Time-kill Assay of Bacterial Pathogens

Time–kill curves were determined as described in [52 (link)] with some modifications. One colony of each bacterium (Psg CFBP 2214 and Cff CFBP 3418) was pre-cultured in 4 mL of King’s B liquid medium for 12 h at 28 °C and incubated on a shaker ES 20 (Biosan, Riga, Latvia) at 200 rpm. The cells were precipitated via centrifugation and titrated to a concentration of 10⁴ CFU/mL with sterile water. Bacterial titer control was carried out spectrophotometrically according to the OD₆₀₀ index measured using a Nanodrop One (Thermo Fisher Scientific, Waltham, MA, USA). The cell suspension was then transferred to 1.5 mL sterile test tubes, and preparations were added to concentrations of 1xMBC. After that, the tubes were placed in a Thermomixer C (Eppendorf, Hamburg, Germany) and cultivated at 27 °C and 350 rpm. After 0, 2, 5 and 30 min and 1, 2 and 24 h, 10 µL of the mixture was taken, diluted in sterile SPS buffer, and dispersed on King’s B agarized medium. Colonies were counted after 48 h of cultivation at 28 °C. A suspension without antimicrobial agents was used as a negative control. The experiment was repeated three times.