4.4. Time–Kill Curve Assay

The synergistic antimicrobial effect was further determined by a time–kill assay, according to a previous method [26]. The influences of subinhibitory concentrations of BDMC and antibiotics (gentamicin, ampicillin, and oxacillin), either as individual drugs or in combination, on the growth of ATCC 33591 were examined. Bacterial growth curves were observed at five different time phases (0, 4, 8, 16, and 24 h). Moreover, 1/2 MIC BDMC combined with 1/2 MIC antibiotic, 1/2 MIC BDMC alone, and 1/2 MIC antibiotic alone were compared with a control (drug-free) to determine the synergistic effect. Bacterial cultures were diluted with sterilized MHB to ~1.5 × 10⁵ CFU/mL and the MHB-containing bacteria were incubated at 37 °C, for 24 h. After incubation for 24 h, the number of viable cells was counted on a drug-free MHA plate to determine the rate and extent of bacterial death. Three independent experiments were performed with the data presented as the mean ± standard deviation.