Minimum inhibitory concentration (MIC) determination

Antibiotic MICs were determined by the broth microdilution method as described previously [19]. Each antibiotic was prepared with serial two-fold working dilutions in deionized water and the final concentration of each antibiotic in one 96-microwell plate ranged between 0.5 and 4096 mg/l. Bacterial strains were cultured twice in TSB and matched a McFarland 0.5 turbidity standard (bioMérieux, March L’Etoile, France). Each suspension was diluted a further 1:100 in cation-adjusted MH broth to achieve an adequate inoculum concentration. The final inoculum density was 5 × 10⁵ colony-forming units (cfu)/ml per well on 96-microwell plates. The MIC of each antibiotic was recorded as the lowest concentration where no growth was observed in the wells after incubation for 18 h or 24 h. MIC results were confirmed by at least three independently performed tests.