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Journal of Clinical Microbiology, September 1999, p. 2952-2961, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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.

P. Kohner, J. Uhl, C. Kolbert, D. Persing, and F. Cockerill III*

Mayo Clinic and Foundation, Rochester, Minnesota

Received 30 November 1998/Returned for modification 31 March 1999/Accepted 21 May 1999

Ninety-nine clinical staphylococcal isolates (58 coagulase-negative Staphylococcus spp. [CoNS] and 41 Staphylococcus aureus isolates) were evaluated for susceptibility to oxacillin. The following susceptibility testing methods, media, and incubation conditions were studied: agar dilution by using Mueller-Hinton (MH) medium (Difco) supplemented with either 0, 2, or 4% NaCl and incubation at 30 or 35°C in ambient air for 24 or 48 h; disk diffusion by using commercially prepared MH medium (Difco) and MH II agar (BBL) and incubation at 35°C in ambient air for 24 or 48 h; and agar screen (spot or swab inoculation) by using commercially prepared agar (Remel) or MH agar (Difco) prepared in-house, each containing 4% NaCl and 6 µg of oxacillin/ml (0.6-µg/ml oxacillin was also studied with MH agar prepared in-house for the agar swab method and CoNS isolates) and incubation at 35°C in ambient air for 24 or 48 h for swab inoculation and at 30 or 35°C in ambient air for 24 or 48 h for spot inoculation. The results 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 susceptibility testing (the breakpoint for susceptible isolates was <= 2 µg/ml), the best methods for CoNS isolates were (i) agar dilution by using MH medium supplemented with 4% NaCl and incubation at 35°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-house supplemented with 4% NaCl and containing 0.6 µg oxacillin/ml and incubation at 35°C for 48 h (one isolate that did not carry the mecA gene did not grow, and the sensitivity was 100%). All but one (agar dilution without added NaCl and incubation at 30°C for 48 h) of the methods tested revealed all oxacillin-resistant S. aureus isolates, and no growth failures occurred with any method. If the breakpoint for susceptibility was lowered to <= 1 µg/ml for agar dilution methods, more CoNS isolates with oxacillin resistance related to the mecA gene were detected when 0 or 2% NaCl agar supplementation was used. Only one CoNS isolate with mecA gene-associated resistance was not detected by using agar dilution and MH medium supplemented with 4% NaCl with incubation for 48 h. When the breakpoint for susceptibility was decreased 10-fold (from 6.0 to 0.6 µg of oxacillin per ml) for the agar swab screen method, fully 100% of the CoNS isolates that carried the mecA gene were identified.

* Corresponding author. Mailing address: Bacteriology Laboratory, Division of Clinical Microbiology, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905. Phone: (507) 284-2901. Fax: (507) 284-4272. E-mail: cockerill.franklin{at}mayo.edu.

Journal of Clinical Microbiology, September 1999, p. 2952-2961, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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