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.

Clofazimine for the treatment of tuberculosis

“…Other proposed mechanisms that may contribute to clofazimine’s bactericidal action include i) direct, non-specific membrane disruption ( Oliva et al, 2004 ), ii) direct interference with bacterial potassium uptake ( De Bruyn et al, 1996 ; Steel et al, 1999 ), iii) selective binding to mycobacterial DNA with blocking of template function ( Morrison and Marley, 1976a ; Morrison and Marley, 1976b ) and iv) reversal of the inhibitory effects of certain mycobacterial proteins on phagocyte activity ( Wadee et al, 1988 ). In summary, clofazimine appears to have multiple mechanisms of antimicrobial activity, possibly with differential importance of specific mechanisms under distinct physiological conditions ( Lu et al, 2011 ; Cholo et al, 2017 ), which may explain the lack of a single dominant, target-specific genetic marker associated with clofazimine resistance ( CRyPTIC Consortium, 2022 ).…”

CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database

“…For the purpose of CARD’s curation efforts mutations found to be non-significant (p > 0.05) were excluded, synonymous mutations were excluded, and mutations graded as no association with resistance or indeterminate have been archived but excluded from being displayed on the CARD website, included in download files, or RGI reporting. Future curation efforts will include incorporation of the large volume of information available in the recent CRyPTIC project ( 23 , 24 ).…”

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of mostMycolicibacterium smegmatismRNAs

“…mRNA metabolism is a critical aspect of mycobacterial biology, as regulation of gene expression facilitates adaptation to stressors both during infection and in the environment, and regulation of mRNA degradation permits energy conservation during severe stress. The roles and regulation of mycobacterial mRNA degradation enzymes remain largely undefined; however, recent reports of interplay between RNA metabolism and drug resistance have highlighted the relevance of these pathways (2–6).…”