Drug Susceptibility, MIC, and Efflux Pump Inhibition Testing

We used the agar proportion method for drug susceptibility testing. A suspension with turbidity equivalent to a 1.0 McFarland standard was prepared from Mtb colonies grown on Lowenstein–Jensen media for <14 days. The suspension was the standard inoculum for all dilutions after confirmation of turbidity by nephelometer. One hundred microliters each of 10⁻² and 10⁻⁴ dilutions of the standard inoculum was plated on 7H10 agar with and without ofloxacin. Phenotypic resistance was defined as >1% colony growth in the presence of 2 mg/L ofloxacin compared with colony growth in the absence of drug [11]. MIC testing was performed using liquid media by the resazurin microtiter assay using an ofloxacin critical concentration of 2 mg/L [11]. The ofloxacin MIC was assessed at 2-fold dilutions between 0.5 and 64 mg/L. If growth was observed at day 7 in the control well, 30 μL of 0.01% resazurin solution was added to each well, followed by 24 hours of incubation. MIC was determined as the lowest ofloxacin concentration preventing color change. The MIC was done in triplicate; the median result was used for analyses. Tests were done in the absence of ofloxacin and separately in the presence of 2 μg/mL ofloxacin.

We performed efflux pump inhibition assays on 41 of 42 ofloxacin-resistant isolates (testing of 1 isolate was unsuccessful) in the presence and absence of the efflux pump inhibitor verapamil as reported previously [11, 13]. We tested each isolate with and without verapamil at all drug concentrations in triplicate; we used the median MIC of triplicate testing results.