Antibiotic susceptibility testing and time kill assays

Rifampicin and daptomycin MIC were measured using E-tests (BioMérieux) on Mueller-Hinton plates supplemented with 50 mg/L Ca²⁺ following the manufacturer’s instructions. For daptomycin time kill assays, 10 mL of brain heart infusion (BHI) broth supplemented with 8 mg/L daptomycin and 50 mg/L Ca²⁺ was inoculated with 10⁶ CFU/mL of an overnight culture. Cultures were incubated at 37 °C with constant shaking, and samples were collected at 3, 6 and 24 h time points. Cell survival after daptomycin exposure was assessed by calculating the ratio of the CFU at 3, 6 and 24 h on the CFU of the initial inoculum (10⁶ CFU/mL) and taking the average colony counts of duplicate BHI agar plates. All daptomycin time kill assays were performed in biological triplicate.