Rifampicin reduces susceptibility to ofloxacin in rifampicin resistant Mycobacterium

Rationale: Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through mutations in the rpoB gene. Physiological changes induced by exposure of rifampicin resistant M. tuberculosis strains to rifampicin has not been studied.Objective: To determine whether rifampicin induces the activation of efflux pumps in rifampicin resistant M. tuberculosis strains thereby defining the level of rifampicin resistance and reducing susceptibility towards ofloxacin.Methods and Results: Rifampicin minimum inhibitory concentrations (MIC’s) varied independently of rpoB mutation and genetic background upon RIF MIC determination in resistant strains (n= 60). Addition of efflux pump inhibitors, reserpine and verapamil significantly restored rifampicin susceptibility in all isolates (p= 0.0000; 95% CI). Quantitative RT-PCR demonstrated that rifampicin (2 µg/ml) induced differential expression of putative efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in 2 µg/ml rifampicin for 7 days induced ofloxacin resistance (MIC> 2 µg/ml) in strains with an rpoB 531 mutation. Susceptibility to ofloxacin was restored by exposure to efflux pump inhibitors. In vivo studies in BALB/c mice showed that verapamil in combination with first-line anti-TB drugs significantly reduced pulmonary colony-forming-units after 1 and 2 months of treatment (p< 0.05)..Conclusion: Exposure of rifampicin resistant M. tuberculosis strains to rifampicin induces activation of efflux pumps and transporter genes with concomitant increase in the MIC’s for