JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2011, p. 1240–1244 0095-1137/11/$12.00 doi:10.1128/JCM.02220-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 4
Genotypic and Phenotypic Characterization of Methicillin-Susceptible Staphylococcus aureus Isolates Misidentified as Methicillin-Resistant Staphylococcus aureus by the BD GeneOhm MRSA Assay䌤 Paul D. Stamper,1* Lisa Louie,2 Henry Wong,2 Andrew E. Simor,2 Jason E. Farley,3,4 and Karen C. Carroll1,3,4 Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland1; Department of Microbiology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada2; Division of Infectious Disease, The Johns Hopkins University School of Medicine, Baltimore, Maryland3; and The Johns Hopkins University School of Nursing, Baltimore, Maryland4 Received 3 November 2010/Returned for modification 26 December 2010/Accepted 1 February 2011
Twenty-three nasal swab samples that tested positive for methicillin-resistant Staphylococcus aureus (MRSA) on initial testing by the BD GeneOhm MRSA assay (BD-MRSA PCR; BD GeneOhm, San Diego, CA) were culture positive only for methicillin-susceptible S. aureus (MSSA) from an enrichment broth. The 23 recovered isolates were confirmed as MSSA by a variety of phenotypic methods, including the BD Phoenix automated microbiology system (BD Diagnostics, Sparks, MD), oxacillin screening agar (BD Diagnostics), BBL CHROMagar MRSA (BD Diagnostics), and a PBP2ⴕ assay (Denka Seiken Co., Tokyo, Japan); susceptibilities were determined by using Mueller-Hinton agar with oxacillin. All were positive by nuc PCR, specific for S. aureus, but negative for mecA with one exception. Isolates were characterized by using multiplex PCR methodology to determine structural types and variants (SCCmec typing); additional PCRs were performed for the detection of the ccr and mec complexes, the junkyard regions as well as the Panton-Valentine leukocidin. Pulsed-field gel electrophoresis was used to determine clonality. One phenotypic MSSA isolate contained an intact SCCmec. Twelve MSSA isolates tested positive for MRSA by the BD-MRSA PCR because of amplification of the mec priming site flanking the SCC insertion point, although these isolates lacked mecA. The 10 remaining isolates were not MRSA and tested as MSSA by phenotypic and genotypic assays. In our patient population, diagnostic and surveillance testing and subsequent infection control practices may be impacted by the frequency of these excision events when using the BD-MRSA PCR for MRSA detection. (MSSA) and other bacteria. The amplified products vary in size, 176 to 278 bp, depending on the SCCmec right extremity sequence amplified and are detected using fluorescently labeled molecular beacon probes (9). Donnio et al. described a number of MSSA isolates (n ⫽ 9) from French hospitals containing partial SCCmec elements without mecA (4) and predicted that PCR assays based on the amplification by primers from the SCCmec-orfX region (i.e., BD-MRSA PCR) would be falsely determined as positive for MRSA. The loss of the methicillin-resistant phenotype due to the partial excision of the SCCmec has been suggested as the mechanism behind the high prevalence of false-positive BDMRSA PCR results observed in some populations (2, 6, 17, 25). In the original description of what is now the BD GeneOhm MRSA assay, Huletsky et al. found that 4.6% of the isolates tested from their worldwide collection displayed this phenomenon (9). This study elucidated the phenomenon of false-positive BD-MRSA PCR results in clinical samples tested for MRSA surveillance. S. aureus isolates from lysates which initially tested “false positive” from which only phenotypic MSSA was recovered were examined for the loss of functional mecA and SCCmec. The phenotype and genotype of the S. aureus isolates were confirmed with susceptibility to oxacillin, the presence or absence of the ccrAB locus, and the arrangement of the SCCmec.
The development of real-time PCR assays that differentiate methicillin-resistant Staphylococcus aureus (MRSA) from other staphylococci by linking a region of the mecA complex to an area specific on the S. aureus chromosome (orfX) (9) enabled molecular MRSA surveillance and diagnosis in a variety of healthcare settings. The utility of these tests, such as the BD GeneOhm MRSA assay (BD-MRSA PCR; BD GeneOhm, San Diego, CA), has been described in numerous publications (2, 3, 6, 17, 25). However, performance issues related to “falsepositive” results have been reported by some investigators (2, 3, 21). The staphylococcal cassette chromosome mec (SCCmec) is a structurally diverse mobile genetic element which varies by structural organization and genetic content (10). Typically, broad-spectrum beta-lactam resistance of S. aureus is carried on the mecA gene encoded within the SCCmec (10). The BDMRSA PCR uses five forward primers that target a region of the SCCmec at the extreme right end near the insertion site and one reverse primer targeting the downstream orfX gene (9) to differentiate MRSA from methicillin-susceptible S. aureus
* Corresponding author. Mailing address: Johns Hopkins Hospital Microbiology Laboratory, Meyer B1-193, 600 N. Wolfe St., Baltimore, MD 21287. Phone: (410) 955-5077. Fax: (410) 614-8087. E-mail:
[email protected]. 䌤 Published ahead of print on 9 February 2011. 1240
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CHARACTERIZATION OF BD-MRSA PCR FALSE POSITIVES
(1135, 1268) (1510) (1372, 1377, 1491) (1422) (1353)
11 (1028, 1033, 1052, 1058, 1075, 1096, 1276, 1432, 1487, 1545, 1571) 1 (1185) 1 (1374)
1 (1393)
NG/NG NG/NG NG/NG NG/NG NG/NG
NG/NG NG/NG
NG/NG
NG/NG
OSA/CHROM
0.5 0.5 0.5 0.5 0.25
0.5 0.5
0.5–1.0
0.5
OXA MIC (g/ml)
–
– – – – –
⫹ –
⫹
⫹
MRSA PCR
–
– – – – –
II –
–
IVa
SCCmec rightjunction PCR
–
– – – – –
– –
–
Nd
ccr complex PCR
–
– – – – –
– –
–
⫹
mecA complex PCR
–
– – – – –
– ⫹
–
⫹
PVL PCR
–
– – – – –
⫹ –
⫹
⫹
Huletsky MPX PCR
EMRSA16-like
USA600-like USA400-like Other EMRSA15-like EMRSA16-like
USA100-like USA300-like
USA400-like
USA300
–
– – – – ⫹
⫹ ⫹
⫹
⫹
MRSA PCR
MSSA
MSSA MSSA MSSA MSSA MSSA
MRSA MRSA
MRSA
MRSA
StaphSR
–/NG
–/NG –/NG –/NG –/NG –/NG
–/NG –/NG
–/NG
–/NG
PBP2⬘/Ox MH
No. of isolates (isolate no.)
2 1 3 1 1
0.25
SHSC test resultb BD test resultc
TABLE 1. Review of test results for S. aureus isolates examined at three separate laboratories
JHH test resulta
PFGE Comment
True positive; gene not expressed False positive
False positive False positive; nonspecific amplification
False positive; nonspecific amplification
Isolates. The results of the testing at BD GeneOhm in Quebec using the BD-MRSA PCR and StaphSR assay, as well as the Huletsky multiplex PCR, are presented in Table 1. Testing of all isolates agreed between JHH and Sunnybrook laboratories. Two isolates tested negative using the BD-MRSA PCR assay at JHH and Sunnybrook but were positive by BD-MRSA PCR and positive for the MRSA portion of the StaphSR when tested at BD GeneOhm. Positivity was attributed to nonspecific amplification or errant amplicons. One isolate, USA300 as determined by PFGE (specimen 1393), was identified by using phenotypic tests (PBP2⬘, CHROM-MRSA, the Phoenix instrument, and OSA) as an MSSA, but genotypic testing was consistent with MRSA revealing that the isolate contained a SCCmec IVa cassette with an intact mecA (11). The isolate was PVL positive. BD-MRSA PCR and the StaphSR assay correctly detected the presence of the SCCmec in specimen 1393; however, even though the isolate contained the mecA gene, the gene product PBP2⬘ was not detectable. The methicillin-resistant gene machinery was present, but the product was not expressed.
NG/NG
RESULTS
1 (1016)
S. aureus isolates. During an epidemiological study performed on adults that compared chromogenic media and the BD-MRSA PCR for detection of MRSA from anterior nasal specimens (5), 23 MSSA isolates were recovered that were initially identified as MRSA positive by the BD-MRSA PCR in which no MRSA was recovered (6). Isolates (n ⫽ 23) were recovered directly from subculture of the enrichment Trypticase soy broth with 6.5% NaCl. S. aureus isolates were identified by use of Gram staining, slide coagulation, and additional biochemicals as required. In addition, the isolates were sent to the research laboratories at BD GeneOhm, Quebec City, Quebec, Canada, and Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada, for separate independent analyses. Phenotypic MSSA confirmation. The BD Phoenix automated microbiology system (Phoenix; BD Diagnostics, Sparks, MD) was used to confirm initial identification and susceptibility to oxacillin. All 23 isolates were confirmed as methicillin-susceptible S. aureus, and the results are summarized in Table 1. Susceptibility testing was performed, and MICs to oxacillin ranged from 0.25 to 1 g/ml. Isolates were subcultured to oxacillin screening agar (OSA; BD Diagnostics, Sparks, MD) and to BBL CHROMagar MRSA (CHROM-MRSA; BD Diagnostics) for growth. At BD GeneOhm, the isolates were independently analyzed for the presence of the penicillin-binding protein 2⬘ (2a) with the PBP2⬘ assay (Denka Seiken Co., Tokyo, Japan), and susceptibility to oxacillin was confirmed by using oxacillin screening agar (BD Diagnostics). Tests were performed according to the manufacturer’s instructions. All 23 isolates failed to grow on CHROM-MRSA and OSA at The Johns Hopkins Hospital (JHH) laboratory. In the BD GeneOhm laboratory, no isolates grew on the oxacillin screening agar plates, and all tested negative with the PBP2⬘ assay. Genotypic confirmation, strain typing, and PVL testing. Isolates were tested by using the BD-MRSA PCR assay at JHH, Baltimore, MD, and at BD GeneOhm using the BD-MRSA PCR, and by using the BD GeneOhm StaphSR assay (BD-SR PCR) (Table 1). At the Sunnybrook Health Sciences Centre, isolates were confirmed as S. aureus (n ⫽ 23) using a species-specific PCR assay to detect the presence of the nuclease gene, nuc (13), and only one isolate contained an amplifiable mecA gene (13). After testing with the original Huletsky multiplex PCR (9), the structural types and variants of the mec element were determined by using the multiplex PCR methodology described by Oliveira et al. (18). Detection of the J1 region, cassette recombinase (ccr), and methicillin resistance (mec) gene complexes was performed using PCR assays described by Kondo et al. (11). Pulsed-field gel electrophoresis (PFGE) was performed, and profiles were manually examined (16). Images were digitized into BioNumerics version 5.10, and a cluster analysis using the Dice coefficient and UPGMA (unweighted pair-group method with arithmetic averages) was generated using the “USA” PFGE clonal designations. The presence of the Panton-Valentine leukocidin (PVL) gene, encoding the cytotoxin causing leukocyte destruction and tissue necrosis, was detected by PCR (12).
b
c
a JHH, Johns Hopkins Hospital. OSA, Oxacillin Screening Agar (BD Diagnostics Sparks, MD); CHROM, BBL CHROMagar MRSA (BD Diagnostics, Sparks, MD); OXA MIC, oxacillin MIC; NG, no growth; ⫹, positive; ⫺, negative. Oxacillin MIC ⫽ 1 for isolates 1033, 1276, 1432, 1487, and 1545; oxacillin MIC ⫽ 0.5 for isolates 1028, 1052, 1058, 1075, 1096, and 1571. SHSC, Sunnybrook Health Sciences Centre. MPX, multiplex; Nd, not done. BD, BD-GeneOhm. Ox MH, oxacillin Mueller-Hinton.
MATERIALS AND METHODS
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and 1268 [USA600-like]) were negative for all SCCmec loci and were negative with the Huletsky multiplex PCR. All were BD-MRSA negative and negative for MSSA by StaphSR. All were mecA negative with the PCR specific for mecA, but one isolate—1353—did test positive as MRSA using the BDMRSA assay at BD GeneOhm in Quebec City. These strains have recognized genetic backgrounds of known MRSA lineages by PFGE except for isolates 1372, 1377, and 1491. Elements of the SCCmec were not detected at Sunnybrook Health Sciences Center, which confirmed the negative result with the BD-MRSA PCR at the JHH. A dendrogram with the cluster analysis of all 23 isolates is shown in Fig. 2. DISCUSSION FIG. 1. PFGE profiles of MSSA isolates resembling USA400. Lane 1, USA400; lanes 2 to 13, MSSA isolates resembling USA400, falsely positive for MRSA by MRSA-PCR. The arrow indicates a DNA fragment harboring the mecA gene (based on Southern hybridization).
Eleven isolates (1028, 1033, 1052, 1058, 1075, 1096, 1276, 1432, 1487, 1545, and 1571; Table 1) tested virtually identical except for the oxacillin MIC: for five of the isolates, the oxacillin MICs were 1 g/ml, and for 6 isolates the MICs were 0.5 g/ml. By PFGE, the 11 isolates appeared to resemble USA400 (17). The Huletsky multiplex PCR was positive as determined using the mecii574 forward primer with the Xsau325 reverse primer in orfX. Although the mecii574 primer targets SCCmec I, II, and IV cassettes, others have elucidated that these strains most likely carry a type IVa cassette prior to excision of mecA (15). The 11 isolates phenotypically tested as MSSA, lacked mecA, and were considered “false positive” by the BD-MRSA and StaphSR assays. Figure 1 shows the PFGE profiles of the MSSA USA400-like isolates compared to MRSA USA400. The S. aureus isolate (i.e., isolate 1185) was identified as MRSA by BD-MRSA, StaphSR, and Hultesky PCR even though the mecA from the cassette was missing. The isolate appears to have remnants of a type II cassette by SCCmec multiplex PCR and was positive only for loci G, D, and B, which targeted the “J” region (left side). (Exact deletion junction would require further sequencing.) As estimated by PFGE, remnants of the SCCmec II cassette remained after the ⬃25-kb deletion. This “false-positive” isolate resembles USA100, and this observation emphasizes that the “false-positive” phenomenon is not restricted to strains resembling the USA400 clone. One isolate, 1374, was PVL positive and was identified by all phenotypic tests as MSSA but had a USA300-like PFGE profile. The strain most likely had a SCCmec type IVa cassette, but it appears that the entire cassette was excised. The Huletsky multiplex PCR was negative, and the isolate was negative by the BD-MRSA PCR at JHH so the isolate should not have tested positive by the BD-MRSA or StaphSR, as tested by BD GeneOhm. This was a false positive from nonspecific amplification or perhaps contamination with stray amplicon. Nine isolates resembling several MRSA pulsed-field clones (1422 [PFGE EMRSA15-like]; 1016 and 1353 [EMRSA16like]; 1372, 1377, and 1491 [other]; 1510 [USA400-like[; 1135
The sensitivity and specificity of the BD-MRSA PCR in our patient population were 89.0 and 91.7%, respectively, when CHROM MRSA and an enrichment broth were used to recover all S. aureus (6). The extremely low-positive predictive value (PPV) of the BD-MRSA PCR, 65.9%, was of concern and prompted this investigation. During the evaluation of the BD-MRSA PCR compared to CHROMagar MRSA (6), we initially assumed that the primary sample contained nonviable MRSA, that heteroresistant MRSA was outcompeted by MSSA, or that nonspecific amplification had occurred. Similar to other findings (2, 6, 17, 19, 21, 25) from varied geographical locations and diverse populations, our data demonstrate that mecA “dropouts” exist with some frequency. We do not believe that the dropout was related to testing or storage conditions since these observations were apparent upon initial testing. According to the study by Veguilla et al. (23), mecA appears to be quite stable with prolonged preservation and multiple freeze-thaws over at least a 2-year period of time. The isolates in our study were of low passage and were tested within a relatively short period of time after they were recovered. Recently, Wong et al. (24) investigated false-positive results from a single-locus PCR assay, the BD GeneOhm MRSA assay (BD-MRSA PCR), targeting the SCCmec/orfX junction and found that isolates of MSSA resembling USA400 and/or MSSA476 may harbor an additional copy of the dcs locus, thereby resulting in a falsely positive signal in the absence of an intact SCCmec element. This may be the case in our collection of isolates, although further sequencing of the chromosomal regions flanking the missing SCC elements would need to be performed. In addition, Wong et al. also described USA100like MSSA isolates that had a partial excision that excised mecA, leaving remnants of a SCCmec II cassette (24), again similar to one of the isolates, isolate 1185, tested in our collection. The results demonstrate that in our population, these problematic MSSA strains appear to resemble certain community-acquired-MRSA clones such as USA400, but the problem is not limited to only USA400 strain types. Our testing supports the suggestion of others (2, 4, 9) that the retained segments of the right extremity-junction sequence of the SCCmec by S. aureus strains missing the mecA gene are amplified. Of the 23 of 42 (55%) initial false-positive BDMRSA PCR specimen lysates that contained MSSA, 12 of 42 (29%) contained phenotypic MSSA but genotypic MRSA. Only one isolate actually contained mecA. The incomplete or partial SCCmec gene may alter the diagnostic reliability of
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FIG. 2. Cluster analysis of isolates (n ⫽ 23). The D locus of the SCCmec was estimated based on the location of the mecA band from previous work (24).
molecular assays based on this target (4, 9), and we believe that the problem is reflected in our reduced specificity (91.7%) (6) and the isolate test results in the present study. In addition, of the 19 PCR-positive, culture-negative samples, 9 lysates were again positive on repeat PCR testing despite the negative enrichment broth results. Several possible scenarios may apply to this situation. One possibility is that these specimens contained nonviable MRSA or nonviable S. aureus that contain a residual fragment containing the target SCCmec region detected with the PCR assay. Alternatively, a Staphylococcus species not S. aureus that contained a homologous target of the SCCmec region (1, 8, 14) could have been detected by the BD-MRSA PCR assay. Unfortunately, we did not examine the role of CoNS or attempt to quantify nonviable S. aureus DNA within the false-positive initial lysates in which no S. aureus was recovered. Huletsky et al. (9) did not see this phenomenon among 286 CoNS isolates; however, we did not investigate the CoNS to rule out this possibility. Gro ¨bner et al. (7) using the StaphSR assay, which relies on the same primers for detection of MRSA as the BD-MRSA PCR test, found three MSSA isolates that tested false positive for MRSA. The prevalence of this occurrence in their isolates that can be attributed to the mecA “dropouts” is similar to the prevalence of false-positive MRSA reported initially by Hu-
letsky et al. (9). Although Snyder et al. (20) did not see this phenomenon during testing (59 S. aureus isolates), reexamination of our false-positive blood culture isolates (n ⫽ 2) (22) leads us to conclude that this can occur not only in nasal surveillance cultures but also in blood cultures. Our two falsepositive MRSA isolates (22) from blood were mecA “dropouts” determined to be false positive by the Huletsky PCR. One blood culture isolate resembled PFGE USA400 but was mecA negative, whereas the other blood culture isolate was a mecA-negative strain resembling PFGE USA100 with a SCCmec II deletion (⬃25 kb). The presence of an amplicon using the Huletsky et al. PCR, the BD-MRSA PCR, and the StaphSR assay with the absence of amplification of the ccr and mecA complexes implies that the SCCmec element is present but that there was a deletion of the mecA gene complex. Similar to the BD-MRSA PCR, the BD StaphSR, relying on very similar primers and the same methodology, will also have false positives due to this phenomenon. When implementing a MRSA screening assay as a surveillance tool in hospitals, long-term care facilities and other institutionalized settings, the strengths and limitations of the diagnostic test must be understood to integrate efficiently with infection control practices. As others have noted (4, 19), positive test results with the MRSA assay should be verified by
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concomitant culture for infection control practices or epidemiological studies. The sensitivity and specificity of the BDMRSA PCR in endemic settings has been adequate but, in our high prevalence population, the large number of false positives due to a remnant portion of the SCCmec necessitates that a positive result by the BD-MRSA PCR should be confirmed by culture. ACKNOWLEDGMENTS Luc Parise performed independent confirmation of the MSSA isolates with PBP2⬘ and Staphaurex at BD-GeneOhm, Quebec City, Quebec, Canada. This study was performed with the assistance of Mian Cai, Tracy Ross, and Sharon Speser at the Johns Hopkins Hospital. This research was supported by a National Research Service Award funded by the National Institute of Nursing Research, by National Institutes of Health (NIH) grant 1 F31 NR009334-01, and by an NIHfunded T-32 Institutional Training grant in Health Disparities. The laboratory work was supported in part by a grant from BD Diagnostics (Sparks, MD) and BD GeneOhm (San Diego, CA).
J. CLIN. MICROBIOL.
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13.
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