Emergence of Canonical and Noncanonical Genomic Variants following In Vitro Exposure of Clinical Mycobacterium tuberculosis Strains to Bedaquiline or Clofazimine

In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected. We hypothesized that in vitro generation of bedaquiline- or clofazimine-resistant mutants could provide insight into additional mechanisms of action. We performed whole-genome sequencing and determined phenotypic MICs for both drugs on progenitor and mutant progenies. Mutants were induced through serial passage on increasing concentrations of bedaquiline or clofazimine. Rv0678 variants were identified in both clofazimine- and bedaquiline-resistant mutants, with concurrent atpE SNPs occurring in the latter. Of concern was the acquisition of variants in the F420 biosynthesis pathway in clofazimine-resistant mutants obtained from either a fully susceptible (fbiD: del555GCT) or rifampicin mono-resistant (fbiA: 283delTG and T862C) progenitor. The acquisition of these variants possibly implicates a shared pathway between clofazimine and nitroimidazoles. Pathways associated with drug tolerance and persistence, F420 biosynthesis, glycerol uptake and metabolism, efflux, and NADH homeostasis appear to be affected following exposure to these drugs. Shared genes affected by both drugs include Rv0678, glpK, nuoG, and uvrD1. Genes with variants in the bedaquiline resistant mutants included atpE, fadE28, truA, mmpL5, glnH, and pks8, while clofazimine-resistant mutants displayed ppsD, fbiA, fbiD, mutT3, fadE18, Rv0988, and Rv2082 variants. These results show the importance of epistatic mechanisms as a means of responding to drug pressure and highlight the complexity of resistance acquisition in M. tuberculosis.In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected.