MIC Determination by the Broth Microdilution Assay

Antibiotic solutions (Sigma-Aldrich, St. Louis, MO, United States) were freshly prepared before each experiment. The minimum inhibitory concentration (MIC) values for E. coli O157:H7 cells cultured under LSMMG and NG were determined by the method currently recommended by CLSI (Watts et al., 2008) in sterile, 96-well, U-bottomed plates. Briefly, each microdilution well-containing 100 μl of the corresponding twofold antibiotic dilution was inoculated with 100 μl of a cell suspension (final concentrations of ca. 5.0 × 10⁵ CFU/ml). The microdilution trays were incubated at 37°C for 18 h, and the MIC was defined as the lowest concentration of antibiotic for which no visible cell growth was observed. In each case, cell suspensions inoculated in the absence of antibiotics served as the positive control. The MIC values determined (Table ​(Table1)1) were used to calculate antibiotic concentrations corresponding to 1/2 × MIC and 4 × MIC. All experiments were repeated in triplicate.

The susceptibility and Minimum Inhibitory Concentrations (MIC) of the tested antibiotics against E. coli O157:H7 ATCC 43895 cultured under LSMMG and NG in MH broth for 24 h.