4.8. Measurement of MPC

MPC measurements were performed as previously described with some modifications [38,39]. Briefly, H. pylori was cultured in BHI broth containing 7% fetal bovine serum and incubated for 24 h. Cultures were centrifuged at 5000× g for 10 min and resuspended in BHI broth to obtain a concentration of 10¹¹ CFU/mL. A series of Columbia blood agar plates supplemented with 7% defibered sheep blood, various known MNZ concentrations and 0.3 mg/mL hLYS were plated with 100 μL of a suspension containing 10¹⁰ CFU of H. pylori and compared with MNZ alone. The inoculated plates were incubated for 72 h under microaerophilic conditions at 37 °C and screened visually to count the CFU. The lowest concentration of drug without visual colony growth was set as a provisional mutation prevention concentration (MPCpr) after culture for 72 h. After repeating the above operation, MPCpr was set as the baseline with a 20% linear decreasing concentration of antibiotic, and agar plates containing different concentrations of drug were prepared. The MPC was defined as the lowest concentration of drug that still did not support visual colony growth after 72 h of culture. The experiments were carried out three times.