Dataset Information

Detecting novel genetic variants associated with isoniazid-resistant Mycobacterium tuberculosis.

ABSTRACT:

Background

Isoniazid (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 findings

INH-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.

Conclusion

The discovery of novel targets associated with INH-resistance described in this study may potentially be important for the development of improved molecular detection strategies.

SUBMITTER: Shekar S

PROVIDER: S-EPMC4099304 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Detecting novel genetic variants associated with isoniazid-resistant Mycobacterium tuberculosis.

Shekar Sandhya S Yeo Zhen Xuan ZX Wong Joshua C L JC Chan Maurice K L MK Ong Danny C T DC Tongyoo Pumipat P Wong Sin-Yew SY Lee Ann S G AS

PloS one 20140715 7

<h4>Background</h4>Isoniazid (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.<h4>Methodology/principal find ...[more]

PMID: 25025225

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Project description:BackgroundTuberculosis (TB) incidence and mortality are declining worldwide; however, poor detection of drug-resistant disease threatens to reverse current progress toward global TB control. Multiple, rapid molecular diagnostic tests have recently been developed to detect genetic mutations in Mycobacterium tuberculosis (Mtb) genes known to confer first-line drug resistance. Their utility, though, depends on the frequency and distribution of the resistance associated mutations in the pathogen population. Mutations associated with rifampicin resistance, one of the two first-line drugs, are well understood and appear to occur in a single gene region in >95% of phenotypically resistant isolates. Mutations associated with isoniazid, the other first-line drug, are more complex and occur in multiple Mtb genes.Objectives/methodologyA systematic review of all published studies from January 2000 through August 2013 was conducted to quantify the frequency of the most common mutations associated with isoniazid resistance, to describe the frequency at which these mutations co-occur, and to identify the regional differences in the distribution of these mutations. Mutation data from 118 publications were extracted and analyzed for 11,411 Mtb isolates from 49 countries.Principal findings/conclusionsGlobally, 64% of all observed phenotypic isoniazid resistance was associated with the katG315 mutation. The second most frequently observed mutation, inhA-15, was reported among 19% of phenotypically resistant isolates. These two mutations, katG315 and inhA-15, combined with ten of the most commonly occurring mutations in the inhA promoter and the ahpC-oxyR intergenic region explain 84% of global phenotypic isoniazid resistance. Regional variation in the frequency of individual mutations may limit the sensitivity of molecular diagnostic tests. Well-designed systematic surveys and whole genome sequencing are needed to identify mutation frequencies in geographic regions where rapid molecular tests are currently being deployed, providing a context for interpretation of test results and the opportunity for improving the next generation of diagnostics.

Project description:Tuberculosis (TB) continues to be an important public health threat worldwide, due in part to drug resistant Mycobacterium tuberculosis (Mtb) strains. The United States recently reported a shortage of isoniazid (INH), which could drive higher INH resistance rates. Changes in the Mtb proteome before and after acquisition of INH resistance in a clean genetic background remain understudied and may elucidate alternate drug targets. Here, we focused on Mtb clonal strains to characterize the consequences of INH resistance on mycobacterial metabolism. Proteomic analysis was conducted by liquid-chromatography tandem mass spectrometry (LC-MS/MS) of cellular and secreted fractions, followed by a normalized spectral counting (NSAF) analysis (data are available via ProteomeXchange with identifier PXD009549). Two different Mtb clonal pairs representing a specific genetic lineage (one clinical and one generated in the laboratory) but sharing a katG mutation associated with INH resistance, were used in our analysis. Overall, we found 26 Mtb proteins with altered abundances after acquisition of INH resistance across both Mtb genetic lineages studied. These proteins were involved in ATP synthesis, lipid metabolism, regulatory events, and virulence, detoxification, and adaptation processes. Proteomic findings were validated by Western blotting analyses whenever possible. Mycolic acid (MA) analysis through LC/MS in the clonal Mtb pairs did not reveal a common trend in the alteration of these fatty acids across both INHr strains but revealed a significant reduction in levels of the two more abundant ?-MA features in the clinical INHr strain. Interestingly, the clinical clonal pair demonstrated more variation in the abundance of the proteins involved in the FAS II pathway. Together, the proteomic and lipidomic data highlight the identification of potential drug targets such as alternative lipid biosynthetic pathways that may be exploited to combat clinically relevant Mtb INHr strains.

Dataset Information

Detecting novel genetic variants associated with isoniazid-resistant Mycobacterium tuberculosis.

Background

Methodology/principal findings

Conclusion

Publications

Detecting novel genetic variants associated with isoniazid-resistant Mycobacterium tuberculosis.

Similar Datasets

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