simultaneously identify a causativepathogen and generate its antimicrobial resistance profileRecent oligonucleotide-based DNA microarrays match thischallenge In a recent study Zhang and coworkers describedthat CapitalBio DNAmicroarray (CapitalBio Corp) could ina mean time of 58 hours simultaneously identify Mycobac-terium species and detect mutations that confer isoniazid(INH) and rifampicin (RMP) resistance in specimens col-lected from spinal tuberculosis patients as compared toconventional culture and drug susceptibility testing whichtook a mean time of 568 days [55] Briefly oligonucleotideprobes which had been designed to identify Mycobacteriumspecies based on 16S rRNA sequences andmutations of rpoBinhA and katG that confer INH and RMP resistance werecovalently linked to the surface of aldehyde-activated slidesDNAwas extracted from specimens PCRwas used to amplifythe resistance genes and amplicons hybridized on the slidesThe emitted fluorescent signals were analyzed Guo andcoworkers evaluated the ability of a biochip which is basedon the same principle to rapidly and simultaneously identifymultidrug-resistant M tuberculosis (MRTB) and mutationsof rpoB inhA and katG that confer INH and RMP resistancein clinical sputum specimens [56] This group found thatthe biochip could in a mean time of 6 hours simultaneouslyidentifyM tuberculosis and detectmutations that confer INHand RMP resistance
Recent reports have also reported the availability ofCheck-Pointrsquos ESBLKPC DNA microarray for the identi-fication and detection of extended-spectrum 120573-lactamases(ESBLs) and Klebsiella pneumoniae carbapenemases (KPCcarbapenemases) [57 58] This array uses a methodologyknown as multiplex ligation detection to identify ESBL-associated or at least partially ESBL-associated genes (blaTEMblaSHV and blaCTX-M) and blaKPC genes (for details see [57])In a study to evaluate the rapidness at which this arraycould identify and detect these genes Naas and coworkersfound that Check-Pointrsquos ESBLKPC DNA microarray couldidentify them in 7-8 hours as compared to conventionalsusceptibility testing that took a mean time of 54 hoursSimilar results were observed by Willemsen and coworkersin a study that was aimed at evaluating the rapidness at
BioMed Research International 5
which this array could identify and detect these ESBLKPCgenes in hospitals in the Netherlands [58] In addition todetecting and identifying ESBLKPC resistance in gastroin-testinal tract infections caused by Enterobacteriaceae Check-Pointrsquos ESBLKPC DNA microarray has also been usedto detect and identify KPC resistance in hospital-acquiredpneumonia caused by Klebsiella pneumoniae [59] Based onthese experiences the Check-MDR CT 102 DNA microarrayfor the detection of the most prevalent carbapenemase genes(blaNDM blaVIM blaKPC blaOXA-48 and blaIMP) and extended-spectrum120573-lactamase- (ESBL-) related gene families (blaSHVblaTEM and blaCTX-M) has been developed The evaluation ofthe rapidness of the Check-MDR CT 102 DNA microarrayto detect these genes has shown that it yields results 5 hoursfaster than Check-Pointrsquos ESBLKPC DNAmicroarray [60]
At present the DNA microarray technology is mostlyused in the routine detection of antimicrobial resistance ofTB and HIV [61ndash66] The routine use of systems such asMVPlex (Genaco Biomedical Products Huntsville USA) andStaphPlex systems (Genaco Biomedical Products HuntsvilleUSA) which combine both qPCR and DNA microarraytechnology suggest that independent DNA microarray tech-nology might find further applications in the routine clinicalmicrobiology [67 68] The MVPlex system detects the nucmecA (SCCmec)-orfX vanA vanB ddl and tuf genes toscreen for MRSA in nasal swabs [69] and the relatedStaphPlex system performs simultaneous species-level iden-tification (nuc versus tuf ) and detection of mecA aacAermA ermC tetM and tetK as well as Panton-Valentineleukocidin (PVL) for the rapid detection and characterizationof staphylococci directly from positive blood culture bottles[70]
33 Luminex xMAP Technology The description of cooc-curring single nucleotide polymorphism (SNP) mutations inantimicrobial resistance associated genes allows for targetedresistance testing For example unequivocally genetic studieshave proven that there are 5 different mutations in quinoloneresistance-determining region (QRDR) of gyrA gyrB andparE within Salmonella typhi [71] Similar studies havealso shown distinct mutations in the quinolone resistance-determining region (QRDR) of gyrA within Campylobacterjejuni and Campylobacter coli [72]
Rapid simultaneous detection of cooccurring singlenucleotide polymorphism (SNP) mutations in antimicrobialresistance associated genes remains however challengingMost molecular assays such as qPCR and pyrosequencinglack the capability to simultaneously detect cooccurringsingle nucleotide polymorphism (SNP)mutations in differentgenes in a given specimen [73] However this challengehas been overcome by Luminex xMAP Technology a multi-plexing technology which allows for simultaneous detectionof multiple nucleic acid sequences in a single reaction[74] During operation microtiter plates are loaded withmicrospheres that is coated and color-coded beads Themicrospheres are mixed with purified nucleic acids of the testorganism and allowed to hybridize emitting monochromaticlight which the Luminex analyzer reads and interprets At
present this technology has been used to simultaneouslydetect 11 mutations in gyrA gyrB and parE of SalmonellaTyphi and Salmonella Paratyphi A [75] Further it has beenused to simultaneously detect mutations in gyrA of C jejuniandC coli [76] In comparison to sequencing andmicroarraytechnology Luminex xMAPTechnology has been found to beflexible rapid and cost effective [74ndash76]
34 Next Generation Sequencing (NGS) Near whole genomesequencing (WGS) or next generation sequencing (NGS)allows for the assessment of bacterial genomes within severalhours A variety of different technological solutions havebeen introduced including laser printer sized benchtopdevices like 454GS Junior (Roche Basel Switzerland)MiSeq(Illumina San Diego CA USA) and Ion Torrent PGM (LifeTechnologies Grand Island NYUSA) In a previous analysisthe MiSeq (Illumina) system scored best regarding boththroughput per run and error rates while both the 454 GSJunior (Roche) and the Ion Torrent PGM (Life Technologies)systems were prone to homopolymer-associated indel errors[77]
Result interpretation of whole bacterial genomes is basedon either allelic comparisons [78] or single nucleotide poly-morphism (SNP) analysis [79] Data assessment and inter-pretation can be facilitated by commercial software packageslike SeqSphere+ (Ridom BIOINFORMATICS Ltd MunsterGermany) or BioNumerics (Applied Maths Sint-Martens-Latem Belgium)
NGS allows for resistance identification by the presenceof the underlying mechanism rather than just in pharma-codynamic terms [80] so it may revolutionize microbialresistance testing on the long termThis comprises the identi-fication and characterization of resistance genes encoding forextended-spectrum 120573-lactamases (eg 119887119897119886CTX-M 119887119897119886TEM and119887119897119886SHV) plasmid-mediated AmpCs (eg 119887119897119886CMY) quinoloneresistance (eg mutations in gyrA parC or qnr elements)aminoglycoside resistance (eg aminoglycosides modifyingenzymes 16S rRNA methylases) or carbapenemases (eg119887119897119886KPC 119887119897119886NDM) [81]
NGS-based resistance testing is of particular interest forslowly growing infectious agents with atypical resistancepatterns like multidrug-resistant (MDR) or extensive-drugresistance (XDR) M tuberculosis for which rapid identi-fication or exclusion of resistance determinants is of highrelevance for the therapeutic approach Ion Torrent full-gene sequencing with consecutive complete genetic analysiswithin 5 days (Table 5) allowed for reliable resistance detec-tion in M tuberculosis isolates of Burmese Hmong andIndian immigrants in the USA [82] Similar WGS data weredescribed for drug-resistant strains from Russia harbouringalmost all known drug-resistance associated mutations [83]In a direct comparison of Ion Torrent sequencing withphenotypic Bactec MGIT 960 (Becton Dickinson FranklinLakes NJ USA) analysis and genotypicHain line-probe assay(LPA) (Hain Lifescience Ltd Nehren Germany) there wascomplete concordance of NGS to phenotypic resistance andgenotypic rpoB and katG results for the analyzed M tuber-culosis isolates Even more Ion Torrent sequencing detected
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uncommon substitutions and previously uncharacterizedresistance mutations in rpoB rrs and pncA [84] FurtherNGS is able to discriminate mixed mycobacterial genotypesin patient isolates based on single nucleotide variations(SNVs) [85] So it might be suitable to identify resistancemutations in genotypes that occur inminor proportions only
HoweverNGS-based resistance testing is not restricted tomycobacteria Recently NGSwas used to identify transmissi-ble plasmids in multidrug-resistant E coli isolates expressingan ESBL phenotype and transferring their cefotaxime resis-tance marker at high frequency in laboratory conjugationexperiments [86] High-throughput sequencing successfullyproved to be a valuable tool for tracing resistance plasmidsin the course of outbreaks as well [87] However a commer-cial NGS assay (Hospital Acquired Infection BioDetectionSystem Pathogenica Boston MA USA) for investigationsof outbreaks with ESBL-positive Enterobacteriaceae showedgood sensitivity (98) but failed to discriminate betweenESBL and non-ESBL TEM and SHV beta-lactamases or tospecify CTX-M genes by group [88]
Current obstacles to a routine use of NGS technologiesin diagnostic microbiology and resistance testing comprisecosts and scarcely available user-friendly bioinformatics plat-forms [89] Nevertheless NGS technologies provide high-resolution genotyping in a short time frame of only two tofive days [89] Therefore NGSWGS in the microbiologicallaboratory will be the logical next step for the routinediagnosis of infection and the prediction of antimicrobialsusceptibility [90] potentially replacing traditional culturalapproaches on the intermediate or long term
4 Fluorescence In Situ Hybridization (FISH)for the Detection of Bacterial Resistance
FISH (fluorescence in situ hybridization) is a cheap andconvenient option for the identification and resistance testingof bacterial pathogens Traditional FISH is based on specifichybridization of short usually 18ndash25 bases long fluorescent-labelled single-stranded oligonucleotide probes to ribosomalRNA (rRNA) of the target organismwith subsequent analysisunder the fluorescence microscope usually allowing forthe identification of microbes at genus or species level Inprinciple each kind of intracellular RNA can be hybridizedwith FISH probes However rRNA is particularly well suitedas a FISH target because ribosomes are numerous in aprotein-synthesizing cell thus allowing for a boostering offluorescence intensity [91]
This traditional FISH method is both rapid and easy tostandardize so it can be applied for molecular rapid testingSmall modifications of the procedure comprise the use ofpatent-protected commercial peptide nucleic acid (PNA)probes or probes containing locked nucleic acids (LNA)instead of simple single-stranded DNA probes PNA-FISHtechnology reduces nonspecific probe attachment due tothe electrically neutral backbone of the oligonucleotides andis recommendable for routine diagnostics due to a higherdegree of standardization However patent-protected PNA
probes are expensive although they are well suited for thediagnostic routine setting [92]
FISH is particularly suitable for the detection of resistancedeterminants if two prerequisites are guaranteed Ribosoma-llymediated resistance for example affecting antibiotic drugslike macrolide or linezolid is well suited because riboso-mal RNA copies are numerous in living cells allowing forbright fluorescence signals Further FISH can be successfullyapplied if only one or few variable bases provide resistanceso there is no need for a large number of probes in the probepanel
These prerequisites are fulfilled in case of clarithromycinresistance testing in Helicobacter pylori Therefore FISH-based resistance testingwas early evaluated for this indication[93] Clarithromycin in H pylori is basically mediated bythree point mutations in the ribosomal 23S rRNA [94]which can be addressed by three described FISH probesClaR1 ClaR2 and ClaR3 [93] (Table 1) While ClaR1 isassociated with a minimum inhibitory concentration (MIC)of gt64mgL ClaR2 and ClaR3 are associated with varyingMICs between 8mgL and 64mgL [94]
The FISH probes for clarithromycin resistance testing inH pylori were successfully applied to bacteria both fromculture and in bioptic material and extensively assessed invarious studies [93 95ndash97] Reliable test results can even beachieved in formalin-fixed paraffin-embedded tissue afteradequate deparaffination [98] The combined use of probeslabelled with different fluorescence molecules allows for theidentification of coinfections with clarithromycin-sensitiveand -resistant H pylori strains by FISH [99]
Commercial test providers distributed the robust andeasy-to-apply procedure In one study with such a com-mercial test kit [100] a sensitivity of 90 and a specificityof 100 were achieved for the detection of clarithromycin-resistant H pylori within bioptic material In another studyoccasional false-positive H pylori detections were generated[101] although the results of FISH-based resistance test-ing of correctly identified H pylori proved to be reliableRecently a PNA probe-based approach for clarithromycinresistance testing in H pylori showed perfect matching withPCRsequencing in a retrospective studywith formalin-fixedparaffin-embedded tissues (Table 2) [102]
Similar to H pylori FISH-based clarithromycin resis-tance testing could be successfully demonstrated for ther-motolerant Campylobacter spp with a wild-type probe anda clarithromycin resistance probe targeting the A2059Gmutation in the 23S rRNA gene (Table 3) The observedsensitivity and specificity with culture material were 100[103]
Comparable to clarithromycin resistance linezolid resis-tance is ribosomally mediated In enterococci it is typicallycaused by a 2567GgtT base substitution in the 23S rRNA(Table 4) In a collection of 106 enterococcal isolates acorresponding linezolid resistance FISH assay succeeded inpredicting phenotypic resistance in 100 of cases [104]Even a single mutated allele was associated with strongfluorescence signals
First successful attempts of FISH-based resistance testingwere described for non-rRNA-based resistance mechanisms
BioMed Research International 7
Table 1 DNA-FISH-probes detecting clarithromycin resistance in H pylori Russmann et al 2001a [93]
Target Probe Probe sequenceWild type ClaWT 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Table 2 PNA-FISH-probes detecting clarithromycin resistance in H pylori Cerqueira et al 2013 [102] shortened versions of the DNA-FISH-probes from Table 1
Target Probe Probe sequenceWild type HpWT 51015840-GGT-CTT-TCC-GTC-T-31015840
Table 3 DNA-FISH-probes detecting clarithromycin resistance in thermotolerantCampylobacter spp Haas et al 2008 [103] Of note probeC wt 23S is identical with probe ClaWT probe C res 23S 2059AgtG with probe ClaR2 (Table 1)
Target Probe Probe sequenceWild type C wt 23S 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Clarithromycin resistance mutation (A2059G) C res 23S 2059AgtG 51015840-CGG-GGT-CTC-TCC-GTC-TT-31015840
Table 4 DNA-FISH-probes detecting linezolid resistance in enterococci Locked nucleic acids (LNA) were used at the mismatch position(bold underlined print) within in probes
Target Probe Probe sequenceWild type LZD-WT 51015840-CCC-AGC-TCG-CGT-GC-31015840
Agglutination assays lt5 minutes mdash lt100C LowFluorescence in situ hybridization 1-2 hours lt1500000C 100ndash800C IntermediateReal-time PCR(including DNA preparation) 4ndash6 hours 3500000ndash6000000C 1500ndash2500C Strongly depending on
the test systemLoop-mediated isothermal amplification(LAMP) assays lt1 hour 200000ndash400000C 1500ndash2500C Intermediate
Next generation sequencing (NGS) 2ndash5 days 35000000ndash75000000C 7500ndash80000C Very highMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry(MALDI-TOF-MS)
lt5 minutes 7500000ndash30000000C lt100C High
as well FISH-based detection of blaSHV-238240 one of thegenes coding for extended-spectrum 120573-lactamases (ESBL) isan example of a non-rRNA-based FISHprotocol for detectinga particular resistance determinant using the probe 51015840-GAC-CGG-AGC-TAG-CAA-GCG-31015840 [105] However the ESBLphenotype can be associated with a variety of different allelesso this particular probe will be of use only in case of a specificsuspicion for example during an outbreakAccordingly such
a procedure will be reserved for very few if any indications inthe diagnostic routine
Further progression of FISH technology comprisessignal-amplified catalyzed reported deposition (CARD)FISH doubly labeled oligonucleotide probe- (DOPE-) basedFISH combinatorial labelling and spectral imaging (CLASI)FISH and the combination of FISH with other diagnosticapproaches aswell as FISHprocedures for gene identification
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requiring in situ amplification of the respective gene as incase of the rolling circle amplification (RCA) FISH [106]RCA-FISH was successfully applied for the identification ofthe mecA gene in Methicillin resistant Staphylococcus aureus(MRSA) based on the mecA-probes MR-1 51015840-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-31015840 andMR-2 51015840-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-31015840 [107]
FISH-based detection of resistance determinants is apromising diagnostic approach due to its rapidity conve-nience and cost effectiveness The associated rapid detectionof antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associ-ated benefits for the patientrsquos health The main advantage ofFISH is its potential use for resistance testing directly fromprimarymaterial including tissuewith low effort So FISHcanalso be applied in resource-limited settings where expensivetechnologies are not available (Figure 1) In contrast to PCRFISH can also attribute a particular resistance mechanism toa microscopically observed bacterium
However so far FISH is restricted to very few indicationsfor which protocols have been described As a furtherdrawback standardization of FISH-based resistance testing iswidely missing If applied from primary samplematerials liketissue tissue autofluorescence has to be considered requiringconsiderable experience to interpret such diagnostic resultsTo reduce potential interpretation errors FISH from tissuefurther requires counterstaining with a pan-eubacterial FISHprobe and nonspecificDNA staining for example withDAPI(410158406-diamidino-2-phenylindole) to confirm the presence ofnucleic acids of the detected pathogens as recently demanded[108]
Given all these limitations FISH for resistance testingwillpresumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market
5 Direct Fluorescent Imaging ofResistance Determinants by FluorescenceResonance Energy Transfer (FRET)
Nonnucleotide probes labelled with reporter and quenchermolecules allowing for fluorescence energy transfer (FRET)can be used to detect enzymatic resistance mechanisms asdescribed for 120573-lactamases [109] After enzymatic hydrolyza-tion of probes to separate the quencher from the reporter thehydrolyzed probes attach the resistance enzymes as reactiveelectrophiles However this mechanism has so far been onlydescribed for 120573-lactamases in a proof-of-principle analysis[109] and broad evaluation studies are missing Its practicalrelevance for the microbiological routine diagnostics willrequire further evaluation
6 Mass Spectrometric Approaches
Matrix-assisted laser desorption ionization time-of-flightmass spectrometry- (MALDI-TOF MS-) based intact cellmass spectrometry (ICMS) has recently advanced to the stan-dard method for species identification for cultured bacteria
and fungi [24 110ndash114] Promising approaches have beenmade using ICMS spectra for subspecies identification [115]This technique bears a high potential for the fast identi-fication of susceptibility associated biomarker ions that islately only marginally realized in clinical routine diagnosticsThus phyloproteomic approaches help to identify indirectlymostly chromosomal encoded resistance genes by identifyingphylogenetic relatedness [116ndash121] MS can be used to detectchanges in the bacterial or fungal proteome induced byexposition to antimicrobials [24 122ndash124] Whole proteomechanges in consequence of exposition to antimicrobials canbe also detected using stable isotope labeled amino acids(SILAC) [125 126] One very promising approach is the so-calledmass spectrometric beta-lactamase (MSBL) assay [127ndash131] which is based on the mass spectrometric detection ofhydrolyzed beta-lactams Finally there is the combination ofgenotypic and mass spectrometric methods PCR ampliconscan be characterized by PCRelectrospray ionization-massspectrometry (PCRESI MS) [132] and minisequencing [133134] and mass spectrometry-based comparative sequenceanalysis [135 136] can be used to detect susceptibility changesassociated with point mutations
61 Prediction of Broad Spectrum Resistant Clonal Groupsby Phyloproteomics MALDI-TOFMS-based intact cell massspectrometry (ICMS) is potentially able to characterizestrains at the subspecies level and could act as useful toolfor taxonomy and epidemiology [137 138] For the discrim-ination of representative strains particular biomarker ionsthat were completely present or absent as well as shiftsin biomarker masses in a particular subset of strains wereconsidered Using different mathematical algorithms it wasfor example feasible to discriminate Salmonella enterica sspenterica serovar Typhi from other less virulent Salmonellaenterica ssp enterica serotypes [139] to distinguish Campy-lobacter jejuni MLST-ST22 and ST45 from other MLSTsequence types [140] or to perform phyloproteomic analysisof Rhodococcus erythropolis [141] Pseudomonas putida [142]or Neisseria menigitidis [143]
Thefirst approaches to associateMSfingerprintswith sus-ceptibility patterns were designed to differentiate methicillinsusceptible Staphylococcus aureus (MSSA) from methicillinresistant Staphylococcus aureus (MRSA) [144ndash148] Thesewere mostly not standardized and hardly reproducible Butrelatively good reproducibility was demonstrated for thediscrimination of the five major MRSA clonal complexesCC5 CC8 CC22 CC30 and CC45 corresponding to thefive major PFGE MRSA types regardless of their methicillinsensitivity [149 150] A study by Lu and coworkers identifieda set of biomarkers that were able to distinguish betweenmethicillin resistant and vancomycin-intermediate S aureus(VISA) strains and vancomycin-susceptible S aureus strainsas well as between SCCmec types IV and V isolates andSCCmec types IndashIII isolates [151] Further studies demon-strated that isogenic S aureus lacking or artificially harboringSCCmec could not be distinguished in a mass range from2000 to 15000119898119911 [152] whereas isogenic MRSA whichspontaneously reverted to MSSA could be discriminated byMALDI-TOF MS [153]
BioMed Research International 9
(a)
(b)
(c)
(d)
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
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[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
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[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
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[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
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[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
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[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
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[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
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[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
which this array could identify and detect these ESBLKPCgenes in hospitals in the Netherlands [58] In addition todetecting and identifying ESBLKPC resistance in gastroin-testinal tract infections caused by Enterobacteriaceae Check-Pointrsquos ESBLKPC DNA microarray has also been usedto detect and identify KPC resistance in hospital-acquiredpneumonia caused by Klebsiella pneumoniae [59] Based onthese experiences the Check-MDR CT 102 DNA microarrayfor the detection of the most prevalent carbapenemase genes(blaNDM blaVIM blaKPC blaOXA-48 and blaIMP) and extended-spectrum120573-lactamase- (ESBL-) related gene families (blaSHVblaTEM and blaCTX-M) has been developed The evaluation ofthe rapidness of the Check-MDR CT 102 DNA microarrayto detect these genes has shown that it yields results 5 hoursfaster than Check-Pointrsquos ESBLKPC DNAmicroarray [60]
At present the DNA microarray technology is mostlyused in the routine detection of antimicrobial resistance ofTB and HIV [61ndash66] The routine use of systems such asMVPlex (Genaco Biomedical Products Huntsville USA) andStaphPlex systems (Genaco Biomedical Products HuntsvilleUSA) which combine both qPCR and DNA microarraytechnology suggest that independent DNA microarray tech-nology might find further applications in the routine clinicalmicrobiology [67 68] The MVPlex system detects the nucmecA (SCCmec)-orfX vanA vanB ddl and tuf genes toscreen for MRSA in nasal swabs [69] and the relatedStaphPlex system performs simultaneous species-level iden-tification (nuc versus tuf ) and detection of mecA aacAermA ermC tetM and tetK as well as Panton-Valentineleukocidin (PVL) for the rapid detection and characterizationof staphylococci directly from positive blood culture bottles[70]
33 Luminex xMAP Technology The description of cooc-curring single nucleotide polymorphism (SNP) mutations inantimicrobial resistance associated genes allows for targetedresistance testing For example unequivocally genetic studieshave proven that there are 5 different mutations in quinoloneresistance-determining region (QRDR) of gyrA gyrB andparE within Salmonella typhi [71] Similar studies havealso shown distinct mutations in the quinolone resistance-determining region (QRDR) of gyrA within Campylobacterjejuni and Campylobacter coli [72]
Rapid simultaneous detection of cooccurring singlenucleotide polymorphism (SNP) mutations in antimicrobialresistance associated genes remains however challengingMost molecular assays such as qPCR and pyrosequencinglack the capability to simultaneously detect cooccurringsingle nucleotide polymorphism (SNP)mutations in differentgenes in a given specimen [73] However this challengehas been overcome by Luminex xMAP Technology a multi-plexing technology which allows for simultaneous detectionof multiple nucleic acid sequences in a single reaction[74] During operation microtiter plates are loaded withmicrospheres that is coated and color-coded beads Themicrospheres are mixed with purified nucleic acids of the testorganism and allowed to hybridize emitting monochromaticlight which the Luminex analyzer reads and interprets At
present this technology has been used to simultaneouslydetect 11 mutations in gyrA gyrB and parE of SalmonellaTyphi and Salmonella Paratyphi A [75] Further it has beenused to simultaneously detect mutations in gyrA of C jejuniandC coli [76] In comparison to sequencing andmicroarraytechnology Luminex xMAPTechnology has been found to beflexible rapid and cost effective [74ndash76]
34 Next Generation Sequencing (NGS) Near whole genomesequencing (WGS) or next generation sequencing (NGS)allows for the assessment of bacterial genomes within severalhours A variety of different technological solutions havebeen introduced including laser printer sized benchtopdevices like 454GS Junior (Roche Basel Switzerland)MiSeq(Illumina San Diego CA USA) and Ion Torrent PGM (LifeTechnologies Grand Island NYUSA) In a previous analysisthe MiSeq (Illumina) system scored best regarding boththroughput per run and error rates while both the 454 GSJunior (Roche) and the Ion Torrent PGM (Life Technologies)systems were prone to homopolymer-associated indel errors[77]
Result interpretation of whole bacterial genomes is basedon either allelic comparisons [78] or single nucleotide poly-morphism (SNP) analysis [79] Data assessment and inter-pretation can be facilitated by commercial software packageslike SeqSphere+ (Ridom BIOINFORMATICS Ltd MunsterGermany) or BioNumerics (Applied Maths Sint-Martens-Latem Belgium)
NGS allows for resistance identification by the presenceof the underlying mechanism rather than just in pharma-codynamic terms [80] so it may revolutionize microbialresistance testing on the long termThis comprises the identi-fication and characterization of resistance genes encoding forextended-spectrum 120573-lactamases (eg 119887119897119886CTX-M 119887119897119886TEM and119887119897119886SHV) plasmid-mediated AmpCs (eg 119887119897119886CMY) quinoloneresistance (eg mutations in gyrA parC or qnr elements)aminoglycoside resistance (eg aminoglycosides modifyingenzymes 16S rRNA methylases) or carbapenemases (eg119887119897119886KPC 119887119897119886NDM) [81]
NGS-based resistance testing is of particular interest forslowly growing infectious agents with atypical resistancepatterns like multidrug-resistant (MDR) or extensive-drugresistance (XDR) M tuberculosis for which rapid identi-fication or exclusion of resistance determinants is of highrelevance for the therapeutic approach Ion Torrent full-gene sequencing with consecutive complete genetic analysiswithin 5 days (Table 5) allowed for reliable resistance detec-tion in M tuberculosis isolates of Burmese Hmong andIndian immigrants in the USA [82] Similar WGS data weredescribed for drug-resistant strains from Russia harbouringalmost all known drug-resistance associated mutations [83]In a direct comparison of Ion Torrent sequencing withphenotypic Bactec MGIT 960 (Becton Dickinson FranklinLakes NJ USA) analysis and genotypicHain line-probe assay(LPA) (Hain Lifescience Ltd Nehren Germany) there wascomplete concordance of NGS to phenotypic resistance andgenotypic rpoB and katG results for the analyzed M tuber-culosis isolates Even more Ion Torrent sequencing detected
6 BioMed Research International
uncommon substitutions and previously uncharacterizedresistance mutations in rpoB rrs and pncA [84] FurtherNGS is able to discriminate mixed mycobacterial genotypesin patient isolates based on single nucleotide variations(SNVs) [85] So it might be suitable to identify resistancemutations in genotypes that occur inminor proportions only
HoweverNGS-based resistance testing is not restricted tomycobacteria Recently NGSwas used to identify transmissi-ble plasmids in multidrug-resistant E coli isolates expressingan ESBL phenotype and transferring their cefotaxime resis-tance marker at high frequency in laboratory conjugationexperiments [86] High-throughput sequencing successfullyproved to be a valuable tool for tracing resistance plasmidsin the course of outbreaks as well [87] However a commer-cial NGS assay (Hospital Acquired Infection BioDetectionSystem Pathogenica Boston MA USA) for investigationsof outbreaks with ESBL-positive Enterobacteriaceae showedgood sensitivity (98) but failed to discriminate betweenESBL and non-ESBL TEM and SHV beta-lactamases or tospecify CTX-M genes by group [88]
Current obstacles to a routine use of NGS technologiesin diagnostic microbiology and resistance testing comprisecosts and scarcely available user-friendly bioinformatics plat-forms [89] Nevertheless NGS technologies provide high-resolution genotyping in a short time frame of only two tofive days [89] Therefore NGSWGS in the microbiologicallaboratory will be the logical next step for the routinediagnosis of infection and the prediction of antimicrobialsusceptibility [90] potentially replacing traditional culturalapproaches on the intermediate or long term
4 Fluorescence In Situ Hybridization (FISH)for the Detection of Bacterial Resistance
FISH (fluorescence in situ hybridization) is a cheap andconvenient option for the identification and resistance testingof bacterial pathogens Traditional FISH is based on specifichybridization of short usually 18ndash25 bases long fluorescent-labelled single-stranded oligonucleotide probes to ribosomalRNA (rRNA) of the target organismwith subsequent analysisunder the fluorescence microscope usually allowing forthe identification of microbes at genus or species level Inprinciple each kind of intracellular RNA can be hybridizedwith FISH probes However rRNA is particularly well suitedas a FISH target because ribosomes are numerous in aprotein-synthesizing cell thus allowing for a boostering offluorescence intensity [91]
This traditional FISH method is both rapid and easy tostandardize so it can be applied for molecular rapid testingSmall modifications of the procedure comprise the use ofpatent-protected commercial peptide nucleic acid (PNA)probes or probes containing locked nucleic acids (LNA)instead of simple single-stranded DNA probes PNA-FISHtechnology reduces nonspecific probe attachment due tothe electrically neutral backbone of the oligonucleotides andis recommendable for routine diagnostics due to a higherdegree of standardization However patent-protected PNA
probes are expensive although they are well suited for thediagnostic routine setting [92]
FISH is particularly suitable for the detection of resistancedeterminants if two prerequisites are guaranteed Ribosoma-llymediated resistance for example affecting antibiotic drugslike macrolide or linezolid is well suited because riboso-mal RNA copies are numerous in living cells allowing forbright fluorescence signals Further FISH can be successfullyapplied if only one or few variable bases provide resistanceso there is no need for a large number of probes in the probepanel
These prerequisites are fulfilled in case of clarithromycinresistance testing in Helicobacter pylori Therefore FISH-based resistance testingwas early evaluated for this indication[93] Clarithromycin in H pylori is basically mediated bythree point mutations in the ribosomal 23S rRNA [94]which can be addressed by three described FISH probesClaR1 ClaR2 and ClaR3 [93] (Table 1) While ClaR1 isassociated with a minimum inhibitory concentration (MIC)of gt64mgL ClaR2 and ClaR3 are associated with varyingMICs between 8mgL and 64mgL [94]
The FISH probes for clarithromycin resistance testing inH pylori were successfully applied to bacteria both fromculture and in bioptic material and extensively assessed invarious studies [93 95ndash97] Reliable test results can even beachieved in formalin-fixed paraffin-embedded tissue afteradequate deparaffination [98] The combined use of probeslabelled with different fluorescence molecules allows for theidentification of coinfections with clarithromycin-sensitiveand -resistant H pylori strains by FISH [99]
Commercial test providers distributed the robust andeasy-to-apply procedure In one study with such a com-mercial test kit [100] a sensitivity of 90 and a specificityof 100 were achieved for the detection of clarithromycin-resistant H pylori within bioptic material In another studyoccasional false-positive H pylori detections were generated[101] although the results of FISH-based resistance test-ing of correctly identified H pylori proved to be reliableRecently a PNA probe-based approach for clarithromycinresistance testing in H pylori showed perfect matching withPCRsequencing in a retrospective studywith formalin-fixedparaffin-embedded tissues (Table 2) [102]
Similar to H pylori FISH-based clarithromycin resis-tance testing could be successfully demonstrated for ther-motolerant Campylobacter spp with a wild-type probe anda clarithromycin resistance probe targeting the A2059Gmutation in the 23S rRNA gene (Table 3) The observedsensitivity and specificity with culture material were 100[103]
Comparable to clarithromycin resistance linezolid resis-tance is ribosomally mediated In enterococci it is typicallycaused by a 2567GgtT base substitution in the 23S rRNA(Table 4) In a collection of 106 enterococcal isolates acorresponding linezolid resistance FISH assay succeeded inpredicting phenotypic resistance in 100 of cases [104]Even a single mutated allele was associated with strongfluorescence signals
First successful attempts of FISH-based resistance testingwere described for non-rRNA-based resistance mechanisms
BioMed Research International 7
Table 1 DNA-FISH-probes detecting clarithromycin resistance in H pylori Russmann et al 2001a [93]
Target Probe Probe sequenceWild type ClaWT 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Table 2 PNA-FISH-probes detecting clarithromycin resistance in H pylori Cerqueira et al 2013 [102] shortened versions of the DNA-FISH-probes from Table 1
Target Probe Probe sequenceWild type HpWT 51015840-GGT-CTT-TCC-GTC-T-31015840
Table 3 DNA-FISH-probes detecting clarithromycin resistance in thermotolerantCampylobacter spp Haas et al 2008 [103] Of note probeC wt 23S is identical with probe ClaWT probe C res 23S 2059AgtG with probe ClaR2 (Table 1)
Target Probe Probe sequenceWild type C wt 23S 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Clarithromycin resistance mutation (A2059G) C res 23S 2059AgtG 51015840-CGG-GGT-CTC-TCC-GTC-TT-31015840
Table 4 DNA-FISH-probes detecting linezolid resistance in enterococci Locked nucleic acids (LNA) were used at the mismatch position(bold underlined print) within in probes
Target Probe Probe sequenceWild type LZD-WT 51015840-CCC-AGC-TCG-CGT-GC-31015840
Agglutination assays lt5 minutes mdash lt100C LowFluorescence in situ hybridization 1-2 hours lt1500000C 100ndash800C IntermediateReal-time PCR(including DNA preparation) 4ndash6 hours 3500000ndash6000000C 1500ndash2500C Strongly depending on
the test systemLoop-mediated isothermal amplification(LAMP) assays lt1 hour 200000ndash400000C 1500ndash2500C Intermediate
Next generation sequencing (NGS) 2ndash5 days 35000000ndash75000000C 7500ndash80000C Very highMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry(MALDI-TOF-MS)
lt5 minutes 7500000ndash30000000C lt100C High
as well FISH-based detection of blaSHV-238240 one of thegenes coding for extended-spectrum 120573-lactamases (ESBL) isan example of a non-rRNA-based FISHprotocol for detectinga particular resistance determinant using the probe 51015840-GAC-CGG-AGC-TAG-CAA-GCG-31015840 [105] However the ESBLphenotype can be associated with a variety of different allelesso this particular probe will be of use only in case of a specificsuspicion for example during an outbreakAccordingly such
a procedure will be reserved for very few if any indications inthe diagnostic routine
Further progression of FISH technology comprisessignal-amplified catalyzed reported deposition (CARD)FISH doubly labeled oligonucleotide probe- (DOPE-) basedFISH combinatorial labelling and spectral imaging (CLASI)FISH and the combination of FISH with other diagnosticapproaches aswell as FISHprocedures for gene identification
8 BioMed Research International
requiring in situ amplification of the respective gene as incase of the rolling circle amplification (RCA) FISH [106]RCA-FISH was successfully applied for the identification ofthe mecA gene in Methicillin resistant Staphylococcus aureus(MRSA) based on the mecA-probes MR-1 51015840-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-31015840 andMR-2 51015840-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-31015840 [107]
FISH-based detection of resistance determinants is apromising diagnostic approach due to its rapidity conve-nience and cost effectiveness The associated rapid detectionof antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associ-ated benefits for the patientrsquos health The main advantage ofFISH is its potential use for resistance testing directly fromprimarymaterial including tissuewith low effort So FISHcanalso be applied in resource-limited settings where expensivetechnologies are not available (Figure 1) In contrast to PCRFISH can also attribute a particular resistance mechanism toa microscopically observed bacterium
However so far FISH is restricted to very few indicationsfor which protocols have been described As a furtherdrawback standardization of FISH-based resistance testing iswidely missing If applied from primary samplematerials liketissue tissue autofluorescence has to be considered requiringconsiderable experience to interpret such diagnostic resultsTo reduce potential interpretation errors FISH from tissuefurther requires counterstaining with a pan-eubacterial FISHprobe and nonspecificDNA staining for example withDAPI(410158406-diamidino-2-phenylindole) to confirm the presence ofnucleic acids of the detected pathogens as recently demanded[108]
Given all these limitations FISH for resistance testingwillpresumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market
5 Direct Fluorescent Imaging ofResistance Determinants by FluorescenceResonance Energy Transfer (FRET)
Nonnucleotide probes labelled with reporter and quenchermolecules allowing for fluorescence energy transfer (FRET)can be used to detect enzymatic resistance mechanisms asdescribed for 120573-lactamases [109] After enzymatic hydrolyza-tion of probes to separate the quencher from the reporter thehydrolyzed probes attach the resistance enzymes as reactiveelectrophiles However this mechanism has so far been onlydescribed for 120573-lactamases in a proof-of-principle analysis[109] and broad evaluation studies are missing Its practicalrelevance for the microbiological routine diagnostics willrequire further evaluation
6 Mass Spectrometric Approaches
Matrix-assisted laser desorption ionization time-of-flightmass spectrometry- (MALDI-TOF MS-) based intact cellmass spectrometry (ICMS) has recently advanced to the stan-dard method for species identification for cultured bacteria
and fungi [24 110ndash114] Promising approaches have beenmade using ICMS spectra for subspecies identification [115]This technique bears a high potential for the fast identi-fication of susceptibility associated biomarker ions that islately only marginally realized in clinical routine diagnosticsThus phyloproteomic approaches help to identify indirectlymostly chromosomal encoded resistance genes by identifyingphylogenetic relatedness [116ndash121] MS can be used to detectchanges in the bacterial or fungal proteome induced byexposition to antimicrobials [24 122ndash124] Whole proteomechanges in consequence of exposition to antimicrobials canbe also detected using stable isotope labeled amino acids(SILAC) [125 126] One very promising approach is the so-calledmass spectrometric beta-lactamase (MSBL) assay [127ndash131] which is based on the mass spectrometric detection ofhydrolyzed beta-lactams Finally there is the combination ofgenotypic and mass spectrometric methods PCR ampliconscan be characterized by PCRelectrospray ionization-massspectrometry (PCRESI MS) [132] and minisequencing [133134] and mass spectrometry-based comparative sequenceanalysis [135 136] can be used to detect susceptibility changesassociated with point mutations
61 Prediction of Broad Spectrum Resistant Clonal Groupsby Phyloproteomics MALDI-TOFMS-based intact cell massspectrometry (ICMS) is potentially able to characterizestrains at the subspecies level and could act as useful toolfor taxonomy and epidemiology [137 138] For the discrim-ination of representative strains particular biomarker ionsthat were completely present or absent as well as shiftsin biomarker masses in a particular subset of strains wereconsidered Using different mathematical algorithms it wasfor example feasible to discriminate Salmonella enterica sspenterica serovar Typhi from other less virulent Salmonellaenterica ssp enterica serotypes [139] to distinguish Campy-lobacter jejuni MLST-ST22 and ST45 from other MLSTsequence types [140] or to perform phyloproteomic analysisof Rhodococcus erythropolis [141] Pseudomonas putida [142]or Neisseria menigitidis [143]
Thefirst approaches to associateMSfingerprintswith sus-ceptibility patterns were designed to differentiate methicillinsusceptible Staphylococcus aureus (MSSA) from methicillinresistant Staphylococcus aureus (MRSA) [144ndash148] Thesewere mostly not standardized and hardly reproducible Butrelatively good reproducibility was demonstrated for thediscrimination of the five major MRSA clonal complexesCC5 CC8 CC22 CC30 and CC45 corresponding to thefive major PFGE MRSA types regardless of their methicillinsensitivity [149 150] A study by Lu and coworkers identifieda set of biomarkers that were able to distinguish betweenmethicillin resistant and vancomycin-intermediate S aureus(VISA) strains and vancomycin-susceptible S aureus strainsas well as between SCCmec types IV and V isolates andSCCmec types IndashIII isolates [151] Further studies demon-strated that isogenic S aureus lacking or artificially harboringSCCmec could not be distinguished in a mass range from2000 to 15000119898119911 [152] whereas isogenic MRSA whichspontaneously reverted to MSSA could be discriminated byMALDI-TOF MS [153]
BioMed Research International 9
(a)
(b)
(c)
(d)
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
[1] WHO Antimicrobial Resistance Global Report on SurveillanceWHO 2014
[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
[20] WHO Global Tuberculosis Report 2013 World Health Organi-zation Geneva Switzerland 2013
[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
BioMed Research International 15
[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
uncommon substitutions and previously uncharacterizedresistance mutations in rpoB rrs and pncA [84] FurtherNGS is able to discriminate mixed mycobacterial genotypesin patient isolates based on single nucleotide variations(SNVs) [85] So it might be suitable to identify resistancemutations in genotypes that occur inminor proportions only
HoweverNGS-based resistance testing is not restricted tomycobacteria Recently NGSwas used to identify transmissi-ble plasmids in multidrug-resistant E coli isolates expressingan ESBL phenotype and transferring their cefotaxime resis-tance marker at high frequency in laboratory conjugationexperiments [86] High-throughput sequencing successfullyproved to be a valuable tool for tracing resistance plasmidsin the course of outbreaks as well [87] However a commer-cial NGS assay (Hospital Acquired Infection BioDetectionSystem Pathogenica Boston MA USA) for investigationsof outbreaks with ESBL-positive Enterobacteriaceae showedgood sensitivity (98) but failed to discriminate betweenESBL and non-ESBL TEM and SHV beta-lactamases or tospecify CTX-M genes by group [88]
Current obstacles to a routine use of NGS technologiesin diagnostic microbiology and resistance testing comprisecosts and scarcely available user-friendly bioinformatics plat-forms [89] Nevertheless NGS technologies provide high-resolution genotyping in a short time frame of only two tofive days [89] Therefore NGSWGS in the microbiologicallaboratory will be the logical next step for the routinediagnosis of infection and the prediction of antimicrobialsusceptibility [90] potentially replacing traditional culturalapproaches on the intermediate or long term
4 Fluorescence In Situ Hybridization (FISH)for the Detection of Bacterial Resistance
FISH (fluorescence in situ hybridization) is a cheap andconvenient option for the identification and resistance testingof bacterial pathogens Traditional FISH is based on specifichybridization of short usually 18ndash25 bases long fluorescent-labelled single-stranded oligonucleotide probes to ribosomalRNA (rRNA) of the target organismwith subsequent analysisunder the fluorescence microscope usually allowing forthe identification of microbes at genus or species level Inprinciple each kind of intracellular RNA can be hybridizedwith FISH probes However rRNA is particularly well suitedas a FISH target because ribosomes are numerous in aprotein-synthesizing cell thus allowing for a boostering offluorescence intensity [91]
This traditional FISH method is both rapid and easy tostandardize so it can be applied for molecular rapid testingSmall modifications of the procedure comprise the use ofpatent-protected commercial peptide nucleic acid (PNA)probes or probes containing locked nucleic acids (LNA)instead of simple single-stranded DNA probes PNA-FISHtechnology reduces nonspecific probe attachment due tothe electrically neutral backbone of the oligonucleotides andis recommendable for routine diagnostics due to a higherdegree of standardization However patent-protected PNA
probes are expensive although they are well suited for thediagnostic routine setting [92]
FISH is particularly suitable for the detection of resistancedeterminants if two prerequisites are guaranteed Ribosoma-llymediated resistance for example affecting antibiotic drugslike macrolide or linezolid is well suited because riboso-mal RNA copies are numerous in living cells allowing forbright fluorescence signals Further FISH can be successfullyapplied if only one or few variable bases provide resistanceso there is no need for a large number of probes in the probepanel
These prerequisites are fulfilled in case of clarithromycinresistance testing in Helicobacter pylori Therefore FISH-based resistance testingwas early evaluated for this indication[93] Clarithromycin in H pylori is basically mediated bythree point mutations in the ribosomal 23S rRNA [94]which can be addressed by three described FISH probesClaR1 ClaR2 and ClaR3 [93] (Table 1) While ClaR1 isassociated with a minimum inhibitory concentration (MIC)of gt64mgL ClaR2 and ClaR3 are associated with varyingMICs between 8mgL and 64mgL [94]
The FISH probes for clarithromycin resistance testing inH pylori were successfully applied to bacteria both fromculture and in bioptic material and extensively assessed invarious studies [93 95ndash97] Reliable test results can even beachieved in formalin-fixed paraffin-embedded tissue afteradequate deparaffination [98] The combined use of probeslabelled with different fluorescence molecules allows for theidentification of coinfections with clarithromycin-sensitiveand -resistant H pylori strains by FISH [99]
Commercial test providers distributed the robust andeasy-to-apply procedure In one study with such a com-mercial test kit [100] a sensitivity of 90 and a specificityof 100 were achieved for the detection of clarithromycin-resistant H pylori within bioptic material In another studyoccasional false-positive H pylori detections were generated[101] although the results of FISH-based resistance test-ing of correctly identified H pylori proved to be reliableRecently a PNA probe-based approach for clarithromycinresistance testing in H pylori showed perfect matching withPCRsequencing in a retrospective studywith formalin-fixedparaffin-embedded tissues (Table 2) [102]
Similar to H pylori FISH-based clarithromycin resis-tance testing could be successfully demonstrated for ther-motolerant Campylobacter spp with a wild-type probe anda clarithromycin resistance probe targeting the A2059Gmutation in the 23S rRNA gene (Table 3) The observedsensitivity and specificity with culture material were 100[103]
Comparable to clarithromycin resistance linezolid resis-tance is ribosomally mediated In enterococci it is typicallycaused by a 2567GgtT base substitution in the 23S rRNA(Table 4) In a collection of 106 enterococcal isolates acorresponding linezolid resistance FISH assay succeeded inpredicting phenotypic resistance in 100 of cases [104]Even a single mutated allele was associated with strongfluorescence signals
First successful attempts of FISH-based resistance testingwere described for non-rRNA-based resistance mechanisms
BioMed Research International 7
Table 1 DNA-FISH-probes detecting clarithromycin resistance in H pylori Russmann et al 2001a [93]
Target Probe Probe sequenceWild type ClaWT 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Table 2 PNA-FISH-probes detecting clarithromycin resistance in H pylori Cerqueira et al 2013 [102] shortened versions of the DNA-FISH-probes from Table 1
Target Probe Probe sequenceWild type HpWT 51015840-GGT-CTT-TCC-GTC-T-31015840
Table 3 DNA-FISH-probes detecting clarithromycin resistance in thermotolerantCampylobacter spp Haas et al 2008 [103] Of note probeC wt 23S is identical with probe ClaWT probe C res 23S 2059AgtG with probe ClaR2 (Table 1)
Target Probe Probe sequenceWild type C wt 23S 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Clarithromycin resistance mutation (A2059G) C res 23S 2059AgtG 51015840-CGG-GGT-CTC-TCC-GTC-TT-31015840
Table 4 DNA-FISH-probes detecting linezolid resistance in enterococci Locked nucleic acids (LNA) were used at the mismatch position(bold underlined print) within in probes
Target Probe Probe sequenceWild type LZD-WT 51015840-CCC-AGC-TCG-CGT-GC-31015840
Agglutination assays lt5 minutes mdash lt100C LowFluorescence in situ hybridization 1-2 hours lt1500000C 100ndash800C IntermediateReal-time PCR(including DNA preparation) 4ndash6 hours 3500000ndash6000000C 1500ndash2500C Strongly depending on
the test systemLoop-mediated isothermal amplification(LAMP) assays lt1 hour 200000ndash400000C 1500ndash2500C Intermediate
Next generation sequencing (NGS) 2ndash5 days 35000000ndash75000000C 7500ndash80000C Very highMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry(MALDI-TOF-MS)
lt5 minutes 7500000ndash30000000C lt100C High
as well FISH-based detection of blaSHV-238240 one of thegenes coding for extended-spectrum 120573-lactamases (ESBL) isan example of a non-rRNA-based FISHprotocol for detectinga particular resistance determinant using the probe 51015840-GAC-CGG-AGC-TAG-CAA-GCG-31015840 [105] However the ESBLphenotype can be associated with a variety of different allelesso this particular probe will be of use only in case of a specificsuspicion for example during an outbreakAccordingly such
a procedure will be reserved for very few if any indications inthe diagnostic routine
Further progression of FISH technology comprisessignal-amplified catalyzed reported deposition (CARD)FISH doubly labeled oligonucleotide probe- (DOPE-) basedFISH combinatorial labelling and spectral imaging (CLASI)FISH and the combination of FISH with other diagnosticapproaches aswell as FISHprocedures for gene identification
8 BioMed Research International
requiring in situ amplification of the respective gene as incase of the rolling circle amplification (RCA) FISH [106]RCA-FISH was successfully applied for the identification ofthe mecA gene in Methicillin resistant Staphylococcus aureus(MRSA) based on the mecA-probes MR-1 51015840-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-31015840 andMR-2 51015840-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-31015840 [107]
FISH-based detection of resistance determinants is apromising diagnostic approach due to its rapidity conve-nience and cost effectiveness The associated rapid detectionof antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associ-ated benefits for the patientrsquos health The main advantage ofFISH is its potential use for resistance testing directly fromprimarymaterial including tissuewith low effort So FISHcanalso be applied in resource-limited settings where expensivetechnologies are not available (Figure 1) In contrast to PCRFISH can also attribute a particular resistance mechanism toa microscopically observed bacterium
However so far FISH is restricted to very few indicationsfor which protocols have been described As a furtherdrawback standardization of FISH-based resistance testing iswidely missing If applied from primary samplematerials liketissue tissue autofluorescence has to be considered requiringconsiderable experience to interpret such diagnostic resultsTo reduce potential interpretation errors FISH from tissuefurther requires counterstaining with a pan-eubacterial FISHprobe and nonspecificDNA staining for example withDAPI(410158406-diamidino-2-phenylindole) to confirm the presence ofnucleic acids of the detected pathogens as recently demanded[108]
Given all these limitations FISH for resistance testingwillpresumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market
5 Direct Fluorescent Imaging ofResistance Determinants by FluorescenceResonance Energy Transfer (FRET)
Nonnucleotide probes labelled with reporter and quenchermolecules allowing for fluorescence energy transfer (FRET)can be used to detect enzymatic resistance mechanisms asdescribed for 120573-lactamases [109] After enzymatic hydrolyza-tion of probes to separate the quencher from the reporter thehydrolyzed probes attach the resistance enzymes as reactiveelectrophiles However this mechanism has so far been onlydescribed for 120573-lactamases in a proof-of-principle analysis[109] and broad evaluation studies are missing Its practicalrelevance for the microbiological routine diagnostics willrequire further evaluation
6 Mass Spectrometric Approaches
Matrix-assisted laser desorption ionization time-of-flightmass spectrometry- (MALDI-TOF MS-) based intact cellmass spectrometry (ICMS) has recently advanced to the stan-dard method for species identification for cultured bacteria
and fungi [24 110ndash114] Promising approaches have beenmade using ICMS spectra for subspecies identification [115]This technique bears a high potential for the fast identi-fication of susceptibility associated biomarker ions that islately only marginally realized in clinical routine diagnosticsThus phyloproteomic approaches help to identify indirectlymostly chromosomal encoded resistance genes by identifyingphylogenetic relatedness [116ndash121] MS can be used to detectchanges in the bacterial or fungal proteome induced byexposition to antimicrobials [24 122ndash124] Whole proteomechanges in consequence of exposition to antimicrobials canbe also detected using stable isotope labeled amino acids(SILAC) [125 126] One very promising approach is the so-calledmass spectrometric beta-lactamase (MSBL) assay [127ndash131] which is based on the mass spectrometric detection ofhydrolyzed beta-lactams Finally there is the combination ofgenotypic and mass spectrometric methods PCR ampliconscan be characterized by PCRelectrospray ionization-massspectrometry (PCRESI MS) [132] and minisequencing [133134] and mass spectrometry-based comparative sequenceanalysis [135 136] can be used to detect susceptibility changesassociated with point mutations
61 Prediction of Broad Spectrum Resistant Clonal Groupsby Phyloproteomics MALDI-TOFMS-based intact cell massspectrometry (ICMS) is potentially able to characterizestrains at the subspecies level and could act as useful toolfor taxonomy and epidemiology [137 138] For the discrim-ination of representative strains particular biomarker ionsthat were completely present or absent as well as shiftsin biomarker masses in a particular subset of strains wereconsidered Using different mathematical algorithms it wasfor example feasible to discriminate Salmonella enterica sspenterica serovar Typhi from other less virulent Salmonellaenterica ssp enterica serotypes [139] to distinguish Campy-lobacter jejuni MLST-ST22 and ST45 from other MLSTsequence types [140] or to perform phyloproteomic analysisof Rhodococcus erythropolis [141] Pseudomonas putida [142]or Neisseria menigitidis [143]
Thefirst approaches to associateMSfingerprintswith sus-ceptibility patterns were designed to differentiate methicillinsusceptible Staphylococcus aureus (MSSA) from methicillinresistant Staphylococcus aureus (MRSA) [144ndash148] Thesewere mostly not standardized and hardly reproducible Butrelatively good reproducibility was demonstrated for thediscrimination of the five major MRSA clonal complexesCC5 CC8 CC22 CC30 and CC45 corresponding to thefive major PFGE MRSA types regardless of their methicillinsensitivity [149 150] A study by Lu and coworkers identifieda set of biomarkers that were able to distinguish betweenmethicillin resistant and vancomycin-intermediate S aureus(VISA) strains and vancomycin-susceptible S aureus strainsas well as between SCCmec types IV and V isolates andSCCmec types IndashIII isolates [151] Further studies demon-strated that isogenic S aureus lacking or artificially harboringSCCmec could not be distinguished in a mass range from2000 to 15000119898119911 [152] whereas isogenic MRSA whichspontaneously reverted to MSSA could be discriminated byMALDI-TOF MS [153]
BioMed Research International 9
(a)
(b)
(c)
(d)
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
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[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
[20] WHO Global Tuberculosis Report 2013 World Health Organi-zation Geneva Switzerland 2013
[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
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[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
Table 2 PNA-FISH-probes detecting clarithromycin resistance in H pylori Cerqueira et al 2013 [102] shortened versions of the DNA-FISH-probes from Table 1
Target Probe Probe sequenceWild type HpWT 51015840-GGT-CTT-TCC-GTC-T-31015840
Table 3 DNA-FISH-probes detecting clarithromycin resistance in thermotolerantCampylobacter spp Haas et al 2008 [103] Of note probeC wt 23S is identical with probe ClaWT probe C res 23S 2059AgtG with probe ClaR2 (Table 1)
Target Probe Probe sequenceWild type C wt 23S 51015840-CGG-GGT-CTT-TCC-GTC-TT-31015840
Clarithromycin resistance mutation (A2059G) C res 23S 2059AgtG 51015840-CGG-GGT-CTC-TCC-GTC-TT-31015840
Table 4 DNA-FISH-probes detecting linezolid resistance in enterococci Locked nucleic acids (LNA) were used at the mismatch position(bold underlined print) within in probes
Target Probe Probe sequenceWild type LZD-WT 51015840-CCC-AGC-TCG-CGT-GC-31015840
Agglutination assays lt5 minutes mdash lt100C LowFluorescence in situ hybridization 1-2 hours lt1500000C 100ndash800C IntermediateReal-time PCR(including DNA preparation) 4ndash6 hours 3500000ndash6000000C 1500ndash2500C Strongly depending on
the test systemLoop-mediated isothermal amplification(LAMP) assays lt1 hour 200000ndash400000C 1500ndash2500C Intermediate
Next generation sequencing (NGS) 2ndash5 days 35000000ndash75000000C 7500ndash80000C Very highMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry(MALDI-TOF-MS)
lt5 minutes 7500000ndash30000000C lt100C High
as well FISH-based detection of blaSHV-238240 one of thegenes coding for extended-spectrum 120573-lactamases (ESBL) isan example of a non-rRNA-based FISHprotocol for detectinga particular resistance determinant using the probe 51015840-GAC-CGG-AGC-TAG-CAA-GCG-31015840 [105] However the ESBLphenotype can be associated with a variety of different allelesso this particular probe will be of use only in case of a specificsuspicion for example during an outbreakAccordingly such
a procedure will be reserved for very few if any indications inthe diagnostic routine
Further progression of FISH technology comprisessignal-amplified catalyzed reported deposition (CARD)FISH doubly labeled oligonucleotide probe- (DOPE-) basedFISH combinatorial labelling and spectral imaging (CLASI)FISH and the combination of FISH with other diagnosticapproaches aswell as FISHprocedures for gene identification
8 BioMed Research International
requiring in situ amplification of the respective gene as incase of the rolling circle amplification (RCA) FISH [106]RCA-FISH was successfully applied for the identification ofthe mecA gene in Methicillin resistant Staphylococcus aureus(MRSA) based on the mecA-probes MR-1 51015840-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-31015840 andMR-2 51015840-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-31015840 [107]
FISH-based detection of resistance determinants is apromising diagnostic approach due to its rapidity conve-nience and cost effectiveness The associated rapid detectionof antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associ-ated benefits for the patientrsquos health The main advantage ofFISH is its potential use for resistance testing directly fromprimarymaterial including tissuewith low effort So FISHcanalso be applied in resource-limited settings where expensivetechnologies are not available (Figure 1) In contrast to PCRFISH can also attribute a particular resistance mechanism toa microscopically observed bacterium
However so far FISH is restricted to very few indicationsfor which protocols have been described As a furtherdrawback standardization of FISH-based resistance testing iswidely missing If applied from primary samplematerials liketissue tissue autofluorescence has to be considered requiringconsiderable experience to interpret such diagnostic resultsTo reduce potential interpretation errors FISH from tissuefurther requires counterstaining with a pan-eubacterial FISHprobe and nonspecificDNA staining for example withDAPI(410158406-diamidino-2-phenylindole) to confirm the presence ofnucleic acids of the detected pathogens as recently demanded[108]
Given all these limitations FISH for resistance testingwillpresumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market
5 Direct Fluorescent Imaging ofResistance Determinants by FluorescenceResonance Energy Transfer (FRET)
Nonnucleotide probes labelled with reporter and quenchermolecules allowing for fluorescence energy transfer (FRET)can be used to detect enzymatic resistance mechanisms asdescribed for 120573-lactamases [109] After enzymatic hydrolyza-tion of probes to separate the quencher from the reporter thehydrolyzed probes attach the resistance enzymes as reactiveelectrophiles However this mechanism has so far been onlydescribed for 120573-lactamases in a proof-of-principle analysis[109] and broad evaluation studies are missing Its practicalrelevance for the microbiological routine diagnostics willrequire further evaluation
6 Mass Spectrometric Approaches
Matrix-assisted laser desorption ionization time-of-flightmass spectrometry- (MALDI-TOF MS-) based intact cellmass spectrometry (ICMS) has recently advanced to the stan-dard method for species identification for cultured bacteria
and fungi [24 110ndash114] Promising approaches have beenmade using ICMS spectra for subspecies identification [115]This technique bears a high potential for the fast identi-fication of susceptibility associated biomarker ions that islately only marginally realized in clinical routine diagnosticsThus phyloproteomic approaches help to identify indirectlymostly chromosomal encoded resistance genes by identifyingphylogenetic relatedness [116ndash121] MS can be used to detectchanges in the bacterial or fungal proteome induced byexposition to antimicrobials [24 122ndash124] Whole proteomechanges in consequence of exposition to antimicrobials canbe also detected using stable isotope labeled amino acids(SILAC) [125 126] One very promising approach is the so-calledmass spectrometric beta-lactamase (MSBL) assay [127ndash131] which is based on the mass spectrometric detection ofhydrolyzed beta-lactams Finally there is the combination ofgenotypic and mass spectrometric methods PCR ampliconscan be characterized by PCRelectrospray ionization-massspectrometry (PCRESI MS) [132] and minisequencing [133134] and mass spectrometry-based comparative sequenceanalysis [135 136] can be used to detect susceptibility changesassociated with point mutations
61 Prediction of Broad Spectrum Resistant Clonal Groupsby Phyloproteomics MALDI-TOFMS-based intact cell massspectrometry (ICMS) is potentially able to characterizestrains at the subspecies level and could act as useful toolfor taxonomy and epidemiology [137 138] For the discrim-ination of representative strains particular biomarker ionsthat were completely present or absent as well as shiftsin biomarker masses in a particular subset of strains wereconsidered Using different mathematical algorithms it wasfor example feasible to discriminate Salmonella enterica sspenterica serovar Typhi from other less virulent Salmonellaenterica ssp enterica serotypes [139] to distinguish Campy-lobacter jejuni MLST-ST22 and ST45 from other MLSTsequence types [140] or to perform phyloproteomic analysisof Rhodococcus erythropolis [141] Pseudomonas putida [142]or Neisseria menigitidis [143]
Thefirst approaches to associateMSfingerprintswith sus-ceptibility patterns were designed to differentiate methicillinsusceptible Staphylococcus aureus (MSSA) from methicillinresistant Staphylococcus aureus (MRSA) [144ndash148] Thesewere mostly not standardized and hardly reproducible Butrelatively good reproducibility was demonstrated for thediscrimination of the five major MRSA clonal complexesCC5 CC8 CC22 CC30 and CC45 corresponding to thefive major PFGE MRSA types regardless of their methicillinsensitivity [149 150] A study by Lu and coworkers identifieda set of biomarkers that were able to distinguish betweenmethicillin resistant and vancomycin-intermediate S aureus(VISA) strains and vancomycin-susceptible S aureus strainsas well as between SCCmec types IV and V isolates andSCCmec types IndashIII isolates [151] Further studies demon-strated that isogenic S aureus lacking or artificially harboringSCCmec could not be distinguished in a mass range from2000 to 15000119898119911 [152] whereas isogenic MRSA whichspontaneously reverted to MSSA could be discriminated byMALDI-TOF MS [153]
BioMed Research International 9
(a)
(b)
(c)
(d)
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
[1] WHO Antimicrobial Resistance Global Report on SurveillanceWHO 2014
[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
[20] WHO Global Tuberculosis Report 2013 World Health Organi-zation Geneva Switzerland 2013
[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
BioMed Research International 15
[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
requiring in situ amplification of the respective gene as incase of the rolling circle amplification (RCA) FISH [106]RCA-FISH was successfully applied for the identification ofthe mecA gene in Methicillin resistant Staphylococcus aureus(MRSA) based on the mecA-probes MR-1 51015840-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-31015840 andMR-2 51015840-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-31015840 [107]
FISH-based detection of resistance determinants is apromising diagnostic approach due to its rapidity conve-nience and cost effectiveness The associated rapid detectionof antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associ-ated benefits for the patientrsquos health The main advantage ofFISH is its potential use for resistance testing directly fromprimarymaterial including tissuewith low effort So FISHcanalso be applied in resource-limited settings where expensivetechnologies are not available (Figure 1) In contrast to PCRFISH can also attribute a particular resistance mechanism toa microscopically observed bacterium
However so far FISH is restricted to very few indicationsfor which protocols have been described As a furtherdrawback standardization of FISH-based resistance testing iswidely missing If applied from primary samplematerials liketissue tissue autofluorescence has to be considered requiringconsiderable experience to interpret such diagnostic resultsTo reduce potential interpretation errors FISH from tissuefurther requires counterstaining with a pan-eubacterial FISHprobe and nonspecificDNA staining for example withDAPI(410158406-diamidino-2-phenylindole) to confirm the presence ofnucleic acids of the detected pathogens as recently demanded[108]
Given all these limitations FISH for resistance testingwillpresumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market
5 Direct Fluorescent Imaging ofResistance Determinants by FluorescenceResonance Energy Transfer (FRET)
Nonnucleotide probes labelled with reporter and quenchermolecules allowing for fluorescence energy transfer (FRET)can be used to detect enzymatic resistance mechanisms asdescribed for 120573-lactamases [109] After enzymatic hydrolyza-tion of probes to separate the quencher from the reporter thehydrolyzed probes attach the resistance enzymes as reactiveelectrophiles However this mechanism has so far been onlydescribed for 120573-lactamases in a proof-of-principle analysis[109] and broad evaluation studies are missing Its practicalrelevance for the microbiological routine diagnostics willrequire further evaluation
6 Mass Spectrometric Approaches
Matrix-assisted laser desorption ionization time-of-flightmass spectrometry- (MALDI-TOF MS-) based intact cellmass spectrometry (ICMS) has recently advanced to the stan-dard method for species identification for cultured bacteria
and fungi [24 110ndash114] Promising approaches have beenmade using ICMS spectra for subspecies identification [115]This technique bears a high potential for the fast identi-fication of susceptibility associated biomarker ions that islately only marginally realized in clinical routine diagnosticsThus phyloproteomic approaches help to identify indirectlymostly chromosomal encoded resistance genes by identifyingphylogenetic relatedness [116ndash121] MS can be used to detectchanges in the bacterial or fungal proteome induced byexposition to antimicrobials [24 122ndash124] Whole proteomechanges in consequence of exposition to antimicrobials canbe also detected using stable isotope labeled amino acids(SILAC) [125 126] One very promising approach is the so-calledmass spectrometric beta-lactamase (MSBL) assay [127ndash131] which is based on the mass spectrometric detection ofhydrolyzed beta-lactams Finally there is the combination ofgenotypic and mass spectrometric methods PCR ampliconscan be characterized by PCRelectrospray ionization-massspectrometry (PCRESI MS) [132] and minisequencing [133134] and mass spectrometry-based comparative sequenceanalysis [135 136] can be used to detect susceptibility changesassociated with point mutations
61 Prediction of Broad Spectrum Resistant Clonal Groupsby Phyloproteomics MALDI-TOFMS-based intact cell massspectrometry (ICMS) is potentially able to characterizestrains at the subspecies level and could act as useful toolfor taxonomy and epidemiology [137 138] For the discrim-ination of representative strains particular biomarker ionsthat were completely present or absent as well as shiftsin biomarker masses in a particular subset of strains wereconsidered Using different mathematical algorithms it wasfor example feasible to discriminate Salmonella enterica sspenterica serovar Typhi from other less virulent Salmonellaenterica ssp enterica serotypes [139] to distinguish Campy-lobacter jejuni MLST-ST22 and ST45 from other MLSTsequence types [140] or to perform phyloproteomic analysisof Rhodococcus erythropolis [141] Pseudomonas putida [142]or Neisseria menigitidis [143]
Thefirst approaches to associateMSfingerprintswith sus-ceptibility patterns were designed to differentiate methicillinsusceptible Staphylococcus aureus (MSSA) from methicillinresistant Staphylococcus aureus (MRSA) [144ndash148] Thesewere mostly not standardized and hardly reproducible Butrelatively good reproducibility was demonstrated for thediscrimination of the five major MRSA clonal complexesCC5 CC8 CC22 CC30 and CC45 corresponding to thefive major PFGE MRSA types regardless of their methicillinsensitivity [149 150] A study by Lu and coworkers identifieda set of biomarkers that were able to distinguish betweenmethicillin resistant and vancomycin-intermediate S aureus(VISA) strains and vancomycin-susceptible S aureus strainsas well as between SCCmec types IV and V isolates andSCCmec types IndashIII isolates [151] Further studies demon-strated that isogenic S aureus lacking or artificially harboringSCCmec could not be distinguished in a mass range from2000 to 15000119898119911 [152] whereas isogenic MRSA whichspontaneously reverted to MSSA could be discriminated byMALDI-TOF MS [153]
BioMed Research International 9
(a)
(b)
(c)
(d)
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
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[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
[20] WHO Global Tuberculosis Report 2013 World Health Organi-zation Geneva Switzerland 2013
[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
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[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
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[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
Figure 1 Little equipmentmdashas here exemplified by material from the Institute for Microbiology Virology and Hygiene University MedicalCenter Rostockmdashis required for performing FISH analyses (a) Glass apparatus for fixing and washing of slides (b) Slide chamber allowingfor a rapid and steady heat transmission (c) Incubator for the washing step (d) Multichannel fluorescence microscope
One study from New Zealand showed that the discrim-ination of vanB positive vancomycin-resistant Enterococcusfaecium (VRE) and vancomycin-susceptible E faecium usingICMS fingerprinting is feasible [121] but these findings werenot reproducible in other areas Thus it was speculated thatthis was just reflecting the specific epidemiological situationin New Zealand [125]
Other studies on Clostridium difficile demonstrated asufficient discriminatory power of MALDI-TOF MS spectraanalysis to recognize the PCR ribotypes 001 027 and 126078[116] Phyloproteomic analysis is a sufficient tool to identifyhigh-virulent or multidrug-resistant strains of particularbacterial species if their virulence or their resistance isassociated with phylogenetic and therewith phyloproteomicrelatedness Thus it is an up-and-coming technique not onlyfor epidemiological surveys but also for individual patientmanagement
Compared to Gram-positive bacteria Gram-negativebacteria are particularly problematic because their resistancegenes are often encoded on plasmids which can be easilyexchanged with other Gram-negative bacteria even acrossspecies boundaries [154] But some of the extended beta-lactamase genes (ESBL) and carbapenemases are associ-ated with particular bacterial clonal complexes Klebsiellapneumoniae ST258 (expressing KPC carbapenemase) and Ecoli ST131 ST69 ST405 and ST393 (expressing ESBL) [155]belong to these clonal complexes
Similar phyloproteomic analysis has been successfullydemonstrated to discriminate between different subsets of Ecoli strains [156] Coupling MALDI-TOF MS with multivari-ate data analysis allows for discriminating ESBL-expressingE coli B2 ST131 and D (ST69 ST393 and ST405) from otherE coli strains [117 118]
One likely problem in the calculated treatment of Bac-teroides fragilis infections is the possibility that some strainsexpress a high-potential metallo-120573-lactamase encoded by thegene cfiA [157]Themicrobial species B fragilis is subdividedinto two divisions (I and II) and usually only isolates ofdivision II harbor cfiA Recently two independent studiesidentified a set of biomarkers or precisely shifts in biomarkermasses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [119 120]
62 Detection of Whole Proteome Changes Induced by Echi-nocandins Echinocandins namely anidulafungin caspo-fungin and micafungin are the treatment of choice forinvasive and systemic infectionswithCandida andAspergillusspecies They also comprise important reserve antimicro-bial agents especially in the case of infections with azole-resistant strains for example Aspergillus species Due tothe increasing use of echinocandins in the treatment offungal infections the prevalence of echinocandin-resistantisolates caused by mutations in the fks1-3 (hypersensitive forthe immunosuppressant FK560) genes increases [158] Thus
10 BioMed Research International
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
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[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
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[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
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[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
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[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
rapid identification of azole and echinocandin susceptibilityare needful for a successful therapy of systemic mycoses
In a pioneer study the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility of Can-dida albicans was shown by Marinach and coworkers [122]During the test procedure Candida cells were incubated for24 hours in liquid medium containing different concentra-tions of fluconazole After harvesting and acid extraction ofthe Candida cell pellets the supernatants were spotted on aMALDI-TOF target plate and mass spectra were recordedComparable to the estimation of minimal inhibitory concen-trations (MIC) the so-called minimal profile changing con-centration (MPCC) the lowest concentration of fluconazoleat which changes in the mass spectrum were recordable wasestimated by comparing the mass spectra of the particularsuspensions of the fluconazole dilution series RemarkablyMPCC differed only in one dilution step from the MIC andtherewith it is a comparably sufficient parameter reflectingantimicrobial susceptibility [122]
de Carolis and coworkers adapted this procedure to testC albicans Candida glabrata Candida parapsilosis Can-dida krusei Aspergillus fumigatus and Aspergillus flavus forechinocandin MICs that are due to mutations in fks1 andin the case of C glabrata also in fks2 [123] Additionallythey accelerated the data analysis by applying compositecorrelation index (CCI) analysis The CCI value was calcu-lated in comparison to reference spectra of the two extremeconcentrations [123]
This procedure was further optimized by Vella andcoworkers [124] They reduced the incubation period downto 3 hours by incubating the yeast cell suspension withoutas well as with two different echinocandin concentrationscorresponding to intermediate and complete resistance [124]
63 Stable Isotope Labeling by Amino Acids in Cell Culture(SILAC) The successful application of mass spectrometry(MS) in the detection of antimicrobial resistance has alsoopened a door for the entry of another quantitative pro-teomics approach known as SILAC into the era of rapiddetection of antibiotic resistance This approach is basedon the principle that proteins are made up of amino acidsHence cells grown in media supplemented with amino acidsincorporate these amino acids into their cellular proteome[125] In addition protein profiles of a metabolically activecell reveal its metabolic activities at a specific time Alreadyestablished SILAC antimicrobial detection protocols to detectantibiotic resistance involve the growth of three cultures ofthe test strain The first culture is grown in medium withnormal (light) essential amino acids the second culture isgrown in media supplemented with labeled (heavy) essentialamino acids and the third culture is grown in media sup-plemented with both labeled (heavy) essential amino acidsand the analyzed antimicrobial drugThese three cultures aremixed their proteomes are extracted and measured by MSand the peaks are compared The test strain is classified assusceptible if its protein peak profile is similar to that of thefirst culture On the other hand it is classified as resistant ifits protein peak profile is similar to the second culture [159]This approach has been successfully used to differentiate
methicillin susceptible S aureus (MSSA) and methicillinresistant S aureus (MRSA) [160] Also it has been success-fully used to test the susceptibility of P aeruginosa to threeantibiotics of different classes with different modes of actionmeropenem (120573-lactam antibiotic) tobramycin (aminogly-coside) and ciprofloxacin (fluoroquinolone) [126] In bothcases the results were assessed after 2 to 4 hours and theresults were comparable to those obtained from minimuminhibitory concentration (MIC) testing In addition to theseadvantages SILAC is easy and straightforward to performFor this reason very soon it may be used to detect antimi-crobial resistance in antiviral antifungal and antiparasiticdrugs
64 Mass Spectrometric 120573-Lactamase Assay In contrast tothe aforementioned mass spectrometric assays the massspectrometric 120573-lactamase assay (MSBL) is not based on theanalysis of the bacterial proteome The MSBL is based on thedirect mass spectrometric detection of 120573-lactamase metabo-lites [127ndash131] The procedure is as follows First bacteriaare suspended in a buffered solution with and for referencewithout a 120573-lactam antibiotic This suspension is incubatedfor 1 to 3 hours After centrifugation the supernatants areanalyzed byMALDI-TOFMS Specific peaks (mass shifts) forintact and hydrolyzed 120573-lactams indicate functional presenceof 120573-lactamases It was demonstrated that the MSBL deliversresults within 25 hours for bacteria inactivating ampicillinpiperacillin cefotaxime ceftazidime ertapenem imipenemand meropenem [131] Thus particularly NDM-1 VIM-12 KPC-1-3 OXA-48 OXA-162 and IMP carbapenemaseexpression by Enterobacteriaceae Acinetobacter baumanniiand Pseudomonas spp was detectable [128 130]
With a total turn-around-time after positive primarybacterial culture of circa 4 hours this method is significantlyfaster than culture-based susceptibility testing [127ndash131]
65 Mass Spectrometric Analysis of PCR Products PCRESIMS PCRelectrospray ionization-mass spectrometry (PCRESIMS) combines nucleic acid amplificationwithmass spec-trometric analysis of the amplicons which are brought into agas phase using electrospray ionizationThemajor advantageof this technique is its highmultiplexing capacity that enablesthe parallel detection of a wide panel of resistance genesIt was demonstrated that PCRESI MS is able to accuratelydetect nine different KPC carbapenemases (blaKPC-2-10) [132]as well as the gyrA and parC point mutations which areassociated with quinolone resistance in A baumannii [161]
Also because of its high multiplexing capacity PCRESIMS is a suitable tool for simultaneous (sub)species identifi-cation and resistance gene detection which is of particularimportance for the treatment of mycobacterial infections Onthe one hand it is necessary to distinguish nontuberculosismycobacteria (NTM) from M tuberculosis on the otherhand multidrug-resistant tuberculosis (MDR-TB) strainsmust be detected PCRESIMS-based assays have been devel-oped to facilitate NTM species identification and paralleldetection of resistance genes associated with rifampicin
BioMed Research International 11
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
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[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
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[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
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[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
isoniazid ethambutol and fluoroquinolone resistance in TBand NTM [162] Moreover there are enormous time savingscompared to traditional mycobacterial culture and resistancetesting via the agar proportion method [162ndash164]
The high sensitivity of PCRESI MS in the detectionof hard-to-culture or even nonculturable bacteria makes ita reliable method for the direct detection of pathogens inhardly acquirable samples like heart valves [165] as well as forsurveillance studies [166 167]
66 Minisequencing-Primer Extension Followed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Analysis(PEXMALDI-TOF) Another method that was also adaptedfor the rapid detection of ganciclovir resistance in HCMV(human cytomegalovirus) by Zurcher and coworkers is singlenucleotide primer extension (also known as minisequencingor PinPoint assay) followed by matrix-assisted laser desorp-tionionization time-of-flight analysis (PEXMALDI-TOF)[134] In general the combination of PEX and MALDI-TOF MS is a cost-efficient high-throughput method for thedetection of single nucleotide polymorphisms (SNPs) [133]The PEXMALDI-TOF workflow using patient plasma is asfollows [134]
For the primer extension reaction the reverse PEXprimer (51015840-CTT-GCC-GTT-CTC-CAA-C-31015840) was added inhigh concentration The 31015840-end of the primer is locateddirectly at the site of mutation (A594V GCGwild typerarr GTGmutant) to be detected The extension reactioncatalyzed by a DNA polymerase is terminated in the case ofa wild-type allele just after one nucleotide complementary tothe mutated nucleotide and in the case of a mutant after twonucleotides by a didesoxynucleotide (ddNTP) Because of themolecular weight difference in consequence of the varyingmass increase of the PEX primer mutant and wild type canbe discriminated using MALDI-TOF MS [133]
According to current standards HCMVresistance testingis performed using Sanger sequencing [168] By monitoringa patient cohort of five individuals using Sanger sequencingand PEXMALDI-TOF Zurcher et al could demonstratethat the PEXMALDI-TOF method is much more sensitivethan the Sanger method PEXMALDI-TOF requires thepresence of only 20ndash30 of the ganciclovir unsusceptibleHCMVquasispecies to reliably detect the resistancemutation[134] In consequence this method was able to detect theappearance of the UL97 resistance mutation already ten daysafter the ldquolast wild-type only constitutionrdquo whereas Sangersequencing detected the appearance of the resistant subpopu-lation at day 20 [134] Consequently a ganciclovir therapy canbe monitored by PEXMALDI-TOF more contemporary Anecessary change in therapy may be done earlier and criticaltime for the preservation of the graft and the patient can besaved
A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae [169] Conversion of TEMpenicillinases to TEM-type ESBL is mostly due to aminoacid substitutions at Amblerrsquos positions Glu104 Arg164 andGly238 [170] To detect these SNPs in the 119887119897119886TEM genes a setof seven internal primers have been designed to bind near
the three codons of Amblerrsquos positions in such a way thatthe masses of all possible reactions products are maximallydistant fromeach other and are easy to distinguish in themassspectrum All primers are used in one multiplex reactionThus it is feasible to detect different types of TEM-type ESBLin one reaction [169]
Other minisequencing protocols have been establishedto detect fluoroquinolone resistance related SNPs in Ngonorrhoeae [171] clarithromycin resistance in Helicobacterpylori [172] and rifampin and isoniazid-resistance in Mtuberculosis [173]
67 MSCSA-Mass Spectrometry-Based Comparative SequenceAnalysis to Detect Ganciclovir Resistance Mass spectrom-etry-based comparative sequence analysis (MSCSA) was ini-tially established by Honisch and coworkers (SEQUENOMSan Diego USA) for the genotyping of bacteria usingmass spectrometric fingerprinting of the standardmultilocussequence typing (MLST) loci [135]
The MSCSA principle was adapted to facilitate the detec-tion of mutations in the UL97 gene to detect ganciclovirresistance of human cytomegalovirus (HCMV) [136]
HCMV reactivation occurs frequently in consequenceof immune suppression especially after stem cell and solidorgan transplantation [174]Thus HCMV infection may leadto graft dysfunction or even rejection To counteract thisantiviral treatment with the analogue of 21015840-deoxy-guanosineganciclovir is indicated [175] Under therapy whichmay spanseveral months it is necessary to monitor the emergence ofresistance and possibly switch to other drugs such as themore toxic foscarnet [176] Ganciclovir resistance is typicallya consequence of single nucleotide polymorphisms in the 31015840-region of theUL97 kinase gene encoding a viral kinase whichactivates ganciclovir by phosphorylation [177]
These UL97 single nucleotide polymorphisms aredetected by MSCSA as follows after DNA isolation fromEDTA-plasma samples the 31015840-region of the UL97 is amplifiedin two amplicons using T7-promotor-tagged forward primersand SP6-tagged reverse primers Both amplicons are in vitrotranscribed in two separate reactions using T7 and SP6RNA polymerase followed by cytosine or uracil specificRNaseA cleavage of plus and minus strand RNA transcriptsAfter this all four obtained RNaseA cleavage products aretransferred to a SpectroCHIP array (SEQUENOM SanDiego USA) MALDI-TOF mass spectra are recorded andin silico compared to calculated MS spectra of referencesequences Based on the obtained data the UL97 sequencecan be assembled and thereby the presence of a ganciclovirresistance associated single nucleotide polymorphism canbe detected [136] Due to the automation of post-PCRprocessing and analysis as well as reduced hands-on timeacceleration of the detection process of ganciclovir resistancecan be achieved
7 Conclusions and Outlook
To solve the increasing problem of a worldwide rising preva-lence of infections due to multidrug- or even pan-drug-resistant bacteria medical microbiology has to establish a
12 BioMed Research International
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
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[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
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[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
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[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
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[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
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[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
new generation of rapid resistance testing assays The keyfeatures of these new assays should be significant reduction ofturn-around-time (Table 5) and a high multiplexing capacitybecause of the already mentioned shift from Gram-positiveto Gram-negative multidrug-resistant bacteria in recentyears with various resistance mechanisms [1ndash4] So MRSAdetection simply means detection of the penicillin bindingprotein 2A (PBP2A) the SCCmec genetic element respec-tively [178] Detection of vancomycin-resistant S aureus(VRSA) as well as vancomycin-resistant enterococci (VRE)means the detection of Van-A Van-B and rarely Van-C[179]
In contrast to this situation in Gram-positive bacteriamultidrug resistance in Gram-negative bacteria is due tothe expression of extended-spectrum 120573-lactamases (ESBLs)carbapenemases aminoglycoside-blocking 16S rRNAmethy-lases and many other mechanisms associated with severalhundreds of gene variantsmutations [4ndash8] The more theseresistance genes can be detected in parallel the higherthe probability of an exact determination of a particularsusceptibility pattern is
But rapid resistance testing is only one key to thesolution of this problem especially because the multiplexingcapacities of the individual assays are limited and the costs aretoo highThus resistance surveillance programs are and havebeen established at different levels hospital-wide regionaland international For example some hospitals introduced ageneral ESBL screening in analogy to the MRSA screening inhigh-risk groups In recent years various studies were carriedout to identify the ESBL-transmission rate in maximum carehospitals and in households with ESBL-colonized individu-als The studies showed that the ESBL-transmission rate of15 to 45 is relatively low if compliance with standardhygiene measures is guaranteed [180 181] In contrast theESBL-transmission rate in households with common foodpreparation was 25 and therewith comparable high asthe MRSA-transmission rate [181 182] A prospective studydemonstrated a relatively high prevalence of 15 for ESBL-producing Enterobacteriaceae on admission but these strainswere involved in only 10 of the infections at admission time[183] Such regional surveillance studies form the basis fornational and international surveillance statistics such as thosepublished by the European Antimicrobial Resistance Surveil-lance Network (EARS-Net) Such surveillance studies on theprevalence of certain ESBL and carbapenemase subtypes cancontribute to the identification of resistance mechanismsof the quantitatively biggest importance which should beincluded in Gram-negative test panels Thus appropriatesurveillance studies contribute to the solution of the problemof limited multiplexing capacity at least partially
As recently predicted next generation sequencing (NGS)with its highmultiplexing capacitywill soonbe part of routinediagnostics more and more replacing cultural approaches asan accurate and cheap procedure in routine clinical micro-biology practice This will include sequence-based resistancetesting and additional detection of particular virulence fac-tors making culture unnecessary on the intermediate or longterm [184] The generation of microbial sequence data for
ldquoshort termrdquo patient management will revolutionize infecti-ology and diagnostic microbiology allowing for deeper andmore rapid insights into the patientsrsquo infectious pathologies[90] As a high-resolution tool high-throughput sequencinghas the potential to optimize both diagnostics and patientcare [185] NGS will affect antibiotic stewardship [80] bydefining resistance by the presence of a mechanism ratherthan just in pharmacodynamic terms as it is performed rightnow Present obstacles include the imperfect correlation ofgenotype and phenotype further technical challenges haveto be overcome [80] However as NGS becomes increasinglycost effective and convenient it bears the potential to replacethe so far multiple and complex procedures in a microbiolog-ical routine laboratory by just a single straightforward andmost efficient workflow [184]
Besides NGS mass spectrometry will be the secondkey technique in rapid medical microbiology The inte-gration of subtype specific mass spectra databases in MSassociated software packages will enable the identificationof high-virulent strains within very short time periodsThe mass spectrometric 120573-lactamase assay (MSBL) as wellas adaptations to other anti-microbiota classes will expec-tantly advance to helpful tools of the diagnostic micro-biologist Finally the combination of both nucleic acidamplification and mass spectrometric analysis for examplein PCRESI MS assays with its high multiplexing capacityhas the potential to enter routine diagnostic in the comingyears
Nevertheless these highly sophisticated and expensivediagnostic solutions will hardly be available in resource-limited countries for example in the sub-Saharan tropicswhere multidrug resistance is nevertheless on the rise [186]Cheap and easy-to-perform rapid molecular techniques likefluorescence in situ hybridization (FISH) might be an optionfor such settings [187] until MALDI-TOF MS or sequence-based approaches become more affordable and easy to applyThe rapid and correct choice of adequate antibiotic therapywill decide on the survival of critically ill patients withinfectious diseases for example sepsis patients [188 189]In times of decreasing susceptibility to antimicrobial drugsthis choice gets increasingly complicated So the words ofthe ancient German infectious disease specialist Robert Kochbecome more and more true ldquoIf a doctor walks behindhisher patientrsquos coffin sometime cause follows consequencerdquo(Original German text of the witticism ldquoWenn ein Arzthinter dem Sarg seines Patienten geht so folgt manchmaldie Ursache der Wirkungrdquo) Reliable information on theresistance patterns of etiologically relevant pathogens hasto be rapidly available to avoid this final consequence asfrequently as possible
Conflict of Interests
The authors declare that there is no conflict of interestsaccording to the guidelines of the International Committeeof Medical Journal Editors
BioMed Research International 13
Acknowledgments
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
[1] WHO Antimicrobial Resistance Global Report on SurveillanceWHO 2014
[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
[20] WHO Global Tuberculosis Report 2013 World Health Organi-zation Geneva Switzerland 2013
[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
14 BioMed Research International
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
BioMed Research International 15
[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
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[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
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[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
This paper was funded by the Open Access Support Programof the Deutsche Forschungsgemeinschaft and the publicationfund of the Georg August Universitat Gottingen
References
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[2] Y-L Lee Y-S Chen H-S Toh et al ldquoAntimicrobial suscep-tibility of pathogens isolated from patients with complicatedintra-abdominal infections at five medical centers in Taiwanthat continuously participated in the Study for MonitoringAntimicrobial Resistance Trends (SMART) from 2006 to 2010rdquoInternational Journal of Antimicrobial Agents vol 40 supple-ment 1 pp S29ndashS36 2012
[3] B Ghebremedhin ldquoExtended-spectrum of beta-lactamases(ESBL) yesterday ESBL and today ESBL carbapenemase-producing and multiresistant bacteriardquo Deutsche MedizinischeWochenschrift vol 137 no 50 pp 2657ndash2662 2012
[4] D M Livermore ldquoCurrent epidemiology and growing resis-tance of Gram-negative pathogensrdquo Korean Journal of InternalMedicine vol 27 no 2 pp 128ndash142 2012
[5] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoThe Lancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009
[6] D J Wolter P M Kurpiel N Woodford M-F I Palepou RV Goering and N D Hanson ldquoPhenotypic and enzymaticcomparative analysis of the novel KPC variant KPC-5 and itsevolutionary variants KPC-2 andKPC-4rdquoAntimicrobial Agentsand Chemotherapy vol 53 no 2 pp 557ndash562 2009
[7] A Endimiani A M Hujer F Perez et al ldquoCharacterizationof blaKPC-containing Klebsiella pneumoniae isolates detectedin different institutions in the Eastern USArdquo The Journal ofAntimicrobial Chemotherapy vol 63 no 3 pp 427ndash437 2009
[8] L Hidalgo K L Hopkins B Gutierrez et al ldquoAssociation of thenovel aminoglycoside resistance determinant RmtF with NDMcarbapenemase in enterobacteriaceae isolated in India and theUKrdquo Journal of Antimicrobial Chemotherapy vol 68 no 7 pp1543ndash1550 2013
[9] M-H Nicolas-chanoine C Gruson S Bialek-Davenet et alldquo10-fold increase (2006ndash11) in the rate of healthy subjectswith extended-spectrum 120573-lactamase-producing Escherichiacoli faecal carriage in a parisian check-up centrerdquoThe Journal ofAntimicrobial Chemotherapy vol 68 no 3 pp 562ndash568 2013
[10] A Birgy R Cohen C Levy et al ldquoCommunity faecal carriageof extended-spectrum beta-lactamase-producing Enterobacte-riaceae in french childrenrdquo BMC Infectious Diseases vol 12article 315 2012
[11] J Tham M Walder E Melander and I Odenholt ldquoDura-tion of colonization with extended-spectrum beta-lactamase-producingEscherichia coli in patients with travellersrsquo diarrhoeardquoScandinavian Journal of Infectious Diseases vol 44 no 8 pp573ndash577 2012
[12] G Birgand L Armand-Lefevre I Lolom E Ruppe AAndremont and J-C Lucet ldquoDuration of colonizationby extended-spectrum 120573-lactamase-producing Enterobac-teriaceae after hospital dischargerdquo The American Journal ofInfection Control vol 41 no 5 pp 443ndash447 2013
[13] I H Lohr S Rettedal O B Natas U Naseer K Oslashymar andA Sundsfjord ldquoLong-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiellapneumoniae following a nosocomial outbreakrdquo The Journal ofAntimicrobial Chemotherapy vol 68 no 5 Article ID dks502pp 1043ndash1048 2013
[14] J L Cottell M A Webber and L J V Piddock ldquoPersistenceof transferable extended-spectrum-120573-lactamase resistance inthe absence of antibiotic pressurerdquo Antimicrobial Agents andChemotherapy vol 56 no 9 pp 4703ndash4706 2012
[15] Y J Ko H W Moon M Hur C M Park S E Cho andY M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[16] U-O Luvsansharav I Hirai A Nakata et al ldquoPrevalenceof and risk factors associated with faecal carriage of CTX-M 120573-lactamase-producing enterobacteriaceae in rural Thaicommunitiesrdquo Journal of Antimicrobial Chemotherapy vol 67no 7 Article ID dks118 pp 1769ndash1774 2012
[17] N H Wickramasinghe L Xu A Eustace S Shabir T Salujaand P M Hawkey ldquoHigh community faecal carriage rates ofCTX-M ESBL-producing Escherichia coli in a specific popula-tion group in Birmingham UKrdquo The Journal of AntimicrobialChemotherapy vol 67 no 5 Article ID dks018 pp 1108ndash11132012
[18] J A J W Kluytmans I T M A Overdevest I Willemsen et alldquoExtended-spectrum 120573-lactamase-producing Escherichia colifrom retail chicken meat and humans comparison of strainsplasmids resistance genes and virulence factorsrdquo ClinicalInfectious Diseases vol 56 no 4 pp 478ndash487 2013
[19] S Bhattacharya ldquoEarly diagnosis of resistant pathogens howcan it improve antimicrobial treatmentrdquo Virulence vol 4 no2 pp 172ndash184 2013
[20] WHO Global Tuberculosis Report 2013 World Health Organi-zation Geneva Switzerland 2013
[21] E Shmueli R Or M Y Shapira et al ldquoHigh rate ofcytomegalovirus drug resistance among patients receivingpreemptive antiviral treatment after haploidentical stem celltransplantationrdquo Journal of Infectious Diseases vol 209 no 4pp 557ndash561 2014
[22] F Baldanti and G Gerna ldquoHuman cytomegalovirus resistanceto antiviral drugs diagnosis monitoring and clinical impactrdquoJournal of Antimicrobial Chemotherapy vol 52 no 3 pp 324ndash330 2003
[23] Y-W Tang and CW StrattonAdvanced Techniques in Diagnos-tic Microbiology Springer New York NY USA 2006
[24] A Van Belkum G DurandM Peyret et al ldquoRapid clinical bac-teriology and its future impactrdquo Annals of Laboratory Medicinevol 33 no 1 pp 14ndash27 2013
[25] G M Trenholme R L Kaplan P H Karakusis et al ldquoClinicalimpact of rapid identification and susceptibility testing of bacte-rial blood culture isolatesrdquo Journal of Clinical Microbiology vol27 no 6 pp 1342ndash1345 1989
[26] R Laxminarayan A Duse C Wattal et al ldquoAntibioticresistance-the need for global solutionsrdquo The Lancet InfectiousDiseases vol 13 no 12 pp 1057ndash1098 2013
[27] S Doron and L E Davidson ldquoAntimicrobial stewardshiprdquoMayo Clinic Proceedings vol 86 no 11 pp 1113ndash1123 2011
[28] M V Ramirez K C Cowart P J Campbell et al ldquoRapiddetection ofmultidrug-resistantMycobacterium tuberculosis byuse of real-time PCR and high-resolutionmelt analysisrdquo Journalof Clinical Microbiology vol 48 no 11 pp 4003ndash4009 2010
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[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
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[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
[29] T C Dingle and S M Butler-Wu ldquoMALDI-TOF mass spec-trometry for microorganism identificationrdquo Clinics in Labora-tory Medicine vol 33 no 3 pp 589ndash609 2013
[30] K Weist A-K Cimbal C Lecke G Kampf H Ruden and R-P Vonberg ldquoEvaluation of six agglutination tests for Staphylo-coccus aureus identification depending upon local prevalenceof meticillin-resistant S aureus (MRSA)rdquo Journal of MedicalMicrobiology vol 55 no 3 pp 283ndash290 2006
[31] P D de Matos R P Schuenck F S Cavalcante R M Cabocloand K R N dos Santos ldquoAccuracy of phenotypic methicillinsusceptibilitymethods in the detection of Staphylococcus aureusisolates carrying different SCCmec typesrdquo Memorias do Insti-tuto Oswaldo Cruz vol 105 no 7 pp 931ndash934 2010
[32] Q Qian L Venkataraman J E Kirby H S Gold andT Yamazumi ldquoDirect detection of methicillin resistance inStaphylococcus aureus in blood culture broth by use of apenicillin binding protein 2a latex agglutination testrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1420ndash1421 2010
[33] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004
[34] G K Paterson F J EMorgan EMHarrison et al ldquoPrevalenceand properties of mecc methicillin-resistant Staphylococcusaureus (mrsa) in bovine bulk tankmilk in great britainrdquo Journalof Antimicrobial Chemotherapy vol 69 no 3 Article ID dkt417pp 598ndash602 2014
[35] K C Chapin and M C Musgnug ldquoEvaluation of penicillinbinding protein 2a latex agglutination assay for identification ofmethicillin-resistant Staphylococcus aureus directly from bloodculturesrdquo Journal of Clinical Microbiology vol 42 no 3 pp1283ndash1284 2004
[36] N Woodford and A Sundsfjord ldquoMolecular detection ofantibiotic resistance when andwhererdquo Journal of AntimicrobialChemotherapy vol 56 no 2 pp 259ndash261 2005
[37] P-E Fournier M Drancourt P Colson J-M Rolain B LScola and D Raoult ldquoModern clinical microbiology newchallenges and solutionsrdquo Nature Reviews Microbiology vol 11no 8 pp 574ndash585 2013
[38] M J Espy J R Uhl L M Sloan et al ldquoReal-time PCRin clinical microbiology applications for routine laboratorytestingrdquo Clinical Microbiology Reviews vol 19 pp 165ndash2562006
[39] M Maurin ldquoReal-time PCR as a diagnostic tool for bacterialdiseasesrdquo Expert Review of Molecular Diagnostics vol 12 no 7pp 731ndash754 2012
[40] D C T Ong T-H Koh N Syahidah P Krishnan and T YTan ldquoRapid detection of the blaNDM-1 gene by real-time PCRrdquoJournal of Antimicrobial Chemotherapy vol 66 no 7 pp 1647ndash1649 2011
[41] S A Cunningham T Noorie D Meunier N Woodford andR Patel ldquoRapid and simultaneous detection of genes encodingKlebsiella pneumoniae carbapenemase (blaKPC) and NewDelhi metallo-beta-lactamase (blaNDM) in Gram-negativebacillirdquo Journal of Clinical Microbiology vol 51 pp 1269ndash12712013
[42] F Zheng J Sun C Cheng and Y Rui ldquoThe establishmentof a duplex real-time PCR assay for rapid and simultaneousdetection of blaNDM and blaKPC genes in bacteriardquo Annals ofClinicalMicrobiology andAntimicrobials vol 12 no 1 article 302013
[43] L Huang X Hu M Zhou et al ldquoRapid detection of new delhimetallo-120573-lactamase gene and variants coding for carbapene-mases with different activities by use of a PCR-based in vitroprotein expression methodrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1947ndash1953 2014
[44] R Nijhuis Oslash Samuelsen P Savelkoul and A van ZwetldquoEvaluation of a new real-time PCR assay (Check-Direct CPE)for rapid detection ofKPCOXA-48VIM andNDMcarbapen-emases using spiked rectal swabsrdquo Diagnostic Microbiology andInfectious Disease vol 77 no 4 pp 316ndash320 2013
[45] A van der Zee L Roorda G Bosman and et al ldquoMulti-centre evaluation of real-time multiplex PCR for detection ofcarbapenemase genes OXA-48 VIM IMP NDM and KPCrdquoBMC Infectious Diseases vol 14 no 1 article 27 2014
[46] C Cheng F Zheng and Y Rui ldquoRapid detection of blaNDMblaKPC blaIMP and blaVIM carbapenemase genes in bacteriaby loop-mediated isothermal amplificationrdquo Microbial DrugResistance 2014
[47] U S W Reischl T Holzmann M Ehrenschwender et alldquoBakterien- und Pilzgenom-Nachweis PCRNAT Auswertungdes Ringversuchs November 2013 von INSTAND eV zur exter-nen Qualitatskontrolle molekularbiologischer Nachweisver-fahren in der bakteriologischen Diagnostikrdquo Der Mikrobiologevol 24 pp 37ndash56 2014
[48] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[49] M Kaase F Szabados LWassill and S G Gatermann ldquoDetec-tion of carbapenemases in Enterobacteriaceae by a commercialmultiplex PCRrdquo Journal of Clinical Microbiology vol 50 no 9pp 3115ndash3118 2012
[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010
[51] N P Pai C Vadnais CDenkinger N Engel andM Pai ldquoPoint-of-care testing for infectious diseases diversity complexity andbarriers in low- and middle-income countriesrdquo PLoS Medicinevol 9 no 9 Article ID e1001306 2012
[52] C C Boehme M P Nicol P Nabeta et al ldquoFeasibilitydiagnostic accuracy and effectiveness of decentralised use of theXpertMTBRIF test for diagnosis of tuberculosis andmultidrugresistance amulticentre implementation studyrdquoTheLancet vol377 no 9776 pp 1495ndash1505 2011
[53] B Strommenger C Kettlitz G Werner and W Witte ldquoMul-tiplex PCR assay for simultaneous detection of nine clinicallyrelevant antibiotic resistance genes in Staphylococcus aureusrdquoJournal of Clinical Microbiology vol 41 no 9 pp 4089ndash40942003
[54] W Jamal E Al Roomi L R AbdulAziz and V O RotimildquoEvaluation of Curetis Unyvero a multiplex PCR-based testingsystem for rapid detection of bacteria and antibiotic resistanceand impact of the assay on management of severe nosocomialpneumoniardquo Journal of Clinical Microbiology vol 52 pp 2487ndash2492 2014
[55] Z Zhang L Li F Luo et al ldquoRapid and accurate detectionof RMP- and INH-resistant Mycobacterium tuberculosis inspinal tuberculosis specimens by CapitalBio DNA microarraya prospective validation studyrdquo BMC Infectious Diseases vol 12article 303 2012
BioMed Research International 15
[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
[56] Y Guo Y Zhou C Wang et al ldquoRapid accurate determinationof multidrug resistance in M tuberculosis isolates and sputumusing a biochip systemrdquo International Journal of Tuberculosisand Lung Disease vol 13 no 7 pp 914ndash920 2009
[57] T Naas G Cuzon H Truong S Bernabeu and P NordmannldquoEvaluation of a DNA microarray the check-points ESBLKPCarray for rapid detection of TEM SHV and CTX-M extended-spectrum 120573-lactamases and KPC carbapenemasesrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 8 pp 3086ndash30922010
[58] I Willemsen I Overdevest N Al Naiemi et al ldquoNew Diagnos-tic microarray (check-KPC ESBL) for detection and identifica-tion of extended-spectrum beta-lactamases in highly resistantEnterobacteriaceaerdquo Journal of ClinicalMicrobiology vol 49 no8 pp 2985ndash2987 2011
[59] A Endimiani K M Hujer A M Hujer et al ldquoAre we readyfor novel detection methods to treat respiratory pathogens inhospital-acquired pneumoniardquoClinical Infectious Diseases vol52 supplement 4 pp S373ndashS383 2011
[60] J C Stuart G Voets J Scharringa A C Fluit and M ALeverstein-Van Hall ldquoDetection of carbapenemase-producingEnterobacteriaceae with a commercial DNA microarrayrdquo Jour-nal of Medical Microbiology vol 61 no 6 pp 809ndash812 2012
[61] V Mikhailovich D Gryadunov A Kolchinsky A A Makarovand A Zasedatelev ldquoDNA microarrays in the clinic Infectiousdiseasesrdquo BioEssays vol 30 no 7 pp 673ndash682 2008
[62] G Zhang F Cai Z Zhou et al ldquoSimultaneous detection ofmajor drug resistance mutations in the protease and reversetranscriptase genes for HIV-1 subtype C by use of a multiplexallele-specific assayrdquo Journal of Clinical Microbiology vol 51 no11 pp 3666ndash3674 2013
[63] P Masimba J Gare T Klimkait M Tanner and I FelgerldquoDevelopment of a simple microarray for genotyping HIV-1drug resistance mutations in the reverse transcriptase gene inrural TanzaniardquoTropicalMedicine and International Health vol19 no 6 pp 664ndash671 2014
[64] Y Linger A Kukhtin J Golova et al ldquoSimplified microarraysystem for simultaneously detecting rifampin isoniazid etham-butol and streptomycin resistance markers in Mycobacteriumtuberculosisrdquo Journal of Clinical Microbiology vol 52 no 6 pp2100ndash2107 2014
[65] R Moure M Espanol G Tudo et al ldquoCharacterization ofthe embB gene in Mycobacterium tuberculosis isolates frombarcelona and rapid detection of main mutations related toethambutol resistance using a low-density DNA arrayrdquo Journalof Antimicrobial Chemotherapy vol 69 no 4 pp 947ndash954 2014
[66] A Chatterjee D Saranath P Bhatter and N Mistry ldquoGlobaltranscriptional profiling of longitudinal clinical isolates ofMycobacterium tuberculosis exhibiting rapid accumulation ofdrug resistancerdquo PLoS ONE vol 8 no 1 Article ID e54717 2013
[67] M B Miller and Y-W Tang ldquoBasic concepts of microarraysand potential applications in clinical microbiologyrdquo ClinicalMicrobiology Reviews vol 22 no 4 pp 611ndash633 2009
[68] A Afshari J Schrenzel M Ieven and S Harbarth ldquoBench-to-bedside review rapid molecular diagnostics for bloodstreaminfectionmdasha new frontierrdquo Critical Care vol 16 no 3 article222 2012
[69] R P Podzorski H Li J Han and Y-W Tang ldquoMVPlex assayfor direct detection of methicillin-resistant Staphylococcusaureus in naris and other swab specimensrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 3107ndash3109 2008
[70] Y-W Tang A Kilic Q Yang et al ldquoStaphPlex system forrapid and simultaneous identification of antibiotic resistancedeterminants and Panton-Valentine leukocidin detection ofstaphylococci from positive blood culturesrdquo Journal of ClinicalMicrobiology vol 45 no 6 pp 1867ndash1873 2007
[71] P Roumagnac F-X Weill C Dolecek et al ldquoEvolutionaryhistory of Salmonella typhirdquo Science vol 314 no 5803 pp 1301ndash1304 2006
[72] TW JesseMD Englen LG Pittenger-Alley andP J Fedorka-Cray ldquoTwo distinct mutations in gyrA lead to ciprofloxacinand nalidixic acid resistance in Campylobacter coli and Campy-lobacter jejuni isolated from chickens and beef cattlerdquo Journal ofApplied Microbiology vol 100 no 4 pp 682ndash688 2006
[73] C F Taylor andG R Taylor ldquoCurrent and emerging techniquesfor diagnostic mutation detection an overview of methods formutation detectionrdquoMethods inMolecularMedicine vol 92 pp9ndash44 2004
[74] S A Dunbar ldquoApplications of Luminex xMAPŮ technologyfor rapid high-throughput multiplexed nucleic acid detectionrdquoClinica Chimica Acta vol 363 no 1-2 pp 71ndash82 2006
[75] Y Song P Roumagnac F-X Weill et al ldquoA multiplex singlenucleotide polymorphism typing assay for detecting muta-tions that result in decreased fluoroquinolone susceptibilityin Salmonella enterica serovars Typhi and Paratyphi Ardquo TheJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq175 pp 1631ndash1641 2010
[76] L Barco A A Lettini M C D Pozza E Ramon M Faso-lato and A Ricci ldquoFluoroquinolone resistance detection incampylobacter coli and campylobacter jejuni by luminex xMAPtechnologyrdquo Foodborne Pathogens and Disease vol 7 no 9 pp1039ndash1045 2010
[77] N J Loman R VMisra T J Dallman et al ldquoPerformance com-parison of benchtop high-throughput sequencing platformsrdquoNature Biotechnology vol 30 no 5 pp 434ndash439 2012
[78] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the german enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011
[79] T A Kohl R Diel D Harmsen et al ldquoWhole-genome-basedMycobacterium tuberculosis surveillance a standardizedportable and expandable approachrdquo Journal of Clinical Micro-biology vol 52 pp 2479ndash2486 2014
[80] D M Livermore and J Wain ldquoRevolutionising bacteriologyto improve treatment outcomes and antibiotic stewardshiprdquoInfection amp Chemotherapy vol 45 no 1 pp 1ndash10 2013
[81] A Lupo K M Papp-Wallace P Sendi R A Bonomo and AEndimiani ldquoNon-phenotypic tests to detect and characterizeantibiotic resistance mechanisms in Enterobacteriaceaerdquo Diag-nosticMicrobiology and Infectious Disease vol 77 no 3 pp 179ndash194 2013
[82] L T Daum G W Fischer J Sromek et al ldquoCharacteriza-tion of multi-drug resistant Mycobacterium tuberculosis fromimmigrants residing in the USA using Ion Torrent full-genesequencingrdquo Epidemiology and Infection vol 142 no 6 pp1328ndash1333 2014
[83] E N Ilina E A Shitikov L N Ikryannikova et al ldquoCom-parative genomic analysis of Mycobacterium tuberculosis drugresistant strains from Russiardquo PLoS ONE vol 8 no 2 ArticleID e56577 2013
16 BioMed Research International
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
[84] L T Daum J D Rodriguez S A Worthy et al ldquoNext-generation ion torrent sequencing of drug resistance muta-tions inMycobacterium tuberculosis strainsrdquo Journal of ClinicalMicrobiology vol 50 no 12 pp 3831ndash3837 2012
[85] S Das T Roychowdhury P Kumar et al ldquoGenetic heterogene-ity revealed by sequence analysis of Mycobacterium tuberculo-sis isolates from extra-pulmonary tuberculosis patientsrdquo BMCGenomics vol 14 no 1 article 404 2013
[86] J Wang R Stephan K Power Q Yan H Hachler and SFanning ldquoNucleotide sequences of 16 transmissible plasmidsidentified in nine multidrug-resistant Escherichia coli isolatesexpressing an ESBL phenotype isolated from food-producinganimals and healthy humansrdquo The Journal of AntimicrobialChemotherapy 2014
[87] A Brolund O Franzen O Melefors K Tegmark-Wiselland L Sandegren ldquoPlasmidome-analysis of ESBL-producingescherichia coli using conventional typing and high-throughputsequencingrdquo PLoS ONE vol 8 no 6 Article ID e65793 2013
[88] J Veenemans I T Overdevest E Snelders et al ldquoNext gen-eration Sequencing for typing and detection of resistance genesperformance of a new commercial method during an outbreakof ESBL-producing Escherichia colirdquo Journal of Clinical Micro-biology vol 52 no 7 pp 2454ndash2460 2014
[89] N L Sherry J L Porter T Seemann A Watkins T PStinear and B P Howden ldquoOutbreak investigation using high-throughput genome sequencing within a diagnostic microbiol-ogy laboratoryrdquo Journal of Clinical Microbiology vol 51 no 5pp 1396ndash1401 2013
[90] W M Dunne L F Westblade and B Ford ldquoNext-generationand whole-genome sequencing in the diagnostic clinical micro-biology laboratoryrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 31 no 8 pp 1719ndash1726 2012
[91] A Moter and U B Gobel ldquoFluorescence in situ hybridization(FISH) for direct visualization of microorganismsrdquo Journal ofMicrobiological Methods vol 41 no 2 pp 85ndash112 2000
[92] H Stender ldquoPNA FISH an intelligent stain for rapid diagnosisof infectious diseasesrdquo Expert Review of Molecular Diagnosticsvol 3 no 5 pp 649ndash655 2003
[93] H Russmann V A J Kempf S Koletzko J Heesemann and IB Autenrieth ldquoComparison of fluorescent in situ hybridizationand conventional culturing for detection of Helicobacter pyloriin gastric biopsy specimensrdquo Journal of Clinical Microbiologyvol 39 no 1 pp 304ndash308 2001
[94] O Yilmaz and E Demiray ldquoClinical role and importance of flu-orescence in situ hybridization method in diagnosis of H pyloriinfection and determination of clarithromycin resistance in Hpylori eradication therapyrdquo World Journal of Gastroenterologyvol 13 no 5 pp 671ndash675 2007
[95] H Russmann K Adler R Haas B Gebert S Koletzko and JHeesemann ldquoRapid and accurate determination of genotypicclarithromycin resistance in culturedHelicobacter pylori by flu-orescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 39 no 11 pp 4142ndash4144 2001
[96] H Russmann A Feydt-Schmidt K Adler D Aust A Fischerand S Koletzko ldquoDetection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluo-rescent in situ hybridizationrdquo Journal of Clinical Microbiologyvol 41 no 2 pp 813ndash815 2003
[97] A Feydt-Schmidt H Russmann N Lehn et al ldquoFluores-cence in situ hybridization vs epsilometer test for detec-tion of clarithromycin-susceptible and clarithromycin-resistantHelicobacter pylori strains in gastric biopsies from childrenrdquo
Alimentary Pharmacology and Therapeutics vol 16 no 12 pp2073ndash2079 2002
[98] S Juttner M Vieth S Miehlke et al ldquoReliable detection ofmacrolide-resistant Helicobacter pylori via fluorescence in situhybridization in formalin-fixed tissuerdquo Modern Pathology vol17 no 6 pp 684ndash689 2004
[99] E Caristo A Parola A Rapa et al ldquoClarithromycin resistanceof Helicobacter pylori strains isolated from childrenrsquo gastricantrum and fundus as assessed by fluorescent in-situ hybridiza-tion and culture on four-sector agar platesrdquoHelicobacter vol 13no 6 pp 557ndash563 2008
[100] A E Vega T Alarcon D Domingo and M Lopez-BrealdquoDetection of clarithromycin-resistant Helicobacter pylori infrozen gastric biopsies from pediatric patients by a commer-cially available fluorescent in situ hybridizationrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 4 pp 421ndash4232007
[101] O Yilmaz E Demiray S Tumer et al ldquoDetection ofHelicobac-ter pylori and determination of clarithromycin susceptibilityusing formalin-fixed paraffin-embedded gastric biopsy speci-mens by fluorescence in situ hybridizationrdquo Helicobacter vol12 no 2 pp 136ndash141 2007
[102] L Cerqueira R M Fernandes R M Ferreira et al ldquoValidationof a fluorescence in situ hybridization method using peptidenucleic acid probes for detection of Helicobacter pylori clar-ithromycin resistance in gastric biopsy specimensrdquo Journal ofClinical Microbiology vol 51 no 6 pp 1887ndash1893 2013
[103] M Haas A Essig E Bartelt and S Poppert ldquoDetectionof resistance to macrolides in thermotolerant Campylobacterspecies by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 46 no 11 pp 3842ndash3844 2008
[104] G Werner M Bartel N Wellinghausen et al ldquoDetection ofmutations conferring resistance to linezolid in Enterococcusspp by fluorescence in situ hybridizationrdquo Journal of ClinicalMicrobiology vol 45 no 10 pp 3421ndash3423 2007
[105] S Palasubramaniam S Muniandy and P Navaratnam ldquoRapiddetection of ESBL-producing Klebsiella pneumoniae in bloodcultures by fluorescent in-situ hybridizationrdquo Journal of Micro-biological Methods vol 72 no 1 pp 107ndash109 2008
[106] M Wagner and S Haider ldquoNew trends in fluorescence insitu hybridization for identification and functional analyses ofmicrobesrdquo Current Opinion in Biotechnology vol 23 no 1 pp96ndash102 2012
[107] I Smolina N S Miller and M D Frank-Kamenetskii ldquoPNA-based microbial pathogen identification and resistance markerdetection An accurate isothermal rapid assay based ongenome-specific featuresrdquo Artificial DNA PNA and XNA vol1 no 2 pp 76ndash82 2010
[108] A Swidsinski ldquoStandards for bacterial identification by fluo-rescence in situ hybridization within eukaryotic tissue usingribosomal rRNA-based probesrdquo Inflammatory Bowel Diseasesvol 12 no 8 pp 824ndash826 2006
[109] Q Shao Y Zheng X Dong K Tang X Yan and B XingldquoA covalent reporter of 120573-lactamase activity for fluorescentimaging and rapid screening of antibiotic-resistant bacteriardquoChemistry vol 19 no 33 pp 10903ndash10910 2013
[110] P Seng M Drancourt F Gouriet et al ldquoOngoing revolutionin bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spec-trometryrdquoClinical Infectious Diseases vol 49 no 4 pp 543ndash5512009
BioMed Research International 17
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
[111] O Bader M Weig L Taverne-Ghadwal R Lugert U Groszligand M Kuhns ldquoImproved clinical laboratory identification ofhuman pathogenic yeasts by matrix-assisted laser desorptionionization time-of-flight mass spectrometryrdquo Clinical Microbi-ology and Infection vol 17 no 9 pp 1359ndash1365 2011
[112] A Wieser L Schneider J Jung and S Schubert ldquoMALDI-TOFMS in microbiological diagnostics-identification of microor-ganisms and beyond (mini review)rdquo Applied Microbiology andBiotechnology vol 93 no 3 pp 965ndash974 2012
[113] O Bader ldquoMALDI-TOF-MS-based species identification andtyping approaches inmedical mycologyrdquo Proteomics vol 13 no5 pp 788ndash799 2013
[114] M L DeMarco and B A Ford ldquoBeyond identification emerg-ing and future uses for maldi-tof mass spectrometry in the clin-ical microbiology laboratoryrdquo Clinics in Laboratory Medicinevol 33 no 3 pp 611ndash628 2013
[115] E Shitikov E Ilina L Chernousova et al ldquoMass spectrometrybasedmethods for the discrimination and typing ofmycobacte-riardquo Infection Genetics and Evolution vol 12 no 4 pp 838ndash8452012
[116] M Reil M Erhard E J Kuijper et al ldquoRecognition ofClostridium difficile PCR-ribotypes 001 027 and 126078 usingan extended MALDI-TOF MS systemrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 30 no 11 pp1431ndash1436 2011
[117] A Novais C Sousa J de Dios Caballero et al ldquoMALDI-TOFmass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131)and D (ST69 ST405 ST393)rdquo European Journal of ClinicalMicrobiology and Infectious Diseases pp 1ndash9 2014
[118] Y Matsumura M Yamamoto M Nagao et al ldquoDetectionof extended-spectrum-120573-lactamase-producing escherichia coliST131 and ST405 clonal groups by matrix-assisted laser des-orption ionization-time of flight mass spectrometryrdquo Journal ofClinical Microbiology vol 52 no 4 pp 1034ndash1040 2014
[119] I Wybo A de Bel O Soetens et al ldquoDifferentiation ofcfiA-negative and cfiA-positive Bacteroides fragilis isolates bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp1961ndash1964 2011
[120] E Nagy S Becker J Soki E Urban and M KostrzewaldquoDifferentiation of division I (cfiA-negative) and division II(cfiA-positive) Bacteroides fragilis strains by matrix-assistedlaser desorptionionization time of-flight mass spectrometryrdquoJournal of Medical Microbiology vol 60 no 11 pp 1584ndash15902011
[121] P M Griffin G R Price J M Schooneveldt et al ldquoUse ofmatrix-assisted laser desorption ionization-time of flight massspectrometry to identify vancomycin-resistant enterococci andinvestigate the epidemiology of an outbreakrdquo Journal of ClinicalMicrobiology vol 50 no 9 pp 2918ndash2931 2012
[122] C Marinach A Alanio M Palous et al ldquoMALDI-TOF MS-based drug susceptibility testing of pathogens the example ofCandida albicans and fluconazolerdquo Proteomics vol 9 no 20 pp4627ndash4631 2009
[123] E de Carolis A Vella A R Florio et al ldquoUse of matrix-assistedlaser desorption ionization-time of flightmass spectrometry forcaspofungin susceptibility testing of Candida and Aspergillusspeciesrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp 2479ndash2483 2012
[124] A Vella E de Carolis L Vaccaro et al ldquoRapid antifun-gal susceptibility testing by matrix-assisted laser desorption
ionization-time of flight mass spectrometry analysisrdquo Journal ofClinical Microbiology vol 51 no 9 pp 2964ndash2969 2013
[125] M Kostrzewa K Sparbier T Maier and S Schubert ldquoMALDI-TOF MS an upcoming tool for rapid detection of antibioticresistance in microorganismsrdquo Proteomics Clinical Applica-tions vol 7 no 11-12 pp 767ndash778 2013
[126] J S Jung T Eberl K Sparbier et al ldquoRapid detection ofantibiotic resistance based on mass spectrometry and stableisotopesrdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 33 pp 949ndash955 2013
[127] J Hrabak R Walkova V Studentova E Chudackova andT Bergerova ldquoCarbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spec-trometryrdquo Journal of Clinical Microbiology vol 49 no 9 pp3222ndash3227 2011
[128] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011
[129] G P Hooff J J A van Kampen R J W Meesters A vanBelkum W H F Goessens and T M Luider ldquoCharacteriza-tion of 120573-lactamase enzyme activity in bacterial lysates usingMALDI-mass spectrometryrdquo Journal of Proteome Research vol11 no 1 pp 79ndash84 2012
[130] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases bymatrix-assisted laser desorption ionization-time of flight massspectrometryrdquo Journal of ClinicalMicrobiology vol 50 no 7 pp2441ndash2443 2012
[131] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012
[132] A Endimiani G Patel K M Hujer et al ldquoIn vitro activityof fosfomycin against bla
isolates including those nonsusceptible to tigecycline andorcolistinrdquo Antimicrobial Agents and Chemotherapy vol 54 no1 pp 526ndash529 2010
[133] C A Wise M Paris B Morar W Wang L Kalaydjieva andA H Bittles ldquoA standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorptionionization time-of-flight mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 17 no 11 pp 1195ndash1202 2003
[134] S Zurcher C Mooser A U Luthi et al ldquoSensitive and rapiddetection of ganciclovir resistance by PCR based MALDI-TOFanalysisrdquo Journal of Clinical Virology vol 54 no 4 pp 359ndash3632012
[135] C Honisch Y Chen C Mortimer et al ldquoAutomated com-parative sequence analysis by base-specific cleavage and massspectrometry for nucleic acid-basedmicrobial typingrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 25 pp 10649ndash10654 2007
[136] C C Posthuma M T van der Beek C S van der Blij-de Brouwer et al ldquoMass spectrometry-based comparativesequencing to detect ganciclovir resistance in the UL97 geneof human cytomegalovirusrdquo Journal of Clinical Virology vol 51no 1 pp 25ndash30 2011
18 BioMed Research International
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
[137] M-F Lartigue ldquoMatrix-assisted laser desorption ionizationtime-of-flight mass spectrometry for bacterial strain character-izationrdquo Infection Genetics and Evolution vol 13 no 1 pp 230ndash235 2013
[138] P R Murray ldquoMatrix-assisted laser desorption ionization time-of-flight mass spectrometry usefulness for taxonomy andepidemiologyrdquo Clinical Microbiology and Infection vol 16 no11 pp 1626ndash1630 2010
[139] M Kuhns A E Zautner W Rabsch et al ldquoRapid discrimina-tion of Salmonella enterica serovar typhi from other serovarsby MALDI-TOF mass spectrometryrdquo PLoS ONE vol 7 no 6Article ID e40004 2012
[140] A E Zautner W O Masanta A M Tareen et al ldquoDiscrim-ination of multilocus sequence typing-based Campylobacterjejuni subgroups by MALDI-TOF mass spectrometryrdquo BMCMicrobiology vol 13 article 247 2013
[141] K Teramoto W Kitagawa H Sato M Torimura T Tamuraand H Tao ldquoPhylogenetic analysis of Rhodococcus erythropo-lis based on the variation of ribosomal proteins as observed bymatrix-assisted laser desorption ionization-mass spectrometrywithout using genome informationrdquo Journal of Bioscience andBioengineering vol 108 no 4 pp 348ndash353 2009
[142] K Teramoto H Sato L Sun et al ldquoPhylogenetic classificationof Pseudomonas putida strains byMALDI-MS using ribosomalsubunit proteins as biomarkersrdquo Analytical Chemistry vol 79no 22 pp 8712ndash8719 2007
[143] S Suarez A Ferroni A Lotz et al ldquoRibosomal proteins asbiomarkers for bacterial identification by mass spectrometry inthe clinical microbiology laboratoryrdquo Journal of MicrobiologicalMethods vol 94 no 3 pp 390ndash396 2013
[144] V Edwards-Jones M A Claydon D J Evason J WalkerA J Fox and D B Gordon ldquoRapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcusaureus by intact cell mass spectrometryrdquo Journal of MedicalMicrobiology vol 49 no 3 pp 295ndash300 2000
[145] J Walker A J Fox V Edwards-Jones and D B Gor-don ldquoIntact cell mass spectrometry (ICMS) used to typemethicillin-resistant Staphylococcus aureus media effects andinter-laboratory reproducibilityrdquo Journal of MicrobiologicalMethods vol 48 no 2-3 pp 117ndash126 2002
[146] K Bernardo N Pakulat M Macht et al ldquoIdentification anddiscrimination of Staphylococcus aureus strains using matrix-assisted laser desorptionionization-time of flight mass spec-trometryrdquo Proteomics vol 2 pp 747ndash753 2002
[147] K A Jackson V Edwards-Jones C W Sutton and A J FoxldquoOptimisation of intact cell MALDI method for fingerprint-ing of methicillin-resistant Staphylococcus aureusrdquo Journal ofMicrobiological Methods vol 62 no 3 pp 273ndash284 2005
[148] H N Shah L Rajakaruna G Ball et al ldquoTracing the transitionof methicillin resistance in sub-populations of Staphylococcusaureus using SELDI-TOF Mass Spectrometry and ArtificialNeuralNetworkAnalysisrdquo Systematic andAppliedMicrobiologyvol 34 no 1 pp 81ndash86 2011
[149] M Wolters H Rohde T Maier et al ldquoMALDI-TOF MSfingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineagesrdquo International Journalof Medical Microbiology vol 301 no 1 pp 64ndash68 2011
[150] M Josten M Reif C Szekat et al ldquoAnalysis of the matrix-assisted laser desorption ionization-time of flight mass spec-trum of Staphylococcus aureus identifies mutations that allowdifferentiation of the main clonal lineagesrdquo Journal of ClinicalMicrobiology vol 51 no 6 pp 1809ndash1817 2013
[151] J-J Lu F-J Tsai C-M Ho Y-C Liu and C-J Chen ldquoPeptidebiomarker discovery for identification of methicillin-resistantand vancomycin-intermediate Staphylococcus aureus strains byMALDI-TOFrdquo Analytical Chemistry vol 84 no 13 pp 5685ndash5692 2012
[152] F Szabados M Kaase A Anders and S G GatermannldquoIdentical MALDI TOF MS-derived peak profiles in a pair ofisogenic SCCmec-harboring and SCCmec-lacking strains ofStaphylococcus aureusrdquo The Journal of Infection vol 65 no 5pp 400ndash405 2012
[153] P A Majcherczyk T McKenna P Moreillon and P VaudauxldquoThe discriminatory power of MALDI-TOFmass spectrometryto differentiate between isogenic teicoplanin-susceptible andteicoplanin-resistant strains of methicillin-resistant Staphylo-coccus aureusrdquo FEMS Microbiology Letters vol 255 no 2 pp233ndash239 2006
[154] R Canton M Akova Y Carmeli et al ldquoRapid evolutionand spread of carbapenemases among Enterobacteriaceae inEuroperdquo Clinical Microbiology and Infection vol 18 no 5 pp413ndash431 2012
[155] NWoodford J F Turton and DM Livermore ldquoMultiresistantGram-negative bacteria the role of high-risk clones in thedissemination of antibiotic resistancerdquo FEMS MicrobiologyReviews vol 35 no 5 pp 736ndash755 2011
[156] G C Conway S C Smole D A Sarracino R D Arbeit and PE Leopold ldquoPhyloproteomics species identification of Enter-obacteriaceae using matrix-assisted laser desorptionionizationtime-of-flight mass spectrometryrdquo Journal of Molecular Micro-biology and Biotechnology vol 3 no 1 pp 103ndash112 2001
[157] M Trevino P Areses M D Penalver et al ldquoSusceptibilitytrends of Bacteroides fragilis group and characterisation ofcarbapenemase-producing strains by automated REP-PCR andMALDI TOFrdquo Anaerobe vol 18 no 1 pp 37ndash43 2012
[158] D S Perlin ldquoResistance to echinocandin-class antifungaldrugsrdquoDrug Resistance Updates vol 10 no 3 pp 121ndash130 2007
[159] P A Demirev N S Hagan M D Antoine J S Lin and A BFeldman ldquoEstablishing drug resistance in microorganisms bymass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 24 no 8 pp 1194ndash1201 2013
[160] K Sparbier C Lange J Jung A Wieser S Schubert and MKostrzewa ldquoMaldi biotyper-based rapid resistance detection bystable-isotope labelingrdquo Journal of Clinical Microbiology vol 51no 11 pp 3741ndash3748 2013
[161] K M Hujer A M Hujer A Endimiani et al ldquoRapid determi-nation of quinolone resistance in Acinetobacter spprdquo Journal ofClinical Microbiology vol 47 no 5 pp 1436ndash1442 2009
[162] C Massire C A Ivy R Lovari et al ldquoSimultaneous identifi-cation of mycobacterial isolates to the species level and deter-mination of tuberculosis drug resistance by PCR followed byelectrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 908ndash917 2011
[163] F Wang C Massire H Li et al ldquoMolecular characterization ofdrug-resistant Mycobacterium tuberculosis isolates circulatingin China by multilocus PCR and electrospray ionization massspectrometryrdquo Journal of ClinicalMicrobiology vol 49 no 7 pp2719ndash2721 2011
[164] P J Simner S P Buckwalter J R Uhl and N L WengenackldquoIdentification of mycobacterium species and Mycobacteriumtuberculosis complex resistance determinants by use of Pcr-electrospray ionization mass spectrometryrdquo Journal of ClinicalMicrobiology vol 51 no 11 pp 3492ndash3498 2013
BioMed Research International 19
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
[165] C L Brinkman P Vergidis J R Uhl et al ldquoPCR-electrosprayionization mass spectrometry for direct detection of pathogensand antimicrobial resistance from heart valves in patients withinfective endocarditisrdquo Journal of Clinical Microbiology vol 51no 7 pp 2040ndash2046 2013
[166] D M Wolk L B Blyn T A Hall et al ldquoPathogen profilingrapid molecular characterization of Staphylococcus aureus byPCRelectrospray ionization-mass spectrometry and correla-tion with phenotyperdquo Journal of Clinical Microbiology vol 47no 10 pp 3129ndash3137 2009
[167] H C Yun R E Kreft M A Castillo et al ldquoComparison ofPCRelectron spray ionization-time-of-flight-mass spectrome-try versus traditional clinical microbiology for active surveil-lance of organisms contaminating high-use surfaces in a burnintensive care unit an orthopedicward andhealthcareworkersrdquoBMC Infectious Diseases vol 12 article 252 2012
[168] W L Drew ldquoCytomegalovirus resistance testing pitfalls andproblems for the clinicianrdquo Clinical Infectious Diseases vol 50no 5 pp 733ndash736 2010
[169] L N Ikryannikova E A Shitikov D G Zhivankova E NIlina M V Edelstein and V M Govorun ldquoA MALDI TOFMS-basedminisequencingmethod for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains ofEnterobacteriaceaerdquo Journal of Microbiological Methods vol 75no 3 pp 385ndash391 2008
[170] M Gniadkowski ldquoEvolution and epidemiology of extended-spectrum 120573-lactamases (ESBLs) and ESBL-producing microor-ganismsrdquo Clinical Microbiology and Infection vol 7 no 11 pp597ndash608 2001
[171] V A Vereshchagin E N Ilina M M Zubkov T V Priput-nevich A A Kubanova andVMGovorun ldquoDetection of fluo-roquinolone resistance SNPs in gyrA and parC GENES of Neis-seria gonorrhoeae using MALDI-TOF Mass-SpectrometryrdquoMolekulyarnaya Biologiya vol 39 no 6 pp 923ndash932 2005
[172] K T Momynaliev O V Selezneva A A Kozlova V AVereshchagin E N Ilrsquoina and V M Govorun ldquoA2144G is themain mutation in the 235 rRNA gene of Helicobacter pyloriassociated with clarithromycin resistancerdquoGenetika vol 41 no10 pp 1338ndash1344 2005
[173] L N Ikryannikova M V Afanasrsquoev T A Akopian et al ldquoMass-spectrometry based minisequencing method for the rapiddetection of drug resistance in Mycobacterium tuberculosisrdquoJournal of Microbiological Methods vol 70 no 3 pp 395ndash4052007
[174] N S Lurain and S Chou ldquoAntiviral drug resistance of humancytomegalovirusrdquo Clinical Microbiology Reviews vol 23 no 4pp 689ndash712 2010
[175] A Humar and D Snydman ldquoCytomegalovirus in solid organtransplant recipientsrdquoTheAmerican Journal of Transplantationvol 9 supplement 4 pp S78ndashS86 2009
[176] C N Kotton D Kumar A M Caliendo et al ldquoInternationalconsensus guidelines on the management of cytomegalovirusin solid organ transplantationrdquo Transplantation vol 89 no 7pp 779ndash795 2010
[177] S Chou R HWaldemer A E Senters et al ldquoCytomegalovirusUL97 phosphotransferase mutations that affect susceptibility toganciclovirrdquo Journal of Infectious Diseases vol 185 no 2 pp162ndash169 2002
[178] R H Deurenberg and E E Stobberingh ldquoThe molecularevolution of hospital- and community-associated methicillin-resistant Staphylococcus aureusrdquo Current Molecular Medicinevol 9 no 2 pp 100ndash115 2009
[179] J Pootoolal J Neu and G D Wright ldquoGlycopeptide antibioticresistancerdquoAnnual Review of Pharmacology and Toxicology vol42 pp 381ndash408 2002
[180] S Tschudin-Sutter R Frei M Dangel A Stranden and AF Widmer ldquoRate of transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae without contact isola-tionrdquo Clinical Infectious Diseases vol 55 no 11 pp 1505ndash15112012
[181] M Hilty B Y Betsch K Bogli-Stuber et al ldquoTransmissiondynamics of extended-spectrum 120573-lactamase-producing enter-obacteriaceae in the tertiary care hospital and the householdsettingrdquo Clinical Infectious Diseases vol 55 no 7 pp 967ndash9752012
[182] J M Nerby R Gorwitz L Lesher et al ldquoRisk factors forhousehold transmission of community-associated methicillin-resistant staphylococcus aureusrdquo Pediatric Infectious DiseaseJournal vol 30 no 11 pp 927ndash932 2011
[183] K Razazi L P G Derde M Verachten P Legrand P Lespritand C Brun-Buisson ldquoClinical impact and risk factors forcolonization with extended-spectrum 120573-lactamaseproducingbacteria in the intensive care unitrdquo Intensive Care Medicine vol38 no 11 pp 1769ndash1778 2012
[184] X Didelot R Bowden D J Wilson T E A Peto and DW Crook ldquoTransforming clinical microbiology with bacterialgenome sequencingrdquoNature Reviews Genetics vol 13 no 9 pp601ndash612 2012
[185] C Bertelli and G Greub ldquoRapid bacterial genome sequencingmethods and applications in clinical microbiologyrdquo ClinicalMicrobiology and Infection vol 19 no 9 pp 803ndash813 2013
[186] P Herindrainy F Randrianirina R Ratovoson et al ldquoRec-tal carriage of extended-spectrum beta-lactamase-producingGram-negative bacilli in community settings in MadagascarrdquoPLoS ONE vol 6 no 7 Article ID e22738 2011
[187] H Frickmann A Hanle A Essig et al ldquoFluorescence in situhybridization (FISH) for rapid identification of Salmonella sppfrom agar and blood culture broth-An option for the tropicsrdquoInternational Journal ofMedicalMicrobiology vol 303 no 5 pp277ndash284 2013
[188] A Kumar D Roberts K E Wood et al ldquoDuration of hypoten-sion before initiation of effective antimicrobial therapy is thecritical determinant of survival in human septic shockrdquo CriticalCare Medicine vol 34 no 6 pp 1589ndash1596 2006
[189] A Kumar N Safdar S Kethireddy and D Chateau ldquoA survivalbenefit of combination antibiotic therapy for serious infectionsassociated with sepsis and septic shock is contingent only on therisk of death a meta-analyticmeta-regression studyrdquo CriticalCare Medicine vol 38 no 8 pp 1651ndash1664 2010
