Phenotypic detection of methicillin resistance in Staphylococcus aureus by disk diffusion testing and Etest on Mueller-Hinton agar

Cefoxitin is increasingly recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) when using disk diffusion testing. In this study, 95 mecA-negative S. aureus isolates and a highly genetically diverse collection of mecA-positive S. aureus types (n = 50) were used to investigate the influence of technical factors such as disk potency, incubation time, and temperature on Mueller-Hinton agar. The use of cefoxitin MIC testing by Etest for the same purpose was investigated under similar conditions. For disk diffusion, the accuracy was high at both 35 degrees C and 36 degrees C using overnight incubation, while incubation at 30 degrees C or 37 degrees C was associated with slightly lower accuracy. Increasing incubation times from 18 to 24 h did not improve accuracy at either temperature. Cefoxitin Etest MICs for mecA-positive strains were 6 mg/liter or higher, while cefoxitin Etest MICs for mecA-negative strains were :54 mg/liter. Our findings suggest that the current CLSI zone diameter breakpoints should be adjusted from resistance (R) <= 19 mm to R <= 21 mm. In conclusion, cefoxitin disk diffusion testing and Etest MIC testing can accurately predict the presence of the mecA gene in S. aureus. Testing can be reliably performed using incubation temperatures of 35 to 36 degrees C and incubation times of 18 to 22 h. We suggest MRSA interpretive criteria of susceptible (S) <= 4 mg/liter and R > 4 mg/liter, corresponding to S >= 22 mm and R <= 21 mm for the 30-mu g disk and S >= 17 mm and R <= 16 mm for the 10-mu g cefoxitin disk. These criteria resulted in only one mecA-positive isolate being misclassified as susceptible.