In vitro monitoring of drug resistance emergence during stepwise induction of bedaquiline and clofazimine, alone and in combination: a phenotypic and genotypic analysis

The co-resistance between bedaquiline and clofazimine raises significant concerns, as they are commonly co-administered as core drugs in drug-resistant TB regimens. The present study aimed to monitor drug resistance-associated gene mutations and the phenotypic change in Mycobacterium tuberculosis (Mtb) under a stepwise drug resistance induction in vitro using bedaquiline, clofazimine or combined drugs. Drug-resistant Mtb strains were gradually induced in vitro on a drug-containing solid medium with a 2-fold increasing concentration of bedaquiline, clofazimine and their combination. The MIC of the induced drug-resistant Mtb strains was determined. The drug resistance-associated genes, including Rv0678, Rv1979c, atpE and pepQ, were sequenced and analysed. Unlike exposure to bedaquiline alone or the combination of these two drugs, clofazimine alone resulted in drug resistance gene mutations occurring later, specifically in the fourth round of induction as opposed to the second round of induction. Besides, nucleotide deletion or insertion in Rv0678 was the main mutation type for induction under the two-drug combination, while single-nucleotide polymorphisms (SNPs) in Rv0678 were the major mutation types when induced by bedaquiline or clofazimine alone. Rv0678 mutation happened at a relatively lower bedaquiline concentration exposure alone, while atpE mutation occurred at a higher bedaquiline concentration. Regardless of the drug exposure manner, a strong correlation between bedaquiline MICs and clofazimine MICs was observed in all drug resistance strains. Combined exposure to bedaquiline and clofazimine developed Rv0678 mutation as early as exposure to bedaquiline alone. However, rather than SNPs, deletion and insertion were the dominant mutation types in dual-drug exposure strain.