OR10

Accurate molecular detection of bedaquiline resistant tuberculosis is challenging as only few bedaquiline resistance markers have been identified. We performed site-directed mutagenesis to investigate the phenotypic effect of the presence of two variants: atpE Ile66Vale and Rv0678 Thr33Ala. We introduced these variants in the Mycobacterium tuberculosis reference strain using homologous recombineering or recombination. The genotype of the resulting strains was confirmed by Sanger sequencing and whole genome sequencing, and minimal inhibitory concentration (MIC) assays were performed to assess bedaquiline susceptibility. While secondary unwanted variants were introduced, we successfully generated the mutants of interest in Mycobacterium tuberculosis H37Rv. Phenotypic analysis showed that the atpE Ile66Val variant did not elevate the MIC above the critical concentration (MIC 0.25 – 0.5 µg/ml), confirming the observation in a clinical strain and contradicting in silico predictions made in a prior study. The MIC of the Rv0678 Thr33Ala mutant strain (>1.0 µg/ml) classifies the strain as resistant, confirming clinical findings and the predicted disruption of the DNA binding affinity of the MmpR protein. We also performed in silico analyses to predict the impact of the atpE Ile66Vale and Rv0678 Thr33Ala on protein stability and interactions. Our analyses confirmed that the atpE Ile66Val variant minimally disrupts the bedaquiline – ATP synthase interaction and predicted that the Rv0678 Thr33Ala variant substantially affects the DNA binding affinity of the MmpR protein. This study demonstrates that both in vitro site-directed mutagenesis of Mycobacterium tuberculosis and structural modelling are important and complementary methods that help improve our understanding of bedaquiline resistance.