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°C and 36°C using overnight incubation, while incubation at 30°C or 37°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 ≤4 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°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-μg disk and S ≥ 17 mm and R ≤ 16 mm for the 10-μg cefoxitin disk. These criteria resulted in only one mecA- positive isolate being misclassified as susceptible.