Detection of a historic reservoir of bedaquiline/clofazimine resistance-associated variants in Mycobacterium tuberculosis

Abstract

Background: Drug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug.

Methods: We compiled a dataset of 3682 Mtb genomes, including 180 carrying variants in mmpR5, and its immediate background (i.e. mmpR5 promoter and adjacent mmpL5 gene), that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We characterised the occurrence of all nonsynonymous mutations in mmpR5 in this dataset and estimated, using time-resolved phylogenetic methods, the age of their emergence.

Results: We identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade, Ile67fs (a RAV when considered in isolation) was estimated to have emerged prior to the antibiotic era, together with a resistance reverting mmpL5 mutation.

Conclusions: The presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.

Keywords: AMR; Bedaquiline; Drug resistance; Phylogenetics; Tuberculosis.

Fig. 1

Compiled global Mtb genomic datasets. Panels a and b provide the geographic location of isolates included in the lineage 2 and lineage 4 datasets respectively. Pies are scaled by the number of samples per country (raw data available in Additional file 1: Table S1) with the colours providing the fraction of genomes with any nonsynonymous/frameshift variants detected in mmpR5 (coloured as per the legend). Countries comprising samples with known RAVs are highlighted with a red asterisk. Genomic data for which no associated metadata on the geographic location of sampling was available are shown in the Pacific Ocean. Panels c and d provide the collection dates associated to each genome in the lineage 2 and lineage 4 datasets respectively highlighting those with any variants in mmpR5 (colour, as per legend). Lineage 4 Mtb obtained from eighteenth century mummies are excluded from this plot but included in all analyses. The vertical dashed lines indicate the dates of the first clinical trials for clofazimine, bedaquiline and FDA approval of bedaquiline for clinical use

Fig. 2

Global time-calibrated Mtb phylogenies. Inferred dated phylogenies (x-axis) for the a lineage 2 and b lineage 4 datasets. Tips are coloured by the geographic region of sampling as given in the legend. The bar provides the assessed phenotype (colour) based on assignment of nonsynonymous/frameshift variants in mmpR5

Fig. 3

Estimated age of emergence of mmpR5 nonsynonymous/frameshift variants. Inferred point estimates for the age of emergence of clades with mmpR5 variants for the lineage 2 (a) and lineage 4 (b) datasets, including a zoomed-in reproduction of the period from 2007 to 2020. Y-axis provides the absolute number of sequences descending from the identified and dated nodes. The mmpR5 RAV status is given by the colour as defined in the legend at left. *indicates phenotypic data available for considered isolates that are supportive of MIC classification (see text). The full mutation timelines are provided in Additional file 2: Fig S13–S14 and Additional file 3: Table S7