Determining MIC and MBC.

MIC and MBC (minimum bactericidal concentration) estimates were obtained according to standard protocols (64). MIC was determined by inoculating 5 × 10⁵ (± 2 × 10⁵) cells into 3 ml Mueller-Hinton broth containing a range of drug concentrations (1, 0.5, 0.25, 0.125, 0.062, 0.031, 0.015, 0.008, or 0.004 mg/liter) and checking turbidity the following day. After establishing the initial MIC range, the procedure was repeated until the MIC was determined within 0.001 mg/liter for the mutants in the MG1655 background and within 0.02 mg/liter for mutants in the MG1655 GyrB S464Y background. For MRSA WCUH29, ampicillin MIC was determined by inoculating 5 × 10⁵ (± 2 × 10⁵) cells into 3 ml of TSB overnight with the following ampicillin concentrations: 1.69, 3.38, 6.75, 12.5, 25, and 50 mg/liter. MIC measurement was then repeated to determine the exact MIC between 1.69 and 3.38 mg/liter. To measure MBC, the same cultures that were used for MIC measurements were washed and plated for survivors. Prior to the addition of the antibiotic, all cultures were plated by serial dilution to determine the starting CFU/ml count. The concentration corresponding to a 3-log drop in viability was recorded as the MBC for the corresponding strain. The MICs and MBCs for each strain were obtained from testing at least 3 independent biological samples. Both MIC and MBC measurements were also carried out in LB medium as in reference 65, resulting in the estimates being indistinguishable within the error from the ones obtained in Mueller-Hinton medium.