4.6. Time-Kill Kinetics Assay

The kinetics of time-kill change of antibacterial agents against three different strains of B. pseudomallei and E. coli (O157:H7) was used to determine the potential of each antibacterial agent in killing, along with the bacterial growth curve following the procedure established by Pankey, et al. [68]. The single colony of B. pseudomallei and E. coli was cultured in Muller Hinton Broth (MHB) overnight at 37 °C. The overnight bacterial solution was then subcultured into 5 mL fresh MHB and further incubated for 1.5–2 h at 200 rpm to yield the mid-log growth phase. The 1% inoculum was adjusted into fresh MHB and 250 µL of the final concentration of 1–5 × 10⁵ CFU/ml of bacterial solution was added into equal volumes of 250 µL of each antibacterial agent. The solution with treated bacteria was incubated at 37 °C and shaken at 180 rpm in a shaking incubator. The time-kill kinetics was determined at the time points of 0, 30 min, 1, 3, 5 and 24 h. Serial 10-fold dilution was used to count the bacteria at each time point. Bactericidal activity was defined as a reduction of 99.9% or ≥ 3 log10 CFU/mL when compared with untreated control.