S85 Bedaquiline resistance in Mycobacterium tuberculosis predates its clinical use

<h3>Background</h3> Bedaquiline has become a key drug for treatment of drug-resistant tuberculosis. Most clinical resistance is conferred by mutations in <i>Rv0678</i>, a negative repressor of the MmpL5 efflux pump, and confer clofazimine cross-resistance. Here, we estimate the date of emergence of several <i>Rv0678</i> variants in global <i>Mycobacterium tuberculosis</i> lineages. <h3>Methods</h3> We constructed global whole genome sequence datasets of thousands of lineage 2 and 4 isolates using newly generated and publicly available data. We enriched these by screening public repositories for sequences containing all previously reported <i>Rv0678</i> variants. We built a whole genome maximum likelihood phylogenetic tree using RAxML-Ng and dated the nodes of this phylogeny using BEAST. The lineage 2 dataset contained 1514 sequences from isolates collected between 1994–2019. The lineage 4 dataset contained 2168 sequences including three from 18th century mummies. <h3>Results</h3> We identified 483 non-synonymous and promoter variants in 439 sequences. 25 sequences were from isolates collected prior to bedaquiline clinical trials in 2007 and 21 of these contained bedaquiline resistance-associated variants. Most <i>Rv0678</i> mutations occurred in sequences carrying other resistance variants. In lineage 2 we identify 58 unique emergences of resistance estimated to have occurred between 1988–2018 (figure 1), of which 40 were represented by a single genome. In lineage 4 we identify 85 unique emergences estimated to have occurred between 1701–2019 (figure 1), of which 59 were represented by a single genome. We also identified a clade of 65 samples carrying the Ile67fs variant that we estimated to have arisen in 1701 (1657–1732). This predates the first use of clofazimine or bedaquiline. <h3>Conclusions</h3> <i>Rv0678</i> mutations conferring bedaquiline/clofazimine resistance have been in circulation since before the antibiotic era, implying non-synonymous mutations in <i>Rv0678</i> have little fitness cost. This pre-existing reservoir of resistant strains is likely to expand with increasing bedaquiline and clofazimine use.