4.7. Time-Kill Assays

The time-kill assays (TKAs) were performed on all the synergistic combinations from the checkboard assays. For different isolates that demonstrated synergy to the same antibiotic combinations, only one was selected for the TKA. The kill rate was determined by enumerating the viable cell counts at specific intervals over 24 ± 2 h. The MICs of each antibiotic alone and the combined antibiotics at 20 mL each were investigated in a 100 mL conical flask. Then, 200 µL of the adjusted 0.5 McFarland inoculum was added to 20 mL of 2× MHB to produce a final concentration of 5 × 10⁵ CFU/mL when added to the antibiotics to be assayed. The cultures were incubated at 35 ± 1 °C with shaking at 120 rpm. Aliquots were removed from the cultures at 0, 1, 2, 4, 6, 8, 12, and 24 h and a 10-fold dilution series was performed in sterile 2× MHB. A 100 µL of each appropriate dilution was spread on MHA plates in triplicates. The plates were incubated at 35 ± 1 °C, and colony counts were recorded after 24 ± 2 h. A growth control was run in parallel with each experiment. The time–kill curves were determined by plotting the mean colony counts (log CFU/mL) against the incubation time (hours). The combination’s efficacy was synergistic when viable bacteria were reduced by ≥2 log10 CFU/mL compared to the most active single antibiotic. The combination therapy’s efficiency was also evaluated as bactericidal when there was a ≥3 log10 CFU/mL reduction compared to the initial inoculum at 24 ± 2 h.