Clinical validation of the molecular BD GeneOhm StaphSR assay for direct detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in positive blood cultures

The rapid detection of Staphylococcus aureus bacteremia and a swift determination of methicillin susceptibility has serious clinical implications affecting patient mortality. This study evaluated the StaphSR assay (BD GeneOhm, San Diego, CA), a real-time PCR assay, for the identification and differentiation of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from 300 positive blood cultures. The BD GeneOhm StaphSR assay was performed and interpreted according to the manufacturer's recommendations. Positive blood cultures (containing predominantly gram-positive cocci in clusters) were subcultured on 5% sheep blood agar plates. After 18 to 24 h of incubation, isolates morphologically consistent with S. aureus were presumptively identified by latex agglutination (Staphaurex Plus; Remel, Lenexa, KS). Susceptibility testing was initially performed with the Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). Additional susceptibility testing of samples with discrepant results was done using BBL oxacillin screen agar (BD Diagnostics, Sparks, MD), oxacillin and cefoxitin Etests (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar, an immunoassay for penicillin binding protein 2' (Denka Seiken Co., Tokyo, Japan), and mecA PCR. The sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm StaphSR assay for MSSA detection were 98.9, 96.7, 93.6, and 99.5%, respectively. For the detection of MRSA, the BD GeneOhm StaphSR assay was 100% sensitive and 98.4% specific; positive and negative predictive values for MRSA detection were 92.6 and 100%, respectively. Inhibition was seen with only one sample, and the issue was resolved upon retesting. The BD GeneOhm StaphSR assay appears to be a valuable diagnostic tool for quickly differentiating bacteremia caused by MSSA and MRSA from that caused by other gram-positive cocci.     

Genotypic and phenotypic characterization of methicillin-susceptible Staphylococcus aureus isolates misidentified as methicillin-resistant Staphylococcus aureus by the BD GeneOhm MRSA assay

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 PBP2' 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.     

mecA-positive Staphylococcus aureus with low-level oxacillin MIC in Taiwan

Although the presence of mecA is the genotypic determinant of methicillin-resistant Staphylococcus aureus (MRSA), certain MRSA strains, especially community-associated MRSA (C-MRSA), can display an oxacillin MIC in the Clinical and Laboratory Standards Institute susceptible breakpoint range (≤ 2 μg/ml). Among 91 and 180 isolates thought to be methicillin-susceptible S. aureus (MSSA) with oxacillin MICs of 2 and 1 μg/ml as determined by the Sensititre broth microdilution test initially, 52 (57.1%) and 6 (3.3%), respectively, were mecA positive. These mecA-positive low-oxacillin-MIC isolates belong to the dominant Taiwan C-MRSA clone (clonal complex [CC] 59), 56 of which carried SCCmec type V and were pvl positive, and 43 of which belonged to spa CC t437. All 271 isolates were retested by Sensititre, as well as by Vitek II and disk diffusion (DD). Based on the oxacillin results, the sensitivities of the Sensititre, Vitek II, and DD methods were 48.3% (28/58), 46.6% (27/58), and 89.6% (52/58), respectively. Although cefoxitin was better at detecting these isolates, 12.1, 10.4, and 5.2% of these isolates were still misidentified as MSSA by Sensititre, Vitek II, and DD, respectively. These results highlight the difficulty in the accurate identification of MRSA with borderline oxacillin MICs in the CC59:SCCmec V clone, which likely has contributed to its spread in the health care and community settings. Since this clone has now been detected in other countries, and since other C-MRSA lineages have also been found to have low-level β-lactam resistance, the findings of the present study may be relevant to other regions. Further studies are warranted to determine the extent and clinical impact of such misidentification.     

The detection and differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus endocarditis by using the BD GeneOhm StaphSR Assay

We use the BD GeneOhm StaphSR Assay (BD Diagnostics, Oakville, Canada) to screen for Staphylococcus aureus nasal colonization and sought to evaluate this assay for the assessment of valve specimens from patients with endocarditis. We examined 23 paired fresh and formalin-fixed, paraffin-embedded cardiac valve tissue samples, 12 of which had S aureus endocarditis, using the BD GeneOhm StaphSR Assay for the detection and differentiation of methicillin-susceptible and methicillin-resistant S aureus. This assay appropriately characterized all specimens with respect to the presence or absence of S aureus. There was an 87.5% correlation between the presence or absence of the mecA gene and the oxacillin susceptibility results for the S aureus isolates studied. The GeneOhm StaphSR assay accurately detected S aureus in cardiac valve tissue samples. Rare discordances were observed between oxacillin susceptibility status and mecA gene detection by this assay.     

Methicillin-Resistant Staphylococcus aureus: Comparison of Susceptibility Testing Methods and Analysis of mecA-Positive Susceptible Strains

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the mecA gene used as the "gold standard" assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecA was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that mecA- and PBP 2a-positive S. aureus isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA.     

Three-Way Comparison of BBL CHROMagar MRSA II,MRSASelect, and Spectra MRSA for Detection of Methicillin-Resistant Staphylococcus aureus Isolates in Nasal Surveillance Cultures

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired and life-threatening infections. Active surveillance programs for MRSA utilize either molecular or culture-based methods. A prospective study was performed to compare the performance of selective and differential chromogenic media, BBL CHROMagar MRSA II (CMRSA II; BD Diagnostics, Sparks, MD), MRSASelect (Bio-Rad Laboratories, Redmond, WA), and Spectra MRSA (Remel, Lenexa, KS), for the detection of MRSA in nasal swab specimens. A total of 515 compliant remnant nasal swab specimens were sequentially used to inoculate BBL Trypticase soy agar with 5% sheep blood (TSA II) and each chromogenic medium. After 24 h of incubation, colony color reactions and morphology on chromogenic media were compared to suspicious colonies on nonselective TSA II. MRSA on TSA II was confirmed by Gram staining, a coagulase test, and a cefoxitin disk test. The overall prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) on TSA II was 12.4% (64/515) and 9.7% (50/515), respectively. When each chromogenic medium was compared to the standard culture method, the sensitivity and specificity, respectively, were as follows: CMRSA II, 87.7% and 98.6%; MRSASelect, 89.0% and 93.4%; and Spectra MRSA, 83.6% and 92.1%. The positive predictive values were highest for CMRSA II (91.4%), followed by MRSASelect (69.1%) and Spectra MRSA (63.5%). False-positive results on chromogenic media were mainly due to color interpretation. The negative predictive values for all three media were greater than 97%. In conclusion, CMRSA II gave the best overall results for detecting MRSA from nasal specimens.     

Molecular epidemiology of community-associated antimicrobial-resistant Staphylococcus aureus in Seoul, Korea (2003): pervasiveness of multidrug-resistant SCCmec type II methicillin-resistant S. aureus

There is an extremely high incidence of antimicrobial resistance of the clinical isolates of Staphylococcus aureus in Korea. This study carried out a molecular investigation to determine the prevalence of the community-associated antimicrobial-resistant S. aureus and methicillin-resistant S. aureus (MRSA). The percentage resistance from the nasal swabs of healthy volunteers in 2003 in Seoul is as follows: penicillin (91%), erythromycin (EM, 14%), gentamicin (GM, 9.3%), tetracycline (TE, 8.2%), cephalothin (4%), oxacillin (OX, MRSA; 3.8%), clindamycin (CC, 2.6%), ciprofloxacin (CIP, 0.8%), and sulfamethoxazole/trimethoprim (0.6%). The community-associated MRSA (C-MRSA) strains were examined by pulsed-field gel electrophoresis (PFGE) analysis of the SmaI macro-fragments, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing using the PCR analysis. The Korean C-MRSA isolates were clustered into three distinct groups. One PFGE group containing the C-MRSA strains showed resistance to CC, EM, and GM, a high level (32-96 microg/ml) of resistance to methicillin, sequence type 5 (ST5), and SCCmec type II, which is the most common hospital associated-MRSA (H-MRSA) isolated in Korea. These results highlight the heterogeneous genetic background of the C-MRSA as well as the pervasiveness of the H-MRSA isolates in this community.     

Improved and rapid detection of methicillin-resistant Staphylococcus aureus nasal carriage using selective broth and multiplex PCR

To improve efficiency in detecting nasal methicillin-resistant Staphylococcus aureus (MRSA), we evaluated a multiplex PCR using pre-enrichment of the specimen in selective broths, and compared it with detection performed by routine tests in hospital laboratories. Nasal swab specimens from 311 patients were inoculated onto mannitol-salt agar (MSA) at the hospital laboratories and in two Mueller-Hinton broths with 7% NaCl containing oxacillin at concentrations of 2 and 4 micro g/ml. Isolates on MSA were identified as MRSA by classical laboratory tests (coagulase and oxacillin disk diffusion tests). Oxacillin broth cultures were subcultured on blood agar and MRSA isolates were identified by coagulase and susceptibility tests, including agar dilution and the oxacillin-screening method (gold standard method). Simultaneously, multiplex-PCR was performed from the selective broths to detect S. aureus species-specific and mecA gene segments (OxMPCR method). Thirty-two S. aureus isolates were recovered: 29 (90.6%) were MRSA strains and 3 (9.4%) were oxacillin-susceptible isolates. Twenty-eight (96.5%) MRSA isolates were detected by OxMPCR, while 17 (58.6%) were identified by routine tests (P=0.002). This new method for detection of MRSA nasal carriers showed higher sensitivity and led to faster reporting--i.e., within 24 h--of results.     

Catheter-associated fungemia caused by Fusarium chlamydosporum in a patient with lymphocytic lymphoma.

We evaluated the use of mannitol salt agar with oxacillin for use as a primary screening medium for the simultaneous detection and identification of methicillin-resistant Staphylococcus aureus in clinical surveillance specimens. Oxacillin agar dilution susceptibility tests with mannitol salt agar and Mueller-Hinton agar were performed in parallel with disk-agar diffusion testing on 95 oxacillin-susceptible and 105 oxacillin-resistant S. aureus stock isolates. MICs were found to be comparable, showing distinct separation of susceptible and resistant isolates into two groups with MICs of less than or equal to 2 and greater than or equal to 32 micrograms/ml, respectively. In accord with these findings, 4 micrograms of oxacillin per ml was selected for use in the screening medium. For performance evaluation, mannitol salt agar with 4 micrograms of oxacillin per ml was compared with mannitol salt agar without oxacillin by performing parallel screening tests on 153 clinical surveillance specimens. For detection of methicillin-resistant S. aureus, mannitol salt agar with 4 micrograms of oxacillin per ml was as sensitive as mannitol salt agar without oxacillin and required significantly fewer confirmatory tests. For primary identification of methicillin-resistant S. aureus, mannitol salt agar with 4 micrograms of oxacillin per ml was 6.4% false-positive and 1.1% false-negative, with a 93.6% positive predictive value. These findings indicate that mannitol salt agar with 4 micrograms of oxacillin per ml can be used as a reliable and cost-effective screening medium for the simultaneous detection and identification of methicillin-resistant S. aureus in clinical surveillance specimens.     

Comparison of the BBL CHROMagar Staph aureus agar medium to conventional media for detection of Staphylococcus aureus in respiratory samples

Screening for Staphylococcus aureus has become routine in certain patient populations. This study is the first clinical evaluation of the BBL CHROMagar Staph aureus agar (CSA) medium (BD Diagnostics, Sparks, Md.) for detection of S. aureus in nasal surveillance cultures and in respiratory samples from cystic fibrosis (CF) patients. S. aureus colonies appear mauve on CSA. Other organisms are inhibited or produce a distinctly different colony color. S. aureus was identified from all media by slide coagulase, exogenous DNase, and mannitol fermentation assays. Susceptibility testing was performed using the agar dilution method. A total of 679 samples were evaluated. All samples were inoculated onto CSA. Nasal surveillance cultures were inoculated onto sheep blood agar (SBA) (BD Diagnostics), and samples from CF patients were inoculated onto mannitol salt agar (MSA) (BD Diagnostics). Of the 679 samples cultured, 200 organisms produced a mauve color on CSA (suspicious for S. aureus) and 180 were positive for S. aureus on SBA or MSA. Of 200 CSA-positive samples 191 were identified as S. aureus. Nine mauve colonies were slide coagulase negative and were subsequently identified as Staphylococcus lugdunensis (one), Staphylococcus epidermidis (three), Staphylococcus haemolyticus (one), and Corynebacterium species (four). CSA improved the ability to detect S. aureus by recovering 12 S. aureus isolates missed by conventional media. Of the 192 S. aureus isolates recovered, 122 were methicillin susceptible and 70 were methicillin resistant. Overall, the sensitivity and specificity of CSA in this study were 99.5 and 98%, respectively. There was no difference in the performance of the slide coagulase test or in susceptibility testing performed on S. aureus recovered from CSA compared to SBA or MSA. Our data support the use of CSA in place of standard culture media for detection of S. aureus in heavily contaminated respiratory samples.     

Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.     

Nationwide surveillance for Staphylococcus aureus with reduced susceptibility to vancomycin in Korea

Methicillin-resistant Staphylococcus aureus (MRSA) accounts for more than 70% of S. aureus isolates from tertiary hospitals in Korea. Clinical isolates of S. aureus were collected from eight provincial, university-affiliated hospitals during the period from June 1999 to January 2001 for nationwide surveillance. All isolates were screened for reduced susceptibility to vancomycin by using brain heart infusion agar containing 4 micro g of vancomycin per milliliter. Population analysis and the determination of the MIC of vancomycin were done for the isolates which grew on the screening agar plates. Of 682 total isolates, MRSA accounted for 64% (439 of 682). Of 27 (4%) isolates that grew on the screening agar plates, none showed the heteroresistance phenotype. No strains with reduced susceptibility to vancomycin were identified.     

Evaluation of a new selective medium for methicillin-resistant Staphyloccocus aureus

The aims of this study were to evaluate the performance of a new medium, desferrioxamine oxacillin tellurite egg-yolk mannitol salt agar (DOTEMSA) in detecting methicillin-resistant Staphylococcus aureus (MRSA) and then to compare this medium against the Public Health Laboratory Service (PHLS) recommendation of mannitol salt agar (Oxoid) with oxacillin (OMSA) and Baird-Parker medium with ciprofloxacin (BPC) for the isolation of MRSA. The individual selective agents contained in DOTEMSA were tested against isolates of coagulase-negative staphylococci (CNS) and the medium with all constituents was challenged with various bacteria. Routine screening specimens were plated out on OMSA, BPC and DOTEMSA and the plates were incubated and examined at 24 and 48 h. Tellurite, desferrioxamine and oxacillin each inhibited the majority of CNS isolates; only three (of 103) grew in the presence of all three agents. Sixty-two of 63 isolates of MRSA grew on DOTEMSA and 59 produced lipase. Most other bacteria were inhibited. In all, 184 MRSA isolates were isolated from 540 screening specimens. The sensitivity of OMSA, BPC and DOTEMSA was 42%, 81% and 51% at 24 h, and 60%, 89% and 89% at 48 h. At 48 h, the combination of BPC and DOTEMSA detected 99% of MRSA isolates. Seventy, 49 and one non-MRSA isolates needed investigation for each of the three media respectively. A proposed strategy for MRSA screening would use BPC and DOTEMSA, examining BPC at 24 h and both media at 48 h. Provisional reports could then be issued at 24 h on the basis of rapid agglutination tests to confirm isolates as S. aureus from BPC and at 48 h on the basis of typical colonies from DOTEMSA.     

Evaluation of the BBL Crystal MRSA ID System for detection of oxacillin resistance in Staphylococcus aureus.

AIMS: To evaluate the BBL Crystal MRSA ID System for detection of oxacillin resistance in Staphylococcus aureus. METHODS: 52 methicillin resistant S aureus (MRSA) from five different countries and 85 methicillin susceptible S aureus (MSSA) were included. The species was confirmed by tube coagulation and detection of the clumping factor using the Staphaurex Plus. Clonal non-identity of the MRSA isolates was shown by pulsed field gel electrophoresis. MIC values (oxacillin) were determined using the Etest. Polymerase chain reaction was carried out to detect the mecA gene. The BBL Crystal MRSA ID System was carried out according to the manufacturer's instructions. RESULTS: The BBL Crystal MRSA ID System showed fluorescence in 45 of 52 MRSA (sensitivity 86.5%; negative predicitive value 92.2%), and the specificity was 97.6% (positive predicitive value 95.7%). Two of seven MRSA that failed to show fluorescence had MIC values (oxacillin) of 4 mg/litre. CONCLUSIONS: The BBL Crystal MRSA ID System is a valuable test for detecting oxacillin resistance in S aureus. Its major advantage is the short time (4-5 hours) required to perform the test when organisms are grown on tryptic soy agar or sheep blood agar. Difficulties may arise in borderline resistant isolates.     

Rapid Slide Latex Agglutination Test for Detection of Methicillin Resistance in Staphylococcus aureus

The MRSA screen test (Denka Seiken Co., Ltd.), a commercially available, rapid (20-min) slide latex agglutination test for the determination of methicillin resistance by detection of PBP 2a in Staphylococcus aureus, was compared with the oxacillin agar screen test and PCR detection of the mecA gene. A total of 563 S. aureus isolates were tested. Two hundred ninety-six of the isolates were methicillin-susceptible isolates from cultures of blood from consecutive patients. Also, 267 methicillin-resistant isolates that comprised 248 different phage types were tested. Methicillin resistance was defined as the presence of the mecA gene. Of the 267 mecA gene-positive isolates, 263 were positive by the MRSA screen test (sensitivity, 98.5%), and all the mecA-gene negative strains were negative by the MRSA screen test (specificity, 100%). The oxacillin agar screen test detected methicillin resistance in 250 of the mecA gene-positive isolates (sensitivity, 93.6%). The sensitivity of the MRSA screen test was statistically significantly higher than the sensitivity of the oxacillin agar screen test (P < 0. 05). The MRSA screen test is a highly sensitive and specific test for the detection of methicillin resistance. Also, it offers results within half an hour and is easy to perform, which makes this test a valuable tool in the ongoing battle against methicillin-resistant S. aureus.     

Methicillin-resistant Staphyloccocus aureus: evaluation of five selective media

This study evaluates the performance in isolating methicillin-resistant Staphylococcus aureus (MRSA) of three media: the reduced salt formulation of mannitol salt agar plus oxacillin (MMSAO); CHROMagar Staph aureus plus ciprofloxacin (CHRAC); and Halifax MRSA medium (HMO), against the previously recommended mannitol salt agar (7% salt) plus oxacillin (OMSAO) and Baird-Parker medium plus ciprofloxacin (BPC). MRSA screening swabs were plated out onto the five selective media and the plates examined at 24 and 48 h. Suspected colonies were confirmed as MRSA by detection of heat-labile DNase, coagulase and/or protein A, and by confirming resistance to methicillin. Of 719 specimens examined, 191 grew MRSA on at least one medium. The relative sensitivities of the five media at 48 h were as follows: BPC, 94%; CHRAC, 70%; OMSAO, 61%; HMO, 56%; and MMSAO, 46%. In addition, BPC gave the least number of unnecessary investigations for non-MRSA isolates. The current advantage of BPC when performing direct culture for MRSA was confirmed. The other ciprofloxacin-containing medium also produced reasonable results. Of the two mannitol salt agar media, the formulation with 7% salt gave better results. HMO proved unreliable at isolating MRSA.     

Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam,the Vitek 2 system,and the MRSA-screen latex agglutination test

Very-low-level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the mecA gene by PCR was considered to be the "gold standard." We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-microg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter(-1) for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of S. aureus studied were divided into 69 MSSA (mecA-negative) and 83 MRSA (mecA-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-microg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-microg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA.     

Mueller–Hinton agar is superior to PDM blood agar for detection of methicillin-resistant Staphylococcus aureus

The aim of this study was to compare the expression of oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) on Paper Disc Method agar supplemented with 5% defibrinated blood (PDM blood agar) and Mueller–Hinton agar supplemented with 2% NaCl (MH NaCl agar) using different susceptibility tests. Fifty mecA- containing isolates of S. aureus, exhibiting 46 different pulsed-field gel electrophoresis patterns, were comparatively tested using the E test, the single disk diffusion test, and the multipoint inoculation technique, under various culture conditions. The E test incubated at 35 °C for 24 h (breakpoint of resistance ≥2.0 mg/L) detected 94% of the isolates on MH NaCl agar, compared with 28% for PDM blood agar ( P  < 0.05). The disk diffusion test (breakpoint ≤ 10 mm in diameter) under these incubation conditions detected resistance in 100% of the isolates on MH NaCl agar and in 80% of the isolates on PDM blood agar ( P  < 0.05). The multipoint technique (breakpoint ≥1 mg/L), applied at 35 °C for 24 h, detected 100% on MH NaCl agar and 46% on PDM blood agar ( P  < 0.05). Irrespective of the method of susceptibility testing evaluated, MH NaCl agar was superior to PDM blood agar for the detection of oxacillin resistance in mecA -containing S. aureus .     

SCREENING FOR METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS CARRIERS AMONG PATIENTS AND HEALTH CARE WORKERS OF A TERTIARY CARE HOSPITAL IN SOUTH INDIA

A total of 200 subjects were screened for carriage of methicillin-resistant Staphylococcus aureus (MRSA) at different sites using oxacillin blood agar and mannitol salt agar with oxacillin. Overall carriage rate was 8.5%, with the highest rate in inpatients (15.6%) while the lowest was seen in health care workers (1.8%). The commonest site of colonization was the anterior nares. Oxacillin blood agar was found to be superior to mannitol salt agar with oxacillin for the isolation of MRSA. Male sex and prolonged hospital stay were found to be the major risk factors for MRSA colonization.     

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.