Complementary use of MALDI-TOF MS and real-time PCR melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE

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1 J Antimicrob Chemother 2015; 70: doi: /jac/dku411 Advance Access publication 21 October 2014 Complementary use of and real-time PCR melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE Wai-Sing Chan 1, Tsz-Ming Chan 1, Tsz-Wan Lai 1, Jasper Fuk-Woo Chan 2, Raymond Wai-Man Lai 3, Christopher Koon-Chi Lai 4 and Bone Siu-Fai Tang 1 * 1 Department of Pathology, Hong Kong Sanatorium & Hospital, Hong Kong, China; 2 State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong, China; 3 Department of Microbiology, Prince of Wales Hospital, Hong Kong, China; 4 Department of Pathology, Queen Elizabeth Hospital, Hong Kong, China *Corresponding author. Tel: ; Fax: ; bonetang@hksh.com Received 28 June 2014; returned 17 July 2014; revised 19 August 2014; accepted 19 September 2014 Objectives: To develop a rapid method for routine screening of methicillin-resistant staphylococci and VRE for clinical isolates and positive blood cultures. Methods: Our method consisted of two parts: was used for identification of staphylococci and enterococci, followed by antibiotic resistance detection by real-time PCR melt curve analysis without DNA extraction. The latter part included a triplex reaction for staphylococcal culture isolates (meca, meca LGA251 and Panton Valentine leucocidin genes), dual PCR of meca/meca LGA251 and nuc genes for staphylococcal blood cultures, and a duplex reaction for enterococci (vana and vanb genes). A total of 124 clinical isolates and 56 positive blood cultures were tested. was performed using Microflex LT (Bruker Daltonik, Bremen, Germany) and Rotor-Gene Q (Qiagen, Hilden, Germany) was used for real-time PCR melt curve analysis. The total assay time was,2.5 h. Results: The results revealed 100% concordance with antibiotic susceptibility testing or other reference methods for all culture isolates and enterococcal blood cultures. The percentage of concordance for staphylococcal blood cultures was 97.5%. Conclusions: The method described herein was fast, economical, reliable and capable of detecting meca LGA251, vanb1 and vanb2 genotypes, which are not included in most commercial assays. Large-scale screening is required to further test the performance of this protocol, especially for genotypes that are infrequently encountered. Keywords: antibiotic resistance, mixed blood cultures, MRSA, multiplex, VRE Introduction Antibiotic resistance is a ceaseless challenge to medical systems worldwide. Early detection of resistant aetiological agents and hence timely prescription of appropriate antibiotics have been shown to correlate with better clinical outcome. 1,2 Clinical microbiology laboratories play a pivotal role in this aspect. The boom of molecular diagnostic techniques in the past 30 years has revolutionized traditional culture-based approaches of pathogen detection. Ideally, the identities and antibiotic resistance traits of aetiological agents are detected directly from clinical specimens with a short turnaround time. 3,4 To fit into busy laboratory schedules, commercial molecular detection assays are designed to be highly automated with minimal hands-on time, and this convenience is reflected on the cost per reaction, rendering these tests performed mostly on an ad hoc basis. In addition, the narrow specimen variety and throughput also limit the use of these state-of-the-art methods for mass screening purposes. In light of this, we tailored a robust yet economical method for routine screening of antibiotic-resistant bacteria, initially focusing on methicillin-resistant staphylococci (MRS) and VRE. Our method combined the species-discriminating power of and the multiplex capability of real-time PCR melt curve analysis (MCA). The screening targets included clinical isolates and positive blood cultures. Materials and methods Procedure overview Our method was eligible for both clinical isolates and positive blood cultures and consisted of two parts. First, was used for # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oup.com 441

2 Chan et al. screening suspected clinical isolates or positive blood cultures. If Staphylococcus or Enterococcus species were identified, the same sample would be subjected to real-time PCR MCA for the detection of related antibiotic resistance/cytotoxin genes, including meca, meca LGA251 and Panton Valentine leucocidin (PVL) genes in a triplex reaction for staphylococcal culture isolates, dual PCR of meca/meca LGA251 and nuc genes for staphylococcal blood cultures, and vana and vanb genes in a duplex reaction for enterococci. A schematic workflow is shown in Figure 1. The total assay time was,2.5 h. Positive control strains Four ATCC (Manassas, VA, USA) strains were used in this study, including ATCC w TM (PVL-positive control), ATCC w TM (meca- and Suspected colony on culture plate Positive blood culture Sepsityper Minute amount of colony + 10 μl of ddh 2 O 2 μl for No Staphylococcus/Enterococcus species? S. aureus or E. faecium/faecalis? No Yes Yes No MS score fulfills the criteria for preliminary ID reporting? Conventional workflow Yes 1 μl of bacterial suspension Mixed staphylococcal/ enterococcal blood culture 4 drops of broth + 4 ml of ddh 2 O 1 μl REAL-TIME PCR MCA Staphylococcal culture isolates: meca/meca LGA251 /PVL triplex PCR Staphylococcal blood cultures: dual PCR of meca/meca LGA251 and nuc genes Enterococci: vana/vanb duplex PCR Preliminary information on antibiotic resistance <2.5 h Figure 1. Schematic workflow of MRS and VRE screening in this study. 442

3 and MCA for rapid identification of methicillin-resistant staphylococci and VRE JAC nuc-positive control), ATCC w BAA-2312 TM (meca LGA251 -positive control) and ATCC w TM (vanb-positive control). A vancomycin-resistant Enterococcus faecium clinical strain was used as a vana-positive control (vana-positive by Xpert w vana/vanb, Cepheid, Sunnyvale, CA, USA). Clinical strains A total of 124 clinical isolates and 56 positive blood cultures were tested. For clinical isolates, 86 were isolated from pure cultures retrieved from Microbank cryovials (Pro-Lab Diagnostics, Richmond Hill, Canada), whereas 38 were isolated from primary culture plates which were mostly mixed with other bacterial species. As the number of positive blood cultures was insufficient (n¼3), 53 mimicked samples were prepared for testing their inhibitory effect on the assay. The details of these strains are summarized in Table 1. The antibiotic susceptibility patterns of these isolates were well characterized with reference to guidelines recommended by the CLSI. The presence of the PVL gene in these isolates was confirmed by a local reference laboratory (Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong, China) or in-house PVL PCR. The presence of vana or vanb genes was confirmed by Xpert w vana/vanb or the corresponding laboratories listed in Table 1. Table 1. Clinical strains used in this study Bacterial species Source No. of cases From culture plate From BC MSSA (PVL+) MB 2 2 NA CA-MRSA MB a Non-CA-MRSA MB a MSSA (PVL2) PC NA MSSA (PVL+) PC 2 2 NA Non-CA-MRSA PC 7 7 NA MSSA+MRSC MB 6 NA 6 a MSSA+MRSE MB 8 NA 8 a MSSA+MRSH MB 3 NA 3 a MSSA+MRSW MB 3 NA 3 a VS E. faecalis PC 7 7 NA VS E. faecalis BC 3 NA 3 VS E. faecium MB 1 1 NA VR E. faecium (vana+) MB a VR E. faecium (vana+) QMH a VR E. faecium (vana+) PC 2 2 NA VR E. faecium (vana+) QEH NA VR E. faecium (vanb+) PWH a VS E. gallinarum MB 1 1 NA VI E. gallinarum MB 3 3 NA Total BC, blood culture; CA-MRSA, community-associated MRSA; MB, Microbank cryovial; MRSC/MRSE/MRSH/MRSW, methicillin-resistant Staphylococcus capitis/staphylococcus epidermidis/staphylococcus hominis/staphylococcus warneri; NA, not applicable; PC, primary culture; VI/VR/VS, vancomycin intermediate/resistant/susceptible; PWH, Prince of Wales Hospital, Hong Kong; QEH, Queen Elizabeth Hospital, Hong Kong; QMH, Queen Mary Hospital, Hong Kong. a Mimicked positive blood culture. Retrieval of cryopreserved strains and preparation of mimicked positive blood cultures The Microbank strains were retrieved from cryovials, inoculated onto Columbia agar supplemented with 5% sheep blood (Oxoid, Cambridge, UK) and then incubated overnight at 358C and 5% CO 2. To prepare mimicked positive blood cultures, 0.5 ml of bacterial suspension (0.5 McFarland) was spiked into leftover negative blood culture vials followed by overnight incubation at 358C. Specimen selection criteria For primary culture plates, all E. faecium/enterococcus faecalis (VRE screening cases only) and Staphylococcus aureus colonies were tested. Discrete colonies were picked from areas with scanty growth of bacteria. The respective top 10 classification results should belong to the same species, with the highest identification score 2.0. For species with reference mass spectrum profiles (MSPs),10, the scores of all their MSPs should be higher than that of other species in the list, and the scores of other species should be,1.7. Mimicked positive blood cultures were prepared with single MRSA or VRE species or combinations of MSSA and methicillin-resistant CoNS (MRCoNS). In routine practice, a preliminary MS identification report will not be issued unless: (i) the highest identification score of the species is 2.0; and (ii) the highest identification score of the second species, if any, is,1.6. Bacterial identification by Bacterial identification was facilitated by (Microflex LT, Bruker Daltonik, Bremen, Germany). Spectra were obtained with an accelerating voltage of 20 kv in linear mode with an m/z range of Da. Spectra were analysed with MALDI Biotyper version 3.0 and Reference Library version (Bruker Daltonik, Bremen, Germany). For bacterial colonies on primary culture plates, a minute amount of isolate was picked with a sterile pipette tip and resuspended in 10 ml of ddh 2 O. A 2 ml aliquot of this suspension was placed on the target plate, air dried and covered with 1 ml of absolute formic acid (Sigma-Aldrich, St Louis, MA, USA). Each dried spot was then covered with 1 ml of 10mg/mLmatrixsolution(a-cyano-4-hydroxycinnamic acid, Bruker Daltonik, Bremen, Germany). The dried samples were then subjected to MS measurement after successful calibration with Bacterial Test Standard (Bruker Daltonik, Bremen, Germany). For positive blood cultures, a Sepsityper Kit TM (Bruker Daltonik, Bremen, Germany) was used according to the manufacturer s instructions. Real-time PCR MCA Three sets of PCR were used in this study, namely meca/meca LGA251 /PVL triplex PCR for staphylococcal culture isolates, dual PCR of meca/ meca LGA251 and nuc genes for staphylococcal blood cultures, and vana/ vanb duplex PCR for enterococci. The primer sequences are shown in Table 2. Besides meca LGA251 and nuc primer pairs and the reverse primer for the PVL gene, all primers were designed by aligning sequences retrieved from the GenBank database of the National Center for Biotechnology Information (NCBI) using the ClustalW alignment tool of Molecular Evolutionary Genetics Analysis software version The specificity of the primers was evaluated by using the NCBI Basic Local Alignment Search Tool. For isolates from primary culture plates, 1 ml of bacterial suspension (the same suspension as for ) was used as DNA template. For positive blood cultures, the quick method described by Adams 6 was 443

4 Chan et al. Table 2. Primers used in this study Primer name Sequence Size (bp) Source meca Fp 5 -AAAGAACCTCTGCTCAACAAGT this study meca Rp 5 -CCATTTACCACTTCATATCTTGTAACG-3 meca LGA251 Fp 5 -GAAAAAAAGGCTTAGAACGCCTC Stegger et al., meca LGA251 Rp 5 -GAAGATCTTTTCCGTTTTCAGC-3 nuc Fp 5 -GCGATTGATGGTGATACGGTT Louie et al., nuc Rp 5 -AGCCAAGCCTTGACGAACTAAAGC-3 PVL Fp 5 -TCAGGTGGAGGTAATGGTTC this study PVL Rp 5 -GATAGGACACCAATAAATTCTGGATTG-3 Stegger et al., vana Fp 5 -GCATGGCAAGTCAGGTGAA this study vana Rp 5 -GATTCAATTGCGTAGTCCAATTCG-3 vanb Fp 5 -CCATGTACGGAATGGGAAGC this study vanb Rp 5 -CTTCACAAAGACAGGGTAGGTAAG-3 Fp, forward primer; Rp, reverse primer. adopted. Briefly, four drops of blood culture broth were added to 4 ml of ddh 2 O and 1 ml of this suspension was used as DNA template. The 24 ml working master mixture contained 12.5 ml of 2X Type-it HRM PCR Master Mix (Qiagen, Hilden, Germany), 0.4 mm (final concentration) of each primer (Tech Dragon Ltd, Hong Kong, China) and an appropriate volume of ddh 2 O. Real-time PCR MCA was performed using Rotor-Gene Q (Qiagen, Hilden, Germany) with the following conditions: initial cell lysis anddenaturationat958c for 10 min; three step cycling for 40 times (958C for 10 s, 558C for 30 s and 728C for 15 s); and MCA settings of a ramp from 70 to 908C, rising by 0.18C in each step. Derivative melt curves (df/dt versus T m ) were generated automatically by using the Melt function of the program. The df/dt threshold was set at 0.5 arbitrary units. The threshold cycle (Ct) of each amplification reaction (staphylococcal blood cultures only) was determined by using the Quantitation tool of the program. Samples with cycle numbers 38 and correct T m values were considered as PCR-positive. Result interpretation The result interpretation guidelines are shown in Figure 2. Forpositive blood cultures, the interpretation of results was more conservative due to limited information on the potential presence of coexistent staphylococcal/enterococcal species (i.e. mixed blood cultures). All preliminary results generated by this rapid method were confirmed by conventional bacterial culture and antibiotic susceptibility testing. Results and discussion Initially, positive control strains were tested to determine the preliminary T m value of each target amplicon. Duplicate samples were tested in three individual runs. Gel electrophoresis confirmed that the amplicons were of expected sizes. The mean T m value, standard deviation (SD) and coefficient of variation (CV), respectively, were as follows: meca LGA251,76.408C, 0.07 and 0.10%; meca, C, 0.03 and 0.04%; nuc, C, 0.08 and 0.1%; PVL, C, 0.07 and 0.001%; vana, C, 0.06 and 0.07%; and vanb, C, 0.10 and 0.12%. As the melt curves of these amplicons were well separated from each other (Figure 3), the same assay conditions were applied on clinical isolates and positive blood cultures. A total of 124 clinical isolates and 56 positive blood cultures were screened. The results revealed 100% concordance (140/140) with antibiotic susceptibility patterns and genotypes determined by reference methods for all culture isolates and enterococcal blood cultures. The degree of PCR inhibition was 0% (0/145). The mean T m value, SD and CV, respectively, including the results of clinical samples, were as follows: meca, C, 0.14 and 0.18% (n¼98); nuc, C, 0.16 and 0.2% (n¼40); PVL, C, 0.06 and 0.08% (n¼33); vana, C, 0.07 and 0.08% (n¼46); and vanb, C, 0.26 and 0.30% (n¼6). meca LGA251 data were not available due to the lack of meca LGA251 -positive clinical strains in our laboratory, though the primer specificity was well evaluated by Stegger et al. 7 In general, these amplicons displayed reproducible T m values, whilst those of vanb amplicons showed minor variation ( C). The variations in genotype and GC content might account for this. 9,10 As a proof of concept, the amplicons of vanb-positive control ATCC w TM (T m : C) and the two vanb-positive vancomycin-resistant E. faecium clinical strains (T m :84.71and C) were sequenced. The GC contents were 48.3%, 49.7% and 49.8%, respectively. The lower GC content of the ATCC w TM amplicon might account for its lower T m value. Further analysis of sequence data confirmed that ATCC w TM contained vanb1 whereas the two clinical strains possessed vanb2 elements. The data demonstrated the potential of this assay for distinguishing between vanb1 and vanb2 genotypes, which requires further evaluation with more clinical isolates. In addition, no false-positive signal was detected for the three vancomycinintermediate Enterococcus gallinarum isolates, whose intrinsic resistance to glycopeptides is usually contributed by the VanC phenotype. 11,12 One challenge for the molecular detection of transmissible antibiotic resistance genes is the possession of such genes by other non-target microbes in polymicrobial samples, resulting in false-positive signals. For instance, the meca gene can be found 444

5 and MCA for rapid identification of methicillin-resistant staphylococci and VRE JAC Culture isolates S. aureus CoNS Positive blood cultures S. aureus CoNS Enterococcus species Figure 2. Result interpretation guidelines. Staphylococci panel MCA meca/meca LGA251 PVL Preliminary results a + + PVL+ MRSA + PVL MRSA + PVL+ MSSA PVL MSSA + MRCoNS MSCoNS MCA meca/meca LGA251 nuc ΔCt Preliminary results a + + <2 Suspected MRSA b (i) MSSA + MRCoNS (ii) MRSA + MRCoNS + / MSSA b + + <2 MSCoNS + MRSA (i) MSSA + MRCoNS (ii) MRSA + MRCoNS + / Suspected MRCoNS + / MSCoNS + MSSA / MSCoNS Enterococci panel MCA Preliminary results a vana/vanb Culture isolates Blood cultures + VRE (species level) VRE (genus level) VSE (species level) VSE (genus level) MSCoNS, methicillin-susceptible CoNS; VSE, vancomycin-susceptible enterococci; ΔCt, difference in threshold cycle between meca/meca LGA251 and nuc amplification reactions (absolute value). a All preliminary results should be confirmed by conventional method. b The presence of MSCoNS could not be excluded. 5.0 nuc df/dt meca LGA251 meca PVL vana vanb1 vanb C Figure 3. Typical derivative melt curves (df/dt versus T m )ofmeca LGA251 (ATCC w BAA-2312 TM, 138 bp, T m : 76.48C), meca (ATCC w TM, 184 bp, T m :77.458C), PVL (ATCC w TM, 305 bp, T m : C), nuc (ATCC w TM, 270 bp, T m : 78.98C); vana (in-house positive control, 294 bp, T m : C), vanb1 (ATCC w TM, 351 bp, T m : C) and vanb2 (clinical strain, 351 bp, T m : C) PCR amplicons. 445

6 Chan et al. Table 3. results and DCt values for staphylococcal blood cultures Clinical strain Bruker MSP library DCt Monomicrobial blood cultures [best match (score)] MRSA (n¼20) S. aureus ( ) categorical % concordance Bimicrobial blood cultures [best match (score)] MSSA 1:1 MRSC S. aureus (2.179) 4.81 MSSA 1:1 MRSE S. aureus (2.04); Staphylococcus epidermidis (1.786) a 5.06 MSSA 1:1 MRSC S. aureus (2.183) 5.78 MSSA 1:1 MRSE S. aureus (2.119); S. epidermidis (1.896) a 4.23 MSSA 1:1 MRSC S. aureus (2.221) 5.25 MSSA 1:1 MRSW S. aureus (2.113) 2.39 MSSA 1:1 MRSC S. aureus (1.867); Staphylococcus capitis (1.823) a 3.04 MSSA 1:1 MRSE S. aureus (1.832); S. epidermidis (1.539) a 3.43 MSSA 1:1 MRSC S. aureus (2.233) 5.43 MSSA 1:1 MRSE S. aureus (2.187) 2 MSSA 5:1 MRSC S. aureus (2.206) 4.71 MSSA 5:1 MRSE S. aureus (2.105) 0.54 MSSA 5:1 MRSE S. aureus (2.15) 6.95 MSSA 5:1 MRSW S. aureus (2.269) 3.66 MSSA 5:1 MRSE S. aureus (2.328) 4.02 MSSA 5:1 MRSE S. aureus (2.294) 3.77 MSSA 5:1 MRSW S. aureus (2.052) 5.69 MSSA 5:1 MRSH S. aureus (2.288) 6.97 MSSA 5:1 MRSH S. aureus (2.018) 7.54 MSSA 5:1 MRSH S. aureus (1.881); Staphylococcus simiae (1.624) a 3.08 categorical % concordance Overall % concordance MRSC/MRSE/MRSH/MRSW, methicillin-resistant S. capitis/s. epidermidis/s. hominis/s. warneri. a For illustration only, preliminary MS identification reports will not be issued in routine practice. in MRCoNS and the presence of the vanb gene in various bacterial species has been reported, e.g. in Streptococcus and Clostridium species. 13 Whilst the latter is more likely to be present in stool samples than in positive blood cultures, the coexistence of MSSA and MRCoNS is not uncommon in positive blood cultures. In this study, we attempted to minimize this false positivity by finetuning the protocol and result interpretation guidelines. Despite the strong microbial identification power of MALDI-TOF MS, its species-resolving capability for polymicrobial blood cultures is not satisfactory. Previous evaluation studies demonstrated that identification was confined to single microbial species for a majority of polymicrobial blood cultures In this study, only 20% of bistaphylococcal blood cultures were correctly identified (Table 3). This phenomenon may be interpreted from two facets. First, each reference spectrum from the MSP library was created with single microbial species. With a reduced number of detectable peaks due to lower microbial concentration, 16 the minority microbial species is usually overlooked by spectra-matching algorithms, leading to the identification of dominant species only. Second, even if the concentrations of two microbes are comparable, the preference of the spectramatching may not be equivalent. This is especially prominent for closely related species with similar MS spectra, e.g. our data revealed that S. aureus was preferentially identified compared with other CoNS in bistaphylococcal blood cultures (Table 3). This phenomenon may also be contributed by the relative abundance and quality of reference spectra in the MSP library. In short, provides us with a general idea on the dominant microbial species in a polymicrobial blood culture sample, yet with limited information on residual microbial species. Despite this limitation, MS results provide crucial direction for the molecular detection of antibiotic resistance and subsequent result interpretation. In the past decade, much effort has been devoted to circumvent false MRSA-positive amplification signals due to coexistence of MSSA and MRCoNS in polymicrobial samples. These strategies can be generally divided into two approaches, namely (i) the detection of the staphylococcal cassette chromosome mec (SCCmec) orfx junction, and (ii) the difference in threshold cycle (DCt) between the amplification of S. aureus-specific genomic sequence and the meca gene. As the sequence diversity of SCCmec complicates primer design and subsequent T m analysis, the latter approach was adopted in this study. Staphylococcal blood cultures were subject to two sets of PCR, namely (i) the amplification of meca and meca LGA251 genes, which are the genetic determinants of methicillin resistance, and (ii) the amplification of the nuc gene, which encodes a thermostable nuclease of S. aureus. As these genes share the same copy number in the 446

7 and MCA for rapid identification of methicillin-resistant staphylococci and VRE JAC MRSA genome, they should have very similar Ct values and the resulting DCt value should be close to zero. For mixed samples containing MSSA and MRCoNS, unless the bacterial loads are in 1:1ratiowiththesamerate/extentofbacteriallysis,theDCt value should be higher than that of MRSA in most circumstances. Initially, the cut-off value of DCt, 2 was adopted. The results revealed an overall concordance of 97.5% (39/40) for all staphylococcal blood cultures tested. We have demonstrated the complementary use of MALDI-TOF MS and real-time PCR MCA for routine screening of MRS and VRE. Our method was rapid (,2.5 h), economical (,2 GBP), reliable (100% concordance for all culture isolates and enterococcal blood cultures; 97.5% concordance for staphylococcal blood cultures and 0% inhibition) and convenient (DNA extraction was not required). The species identification power of allowed accurate identification of Staphylococcus and Enterococcus species. The multiplex capability of real-time PCR MCA allowed simultaneous detection of multiple clinically significant antibiotic resistance/cytotoxin genes. Upon careful design of primers and optimization of assay conditions, this in-house method could be adjusted for the detection of emerging antibiotic resistance/ virulence-associated genes. Our method was especially useful for mixed bacterial cultures in which the amount of target organism is insufficient for rapid susceptibility assay. Before the maturation of emerging rapid susceptibility testing technologies, the molecular detection of antibiotic resistance genes will continue to provide timely information for appropriate prescription and infection control measures. Conclusions We have demonstrated that the complementary use of and real-time PCR MCA is feasible for routine screening of MRS and VRE. Future large-scale testing is required to further evaluate the performance of this method and streamline the procedure for routine use. Acknowledgements We would like to thank Mr Ho-Yin Lam for sequencing the PCR amplicons. Funding This study and the data generated from it were part of the routine work of the Hong Kong Sanatorium & Hospital. Transparency declarations None to declare. References 1 Nguyen DT, Yeh E, Perry S et al. Real-time PCR testing for meca reduces vancomycin usage and length of hospitalization for patients infected with methicillin-sensitive staphylococci. J Clin Microbiol 2010; 48: Lodise TP, McKinnon PS, Swiderski L et al. Outcomes analysis of delayed antibiotic treatment for hospital-acquired Staphylococcus aureus bacteremia. Clin Infect Dis 2003; 36: Wolk DM, Picton E, Johnson D et al. Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares. J Clin Microbiol 2009; 47: Altun O, Almuhayawi M, Ullberg M et al. Clinical evaluation of the FilmArray blood culture identification panel in identification of bacteria and yeasts from positive blood culture bottles. J Clin Microbiol 2013; 51: Tamura K, Dudley J, Nei M et al. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007; 24: Adams DN. Shortcut method for extraction of Staphylococcus aureus DNA from blood cultures and conventional cultures for use in real-time PCR assays. J Clin Microbiol 2005; 43: Stegger M, Andersen PS, Kearns A et al. Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either meca or the new meca homologue meca LGA251. Clin Microbiol Infect 2012; 18: Louie L, Goodfellow J, Mathieu P et al. Rapid detection of methicillinresistant staphylococci from blood culture bottles by using a multiplex PCR assay. J Clin Microbiol 2002; 40: Mendes RE, Kiyota KA, Monteiro J et al. Rapid detection and identification of metallo-b-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol 2007; 45: Monteiro J, Widen RH, Pignatari ACC et al. Rapid detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother 2012; 67: Leclercq R, Dutka-Malen S, Duval J et al. Vancomycin resistance gene vanc is specific to Enterococcus gallinarum. Antimicrob Agents Chemother 1992; 36: Hollenbeck BL, Rice LB. Intrinsic and acquired resistance mechanism in Enterococcus. Virulence 2012; 3: Mak A, Miller MA, Chong G et al. Comparison of PCR and culture for screening of vancomycin-resistant enterococci: highly disparate results for vana and vanb. J Clin Microbiol 2009; 47: Chen JHK, Ho PL, Kwan GSW et al. Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. J Clin Microbiol 2013; 51: Kok J, Thomas LC, Olma T et al. Identification of bacteria in blood culture broths using matrix-assisted laser desorption-ionization Sepsityper TM and time of flight mass spectrometry. PLoS One 2011; 6: e Christner M, Rohde H, Wolters M et al. Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. J Clin Microbiol 2010; 48: