Identification of novel mutations associated with clofazimine resistance in Mycobacterium tuberculosis

Objectives: Although clofazimine has been traditionally used to treat leprosy, there is recent interest in using clofazimine for the treatment of MDR-TB and drug-susceptible TB. However, the mechanisms of resistance to clofazimine are poorly understood. Here, we investigated the molecular basis of clofazimine resistance using resistant mutants isolated in vitro. Methods: We isolated 96 mutants of Mycobacterium tuberculosis resistant to clofazimine and performed WGS and Sanger sequencing to identify possible mutations associated with clofazimine resistance. Results: We found that 97% (93/96) of clofazimine-resistant mutants had a mutation in rv0678 encoding a transcription repressor for efflux pump MmpL5. Two mutational hot spots at nucleotide positions 193 and 466 in rv0678 accounted for 43.8% (42/96) and 11.5% (11/96) of the mutations, respectively. The previously reported A202G mutation (S68G) in rv0678 occurred less frequently, in 5 of 96 mutants. The remaining 34 mutations were scattered along the entire rv0678 gene. We discovered two new genes (rv1979c and rv2535c) associated with clofazimine resistance in mutants without rv0678 mutations. Conclusions: Mutations in rv0678 are a major mechanism of clofazimine resistance. Our findings provide useful information for the design of new molecular tests for rapid detection of clofazimine resistance. Further studies are needed to address the role of rv1979c and rv2535c in clofazimine resistance and mechanisms of action.