In vitro approaches for generation of Mycobacterium tuberculosis mutants resistant to bedaquiline, clofazimine or linezolid and identification of associated genetic variants.

Bedaquiline, clofazimine and linezolid are pertinent drugs for drug-resistant tuberculosis. Drug-resistant mutants provide insight into important resistance acquisition mechanisms. Methods for in vitro Mycobacterium tuberculosis mutant generation are poorly described. Induction (serial passaging) and spontaneous (adapted Luria-Delbrück assay) approaches using M. tuberculosis ATCC reference strains (one fully-susceptible, four unique mono-resistant) were performed. Mutant MIC values were confirmed (MGIT960) and resultant RAVs compared between approaches and to a catalog of previously published RAVs. Mutant MIC values showed a 3-4-fold (induced) and a 1-4-fold (spontaneous) increase compared to baseline. The pyrazinamide-resistant strain had higher baseline MIC values and acquired resistance (≥4-fold) in fewer passages than other strains (induction approach) for bedaquiline. Previously described and novel RAVs in atpE (8 vs. 1) and rv0678 (4 vs. 12) genes were identified in bedaquiline- and clofazimine-resistant mutants. No rv1979c and rv2535c RAVs were identified. Previously described RAVs were identified in rplC and rrl genes for linezolid-resistant mutants. Both approaches successfully led to in vitro mutants with novel RAVs being described in atpE and rv0678 genes. It was observed that pre-existing resistance may influence mutant phenotypic and genotypic characteristics and warrants further attention.