Whole Genome Sequencing Identifies Novel Mutations Associated With Bedaquiline Resistance in Mycobacterium tuberculosis.

Bedaquiline (BDQ), a new antitubercular agent, has been used to treat drug-resistant tuberculosis (TB). Although mutations in atpE, rv0678, and pepQ confer major resistance to BDQ, the mechanisms of resistance to BDQ in vitro and in clinical settings have not been fully elucidated. We selected BDQ-resistant mutants from 7H10 agar plates containing 0.5 mg/L BDQ (the critical concentration) and identified mutations associated with BDQ resistance through whole genome sequencing and Sanger sequencing. A total of 1,025 mutants were resistant to BDQ. We randomly selected 168 mutants for further analysis and discovered that 157/168 BDQ-resistant mutants harbored mutations in rv0678, which encodes a transcriptional regulator that represses the expression of the efflux pump, MmpS5-MmpL5. Moreover, we found two mutations with high frequency in rv0678 at nucleotide positions 286-287 (CG286-287 insertion; accounting for 26.8% [45/168]) and 198-199 (G198, G199 insertion, and G198 deletion; accounting for 14.3% [24/168]). The other mutations were dispersed covering the entire rv0678 gene. Moreover, we found that one new gene, glpK, harbors a G572 insertion; this mutation has a high prevalence (85.7%; 144/168) in the isolated mutants, and the minimum inhibitory concentration (MIC) assay demonstrated that it is closely associated with BDQ resistance. In summary, we characterized 168/1,025 mutants resistant to BDQ and found that mutations in rv0678 confer the primary mechanism of BDQ resistance. Moreover, we identified a new gene (glpK) involved in BDQ resistance. Our study offers new insights and valuable information that will contribute to rapid identification of BDQ-resistant isolates in clinical settings.