Validation of Novel Mycobacterium tuberculosis Isoniazid Resistance Mutations Not Detectable by Common Molecular Tests

Abstract

Resistance to the first-line antituberculosis (TB) drug isoniazid (INH) is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole-genome sequencing (WGS) to analyze 52 phenotypically INH-R complex (MTBC) clinical isolates that lacked the common S315T or promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered mutations conferred resistance to INH at a MIC of ≥0.25 μg/ml and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods and suggest that its use (or use of expanded conventional or next-generation-based targeted sequencing) may provide earlier diagnosis of INH-R TB.

<p>Association Between the Phenotype and Genotype of Isoniazid Resistance Among <em>Mycobacterium tuberculosis</em> Isolates in Thailand</p>

“…Previous studies had more than one gene mutations exhibiting a higher MIC range. 5,18,25,26 However, in our study, we detected only a single gene mutation.…”

“…Isoniazid gene mutations should be confirmed by whole-genome sequencing, which can detect uncommon isoniazid-resistance mutations such as katG S315N/G, ahpC promoter, or fabG1. 26 Recent studies have shown that these uncommon gene mutations may be susceptible or have low-/high-level resistance to isoniazid. 5,26 WHO recommends that DSTs of both phenotypes and genotypes should be used for all isolates.…”

Molecular Detection of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Isolates from Lorestan Province, Iran from 2014 to 2017

“…In this study, the frequency of MTB was similar to other Iranian studies with 3.8% frequency. However, our data are contrary to previous studies, which were reported mutations of the codon315 of the katG gene are associated with INH-resistant MT(31,32).Sadrnia et al, have shown that among of 87 isolates, 33 INH-resistant isolates had a mutation in Ser315Thr of KatG gene, while 21 susceptible isolates had no mutation in codon315 of katG gene (33). Bostanabad et al, with a study on the frequency, location and type of mutations in katG gene of MTB isolate from Belarus that the most mutations were formed in codons315, 309 and 316 of katG gene (34).…”

Global Distribution and Evolution of Mycobacterium bovis Lineages

“…Thirteen out of the 44 M. bovis genomes (29.54%) described in the paragraph above originated from isolates obtained from humans, being seven from Africa (Ghana = 3; Tunisia = 1; Malawi = 3), four from Europe (Germany = 2; Italy = 2), one from Russia, and one from the United States. Unfortunately, demographic characteristics of these patients are not described in the related literature (Casali et al, 2014;Walker et al, 2015;Brites et al, 2018;Kandler et al, 2018). With current bovine TB control, the zoonotic transmission of M. bovis in Europe and the USA is considered rare.…”