Kill-curve studies

For the 10⁸ CFU/mL kill-curve assay, 51, 10, and 0.2 μL of 2.5 mg/mL cecropin B (final concentrations, 512, 100, and 2 μg/mL, respectively) were mixed with 100 μL of 10⁹ CFU/mL H. parasuis SH0165 at the mid-logarithmic phase, and fresh TSB medium was added to obtain 250 μL. The mixture was then incubated anaerobically at 37°C. For the 10⁵ CFU/mL kill-curve assay, 0.8, 0.4,and 0.2 of 2.5 mg/mL cecropin B (final concentrations, 8, 4, and 2 μg/mL, respectively) were mixed with 100 μL of 10⁶ CFU/mL H. parasuis SH0165 at the mid-logarithmic phase and fresh TSB medium added to obtain 250 μL. The mixture was then incubated anaerobically at 37°C. Untreated bacteria were used as a negative control. At different indicated times (5, 10, 20, 30, 60, 90, and 120 min), 10 μL bacterial suspensions were collected and diluted in 490 μL TSB medium. The samples were spread onto TSA plates with appropriate dilution, incubated at 37°C for 24 h, and the bacteria colonies counted. The experiments were repeated three times independently.