MIC and minimum bactericidal concentration (MBC) measurement.

The MICs and MBCs were determined as previously described (34). Overnight bacteria cultures were diluted 1:100 into fresh LB or cation-adjusted Mueller-Hinton broth (CA-MHB) and cultured to an OD₆₀₀ of 1.0. Then the bacteria were diluted with LB or CA-MHB and added to each well of a 96-well plate, resulting in 1 × 10⁵ CFU per well. Antibiotic with appropriate concentration was added into the first row, and a serial 2-fold dilution was performed from top to bottom. The plates were incubated at 37°C for 24 h. The lowest antibiotic concentrations in wells with no visible bacterial growth were recorded as MICs. To determine the MBC, the dilution at the MIC and at least two of the higher concentrated dilutions in the MIC test were plated to determine viable bacterial concentrations. The MBC is the lowest concentration that results in 99.9% killing compared to the MIC dilution.