Whole Genome Sequencing Identifies Novel Mutations Associated With Bedaquiline Resistance in <i>Mycobacterium tuberculosis</i>

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.