Genetic diversity of candidate loci linked to Mycobacterium tuberculosis resistance to bedaquiline, delamanid and pretomanid

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the deadliest infectious diseases worldwide. Multidrug and extensively drug-resistant strains are making disease control difficult, and exhausting treatment options. New anti-TB drugs bedaquiline (BDQ), delamanid (DLM) and pretomanid (PTM) have been approved for the treatment of multi-drug resistant TB, but there is increasing resistance to them. Nine genetic loci strongly linked to resistance have been identified (mmpR5, atpE, and pepQ for BDQ; ddn, fgd1, fbiA, fbiB, fbiC, and fbiD for DLM/PTM). Here we investigated the genetic diversity of these loci across >33,000 M. tuberculosis isolates. In addition, epistatic mutations in mmpL5-mmpS5 as well as variants in ndh, implicated for DLM/PTM resistance in M. smegmatis, were explored. Our analysis revealed 1,227 variants across the nine genes, with the majority (78%) present in isolates collected prior to the roll-out of BDQ and DLM/PTM. We identified phylogenetically-related mutations, which are unlikely to be resistance associated, but also high-impact variants such as frameshifts (e.g. in mmpR5, ddn) with likely functional effects, as well as non-synonymous mutations predominantly in MDR-/XDR-TB strains with predicted protein destabilising effects. Overall, our work provides a comprehensive mutational catalogue for BDQ and DLM/PTM associated genes, which will assist with establishing associations with phenotypic resistance; thereby, improving the understanding of the causative mechanisms of resistance for these drugs, leading to better treatment outcomes.

Figure 1

(A), (B) Frequency of mutations identified across data set. The vertical axis is the number of mutations that are found in 1 to 10 or more isolates (horizontal axis). Colours represent the different genes, each bar showing the distribution of those mutations in the candidate genes for each drug (A = Bedaquiline (BDQ), B = Delamanid (DLM)/Pretomanid (PTM)). (C), (D) Intersection of mutations in the different genes by sample. Bars represent the number of samples that hold mutations in each gene, or combination of them (horizontal bars show total samples with mutations in each gene); C = BDQ, D = DLM/PTM.

Figure 2

Phylogenetic tree of high frequency (≥ 10 isolates) mutations in bedaquiline candidate genes. The outer track (c) shows the resistance phenotype; the second track (b) shows the convergent mutations that have arisen in more than one clade; the third track (a) shows the clades formed by isolates harbouring the same phylogenetic-related mutations. Branches are coloured by lineage as per legend.

Figure 3

Phylogenetic tree of high frequency mutations (≥ 10 isolates) in delamanid and pretomanid candidate genes (fgd1 K270M and R64S, fbiC −32A > G and T273A, fbiA T302M and fbiB K448R found in > 290 isolates not represented). Clades formed by isolates harbouring the same mutations are differentiated by colour. The outer (c) track shows the resistance phenotype; the second track (b) shows the convergent mutations that have arisen in more than one clade; the third track (a) shows the clades formed by isolates harbouring the same phylogenetic-related mutations. Branches are coloured by lineage as per legend.