Time-Kill Assay

This assay was performed according to a previously described protocol with small modification (Ling et al., 2015). An overnight culture of either Staphylococcus aureus 533R4 or X. translucens pv. graminis LMG587 was diluted 50-fold in MHB and incubated at 30°C with the shaker at 220rpm. Bacteria were grown to an OD₆₀₀ of 0.5, and then, the concentration of cells was adjusted to ≈5×10⁵ cells per ml for X. translucens and ≈2×10⁸ for S. aureus. Bacterial cultures were then exposed to 10X MIC antimicrobials at 30°C and 220rpm. Bacteria not treated with medipeptin A were used as a negative control. Bacteria treated with vancomycin or colistin were used as a positive control. At desired time points (0, 1, 2, 3, 4, 5, and 6h), 10μl culture was taken and tenfold serially diluted. Subsequently, each sample was plated on MHA plates. After incubation at 30°C for 48h, colonies were counted and the number of CFU per ml was calculated. Each experiment was performed in triplicate.