Repeated evolution of bedaquiline resistance in Mycobacterium tuberculosis is driven by truncation of mmpR5

The antibiotic Bedaquiline (BDQ) is a key component of new WHO regimens for drug resistant tuberculosis (TB) but predicting BDQ resistance (BDQ-R) from genotypes remains challenging. We analysed a collection (n=505) of Mycobacterium tuberculosis from two high prevalence areas in South Africa (Cape Town and Johannesburg, 2019-2020), and found 53 independent acquisitions of 31 different mutations within the mmpR5 regulatory gene, with a particular enrichment of truncated MmpR5 in BDQ-R isolates by either frameshift or introduction of an insertion element. Truncations occurred across three M. tuberculosis lineages, impacting 66% of BDQ-R isolates. Extending our analysis to 1,961 isolates with minimum inhibitory concentrations (MICs) revealed that mmpR5-disrupted isolates had a median BDQ MIC of 0.25 mg/L, compared to the wild-type median of 0.06 mg/L. By matching mmpR5-disrupted isolates with phylogenetically close control isolates without the disruption, we were able to estimate the impact on MIC of individual mutations. In conclusion, as the MIC increase borders the ECOFF threshold for BDQ-R, we recommend the continued use of MICs and detection of MmpR5 truncations to identify modest shifts in BDQ-R.