|Year : 2017 | Volume
| Issue : 2 | Page : 243-246
Is there a need to revise the antibiotic concentration in Clinical and Laboratory Standards Institute-Recommended Oxacillin Screen Agar?
Niveditha Nagasundaram, Sujatha Sistla
Department of Microbiology, JIPMER, Puducherry, India
|Date of Web Publication||5-Jul-2017|
Department of Microbiology, JIPMER, Puducherry - 605 006
Source of Support: None, Conflict of Interest: None
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.
Keywords: Cefoxitin disc diffusion, methicillin-resistant Staphylococcus aureus, oxacillin screen agar
|How to cite this article:|
Nagasundaram N, Sistla S. Is there a need to revise the antibiotic concentration in Clinical and Laboratory Standards Institute-Recommended Oxacillin Screen Agar?. Indian J Med Microbiol 2017;35:243-6
|How to cite this URL:|
Nagasundaram N, Sistla S. Is there a need to revise the antibiotic concentration in Clinical and Laboratory Standards Institute-Recommended Oxacillin Screen Agar?. Indian J Med Microbiol [serial online] 2017 [cited 2018 Feb 25];35:243-6. Available from: http://www.ijmm.org/text.asp?2017/35/2/243/209588
| ~ Introduction|| |
Labelling isolates of Staphylococcus aureus as methicillin susceptible S. aureus (MSSA) or methicillin-resistant S. aureus (MRSA) by clinical microbiology laboratories has significant therapeutic implications. The Clinical and Laboratory Standards Institute (CLSI) recommends the use of cefoxitin disc (30 μg) or oxacillin screen agar (OSA) with the concentration of 6 μg/ml for the detection of MRSA. Although cefoxitin disc diffusion (CDD) has currently become the most widely used method for detecting MRSA, there is a scope for misidentification as a single millimetre (mm) difference in zone diameter can change the interpretation from susceptible to resistant (≤21 mm is resistant; ≥22 mm is susceptible). In addition, this test might miss the rare isolates of MRSA with non-mecA/mecC-mediated resistance (CLSI guidelines, 2014). These drawbacks can be overcome by the use of OSA (6 μg/ml), which can detect MRSA isolates irrespective of resistance mechanisms. Therefore, a combination of CDD and OSA would minimise the chance of missing MRSA.
According to CLSI, the susceptible breakpoint of oxacillin is 2 μg/ml whereas the concentration of oxacillin in OSA is 6 μg/ml. We hypothesised that OSA of 6 μg/ml may fail to detect MRSA isolates which have lower oxacillin minimum inhibitory concentration (MIC) values. Hence, we evaluated the ability of OSA with 2 and 4 μg/ml concentration to detect the isolates of MRSA and compared this with the results of OSA 6 μg/ml, CDD, latex agglutination test (LAT) for penicillin-binding protein 2a (PBP2a) (Denka Seiken, Japan) and polymerase chain reaction (PCR) for mecA/mecC gene. The isolate was confirmed as MRSA if it was positive for either mecA gene and/or its product, PBP2a (gold standard).
| ~ Materials and Methods|| |
Six hundred and seventy-six consecutive, non-duplicate clinical isolates of S. aureus from various specimens submitted to our laboratory between September and November 2013 were screened for methicillin resistance using OSA (6 μg/ml) (Oxacillin from Sigma-Aldrich, USA) and cefoxitin disc (Oxoid) following the CLSI guidelines, 2013 for performance and interpretation. For quality control, ATCC S. aureus 29213 (methicillin susceptible) and ATCC S. aureus 43300 (methicillin resistant) strains were included with each batch.
OSA (2 μg/ml and 4 μg/ml) was prepared and results were also interpreted in accordance with the CLSI guidelines, 2013, except for the change in antibiotic concentration.
Out of 676 test isolates of S. aureus, 460 isolates which did not grow on OSA (6 μg/ml), regardless of their cefoxitin susceptibility, were further tested on OSA (2 and 4 μg/ml), presuming that, those 216 MRSA isolates which grew on OSA 6 μg/ml would have grown on lower concentrations of OSA 2 and 4 μg/ml.
PCR for mecA gene was carried out for all isolates which grew on OSA 2, 4 and 6 μg/ml regardless of their cefoxitin susceptibility. LAT for PBP2a was performed for the isolates which grew on OSA 2 and or 4 μg/ml but not on OSA 6 μg/ml.
Sensitivity, specificity, positive predictive value (PPV), negative predictive value for various detection methods of MRSA were calculated using GraphPad Prism 5 software (Graphpad Software, San Diego, California).
Results were also analysed for determining category agreement rate, major and very major discrepancy rates according to FDA criteria.
| ~ Results|| |
Of the total 676 isolates tested, 216 grew on OSA 6 μg/ml; cefoxitin resistant; positive for mecA PCR and hence identified as MRSA. Among the remaining 460 isolates, 441 did not grow on OSA 2, 4 and 6 μg/ml and were cefoxitin susceptible and hence identified as MSSA. Nineteen isolates which did not grow on OSA 6 μg/ml and grew on OSA 2 and or 4 μg/ml were also identified as MRSA as they were positive for either LAT for PBP2a and/or positive for mecA gene by PCR.
Eight isolates (4.1%) grew only on OSA (2 μg/ml) while 11 grew both on 2 μg/ml and 4 μg/ml indicating that a total of 19 (8%) of MRSA isolates were missed on 6 μg/ml OSA. Out of these 19 isolates, six were also found to be susceptible to cefoxitin (tests performed in triplicate). MIC values of oxacillin for these 19 isolates were determined by performing E-test (bioMérieux, France). The values ranged from 1 μg/ml to 8 μg/ml [Table 1].
|Table 1: Results of various detection methods of methicillin-resistant Staphylococcus aureus for the 19 isolates which failed to grow on oxacillin screen agar 6 μg/ml|
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Sensitivity of OSA 2 μg/ml, OSA 6 μg/ml and CDD were found to be 100%, 92.5% and 97.5% and specificity was found to be 100%, 100% and 98% respectively. PPV for OSA 2 μg/ml, OSA 6 μg/ml and CDD were found to be 100%, 100% and 96.3% respectively [Table 2].
|Table 2: Performance characteristics of various detection methods of methicillin-resistant Staphylococcus aureus (n=676)|
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As per FDA criteria (2), 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 [Table 3].
|Table 3: Calculation of agreement and errors of different test methods in comparison to the reference method as per Food and Drug Administration criteria|
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| ~ Discussion|| |
Six isolates, which were reported as MSSA in routine laboratory tests by CDD and OSA (6 μg/ml) and turned out to be MRSA with OSA (2 μg/ml), were isolated from pus samples (4), wound swab (1) and synovial fluid (1). Although treatment details were not available for these patients, they might have received cloxacillin (since it is the drug of choice for MSSA infections) as the original report was MSSA. This would have disastrous consequences especially for isolates from blood further emphasising the importance of correctly identifying isolates as MRSA.
Although cefoxitin is considered as a surrogate marker for the detection of MRSA, a few MRSA isolates which were cefoxitin susceptible in our study had zone diameters of 22 mm and 23 mm. Those isolates were positive for mecA gene and LAT for PBP2a. Hence, it is evident that those isolates which show borderline zone diameters could be misidentified.
The only drawback of using screen agar with lower concentration of antibiotic is the misidentification of susceptible isolates as resistant. Fortunately, we did not encounter any such isolate in our study thereby indicating 100% specificity of OSA (2 μg/ml).
PCR for mecA gene was repeatedly negative for 3 isolates. Performing PCR with isolates stored for a year in 80% glycerol broth at −80° C could be the possible explanation for the negative results as mecA gene is known to be lost during prolonged storage. For the isolates which gave discrepant results with MIC and cefoxitin susceptibility, the tests were repeated in triplicate and the average readings were taken.
In this study, PCR for mecA/mecC genes and LAT for the detection of PBP 2a was carried out in different periods of the study which could explain the negative PCR results in some PBP2a positive isolates. So far, no studies have reported the performance of OSA with varying concentration to detect MRSA. Although the study was limited to a single centre, the findings are significant enough to warrant repeating this study on a larger and more heterogeneous collection of isolates.
| ~ Conclusion|| |
Our study results proved the better performance of OSA (2 μg/ml) when compared to OSA (6 μg/ml). If similar findings are encountered elsewhere, then it may indicate the need to revise the concentration of OSA to 2 μg/ml.
Financial support and sponsorship
This work was supported by Intramural Research Fund, JIPMER, Puducherry.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
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[Table 1], [Table 2], [Table 3]