Comparison of susceptibility testing methods with mecA gene analysis for determining oxacillin (methicillin) resistance in clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus spp.

Ninety-nine clinical staphylococcal isolates (58 coagulase-negative Staphyl ococcus spp, [CoNS] and 41 Staphylococcus aureus isolates) were evaluated f or susceptibility to oxacillin, The following susceptibility testing method s, media, and incubation conditions mere studied: agar dilution by using Mu eller-Hinton (MH) medium (Difco) supplemented with either 0, 2, or 4% NaCl and incubation at 30 or 35 degrees C in ambient air for 24 or 48 hf dish di ffusion by using commercially prepared MH medium (Difco) and MN II, agar (B BL) and incubation at 35 degrees C in ambient air for 24 or 48 h; and agar screen (spot or swab inoculation) by using commercially prepared agar (Reme l) or MH agar (Difco) prepared in-house, each containing 4% NaCl and 6 mu g of oxacillin/ml (0.6-mu g/ml oxacillin was also studied with MH agar prepa red in-house for the agar swab method and CoNS isolates) and incubation at 35 degrees C in ambient air for 24 or 48 h for swab inoculation and at 30 o r 35 degrees C in ambient air for 24 or 48 h for spot inoculation. The resu lts for these methods were compared to the results for mecA gene detection by a PCR method. Given the ability to support growth and the results for su sceptibility testing (the breakpoint for susceptible isolates was less than or equal to 2 mu g/ml), the best methods for CoNS isolates were (i) agar d ilution by using MH medium supplemented with 4% NaCl and incubation at 35 d egrees C for 48 h (no growth failures were noted, and sensitivity was 97.6% ) and (ii) agar screen (swab inoculation) by using MH medium prepared in-ho use supplemented with 4% NaCl and containing 0.6 mu g oxacillin/ml and incu bation at 35 degrees C for 48 h tone isolate that did not carry the mecA ge ne did not grow, and the sensitivity was 100%), All but one (agar dilution without added NaCl and incubation at 30 degrees C for 48 h) of the methods tested revealed all oxacillin-resistant S. aureus isolates, and no growth f ailures occurred with any method. If the breakpoint For susceptibility was lowered to less than or equal to 1 mu g/ml for agar dilution methods, more CoNS isolates with oxacillin resistance related to the mecA gene were detec ted when 0 or 2% NaCl agar supplementation was used, Only one CoNS isolate with mecA gene-associated resistance was not detected by using agar dilutio n and MH medium supplemented with 4% NaCl with incubation for 48 h. When th e breakpoint for susceptibility was decreased 10-fold (from 6,0 to 0.6 mu g of oxacillin per mi) for the agar swab screen method, fully 100% of the Co NS isolates that carried the mecA gene were identified.