Detecting Novel Genetic Variants Associated with Isoniazid-Resistant Mycobacterium tuberculosis

Abstract

BackgroundIsoniazid (INH) is a highly effective antibiotic central for the treatment of Mycobacterium tuberculosis (MTB). INH-resistant MTB clinical isolates are frequently mutated in the katG gene and the inhA promoter region, but 10 to 37% of INH-resistant clinical isolates have no detectable alterations in currently known gene targets associated with INH-resistance. We aimed to identify novel genes associated with INH-resistance in these latter isolates.Methodology/Principal FindingsINH-resistant clinical isolates of MTB were pre-screened for mutations in the katG, inhA, kasA and ndh genes and the regulatory regions of inhA and ahpC. Twelve INH-resistant isolates with no mutations, and 17 INH-susceptible MTB isolates were subjected to whole genome sequencing. Phylogenetically related variants and synonymous mutations were excluded and further analysis revealed mutations in 60 genes and 4 intergenic regions associated with INH-resistance. Sanger sequencing verification of 45 genes confirmed that mutations in 40 genes were observed only in INH-resistant isolates and not in INH-susceptible isolates. The ratios of non-synonymous to synonymous mutations (dN/dS ratio) for the INH-resistance associated mutations identified in this study were 1.234 for INH-resistant and 0.654 for INH-susceptible isolates, strongly suggesting that these mutations are indeed associated with INH-resistance.ConclusionThe discovery of novel targets associated with INH-resistance described in this study may potentially be important for the development of improved molecular detection strategies.

Redefining MTBDR<I>plus</I> test results: what do indeterminate results actually mean?

“…The slightly lower sensitivity of the MTBDR plus test for INH, as compared to conventional methods, is likely due to the presence of genetic mutations conferring INH resistance that are located outside the katG and inhA promoter gene regions included in the LPA [26, 27, 33, 35]. In the present study, we did not identify any ahpC promoter mutations via PSQ, but other gene regions have been implicated in conferring resistance to INH that were not included in the assay, such as kasA [41]. An alternative explanation for the persistence of discordant results may be the presence of mixed populations of bacteria, consisting of both susceptible and resistant strains, and hetero-resistance [29, 30, 34].…”

Deciphering Within-Host Microevolution of Mycobacterium tuberculosis through Whole-Genome Sequencing: the Phenotypic Impact and Way Forward

“…The genes pks4, pks5, and fadD15 are involved in the biosynthesis of fatty acids (pks4, pks5) and in their activation as acyl-coenzyme A (fadD15) (18). Mutations in genes of these gene families have been associated with compensatory mechanisms (Table 2) or INH resistance (pks5), based on the absence of such mutations in susceptible strains and association studies (70). mviN encodes a probable conserved transmembrane protein which has been found to be essential for in vitro growth (18).…”

Recent advances for identification of new scaffolds and drug targets for Mycobacterium tuberculosis

“…INH, a member of the current regimen is a pro‐drug, activated by KatG (catalase peroxidase) and inhibits InhA, an enzyme involved in mycolic acid biosynthesis . Mutations in several M. tuberculosis enzymes such as KatG, InhA, AhpC (alkyl hydroperoxide reductase C), KasA (β‐ketoacyl‐ACP synthase A), or Ndh (NADH dehydrogenase) have been associated with INH resistance . Recently, direct inhibitors of InhA have also been identified.…”