Characterizing the novel mutations associated with bedaquiline resistance in Mycobacterium tuberculosis

Bedaquiline (BDQ), one of the new antitubercular agents, has been used to treat drug-resistant tuberculosis (TB). Although mutations in atpE, rv0678 and pepQ confer the major resistance to BDQ, the mechanism resistance to BDQ for a portion of isolates identified in vitro and in clinical remain unknown. Here, BDQ-resistant mutants were selected from 7H10 agar plates containing 0.5 mg/L BDQ (the critical concentration). Mutations associated with BDQ resistance were identified through whole genome sequence, PCR and Sanger sequencing analysis. A total of 1025 mutants were found to resistance to BDQ. We randomly picked 168 mutants for further analysis and discovered that 157/168 BDQ-resistant mutants harbored mutations in rv0678 encoding a transcriptional regulator repressing the expression of efflux pump MmpS5-MmpL5. Moreover, we found two mutations with high frequency in rv0678 gene, including nucleotide positions 286-287 (CG286-287 insertion; accounted for 26.8% (45/168)) and 198-199 (G198, G199 insertion, G198 deletion; accounted for 14.3 (24/168)). The remains were dispersed covering the entire rv0678 gene. Moreover, we found that one new gene, glpK harboring G572 insertion with high prevalence (85.7%, 144/168) in the isolated mutants and MIC assay demonstrated that G572 insertion mutation in glpK was closely associated with BDQ resistance. In summary, our study characterized 168/1025 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 the rapid identification of BDQ resistant isolates in the clinical setting.