P64

Drug-resistant tuberculosis is a serious global health threat. Bedaquiline (BDQ) is a new promising core drug, targeting the respiratory chain in Mycobacterium tuberculosis (Mtb). While mutations in the BDQ target gene, atpE, are still rare in clinical isolates, mutations in the Rv0678 gene, a transcriptional repressor responsible for the down regulation of efflux pump MmpS5-MmpL5, are increasingly observed, and treatment failure has already been reported. Nevertheless, underlying mechanisms of (cross)-resistance remain unresolved, hampering the development of molecular based diagnostic tests.

To distinguish resistance associated variants from other polymorphisms, by assessing the in vitro onset of mutations under drug pressure, combined with their impact on minimum inhibitory concentrations (MIC) and on protein stability. Isolates were exposed in vitro to sub-lethal concentration of BDQ or clofazimine (CFZ). Selected colonies had BDQ- and CFZ-MICs determined on 7H10 agar. Sanger sequencing for all isolates and additional Whole Genome Sequencing for a subset of isolates were used to search for mutations in Rv0678, atpE and pepQ. In silico characterization of relevant mutations was performed using FoldX and Alpha Fold computational tools.

Colonies that grew on BDQ medium had mutations in Rv0678, atpE or pepQ, while CFZ-exposed isolates presented mutations in Rv0678 and pepQ mutation, but none in atpE. Mutations were scattered across the whole Rv0678 gene and no single hotspot could be described. Most Rv0678 mutations led to increased BDQ- and/or CFZ-MIC. Finally, in silico characterization of Rv0678 mutations suggests that the C46R mutant may have an important impact on Rv0678 protein stability.