Rifampicin reduces susceptibility to ofloxacin in rifampicin-resistant Mycobacterium tuberculosis through efflux

Abstract

Rationale: Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.

Objective: To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility.

Methods: Rifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RT-qPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil.

Measurements and main findings: Rifampicin MICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC > 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05).

Conclusion: Exposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment.

Figure 1

Normalized growth curves of multidrug-resistant tuberculosis (MDR-TB) isolates cultured in the presence or absence of rifampicin and/or reserpine or verapamil. MDR-TB isolates (n = 15) were cultured in enriched BACTEC 12B medium in the absence rifampicin (open circles), in the presence of rifampicin (2 μg/ml) (open squares), in the presence of verapamil (50 μg/ml) (open diamonds), in the presence of reserpine (80 μg/ml) (solid circles), in the presence of rifampicin (2 μg/ml) and verapamil (50 μg/ml) (solid squares), and in the presence of rifampicin (2 μg/ml) and reserpine (80 μg/ml) (open triangles). Growth curves were normalized using STATISTICA software and the percent susceptibility restored was calculated using the formula: $$\frac{\left(\right({\mathrm{GI}}_{\mathrm{RIF}}-{\mathrm{GI}}_{\mathrm{EPI}+\mathrm{RIF}})-({\mathrm{GI}}_{\text{CONTROL}}-{\mathrm{GI}}_{\mathrm{EPI}}\left)\right)}{{\mathrm{GI}}_{\mathrm{RIF}}},$$ where GI_RIF = growth index in the presence of 2 μg/ml rifampicin, GI_EPI = growth index in the presence of either 50 μg/ml verapamil or 80 μg/ml reserpine, GI_EPI+1RIF = growth index in the presence of 2 μg/ml rifampicin and either 50 μg/ml verapamil or 80 μg/ml reserpine, and GI_CONTROL = growth index in the absence of any drug and diluted 1:100. The percent susceptibility restored ranged from 55 to 81% for reserpine and from 61 to 86% for verapamil (Table 1) irrespective of the genetic background. Restoration of rifampicin susceptibility was statistically significantly (P = 0.0000; 95% CI) when compared with treatment in the absence of the efflux pump inhibitor.

Figure 2

Induction of ofloxacin (OF) resistance (increased minimum inhibitory concentration [MIC]) by exposure of rifampicin mono-resistant isolates with different genetic backgrounds and rpoB mutations to rifampicin (2 μg/ml) for varying time intervals. Medium shaded bars = OFL MIC without exposure to rifampicin; darkly shaded bars = OFL MIC after exposure to rifampicin for 24 hours; and lightly shaded bars = OFL MIC after exposure to rifampicin for 7 days.

Figure 3

Pulmonary bacillary loads (cfu) in mice infected with multidrug-resistant tuberculosis (MDR-TB) strain. Mice infected with the MDR-TB strain were treated with verapamil alone (bars 3 and 4), first-line anti-TB drugs alone (bars 5 and 6), a combination of verapamil and first-line anti-TB drugs (bars 1 and 2), or a saline solution (bars 7 and 8). Verapamil in combination with first-line anti-TB drugs (isoniazid, rifampicin, pyrazinamide, and verapamil) significantly reduced the cfu at 30 (open bars) and 60 (solid bars) days after initiation of treatment. Data are expressed as means ± SD, five mice per time point. * Represents statistical significance (P < 0.05).