MIC testing and efflux pump inhibition

MIC testing was performed using liquid medium by the resazurin microtitre assay (REMA). This method has been validated for ofloxacin resistance testing in M. tuberculosis, using a critical concentration of 2 mg/L.^18,19 The ofloxacin (Sigma-Aldrich, St Louis, MO, USA) 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 h of incubation. Colour change indicated bacterial growth and MIC was determined as the lowest ofloxacin concentration preventing colour change. The critical concentration of ofloxacin was 2 mg/L.

We performed efflux pump inhibition assays on 27 ofloxacin-resistant isolates and one susceptible isolate. Isolates were tested both in the presence and absence of three different efflux inhibitors: CCCP, reserpine and verapamil. Final concentrations used in the assays were 0.25 mg/L CCCP, 10 mg/L reserpine and 50 mg/L verapamil.^20–22 Each isolate was tested without efflux inhibitor and separately with each efflux pump inhibitor at all drug concentrations; testing was performed in triplicate under all conditions.