Site-directed mutagenesis of Mycobacterium tuberculosis and functional validation to investigate potential bedaquiline resistance-causing mutations.

Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays.