Determination of Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) of cefoxitin was determined for all isolates by broth microdilution reference method according to ISO standard 20776-1 (ISO, 2006) corresponding to the European Committee on AST (EUCAST, 2019a) and the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2018). Using cation-adjusted Mueller–Hinton broth (CAMHB, BD Diagnostic, Heidelberg, Germany), a final inoculum size of approximately 5 × 10⁵ cfu/ml was adjusted and confirmed by vital cell counting of serial dilutions onto tryptic soy agar (TSA) plates in triplicates after overnight incubation. Cefoxitin (TCI Deutschland, Eschborn, Germany) concentrations in a range of 0.25–128 mg/L were tested in double dilution steps. After 18 ± 2 h incubation at 35 ± 1°C the results were read. All tests were performed in triplicates and the median MIC was determined. MIC results were interpreted according to the EUCAST breakpoints (EUCAST, 2019a). MIC₅₀, MIC₉₀, and MIC ranges of consecutively collected clinical isolates were calculated for analysis. S. aureus ATCC 29213 reference strain was used as quality control (QC).