Anthelmintic avermectins kill Mycobacterium tuberculosis, including multidrug-resistant clinical strains

Abstract

Avermectins are a family of macrolides known for their anthelmintic activities and traditionally believed to be inactive against all bacteria. Here we report that members of the family, ivermectin, selamectin, and moxidectin, are bactericidal against mycobacterial species, including multidrug-resistant and extensively drug-resistant clinical strains of Mycobacterium tuberculosis. Avermectins are approved for clinical and veterinary uses and have documented pharmacokinetic and safety profiles. We suggest that avermectins could be repurposed for tuberculosis treatment.

Fig 1

Avermectins used in this study. Images were obtained from ChemSpider.

Fig 2

Time-kill kinetics of avermectins against M. tuberculosis. Two experiments were performed independently, at two different locations, under similar but not identical growth conditions. (A) Dose titration. Frozen stocks of M. tuberculosis H37Rv were cultured in 15 ml of 7H9 broth supplemented with 10% albumin-dextrose-catalase, 0.2% glycerol in standing 25-cm² tissue culture flasks at 37°C and 5% CO₂ for 3 days before the addition of avermectins. Cultures were agitated only during sampling. (B) Fixed dose strain comparison. Cultures (5 ml) of M. tuberculosis H37Rv and mc²5857 (an MDR isolate) were pregrown in shaking 35-ml bottles in 7H9 broth supplemented with 10% ADC, 0.2% glycerol, and 0.05% tyloxapol to an optical density at 600 nm (OD₆₀₀) of 0.8. To assess drug activity, cultures were washed once in phosphate-buffered saline (PBS), diluted (1/150) in the same medium (without tyloxapol), and grown at 37°C in shaking 15-ml conical tubes containing ivermectin, selamectin, or moxidectin at 20 μg/ml. Prior to plating, cell clumps were disrupted by sonication and viability was quantified by plating on 7H10 agar supplemented with 10% oleic acid-albumin-dextrose-catalase and 0.2% glycerol. Plates were incubated at 37°C, and colonies were counted after 2 weeks of incubation (microscopically) and reassessed after 4 weeks. Concentrations of avermectins are expressed in μg/ml. IVM, ivermectin; SEL, selamectin; and MXD, moxidectin.

Fig 3

Effect of concentration and exposure on the bactericidal activities of avermectins against M. tuberculosis. Comparison of concentration and area under the curve over MIC ratios (C/MIC and AUC/MIC, respectively) on the bactericidal activities of the avermectins. The bactericidal effect was calculated on the basis of the initial inoculum prior to the addition of avermectins. Analysis of antimycobacterial activity was performed by comparing the rates of bacterial killing determined by nonlinear regression analysis with 95% confidence limits. The r² values on days 7 (filled circles), 14 (filled squares), and 21 (filled triangles) were 0.9959, 0.9766, and 0.9989 for ivermectin, 0.9591, 0.9827, and not available for moxidectin, and 0.88, 0.9949, and 0.9992 for selamectin, respectively. Cells were processed as described in the Fig. 2 legend for panel B but grown at 37°C in shaking 96-well plates containing 2-fold serial drug dilutions. The MIC values used for calculations were 6, 3, and 3 μg/ml for ivermectin, moxidectin, and selamectin, respectively, as calculated by the MTT assay.