Time-Kill Kinetic Assays

Time-kill assays were performed following CLSI guidelines and previously described methods (Wang et al., 2015; Clinical Laboratory Standard Institute [CLSI], 2017; Zheng et al., 2017). Overnight bacterial cell cultures were suspended in CAMHB and adjusted to an absorbance of approximately 10⁶ CFU/ml. Varying concentrations of the test compounds were added to the inoculum suspensions, with final concentrations corresponding to 1x MIC, 2x MIC, and/or 4x MIC, and incubated at 37°C. Aliquots were removed from the inoculum cultures after 0, 1, 2, 4, 6, 8, and 24 h of incubation. They were then serially diluted, plated on MH agar and incubated for 24 h at 37°C. Bacterial cell viability was determined by colony count. The assays were performed in triplicate. Data was presented as mean and standard deviation of three independent replicates, analyzed with one-way ANOVA followed by Dunnett’s test to determine the significance relative to the untreated bacteria (P < 0.05).