Genome-wide association studies of global Mycobacterium tuberculosis resistance to 13 antimicrobials in 10,228 genomes identify new resistance mechanisms

Abstract

The emergence of drug-resistant tuberculosis is a major global public health concern that threatens the ability to control the disease. Whole-genome sequencing as a tool to rapidly diagnose resistant infections can transform patient treatment and clinical practice. While resistance mechanisms are well understood for some drugs, there are likely many mechanisms yet to be uncovered, particularly for new and repurposed drugs. We sequenced 10,228 Mycobacterium tuberculosis (MTB) isolates worldwide and determined the minimum inhibitory concentration (MIC) on a grid of 2-fold concentration dilutions for 13 antimicrobials using quantitative microtiter plate assays. We performed oligopeptide- and oligonucleotide-based genome-wide association studies using linear mixed models to discover resistance-conferring mechanisms not currently catalogued. Use of MIC over binary resistance phenotypes increased sample heritability for the new and repurposed drugs by 26% to 37%, increasing our ability to detect novel associations. For all drugs, we discovered uncatalogued variants associated with MIC, including in the Rv1218c promoter binding site of the transcriptional repressor Rv1219c (isoniazid), upstream of the vapBC20 operon that cleaves 23S rRNA (linezolid) and in the region encoding an α-helix lining the active site of Cyp142 (clofazimine, all p < 10-7.7). We observed that artefactual signals of cross-resistance could be unravelled based on the relative effect size on MIC. Our study demonstrates the ability of very large-scale studies to substantially improve our knowledge of genetic variants associated with antimicrobial resistance in M. tuberculosis.

Fig 1

(A) Phylogeny of 10,228 isolates sampled globally by CRyPTIC used in the GWAS analyses. Lineages are coloured blue (lineage 1), green (2), orange (3), and yellow (4). Branch lengths have been square root transformed to visualise the detail at the tips. (B) Distributions of the log₂ MIC measurements for all 13 drugs in the GWAS analyses, AMI, BDQ, CFZ, DLM, EMB, ETH, INH, KAN, LEV, LZD, MXF, RFB, and RIF. The red line indicates the ECOFF breakpoint for binary resistance versus sensitivity calls [31]. AMI, amikacin; BDQ, bedaquiline; CFZ, clofazimine; DLM, delamanid; ECOFF, epidemiological cutoff; EMB, ethambutol; ETH, ethionamide; INH, isoniazid; KAN, kanamycin; LEV, levofloxacin; LZD, linezolid; MIC, minimum inhibitory concentration; MXF, moxifloxacin; RFB, rifabutin; RIF, rifampicin.

Fig 2. Sample heritability for MIC (orange) versus binary resistance/sensitivity (blue) assuming additive genetic variation in oligopeptide presence/absence across 13 drugs, DLM, CFZ, LZD, BDQ, MXF, LEV, KAN, EMB, ETH, AMI, RIF, INH, and RFB.

Lines depict 95% CIs. MIC heritability was at least 26% higher than binary heritability for the new and repurposed drugs BDQ, CFZ, DLM, and LZD. AMI, amikacin; BDQ, bedaquiline; CFZ, clofazimine; CI, confidence interval; DLM, delamanid; EMB, ethambutol; ETH, ethionamide; INH, isoniazid; KAN, kanamycin; LEV, levofloxacin; LZD, linezolid; MIC, minimum inhibitory concentration; MXF, moxifloxacin; RFB, rifabutin; RIF, rifampicin.

Fig 3. Manhattan plots of regions containing oligopeptide variants associated with MIC across 13 drugs.

Significant oligopeptides are coloured by the direction (orange = increase, blue = decrease) and magnitude of their effect size on MIC, estimated by LMM [32]. Bonferroni-corrected significance thresholds are shown by the black dashed lines. The top 20 genes ranked by their most significant oligopeptides are annotated alphabetically. Gene names separated by colons indicate intergenic regions. Gene names for those annotated with letters can be found in Table 1. Oligopeptides were aligned to the H37Rv reference; unaligned oligopeptides are plotted to the right in light grey. LMM, linear mixed model; MIC, minimum inhibitory concentration.

Fig 4. Interpreting significant oligopeptide variants for levofloxacin and moxifloxacin MIC in gyrB.

Oligopeptide Manhattan plots are shown for (A) levofloxacin and (B) moxifloxacin. Oligopeptides are coloured by the reading frame that they align to, black for in frame and grey for out of frame in gyrB. Oligopeptides aligned to the region by nucmer but not realigned by BLAST are shown in grey on the right-hand side of the plots. The black dashed lines indicate the Bonferroni-corrected significance thresholds—all oligopeptides above the line are genome-wide significant. Alignment is shown of oligopeptides significantly associated with (C) levofloxacin and (D) moxifloxacin. The H37Rv reference codons are shown at the bottom of the figure, grey for an invariant site, coloured at variant site positions. The background colour of the oligopeptides represents the direction of the β estimate, light grey when β < 0 (associated with lower MIC), dark grey when β> 0 (associated with higher MIC). Oligopeptides are coloured by their amino acid residue at variant positions only. MIC, minimum inhibitory concentration.