Is There a Need to Revise the Antibiotic Concentration in Clinical and Laboratory Standards Institute-Recommended Oxacillin Screen Agar?

Introduction: In routine diagnostic microbiology laboratories, Clinical and Laboratory Standards Institute (CLSI) recommends the use of cefoxitin disc, in addition to oxacillin screen agar (OSA) of 6 μg/ml for the detection of methicillin-resistant Staphylococcus aureus (MRSA), whereas minimum inhibitory concentration values of oxacillin for S. aureus are ≤2 μg/ml (susceptible) and ≥4 μg/ml (resistant). Hence, the study was carried out to evaluate the ability of screen agar with lower concentrations of oxacillin to identify the isolates of MRSA and to compare this with cefoxitin disc diffusion (CDD). Materials and Methods: Six hundred and seventy-six isolates of S. aureus were screened for methicillin resistance by OSA with 2 μg/ml and 4 μg/ml and 6 μg/ml of oxacillin concentration as well as CDD. Polymerase chain reaction for mecA gene was carried out for all isolates which grew on OSA 2, 4 and 6 μg/ml regardless of their cefoxitin susceptibility. Latex agglutination test for penicillin-binding protein 2a was performed for the isolates which grew on OSA 2 and or 4 μg/ml but not on OSA 6 μg/ml. Results: Eight per cent of MRSA isolates was missed by using OSA 6 μg/ml, when compared with other methods. Sensitivities of OSA 2 μg/ml, OSA 6 μg/ml and CDD were found to be 100%, 92.5% and 97.5%, respectively, and specificities for the same were found to be 100%, 100% and 98%, respectively. As per FDA criteria, categorical agreement for OSA 2 μg/ml was found to be 100% in comparison with the reference broth microdilution method. No major and very major discrepancies were documented. Conclusion: Similar findings on a larger and more heterogeneous collection of isolates may indicate the need to revise the concentration of OSA to 2 μg/ml for the detection of MRSA.     

Detection of Methicillin-Resistant Coagulase-Negative Staphylococci by the Vitek 2 System

The accurate performance of the Vitek 2 GP67 card for detecting methicillin-resistant coagulase-negative staphylococci (CoNS) is not known. We prospectively determined the ability of the Vitek 2 GP67 card to accurately detect methicillin-resistant CoNS, with mecA PCR results used as the gold standard for a 4-month period in 2012. Included in the study were 240 consecutively collected nonduplicate CoNS isolates. Cefoxitin susceptibility by disk diffusion testing was determined for all isolates. We found that the three tested systems, Vitek 2 oxacillin and cefoxitin testing and cefoxitin disk susceptibility testing, lacked specificity and, in some cases, sensitivity for detecting methicillin resistance. The Vitek 2 oxacillin and cefoxitin tests had very major error rates of 4% and 8%, respectively, and major error rates of 38% and 26%, respectively. Disk cefoxitin testing gave the best performance, with very major and major error rates of 2% and 24%, respectively. The test performances were species dependent, with the greatest errors found for Staphylococcus saprophyticus. While the 2014 CLSI guidelines recommend reporting isolates that test resistant by the oxacillin MIC or cefoxitin disk test as oxacillin resistant, following such guidelines produces erroneous results, depending on the test method and bacterial species tested. Vitek 2 cefoxitin testing is not an adequate substitute for cefoxitin disk testing. For critical-source isolates, mecA PCR, rather than Vitek 2 or cefoxitin disk testing, is required for optimal antimicrobial therapy.     

BD Phoenix and Vitek 2 Detection of mecA-Mediated Resistance in Staphylococcus aureus with Cefoxitin

The BD Phoenix (BD Diagnostics, Sparks, MD) and Vitek 2 (bioMérieux, Durham, NC) automated susceptibility testing systems have implemented the use of cefoxitin to enhance the detection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). To assess the impact of this change, 620 clinically significant S. aureus isolates were tested in parallel on Phoenix PMIC/ID-102 panels and Vitek 2 AST-GP66 cards. The results for oxacillin and cefoxitin generated by the automated systems were compared to those generated by two reference methods: mecA gene detection and MICs of oxacillin previously determined by broth microdilution according to CLSI guidelines. Testing of isolates with discordant results was repeated to attain a majority or consensus final result. There was 100% final agreement between the results of the two reference methods. For the 448 MRSA and 172 methicillin-susceptible S. aureus isolates tested, the rates of categorical agreement of the results obtained with the automated systems with those obtained by the reference methods were 99.8% for the Phoenix panels and 99.7% for the Vitek 2 cards. A single very major error occurred on each instrument (0.2%) with different MRSA isolates. The only major error was attributed to the Vitek 2 system overcalling oxacillin resistance. In 16 instances (9 on the Phoenix system, 7 on the Vitek 2 system), an oxacillin MIC in the susceptible range was correctly changed to resistant by the expert system on the basis of the cefoxitin result. The inclusion of cefoxitin in the Phoenix and Vitek 2 panels has optimized the detection of MRSA by both systems.The accurate detection of mecA-mediated ß-lactam resistance in Staphylococcus aureus is essential for the treatment of overt infections and the implementation of infection control practices. Although FDA-cleared PCR assays for the rapid detection of methicillin (meticillin)-resistant S. aureus (MRSA) are available for use for surveillance and testing of clinical specimens, isolates causing infections continue to require susceptibility testing to guide therapy.The phenotypic detection of mecA-mediated resistance has presented ongoing challenges due to variable gene expression that is modulated by many factors (1). Variables such as temperature, incubation time, growth medium, and sodium chloride concentrations have been considered in the development of Clinical Laboratory Standards Institute (CLSI) reference susceptibility test methods (3). Among the penicillinase-resistant penicillins, oxacillin is the most stable and sensitive for the detection of mecA-mediated resistance. However, heterogeneously resistant populations may have oxacillin test results indicating susceptibility (1).Recognition that cefoxitin is a stronger inducer of mecA expression than oxacillin led to studies that assessed this agent as a surrogate marker for methicillin resistance (2, 6, 9, 15, 16). For disk diffusion testing of staphylococci, cefoxitin (30 μg) provides more accurate results than oxacillin and zones that are easier to read (2, 3, 6). While cefoxitin has replaced oxacillin in the CLSI disk diffusion test, laboratories may use oxacillin or cefoxitin to predict mecA-mediated resistance by use of the CLSI broth microdilution (BMD) method (4). A resistant oxacillin or cefoxitin MIC test result indicates resistance to penicillins, cephems, carbapenems, and ß-lactams and ß-lactamase inhibitors (4).Manufacturers of automated susceptibility testing instruments have also adapted their products to optimize the detection of mecA-mediated resistance. The BD Phoenix (BD Diagnostics, Sparks, MD) and the Vitek 2 (bioMérieux, Durham, NC) systems now offer panels that include oxacillin and cefoxitin. The instruments'' expert systems interpret any S. aureus isolate that tests positive by the cefoxitin screen (MIC > 4 μg/ml on the Phoenix system, MIC > 6 μg/ml on the Vitek 2 system) as oxacillin resistant.This purpose of this study was to examine the accuracies of the Phoenix and the Vitek 2 instruments for the detection of mecA-mediated resistance in S. aureus. The oxacillin, cefoxitin, and expert system results generated by the Phoenix and Vitek 2 instruments were compared to the results generated by two reference methods: the oxacillin MICs determined by the CLSI BMD method and mecA gene detection by PCR.(This study was presented in part at the 108th General Meeting of the American Society for Microbiology, 2 June 2008, Boston, MA [abstr. C-009].)     

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.     

Nasal carriage of Methicillin-resistant Staphylococcus aureus among healthy population of Kashmir,India

Background: Nasal colonisation with community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) is being increasingly reported, especially in places where people are in close contact and where hygiene is compromised. The aim of this study was to find out prevalence of methicillin resistant S.aureus (MRSA) colonising anterior nares of healthy subjects. Materials and Methods: Nasal swabs of healthy subjects were collected aseptically and cultured using standard microbiological protocols. Antibiotic susceptibility was done by Kirby-Bauer disc diffusion method according to CLSI guidelines. Methicillin resistance was detected by cefoxitin disc diffusion method and confirmed by minimum inhibitory concentration (MIC) and amplification of mecA gene by PCR. Strain typing of MRSA strains was done by PFGE. Results: Out of 820 samples, S.aureus was isolated from 229 (27.92%) subjects. Of the 229 isolates, 15 were methicillin resistant. All S. aureus isolates were susceptible to vancomycin. Nasal carriage of MRSA was found to be 1.83% among healthy population. The isolates were found to be polyclonal by PFGE analysis. Conclusion: High prevalence of MRSA is a cause of concern and strategies to interrupt transmission should be implemented.     

Evaluation of a disk diffusion method with cefoxitin (30 μg) for detection of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The emergence of heterogeneous populations of methicillin-resistant Staphylococcus aureus (MRSA) causes major problems in routine screening for MRSA. In heterogeneous MRSA populations, a proportion of bacterial cells show low-level resistance to oxacillin, with minimal inhibitory concentrations (MICs) of oxacillin ranging between 1 and 100 mg/l, while in homogeneous MRSA populations, the MIC of oxacillin for all cells is >100 mg/l. Routine oxacillin disk diffusion tests often fail to detect heterogeneous MRSA populations. In the present study, a recently proposed disk diffusion method that employs a cephamycin antibiotic (cefoxitin 30 g; BD Sensi-disc, Becton Dickinson, Germany) was evaluated using 155 clinical isolates of S. aureus (73 mecA positive and 82 mecA negative). The results were compared with those of other MRSA screening techniques: a disk diffusion test with oxacillin 1 g and cefoxitin 30 g (BD Sensi-disc; Becton Dickinson), an MRSA latex agglutination test (Denka Seiken, Japan), and an oxacillin screen agar test (6 g/ml; Becton Dickinson). Detection of the mecA gene by polymerase chain reaction was considered the gold standard. The performances of the different methods were determined and compared. The results showed that the cefoxitin disk diffusion test is preferable to the oxacillin disk diffusion method for routine screening to detect MRSA.     

EUCAST disc diffusion criteria for the detection of mecA-Mediated β-lactam resistance in Staphylococcus pseudintermedius: oxacillin versus cefoxitin

ObjectivesUntil recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated β-lactam resistance in Staphylococcus pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius.MethodsWe included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-μg cefoxitin and 1-μg oxacillin discs from three manufacturers and Mueller–Hinton agar from two manufacturers.ResultsCefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6–20 mm and 19–30 mm. For cefoxitin 16% (95% CI 14.8–18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6–2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) <20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively.ConclusionsThis investigation confirms that the 1-μg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-μg cefoxitin disc. For a 1-μg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter <20 mm are resistant to all β-lactam antibiotics except those with activity against methicillin-resistant staphylococci.     

Comparison of Cefoxitin and Oxacillin Disk Diffusion Methods for Detection of mecA-Mediated Resistance in Staphylococcus aureus in a Large-Scale Study

The recommended breakpoints for the cefoxitin disk diffusion test for Staphylococcus aureus were recently modified. In this large-sample study, cefoxitin sensitivity and specificity compared to those of oxacillin were 97.3% and 100%, respectively. This study validated the new cefoxitin breakpoints for the detection of mecA-mediated resistance in S. aureus.     

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group

Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection of mecA- or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/mecC PCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.     

Correlation between the VITEK2 system and cefoxitin disk diffusion for the daily detection of oxacillin resistance in a large number of clinical Staphylococcus aureus isolates

The aim of the present study was to compare the performance of the new VITEK2 AST-P551 card with the cefoxitin disk diffusion method for the daily detection of methicillin resistance with a high number of Staphylococcus aureus clinical isolates. Detection of the PBP2a protein or mecA gene was performed for each discordant case. Seventy (3.3%) isolates out of 2,107 clinical strains showed discordant results, two very major errors, four major errors and 64 minor errors. Fifty-nine (84%) discordant results were resolved, with a final overall agreement of 99.5%. Eleven (0.5%) strains remained discordant (minor error [mE]). Four of 370 MRSA strains were misclassified as susceptible in daily practice by the cefoxitin disk diffusion method. All of these strains were resistant to aminoglycosides and/or fluoroquinolones. The VITEK2 system is highly reliable for methicillin resistance detection at the routine level. Oxacillin-susceptible classified clinical strains with associated resistance patterns required attention.     

Evaluation of Mannitol Salt Agar for Detection of Oxacillin Resistance in Staphylococcus aureus by Disk Diffusion and Agar Screening

Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-μg disk; zone diameter, <16 mm); the specificity was 97.6%. Agar screening (2 μg of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.     

Comparison of screening methods to identify methicillin-resistantStaphylococcus aureus

Screening methods to identify methicillin-resistantStaphylococcus aureus (MRSA) were compared using 96 isolates representing 17 distinct clones. The sensitivity of four commercial agglutination tests was determined in comparison to the tube coagulation test, and the results related to the presence of the coagulase gene. The broth screening test, agar dilution test and disc diffusion test were carried out, and the results related to the presence of themecA gene. Mannitol salt agar and Iso-Sensitest agar with varying salt supplements were used. All agglutination tests had high rates of detection ofStaphylococcus aureus (95.8–99.0%). Resistance in mecA gene-positiveStaphylococcus aureus isolates was correctly detected by the oxacillin broth test, the agar dilution test and the disc diffusion test on mannitol salt agar, whereas on Iso-Sensitest agar detection rates were lower (between 68.5% and 94.4%, depending on the salt supplement). Incubation of the Iso-Sensitest plates for 48 hours significantly improved the rate of detection of resistance, but increased the major error rate up to 71.4%.MecA genepositiveStaphylococcus aureus isolates not detected by the disc diffusion test on Iso-Sensitest agar had significantly lower oxacillin minimal inhibitory concentration values and were significantly less resistant to a variety of antibiotics. Thus, mannitol salt agar might be a suitable medium for use in the disc diffusion and agar dilution test to detect resistance to oxacillin inStaphylococcus aureus.     

Evaluation of Screening and Commercial Methods for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci

The National Committee for Clinical Laboratory Standards recommends 48 h of incubation by the oxacillin salt agar screen (OSAS) method for the detection of methicillin-resistant coagulase-negative staphylococci (CoNS). An earlier identification of methicillin resistance is desirable. The time to detection of the mecA gene by PCR was compared with the times to detection by OSAS, by the oxacillin disk diffusion (ODD) method, and with MicroScan Gram Positive Combo type 6 panels (MicroScan Inc. Sacramento, Calif.) and Vitek GPS-SA cards (bioMérieux Vitek Inc., Hazelwood, Mo.). The combination of the Vitek card and the ODD method detected 92 of 99 methicillin-resistant strains of CoNS at 24 h; however, 6 mecA-positive strains were phenotypically methicillin susceptible. We conclude that most methicillin-resistant CoNS can be detected and the results can be reported after overnight incubation by a combination of methods.     

Comparison of Xpert MRSA/SA Nasal and MRSA/SA ELITe MGB Assays for Detection of the mecA Gene with Susceptibility Testing Methods for Determination of Methicillin Resistance in Staphylococcus aureus Isolates

In a series of 82 Staphylococcus strains isolated from culture, 100% were identified as Staphylococcus aureus by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS); 99.9% (77/82) of them were resistant to benzylpenicillin, oxacillin, and cefoxitin, and 6.1% (5/82) were susceptible to methicillin. Xpert MRSA/SA assay results were concordant with the phenotypic results in 76.8% (63/82) of cases and discordant in 23.2% (19/82) of cases. The MRSA/SA ELITe MGB kit results were concordant with phenotypic results in 100% of the cases. When comparing the Xpert MRSA/SA assay results with the MRSA/SA ELITe MGB kit results, 78% (64/82) of the cases were concordant, while 22% (18/82) of the cases were discordant. No statistically significant differences were observed between the two techniques. The PCR protocol that was used to validate the results of these two methods gave the following results: 49 were conventional methicillin-resistant S. aureus (MRSA) isolates (mecA positive and mecA_LGA251 negative), and 25 were phenotypic MRSA isolates (mecA negative and mecA_LGA251 positive).     

Comparison of phenotypic methods with PCR to screening methicillin resistance in coagulase negative staphylococci

THE AIM OF STUDY: Appreciation of the frequency, the level and the genetic support of methicillin resistance. MATERIAL AND METHODS: Seventy-three strains of coagulase negative staphylococci isolated from various specimens, from January to June 2004, were studied. The phenotypic detection was carried out by disk diffusion test using oxacillin and cefoxitin disks, by the determination of oxacillin Minimal Inhibitor Concentration (E-test), by the oxacillin screening test at a concentration of 4 mug/ml and by the search of the penicillin binding protein PBP2a using the slide latex agglutination test. The results of these methods were compared to PCR of mecA gene. RESULTS: Forty-eight strains carried mecA gene whose 30 were detected by the oxacillin disk, the cefoxitin disk, the oxacillin screening test, the slide latex agglutination test and had a MIC from 24 to 256 mug/ml. Seventeen strains were not detected by oxacillin disk but by cefoxitin disk and the slide latex agglutination test. Among these strains, 13 (76%) had oxacillin MIC from 0.5 to 1,5 mug/ml and not grew on oxacillin agar screening, while 4 (24%) had oxacillin MIC from 6 to 16 mug/ml and grew on this agar. One strain had oxacillin MIC of 0,19 mug/ml and was not detected with any phenotypic method. CONCLUSION: The determination of oxacillin MIC, the search of the PBP2a or more simply the cefoxitin disk had permitted to detect the strains mecA gene (+) with resistant and pre-resistant phenotype but not the strain with sensible phenotype (2.1%).     

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.     

Use of a DNA hybridization method to verify results of screening for methicillin resistance in Staphylococci

Tests were performed by the disk diffusion method, agar dilution method and the E test to determine the susceptibility to methicillin and oxacillin of clinical isolates and control strains ofStaphylococcus aureus (n=106) and coagulase-negative species (n=131). Results were compared with those of a dot blot DNA hybridization test, in which themecA gene was detected using an oligonucleotide probe selected from themecA gene. Among theStaphylococcus aureus strains themecA gene was found in all but two strains inhibited by 8 mg/l of methicillin and all but two strains inhibited by 4 mg/l of oxacillin. A disk test using either 1 µg oxacillin or 10 µg methicillin and a tentative resistance breakpoint of 10 mm gave the best agreement with the hybridization test. For coagulase-negative staphylococci 34 of 35 strains inhibited by 8 mg/l methicillin hybridized with the probe as well as 58 of 82 strains inhibited by 1–4 mg/l; 93 of 97 strains inhibited by 0.5 mg/l oxacillin were also positive in the probe test. Using the 1 µg oxacillin disk and a resistance breakpoint of 10 mm good agreement was obtained between results of the disk diffusion and DNA hybridization tests. It is suggested that this genotypic method for detection of methicillin resistance is used as a reference method for routine methods.     

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.     

The non-association of Panton-Valentine leukocidin and mecA genes in the genome of Staphylococcus aureus from hospitals in South Western Nigeria

Purpose: Virulence genes play important roles in pathogenesis of infections caused by S. aureus. The aim of this study was to determine the prevalence of PVL, eta and mecA genes in S. aureus isolated from patients in South-Western Nigeria.Materials and Methods:In this study, a total of 116 S. aureus isolates from the clinical specimens submitted to laboratories in tertiary hospitals in the South Western Nigeria were used. Antibiotic susceptibility test was carried out to determine the susceptibility pattern of the isolates using multiple antibiotics disc. Minimum inhibitory concentration (MIC) was also carried out to determine the degree of resistant of the isolates to methicillin. PCR was used to screen for the presence of PVL, eta, and mecAgenes. Results:mecA gene was detected in 48 (41.4%) of 116 strains of S. aureus. The MIC₅₀ and MIC₉₀ for mecA negative strains were 1 and 8 μg/ml, respectively while the MIC₅₀ and MIC₉₀ for mecA positive were >256 μg/ml. Twenty eight (24.1%) of 116 isolates were PVL gene positive with none of them mecA+. The prevalence of community acquired MRSA (CA-MRSA) was estimated to be 6.9% using molecular techniques. No localization of mecA gene and PVL gene on the genome of the entire S. aureus strains studied. Site of isolation of organism/specimen type was found to be associated with the prevalence of PVL+ and mecA+ S. aureus (P< 0.01). Conclusion: This study concludes that the PVL+ MRSA is rare and the prevalence of CA-MRSA is low in South-Western, Nigeria.     

Evaluation of the BBL® CrystalTM MRSA ID System for Rapid Detection of Methicillin Resistance in Staphylococcus aureus

Twenty-four clinical isolates of Staphylococcus aureus collected from various geographic areas and four reference strains were studied by (i) agar diffusion with disks impregnated with 5 μg oxacillin and reading after incubation at 30°C for 24 hours, (ii) Southern hybridization with a probe specific for the mecA gene, and (iii) the BBL® Crystal^TM MRSA ID system. There was perfect correlation between the three methods: the BBL® Crystal^TM MRSA ID system detected methicillin resistance in the fifteen strains hybridizing with the mecA probe and classified as resistant by the oxacillin disk diffusion test; the thirteen remaining strains were susceptible by agar diffusion and by the BBL® test and did not hybridize with the mecA probe. The BBL® Crystal^TM MRSA ID System, therefore, appears to be an accurate method for rapid detection of Staphylococcus aureus exhibiting homogeneous resistance to methicillin.