Determining Minimum Inhibitory Concentration

The detection of MIC was performed by the microbroth dilution method (Wang et al., 2020 (link)) with reference to Clinical and Laboratory Standards Institute [CLSI] (2012) . The bacterial suspension was adjusted to an equivalent of 0.5 McFarland standard (1.5 × 10⁸ CFU/ml) and then diluted 100 times. Bacterial suspension (50 μl) and equal volume of serial diluted drugs (1–128 μg/ml) were added into a 96-well cell plate (Corning Costar, United States) and incubated for 16–18 h. The bacterial suspension without compound was regarded as negative control. The lowest concentration of drug that inhibited the growth of visible bacteria was deemed as MIC. As for MBC, the suspension with a concentration higher than or equal to MIC was spotted on an agar plate and incubated for 24 h. The lowest concentration where no bacterial colonies grew on the plate was defined as MBC (She et al., 2020a (link)). The concentration of Ca²⁺ was maintained at 50 μg/ml with daptomycin.