research article usefulness of multiplex real-time pcr for...

Research ArticleUsefulness of Multiplex Real-Time PCR forSimultaneous Pathogen Detection and ResistanceProfiling of Staphylococcal Bacteremia

Yousun Chung1 Taek Soo Kim2 Young Gi Min2 Yun Ji Hong3

Jeong Su Park3 Sang Mee Hwang3 Kyoung-Ho Song4 Eu Suk Kim4 Kyoung Un Park13

Hong Bin Kim4 Junghan Song13 and Eui-Chong Kim12

1Department of Laboratory Medicine Seoul National University College of Medicine 101 Daehak-ro Jongno-guSeoul 03080 Republic of Korea2Department of Laboratory Medicine Seoul National University Hospital 101 Daehak-ro Jongno-gu Seoul 03080 Republic of Korea3Department of Laboratory Medicine Seoul National University Bundang Hospital 82 Gumi-ro 173 Beon-gil Bundang-guSeongnam-si Gyeonggi-do 13620 Republic of Korea4Department of Internal Medicine Seoul National University Bundang Hospital 82 Gumi-ro 173 Beon-gil Bundang-guSeongnam-si Gyeonggi-do 13620 Republic of Korea

Correspondence should be addressed to Kyoung Un Park m91w95pfsnuackr

Received 24 March 2016 Accepted 23 May 2016

Academic Editor Giulio Mengozzi

Copyright copy 2016 Yousun Chung et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Staphylococci are the leading cause of nosocomial blood stream infections Fast and accurate identification of staphylococci andconfirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics A multiplex real-timePCR assay that targets mecA femA specific for S aureus femA specific for S epidermidis 16S rRNA for universal bacteria and16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC) For the 262 blood cultures identified to the species level with the MicroScan WalkAway system(Siemens Healthcare Diagnostics USA) the direct real-time PCR assay of positive blood cultures showed very good agreementfor the categorization of staphylococci into methicillin-resistant S aureus (MRSA) methicillin-susceptible S aureus (MSSA)methicillin-resistant S epidermidis (MRSE)methicillin-susceptible S epidermidis (MSSE) methicillin-resistant non-S epidermidisCoNS (MRCoNS) and methicillin-susceptible non-S epidermidis CoNS (MSCoNS) (120581 = 09313) The direct multiplex real-timePCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with aturnaround time of no more than 4 h Further studies should evaluate the clinical outcome of using this rapid real-time PCR assayin glycopeptide antibiotic therapy in clinical settings

1 Introduction

Staphylococci are the most commonly isolated organismsin clinical laboratories accounting for almost 30 of allnosocomial infections and 50 of nosocomial bloodstreaminfections [1] Staphylococcus aureus is the leading cause ofnosocomial infections [2] and methicillin-resistant S aureus(MRSA) infections result in significant morbidity mortalityand longer hospital stays if not treated early with effectiveantibiotics [3] Coagulase-negative staphylococci (CoNS) arethemost common isolates from blood culture andmore than

70 are resistant to oxacillin [4] Although they are known tocontaminate blood cultures as a result of their colonization onthe skin and mucous membranes they have recently becomeimportant pathogens causing nosocomial infections with theincreasing use of invasive procedures and prosthetic devices[5 6] Fast and accurate identification of staphylococci andconfirmation of their methicillin resistance are crucial forimmediate treatment with effective antibiotics which willresult in decreased morbidity and mortality rates [7]

The conventional culture method for the identificationand susceptibility testing of positive blood cultures has

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 6913860 7 pageshttpdxdoiorg10115520166913860

2 BioMed Research International

Table 1 PCR primers and TaqMan probes formecA femA specific for S aureus (femA-SA) femA specific for S epidermidis (femA-SE) anduniversal 16S rRNA and PCR primers for staphylococcal 16S rRNA

Target genes Sequence

mecA51015840-CATTGATCGCAACGTTCAATTT-31015840

51015840-TGGTCTTTCTGCATTCCTGGA-31015840

51015840-FAM-TGGAAGTTAGATTGGGATCATAGCGTCAT-TAMRA-31015840

femA-SA51015840-TGCCTTTACAGATAGCATGCCA-31015840

51015840-AGTAAGTAAGCAAGCTGCAATGACC-31015840

51015840-JOE-TCATTTCACGCAAACTGTTGGCCACTATG-BHQ1-31015840

femA-SE51015840-CAACTCGATGCAAATCAGCAA-31015840

51015840-GAACCGCATAGCTCCCTGC-31015840

51015840-JOE-TACTACGCTGGTGGAACTTCAAATCGTTATCG-BHQ1-31015840

Universal 16S rRNA51015840-TCCTACGGGAGGCAGCAGT-31015840

51015840-GGACTACCAGGGTATCTAATCCTGTT-31015840

51015840-FAM-CGTATTACCGCGGCTGCTGGCAC-TAMRA-31015840

Staphylococcal 16S rRNA 51015840-GCAAGCGTTATCCGGATTT-31015840

51015840-CTTAATGATGGCAACTAAGC-31015840

some disadvantages including long turnaround time andpotential false-negative results when samples are obtainedafter antimicrobial therapy Real-time PCR is significantlyfaster than conventional PCR and other detection methodsand its excellent sensitivity and specificity low contaminationrisk ease of use and high speed have made real-time PCRtechnology appealing to clinical microbiology laboratories[8]

The aim of this study was to develop and evaluate a mul-tiplex real-time PCR assay for the rapid detection and identi-fication of MRSA methicillin-susceptible S aureus (MSSA)methicillin-resistant S epidermidis (MRSE) methicillin-susceptible S epidermidis (MSSE) methicillin-resistant non-S epidermidisCoNS (MRCoNS) andmethicillin-susceptiblenon-S epidermidis CoNS (MSCoNS) directly from positiveblood cultures containing Gram-positive cocci in clusters(GPCC) by targetingmecA for determining methicillin resis-tance femA specific for S aureus (femA-SA) femA specificfor S epidermidis (femA-SE) 16S rRNA for universal bacteriaand 16S rRNA specific for staphylococci

2 Materials and Methods

21 Blood Culture This study evaluated 290 blood culturescontaining GPCC obtained from March 2013 to December2013 at Seoul National University Bundang Hospital Two ormore pairs of culture bottles for aerobes or anaerobes wereincubated in BacTAlert 3D (bioMerieux Inc Durham NCUSA) or BACTEC FX (BD Diagnostics Sparks MD USA)blood culture systems for 5 days after inoculation for blooddrawn from the patient If bacterial growth was not detectedwithin 5 days then the blood culture result was considerednegative When bacterial growth was noted blood from thepositive bottles was Gram-stained and samples containingGPCC (230 specimens in BacTAlert 3D (bioMerieux Inc)and 60 specimens in BACTEC FX (BD Diagnostics)) wereinoculated onto blood agar plates and cultured overnight at35∘C in a 5CO

2incubator Isolates were identified by colony

morphology Gram-staining catalase and coagulase tests

Final identification according to phenotypic characteristicsand antimicrobial susceptibility tests was performed usingthe MicroScanWalkAway system (Siemens Healthcare Diag-nostics Deerfield IL USA) with Pos Combo Panel Type 1A

22 DNA Extraction A 100 120583L aliquot of blood was drawndirectly from the positive blood culture bottles collected onfilter paper and dried for 15min at room temperature Theblood spot was lysed with 2mL of lysis buffer for 30min atroom temperature Next the paper was removed and 2 120583L oflysozyme-Tris-EDTA buffer was added to 500120583L of the eluateand incubated for 30min at 37∘C The sample was incubatedagain for 10min in 2mL of lysis buffer and the final eluatewas used for nucleic acid extraction with the NucliSENSeasyMAG platform (bioMerieux Inc)

23 Multiplex Real-Time PCR Assay Targeting mecA femA-SA femA-SE and Universal 16S rRNA The double duplexreal-time PCR TaqMan assay was performed with two tubesin one reaction One tube corresponded to the targets femA-SA and mecA and the other to the targets femA-SE anduniversal 16S rRNA The primers and probes for each targetwere designed as described by previously published studies[9 10] (Table 1) Real-time PCR was conducted in a totalvolume of 20 120583L including 20120583L of 10x LightCycler FastStartDNA Master HybProbe (Roche Diagnostics MannheimGermany) 24 120583L of 15mMMgCl

2(Takara Bio Shiga Japan)

02 120583M of each primer and 01 120583M of each probe with 30 120583Lof DNA template The amplification conditions used by them2000rt instrument (Abbott Diagnostic Chicago IL USA)were as follows 95∘C for 10min followed by 35 cycles of95∘C for 15 sec and 60∘C for 1min in a single real-time PCRassay Positive controls with MRSA MSSA and MRSE anda negative control with sterile distilled water (DW) wereincluded throughout the procedures

24 Additional Real-Time PCR Assay Targeting Staphylococcal16S rRNA A real-time PCR assay targeting staphylococcal16S rRNA was performed using a LightCycler 20 system

BioMed Research International 3

Table 2 Interpretation of results by real-time PCR assays

mecA femA-SAa femA-SEb Universal16S rRNA

Staphylococcal16S rRNA

MRSA Pc P Nd P PMSSA N P N P PMRSE P N P P PMSSE N N P P PMRCoNS P N N P PMSCoNS N N N P PNonstaphylococci P or N N N P NafemA specific for S aureusbfemA specific for S epidermidiscPositivedNegative

(Roche Diagnostics) for the detection of staphylococciPrimers targeting staphylococcal 16S rRNA were designedas described in a previously published study [11] (Table 1)Amplification reactions were performed in a 20 120583L volumecontaining 2 120583L of DNA template 025120583M of each primerand 10 120583L of 2x SYBR Premix Ex Taq (Takara Bio) Theconditions consisted of an initial denaturation at 95∘C for10min followed by amplification program for 30 cycles of10 sec at 95∘C 20 sec at 58∘C and 20 sec at 74∘C with fluores-cence acquisition at the end of each cycle The amplificationprogram was followed by a melting program consisting ofheating to 95∘Cwith a 0 sec hold and 15 sec at 60∘Cand a grad-ual increase to 99∘C at a rate of 01∘Csec with fluorescenceacquisition at each temperature transition Positive controlswith MRSA MSSA and MRSE and a negative control withsterile DW were included throughout the procedure Theexistence of the target was confirmed by melting curveanalysis If the melting temperature (Tm) of the samples waswithin 05∘C of the Tm of the positive controlrsquos product theywere regarded as positive

All the above-mentioned real-time PCR assays wererepeated with DNA extracted with subcultured colonies forcomparison with the direct specimens

25 Categorization of Real-Time PCR Results Using a dou-ble duplex real-time PCR assay the detection of femA-SAand universal 16S rRNA indicated the presence of MSSAwhile the detection of femA-SA mecA and universal 16SrRNA indicated the presence of MRSA If femA-SE anduniversal 16S rRNA were detected the presence of MSSEwas inferred while the detection of femA-SE mecA anduniversal 16S rRNA indicated the presence of MRSE Thedetection of mecA and universal 16S rRNA was interpretedas indicating the presence of MRCoNS or methicillin-resistant nonstaphylococci while the detection of universal16S rRNA alone was interpreted as indicating the presenceofMSCoNS ormethicillin-susceptible nonstaphylococciTheadditional PCR test targeting staphylococcal 16S rRNA con-firmed whether the isolate was staphylococci The detectionof universal 16S rRNA but not staphylococcal 16S rRNAwas interpreted as indicating the presence of bacteria otherthan staphylococci (Table 2) The real-time PCR results were

compared with MicroScan identification and susceptibilityresults as a reference

3 Results

31 Identification Results Obtained with the MicroScan Walk-Away System (Siemens Healthcare Diagnostics) Of the 290positive blood cultures with GPCC 262 cultures were iden-tified to the species level by MicroScan as follows 89 Saureus 96 S epidermidis 27 S hominis 26 S capitis 9 Shaemolyticus 5 S capitis subsp urealyticus 2 Staphylococcussaprophyticus 2 S lugdunensis 2 S hominis subsp hominis 1S cohnii 1 S auricularis 1 S schleiferi and 1 S schleiferi subspcoagulans Fifteen isolates showed low-probability identifica-tion with multiple possible staphylococcal species and theremaining 13 isolates were nonstaphylococci including 12Micrococcus species and 1 Enterococcus faecalis

32 Comparison of Results Obtained by Real-Time PCR withDirect Specimens and the Results of the MicroScan WalkAwaySystem (Siemens Healthcare Diagnostics) The real-time PCRidentification ofMRSA correlated with theMicroScan resultsfor 46 out of 47 specimensThe discordant one was identifiedas MSSA by MicroScan The real-time PCR identificationof MSSA correlated with the MicroScan results for all 42isolates

Eighty-five out of 96 blood cultures identified as contain-ing MRSE by real-time PCR were confirmed by MicroScanwhile four isolates were identified as MRCoNS and two asMSSE The MicroScan results for the remaining five isolateswere low-probability identifications with multiple possiblestaphylococcal species that were all resistant to methicillinThe real-time PCR identification of MSSE correlated withthe MicroScan results for seven out of eight isolates thediscordant isolate was identified as MSCoNS by MicroScan

A total of 50 out of 58 blood cultures containingMRCoNS as determined by real-time PCR were confirmedto be MRCoNS with MicroScan while one isolate wasidentified as MRSE and another as MSCoNSTheMicroScanresults for the remaining six isolates were low-probabilityidentifications with multiple possible staphylococcal speciesthat were all resistant to methicillin Eighteen out of 26


Table 3 Comparison of results by real-time PCR with direct specimen and results by MicroScan WalkAway system (thirteen isolates ofnonstaphylococci are excluded)

MicroScan WalkAway system Real-time PCR assays with direct specimenMRSAa MSSAb MRSEc MSSEd MRCoNSe MSCoNSf

MRSA 46 mdash mdash mdash mdash mdashMSSA 1 42 mdash mdash mdash mdashMRSE mdash mdash 85 mdash 1 mdashMSSE mdash mdash 2 7 mdash 1MRCoNS mdash mdash 4 mdash 50 3MSCoNS mdash mdash mdash 1 1 18Unidentified staphylococcilowast mdash mdash 5 mdash 6 4lowastAll the results ofmecA were in concordance with phenotypic methicillin resistanceaMethicillin-resistant S aureusbMethicillin-susceptible S aureuscMethicillin-resistant S epidermidisdMethicillin-susceptible S epidermidiseMethicillin-resistant non-S epidermidis CoNSfMethicillin-susceptible non-S epidermidis CoNS

blood cultures containing MSCoNS by real-time PCR wereconfirmed by MicroScan while three were identified asMRCoNS and one as MSSE The MicroScan results for theremaining four isolates were low-probability identificationswith multiple possible staphylococcal species that were allsensitive to methicillin Thirteen isolates that were inter-preted as nonstaphylococci by real-time PCRwere confirmedas 12 Micrococcus species and 1 Enterococcus faecalis byMicroScan system

For the 262 blood cultures identified to the specieslevel by MicroScan the results agreed very well with thoseobtained by real-time PCR for staphylococcal species cate-gorization into MRSA MSSA MRSE MSSE MRCoNS andMSCoNS (Cohenrsquos unweighted kappa coefficient 120581 = 09313)(Table 3) The sensitivity and specificity of mecA femA-SAfemA-SE universal 16S rRNA and staphylococcal 16S rRNAwere evaluated with the 262 blood cultures according tothe MicroScan results The sensitivity and specificity wererespectively 984 and 945 for themecA gene 1000 and1000 for femA-SA 979 and 970 for femA-SE 1000and 1000 for universal 16S rRNA and 1000 and 1000for staphylococcal 16S rRNA (Table 4)

33 Comparison of the Results Obtained by Real-Time PCRwith Direct Specimens and Real-Time PCR with SubculturedColonies The real-time PCR results of direct specimenscorrelated with the PCR results of subcultured coloniesfor 282 of 290 samples The results for 14 out of the 282concordant samples were discordant with the MicroScanresults Of the seven samples that were interpreted as MRSEby both real-time PCR techniques three were identified byMicroScan asMSCoNS three asmethicillin-resistant staphy-lococci (not identified to the species level) and one as MSSEIn two samples identified as MRCoNS by both real-timePCR techniques the MicroScan identifications were MRSEand MSCoNS Of the four samples identified as MSCoNSby both real-time PCR techniques one was identified byMicroScan as MSSE and the other three were identified

Table 4 Sensitivity and specificity of mecA femA specific forS aureus (femA-SA) femA specific for S epidermidis (femA-SE)universal 16S rRNA and staphylococcal 16S rRNA

Target genes Sensitivity () Specificity ()mecA 984 945femA-SA 1000 1000femA-SE 979 970Universal 16S rRNA 1000 1000Staphylococcal 16S rRNA 1000 1000

as MRCoNS The remaining sample identified as MSSE byboth real-time PCR techniques was identified as MSCoNS byMicroScan

There were eight discordant results between real-timePCR with direct specimens and real-time PCR with sub-cultured colonies In two samples that were identified asMSSA and MSSE by MicroScan only real-time PCR withdirect specimens detected mecA In one sample identifiedas MRCoNS by MicroScan only real-time PCR with directspecimens detected femA-SE In another sample identified asMRSA byMicroScan only real-time PCRwith colonies failedto detect femA-SA Finally in the remaining four sampleswhich were identified as MRSE by MicroScan only real-timePCR with colonies failed to detect femA-SE The discordantresults for the three methods are shown in Table 5

4 Discussion

In this study a multiplex real-time PCR assay that targetsmecA femA-SA femA-SE universal bacterial 16S rRNAand staphylococci-specific 16S rRNA was developed andevaluated with clinical samples for the rapid identificationof GPCC and determination of methicillin susceptibilityOverall there was very good agreement between the real-time PCR with direct specimens and the MicroScan systemfor the 290 blood cultures indicating reliable categorization


Table 5 The discordant results between real-time PCR with direct specimen real-time PCR with subcultured colonies and MicroScanWalkAway system

Real-time PCR withdirect specimen

Real-time PCR with subculturedcolonies

MicroScanWalkaway system Number

MRSEa MRSE MRCoNSb 3MRSE MRSE MSSEc 1MRSE MRSE MR staphylococci 3MSSE MSSE MSCoNSd 1MRCoNS MRCoNS MRSE 1MRCoNS MRCoNS MSCoNS 1MSCoNS MSCoNS MSSE 1MSCoNS MSCoNS MRCoNS 3MRSAe MSSAf MSSA 1MRSA MRCoNS MRSA 1MRSE MSSE MSSE 1MRSE MRCoNS MRCoNS 1MRSE MRCoNS MRSE 4aMRSE methicillin-resistant S epidermidisbMRCoNS methicillin-resistant non-S Epidermidis CoNScMSSE methicillin-susceptible S epidermidisdMSCoNS methicillin-susceptible non-S epidermidis CoNSeMRSA methicillin-resistant S aureusfMSSA methicillin-susceptible S aureus

as MRSA MSSA MRSE MSSE MRCoNS MSCoNS andnonstaphylococci

Real-time PCR correctly identified all 46 positive bloodcultures which were confirmed as MRSA by MicroScanOf the 43 blood cultures identified as MSSA by MicroScanall but one were identified as MSSA by real-time PCR thediscordant culture was identified as MRSA by real-time PCRand as MSSA by additional PCR with subcultured coloniesThis result could be due to nonspecific amplification of themecA gene but the presence of the mecA gene at a very lowlevel in the positive blood bottle is also a potential expla-nation There were no S aureus isolates that were identifiedas mecA negative by real-time PCR but methicillin-resistantby MicroScan It would be more troublesome if methicillin-resistant strains were not detected by PCRbecause these casesare likely to result in treatment failures

In two out of 96 cultures identified as S epidermidisby MicroScan the femA-SE gene was not detected by real-time PCR with direct specimens or real-time PCR withsubcultured colonies As suggested by the manufacturers ofMicroScan we used a cut-off of 85 for identification at thespecies level as indicating a high probability andno additionaltests required However previous studies have reportedthe misidentification of staphylococci by MicroScan andmisidentification by MicroScan is also a possibility in thesecases [12 13]

The mecA gene was detected by real-time PCR in all 86blood cultures that were identified as MRSE by MicroScanIn two samples out of 10 blood cultures identified as MSSEby MicroScan mecA was detected by real-time PCR PCRtesting usingDNA from the subcultured colonieswas positivefor mecA in one sample but negative in the other sample In

the case of positive mecA identification by both PCR assays(direct specimen and subcultured colonies) but identificationas methicillin-susceptible by MicroScan there is a possibilityof false oxacillin susceptibility results due to the heteroresis-tance phenomenon which is a consequence of the complexregulation of the phenotypic expression of the mecA gene[14 15]

Among the 57 isolates identified as MRCoNS byMicroScan three were negative inmecA using real-time PCRdirectly from the blood culture bottles and additional PCRwith subcultured colonies This result could be attributedto false-negatives due to the possible limitations of PCRassays such as the presence of PCR inhibitors Howeveranother possible explanation is a resistance mechanism otherthan mecA Strains without the mecA gene can acquiremethicillin resistance modification of normal PBP genes oroverproduction of staphylococcal 120573-lactamase resulting inmethicillin resistance [16ndash18] There were two discordantresults out of 20 blood cultures identified as MSCoNS byMicroScan In one sample mecA was detected by both real-time PCR assays (direct specimen and subcultured colonies)while femA-SE genes were detected in the other sample byboth real-time PCR assays

For the fifteen staphylococci that were not identified tothe species level by MicroScan the mecA results were all inconcordance with phenotypic methicillin resistance And forthe thirteen isolates identified as Micrococci and Enterococciby MicroScan only universal 16S rRNA genes were detectedby real-time PCR assays

One limitation of this assay is that it cannot determinemethicillin resistance of S aureus when staphylococci otherthan S aureus are also detected asmixed culture with positive


mecA Unlike conventional identification and susceptibilitytesting multiplex real-time PCR assay is unable to indicatemethicillin resistance to each of the staphylococci when itcomes to mixed culture [19 20] It would be importantto incorporate the multiplex real-time PCR assay alongsideconventional identification and susceptibility testing

In a clinical laboratory setting the rapid and reliableidentification of staphylococci and the determination oftheir methicillin susceptibility are important for effectiveantibiotic therapy and avoidance of the inappropriate use ofglycopeptides [21] Conventional identification and suscepti-bility testing of positive blood cultures based on phenotypiccharacteristics can take up to 48 h after the GPCC arerecognized by Gram-staining For prompt initiation withoptimal antibiotic therapy clinical laboratories are incor-porating molecular diagnostic methods such as real-timePCR assays which provide rapid sensitive and specificdetection of microbial pathogens within a few hours [22ndash24] Recently real-time PCR assays have been developedfor the detection of staphylococci directly from positiveblood culture bottles in clinical microbiology laboratories[25ndash33] However these assays are limited in that althoughthey are able to rapidly identify staphylococci and determinemethicillin susceptibility they only allow the identification ofstaphylococci and cannot discriminate Gram-positive cocciother than staphylococci We hypothesized that the additionof a PCR assay targeting staphylococcal 16S rRNA mightmitigate this limitation and target both universal 16S rRNAand staphylococcal 16S rRNA In this study thirteen isolatesthat were recognized as GPCC by preliminary Gram-stainingwere confirmed as Micrococci and Enterococci by MicroScansystem without an assay targeting the staphylococci-specific16S rRNA they would have been interpreted as MSCoNS byreal-time PCR

5 Conclusions

The direct multiplex real-time PCR assay of positive bloodcultures containing GPCC can provide essential informationfor prompt initiation of appropriate antibiotic treatment atthe critical point of infection with a turnaround time of nomore than 4 h which includes 2 h for DNA extraction and15 h for PCR Further studies should evaluate the clinicaloutcome and benefit of using this rapid real-time PCRassay incorporated with conventional culture methods forglycopeptide antibiotic therapy in the case of positive bloodcultures growing GPCC in clinical settings

Additional Points

(i) We developed multiplex real-time PCR assay for rapidcategorization of staphylococci (ii) The targets were mecAfemA universal 16S rRNA and staphylococcal 16S rRNA (iii)The results directly from positive blood culture bottles werecompared with MicroScan (iv) The assay rapidly detectedmethicillin-resistant staphylococci with good agreement

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

The first two authors Yousun Chung and Taek Soo Kimcontributed equally to this work

References

[1] A Skow K A Mangold M Tajuddin et al ldquoSpecies-levelidentification of staphylococcal isolates by real-time PCR andmelt curve analysisrdquo Journal of ClinicalMicrobiology vol 43 no6 pp 2876ndash2880 2005

[2] E Gradelski L Valera L Aleksunes D Bonner and J Fung-Tomc ldquoCorrelation between genotype and phenotypic catego-rization of staphylococci based onmethicillin susceptibility andresistancerdquo Journal of Clinical Microbiology vol 39 no 8 pp2961ndash2963 2001

[3] T P Lodise P S McKinnon L Swiderski and M J RybakldquoOutcomes analysis of delayed antibiotic treatment for hospital-acquired Staphylococcus aureus bacteremiardquo Clinical InfectiousDiseases vol 36 no 11 pp 1418ndash1423 2003

[4] R B R Ferreira N L P Iorio K L Malvar et al ldquoCoagulase-negative staphylococci comparison of phenotypic and geno-typic oxacillin susceptibility tests and evaluation of the agarscreening test by using different concentrations of oxacillinrdquoJournal of Clinical Microbiology vol 41 no 8 pp 3609ndash36142003

[5] T G Krediet E M Mascini E Van Rooij et al ldquoMolecular epi-demiology of coagulase-negative staphylococci causing sepsisin a neonatal intensive care unit over an 11-year periodrdquo Journalof Clinical Microbiology vol 42 no 3 pp 992ndash995 2004

[6] B Favre SHugonnet L CorreaH Sax P Rohner andD PittetldquoNosocomial bacteremia clinical significance of a single bloodculture positive for coagulase-negative staphylococcirdquo InfectionControl and Hospital Epidemiology vol 26 no 8 pp 697ndash7022005

[7] E LMunson D J Diekema S E Beekmann K C Chapin andG V Doern ldquoDetection and treatment of bloodstream infec-tion laboratory reporting and antimicrobial managementrdquoJournal of Clinical Microbiology vol 41 no 1 pp 495ndash497 2003

[8] 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 no 1 pp 165ndash256 2006

[9] P Francois D Pittet M Bento et al ldquoRapid detection ofmethicillin-resistant Staphylococcus aureus directly from sterileor nonsterile clinical samples by a newmolecular assayrdquo Journalof Clinical Microbiology vol 41 no 1 pp 254ndash260 2003

[10] M A Nadkarni F E Martin N A Jacques and N HunterldquoDetermination of bacterial load by real-time PCR using abroad-range (universal) probe and primers setrdquo Microbiologyvol 148 no 1 pp 257ndash266 2002

[11] H Al-Talib C Y Yean A Al-Khateeb et al ldquoA pentaplexPCR assay for the rapid detection of methicillin-resistantStaphylococcus aureus and Panton-Valentine Leucocidinrdquo BMCMicrobiology vol 9 article 113 2009

[12] M Kim S R Heo S H Choi et al ldquoComparison of theMicroScan VITEK 2 and Crystal GP with 16S rRNA sequenc-ing and MicroSeq 500 v20 analysis for coagulase-negativestaphylococcirdquo BMCMicrobiology vol 8 article 233 2008

[13] A N Olendzki E M Barros M S Laport K R N Dos Santosand M Giambiagi-Demarval ldquoReliability of the MicroScan


WalkAway PC21 panel in identifying and detecting oxacillinresistance in clinical coagulase-negative staphylococci strainsrdquoEuropean Journal of Clinical Microbiology amp Infectious Diseasesvol 33 no 1 pp 29ndash33 2014

[14] G L Archer D M Niemeyer J A Thanassi and M JPucci ldquoDissemination among staphylococci of DNA sequencesassociated with methicillin resistancerdquo Antimicrobial Agentsand Chemotherapy vol 38 no 3 pp 447ndash454 1994

[15] F Martineau F J Picard N Lansac et al ldquoCorrelation betweenthe resistance genotype determined by multiplex PCR assaysand the antibiotic susceptibility patterns of Staphylococcusaureus and Staphylococcus epidermidisrdquo Antimicrobial Agentsand Chemotherapy vol 44 no 2 pp 231ndash238 2000

[16] E Galdiero G Liguori M DrsquoIsanto N Damiano and LSommese ldquoDistribution of mecA among methicillin-resistantclinical staphylococcal strains isolated at hospitals in NaplesItalyrdquo European Journal of Epidemiology vol 18 no 2 pp 139ndash145 2003

[17] J L Gerberding C Miick H H Liu and H F Cham-bers ldquoComparison of conventional susceptibility tests withdirect detection of penicillin-binding protein 2a in borderlineoxacillin-resistant strains of Staphylococcus aureusrdquo Antimicro-bial Agents and Chemotherapy vol 35 no 12 pp 2574ndash25791991

[18] A Jain A Agarwal and R K Verma ldquoCefoxitin disc diffusiontest for detection of meticillin-resistant staphylococcirdquo Journalof Medical Microbiology vol 57 no 8 pp 957ndash961 2008

[19] L Jukes J Mikhail N Bome-Mannathoko et al ldquoRapid differ-entiation of Staphylococcus aureus Staphylococcus epidermidisand other coagulase-negative staphylococci and meticillin sus-ceptibility testing directly from growth-positive blood culturesby multiplex real-time PCRrdquo Journal of Medical Microbiologyvol 59 no 12 pp 1456ndash1461 2010

[20] A Kilic K L Muldrew Y-W Tang and A C BasustaogluldquoTriplex real-time polymerase chain reaction assay for simul-taneous detection of Staphylococcus aureus and coagulase-negative staphylococci and determination of methicillin resis-tance directly from positive blood culture bottlesrdquo DiagnosticMicrobiology and Infectious Disease vol 66 no 4 pp 349ndash3552010

[21] 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

[22] S Klaschik L E Lehmann A Raadts et al ldquoDetection anddifferentiation of in vitro-spiked bacteria by real-time PCR andmelting-curve analysisrdquo Journal of ClinicalMicrobiology vol 42no 2 pp 512ndash517 2004

[23] T Mohammadi H W Reesink C M J E Vandenbroucke-Grauls and P H M Savelkoul ldquoOptimization of real-timePCR assay for rapid and sensitive detection of eubacterial 16Sribosomal DNA in platelet concentratesrdquo Journal of ClinicalMicrobiology vol 41 no 10 pp 4796ndash4798 2003

[24] H Sakai G W Procop N Kobayashi et al ldquoSimultaneousdetection of Staphylococcus aureus and coagulase-negativestaphylococci in positive blood cultures by real-time PCR withtwo fluorescence resonance energy transfer probe setsrdquo Journalof Clinical Microbiology vol 42 no 12 pp 5739ndash5744 2004

[25] A Huletsky P Lebel F J Picard et al ldquoIdentification ofmethicillin-resistant Staphylococcus aureus carriage in less than

1 hour during a hospital surveillance programrdquo Clinical Infec-tious Diseases vol 40 no 7 pp 976ndash981 2005

[26] W C Lindsey E S Woodruff D Weed D C Ward andR D Jenison ldquoDevelopment of a rapid diagnostic assay formethicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococcusrdquo Diagnostic Micro-biology and Infectious Disease vol 61 no 3 pp 273ndash279 2008

[27] N K Shrestha M J Tuohy G S Hall C M Isada and G WProcop ldquoRapid identification of Staphylococcus aureus and themecA gene from BacTALERT blood culture bottles by usingthe LightCycler systemrdquo Journal of Clinical Microbiology vol40 no 7 pp 2659ndash2661 2002

[28] T Y Tan S Corden R Barnes and B Cookson ldquoRapid iden-tification of methicillin-resistant Staphylococcus aureus frompositive blood cultures by real-time fluorescence PCRrdquo Journalof Clinical Microbiology vol 39 no 12 pp 4529ndash4531 2001

[29] C Palomares M J Torres A Torres J Aznar and J C Palo-mares ldquoRapid detection and identification of Staphylococcusaureus from blood culture specimens using real-time fluores-cence PCRrdquoDiagnostic Microbiology and Infectious Disease vol45 no 3 pp 183ndash189 2003

[30] S M Paule A C Pasquariello R B Thomson Jr K LKaul and L R Peterson ldquoReal-time PCR can rapidly detectmethicillin-susceptible andmethicillin-resistant Staphylococcusaureus directly from positive blood culture bottlesrdquo AmericanJournal of Clinical Pathology vol 124 no 3 pp 404ndash407 2005

[31] S Grobner and V A J Kempf ldquoRapid detection of methicillin-resistant staphylococci by real-time PCR directly from positiveblood culture bottlesrdquo European Journal of ClinicalMicrobiologyamp Infectious Diseases vol 26 no 10 pp 751ndash754 2007

[32] L C Thomas H F Gidding A N Ginn T Olma and JIredell ldquoDevelopment of a real-time Staphylococcus aureus andMRSA (SAM-) PCR for routine blood culturerdquo Journal ofMicrobiological Methods vol 68 no 2 pp 296ndash302 2007

[33] J Stratidis F J Bia and S C Edberg ldquoUse of real-timepolymerase chain reaction for identification of methicillin-resistant Staphylococcus aureus directly from positive bloodculture bottlesrdquo Diagnostic Microbiology and Infectious Diseasevol 58 no 2 pp 199ndash202 2007

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Table 1 PCR primers and TaqMan probes formecA femA specific for S aureus (femA-SA) femA specific for S epidermidis (femA-SE) anduniversal 16S rRNA and PCR primers for staphylococcal 16S rRNA

Target genes Sequence

mecA51015840-CATTGATCGCAACGTTCAATTT-31015840

51015840-TGGTCTTTCTGCATTCCTGGA-31015840

51015840-FAM-TGGAAGTTAGATTGGGATCATAGCGTCAT-TAMRA-31015840

femA-SA51015840-TGCCTTTACAGATAGCATGCCA-31015840

51015840-AGTAAGTAAGCAAGCTGCAATGACC-31015840

51015840-JOE-TCATTTCACGCAAACTGTTGGCCACTATG-BHQ1-31015840

femA-SE51015840-CAACTCGATGCAAATCAGCAA-31015840

51015840-GAACCGCATAGCTCCCTGC-31015840

51015840-JOE-TACTACGCTGGTGGAACTTCAAATCGTTATCG-BHQ1-31015840

Universal 16S rRNA51015840-TCCTACGGGAGGCAGCAGT-31015840

51015840-GGACTACCAGGGTATCTAATCCTGTT-31015840

51015840-FAM-CGTATTACCGCGGCTGCTGGCAC-TAMRA-31015840

Staphylococcal 16S rRNA 51015840-GCAAGCGTTATCCGGATTT-31015840

51015840-CTTAATGATGGCAACTAAGC-31015840

some disadvantages including long turnaround time andpotential false-negative results when samples are obtainedafter antimicrobial therapy Real-time PCR is significantlyfaster than conventional PCR and other detection methodsand its excellent sensitivity and specificity low contaminationrisk ease of use and high speed have made real-time PCRtechnology appealing to clinical microbiology laboratories[8]

The aim of this study was to develop and evaluate a mul-tiplex real-time PCR assay for the rapid detection and identi-fication of MRSA methicillin-susceptible S aureus (MSSA)methicillin-resistant S epidermidis (MRSE) methicillin-susceptible S epidermidis (MSSE) methicillin-resistant non-S epidermidisCoNS (MRCoNS) andmethicillin-susceptiblenon-S epidermidis CoNS (MSCoNS) directly from positiveblood cultures containing Gram-positive cocci in clusters(GPCC) by targetingmecA for determining methicillin resis-tance femA specific for S aureus (femA-SA) femA specificfor S epidermidis (femA-SE) 16S rRNA for universal bacteriaand 16S rRNA specific for staphylococci

2 Materials and Methods

21 Blood Culture This study evaluated 290 blood culturescontaining GPCC obtained from March 2013 to December2013 at Seoul National University Bundang Hospital Two ormore pairs of culture bottles for aerobes or anaerobes wereincubated in BacTAlert 3D (bioMerieux Inc Durham NCUSA) or BACTEC FX (BD Diagnostics Sparks MD USA)blood culture systems for 5 days after inoculation for blooddrawn from the patient If bacterial growth was not detectedwithin 5 days then the blood culture result was considerednegative When bacterial growth was noted blood from thepositive bottles was Gram-stained and samples containingGPCC (230 specimens in BacTAlert 3D (bioMerieux Inc)and 60 specimens in BACTEC FX (BD Diagnostics)) wereinoculated onto blood agar plates and cultured overnight at35∘C in a 5CO

2incubator Isolates were identified by colony

morphology Gram-staining catalase and coagulase tests

Final identification according to phenotypic characteristicsand antimicrobial susceptibility tests was performed usingthe MicroScanWalkAway system (Siemens Healthcare Diag-nostics Deerfield IL USA) with Pos Combo Panel Type 1A

22 DNA Extraction A 100 120583L aliquot of blood was drawndirectly from the positive blood culture bottles collected onfilter paper and dried for 15min at room temperature Theblood spot was lysed with 2mL of lysis buffer for 30min atroom temperature Next the paper was removed and 2 120583L oflysozyme-Tris-EDTA buffer was added to 500120583L of the eluateand incubated for 30min at 37∘C The sample was incubatedagain for 10min in 2mL of lysis buffer and the final eluatewas used for nucleic acid extraction with the NucliSENSeasyMAG platform (bioMerieux Inc)

23 Multiplex Real-Time PCR Assay Targeting mecA femA-SA femA-SE and Universal 16S rRNA The double duplexreal-time PCR TaqMan assay was performed with two tubesin one reaction One tube corresponded to the targets femA-SA and mecA and the other to the targets femA-SE anduniversal 16S rRNA The primers and probes for each targetwere designed as described by previously published studies[9 10] (Table 1) Real-time PCR was conducted in a totalvolume of 20 120583L including 20120583L of 10x LightCycler FastStartDNA Master HybProbe (Roche Diagnostics MannheimGermany) 24 120583L of 15mMMgCl

2(Takara Bio Shiga Japan)

02 120583M of each primer and 01 120583M of each probe with 30 120583Lof DNA template The amplification conditions used by them2000rt instrument (Abbott Diagnostic Chicago IL USA)were as follows 95∘C for 10min followed by 35 cycles of95∘C for 15 sec and 60∘C for 1min in a single real-time PCRassay Positive controls with MRSA MSSA and MRSE anda negative control with sterile distilled water (DW) wereincluded throughout the procedures

24 Additional Real-Time PCR Assay Targeting Staphylococcal16S rRNA A real-time PCR assay targeting staphylococcal16S rRNA was performed using a LightCycler 20 system










3 Results
















4 Discussion



















5 Conclusions


Additional Points


Competing Interests




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










3 Results
















4 Discussion



















5 Conclusions


Additional Points


Competing Interests




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












4 Discussion



















5 Conclusions


Additional Points


Competing Interests




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















5 Conclusions


Additional Points


Competing Interests




References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article usefulness of multiplex real-time pcr for...

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