Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S ribosomal RNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant.These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

FIG 1

Inhibition of Rv1258c-mediated efflux of SPT by addition of CPZ. Combinations of CPZ and SPT were applied in checkerboard assays against wild-type M. tuberculosis H37Rv (a), the ΔRv1258c mutant (b), the ΔRv1258c pCRS4 complemented mutant (c), and the SPT^r strain (d). Bacterial growth inhibition was assessed in two independent experiments by fluorescence-based resazurin assay. The dashed horizontal line indicates 90% inhibition, and data are the means and standard deviations of two independent biological replicates.

FIG 2

In vitro interaction between SPT and FA. Combinations of SPT and FA were applied in checkerboard assays against wild-type M. tuberculosis H37Rv (a), the SPT^r mutant (b), and the FA^r mutant (c). Bacterial viability was assessed by fluorescence-based resazurin assay. Dashed horizontal lines indicate 90% inhibition, and data are the means and standard deviations of two independent biological replicates.

FIG 3

Activity against pre-MDR M. tuberculosis strains. In vitro activity of RIF-INH against RIF^r M. tuberculosis rpoB^S531L mutant in the absence (a) or presence (b) of 1/2× MIC SPT and against the INH^r M. tuberculosis inhA mutant in the absence (c) or presence (d) of 1/2× MIC SPT. The dashed horizontal line indicates 90% inhibition, and data are the means and standard deviations of two independent biological replicates.