Dataset Information

Whole Genome Sequencing of Mycobacterium tuberculosis Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility.

ABSTRACT: Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been constructive in understanding its evolution, genetic diversity and the mechanisms involved in drug resistance. A large number of sequencing efforts from across the globe have revealed genetic diversity among clinical isolates and the genetic determinants for their resistance to anti-tubercular drugs. Considering the high TB burden in India, the availability of WGS studies is limited. Here we present, WGS results of 200 clinical isolates of M. tuberculosis from North India which are categorized as sensitive to first-line drugs, mono-resistant, multi-drug resistant and pre-extensively drug resistant isolates. WGS revealed that 20% of the isolates were co-infected with M. tuberculosis and non-tuberculous mycobacteria species. We identified 12,802 novel genetic variations in M. tuberculosis isolates including 343 novel SNVs in 38 genes which are known to be associated with drug resistance and are not currently used in the diagnostic kits for detection of drug resistant TB. We also identified M. tuberculosis lineage 3 to be predominant in the northern region of India. Additionally, several novel SNVs, which may potentially confer drug resistance were found to be enriched in the drug resistant isolates sampled. This study highlights the significance of employing WGS in diagnosis and for monitoring further development of MDR-TB strains.

SUBMITTER: Advani J

PROVIDER: S-EPMC6399466 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Whole Genome Sequencing of <i>Mycobacterium tuberculosis</i> Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility.

Advani Jayshree J Verma Renu R Chatterjee Oishi O Pachouri Praveen Kumar PK Upadhyay Prashant P Singh Rajesh R Yadav Jitendra J Naaz Farah F Ravikumar Raju R Buggi Shashidhar S Suar Mrutyunjay M Gupta Umesh D UD Pandey Akhilesh A Chauhan Devendra S DS Tripathy Srikanth Prasad SP Gowda Harsha H Prasad T S Keshava TSK

Frontiers in microbiology 20190226

Whole genome sequencing (WGS) of <i>Mycobacterium tuberculosis</i> has been constructive in understanding its evolution, genetic diversity and the mechanisms involved in drug resistance. A large number of sequencing efforts from across the globe have revealed genetic diversity among clinical isolates and the genetic determinants for their resistance to anti-tubercular drugs. Considering the high TB burden in India, the availability of WGS studies is limited. Here we present, WGS results of 200 c ...[more]

PMID: 30863380

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Project description:Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis.Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline.Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30% to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event.Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.

Project description:The increasing threat of drug resistance and a stagnated pipeline of novel therapeutics endanger the eradication of tuberculosis. Beta-lactams constitute promising additions to the current therapeutic arsenal and two carbapenems are included in group C of medicines recommended by the WHO for use in longer multidrug-resistant tuberculosis regimens. However, the determinants underlining diverse Mycobacterium tuberculosis phenotypes to beta-lactams remain largely undefined. To decipher these, we present a proof-of-concept study based on a large-scale beta-lactam susceptibility screening for 172 M. tuberculosis clinical isolates from Portugal, including 72 antimycobacterial drug-resistant strains. MICs were determined for multiple beta-lactams and strains were subjected to whole-genome sequencing to identify core-genome single-nucleotide variant-based profiles. Global and cell wall-targeted approaches were then followed to detect putative drivers of beta-lactam response. We found that drug-resistant strains were more susceptible to beta-lactams, but significant differences were not observed between distinct drug-resistance profiles. Sublineage 4.3.4.2 strains were significantly more susceptible to beta-lactams, while the contrary was observed for Beijing and 4.1.2.1 sublineages. While mutations in beta-lactamase or cell wall biosynthesis genes were uncommon, a rise in beta-lactam MICs was detected in parallel with the accumulation of mutations in peptidoglycan cross-linking or cell division genes. Finally, we exposed that putative beta-lactam resistance markers occurred in genes for which relevant roles in cell wall processes have been ascribed, such as rpfC or pknA. Genetic studies to validate the relevance of the identified mutations for beta-lactam susceptibility and further improvement of the phenotype-genotype associations are needed in the future. IMPORTANCE Associations between differential M. tuberculosis beta-lactam phenotypes and preexisting antimycobacterial drug resistance, strain sublineage, or specific mutational patterns were established. Importantly, we reveal that highly drug-resistant isolates of sublineage 4.3.4.2 have an increased susceptibility to beta-lactams compared with other strains. Thus, directing beta-lactams to treat infections by specific M. tuberculosis strains and refraining its use from others emerges as a potentially important strategy to avoid resistance development. Individual mutations in blaC or genes encoding canonical beta-lactam targets, such as peptidoglycan transpeptidases, are infrequent and do not greatly impact the MICs of potent carbapenem plus clavulanic acid combinations. An improved understanding of the global effect of cumulative mutations in relevant gene sets for peptidoglycan and cell division processes on beta-lactam susceptibility is also provided.

Project description:Tuberculosis (TB), caused by Mycobacterium tuberculosis bacteria, is a leading infectious cause of mortality worldwide, including in Pakistan. Drug resistant M. tuberculosis is an emerging threat for TB control, making it important to detect the underlying genetic mutations, and thereby inform treatment decision making and prevent transmission. Whole genome sequencing has emerged as the new diagnostic to reliably predict drug resistance within a clinically relevant time frame, and its deployment will have the greatest impact on TB control in highly endemic regions. To evaluate the mutations leading to drug resistance and to assess for evidence of the transmission of resistant strains, 81 M. tuberculosis samples from Khyber Pakhtunkhwa province (North West Pakistan) were subjected to whole genome sequencing and standard drug susceptibility testing for eleven anti-TB drugs. We found the majority of M. tuberculosis isolates were the CAS/Delhi strain-type (lineage 3; n = 57; 70.4%) and multi-drug resistant (MDR; n = 62; 76.5%). The most frequent resistance mutations were observed in the katG and rpoB genes, conferring resistance to isoniazid and rifampicin respectively. Mutations were also observed in genes conferring resistance to other first and second-line drugs, including in pncA (pyrazinamide), embB (ethambutol), gyrA (fluoroquinolones), rrs (aminoglycosides), rpsL, rrs and giB (streptomycin) loci. Whilst the majority of mutations have been reported in global datasets, we describe unreported putative resistance markers in katG, ethA (ethionamide), gyrA and gyrB (fluoroquinolones), and pncA. Analysis of the mutations revealed that acquisition of rifampicin resistance often preceded isoniazid in our isolates. We also observed a high proportion (17.6%) of pre-MDR isolates with fluoroquinolone resistance markers, potentially due to unregulated anti-TB drug use. Our isolates were compared to previously sequenced strains from Pakistan in a combined phylogenetic tree analysis. The presence of lineage 2 was only observed in our isolates. Using a cut-off of less than ten genome-wide mutation differences between isolates, a transmission analysis revealed 18 M. tuberculosis isolates clustering within eight networks, thereby providing evidence of drug-resistant TB transmission in the Khyber Pakhtunkhwa province. Overall, we have demonstrated that drug-resistant TB isolates are circulating and transmitted in North West Pakistan. Further, we have shown the usefulness of whole genome sequencing as a diagnostic tool for characterizing M. tuberculosis isolates, which will assist future epidemiological studies and disease control activities in Pakistan.

Project description:BackgroundPatients with multidrug-resistant (MDR) tuberculosis may have phenotypic heterogeneity in results of drug-susceptibility tests (DSTs). However, the impact of this on clinical outcomes among patients treated for MDR tuberculosis is unknown.MethodsPhenotypic DST heterogeneity was defined as presence of at least 1 Mycobacterium tuberculosis isolate susceptible to rifampicin and isoniazid recovered <3 months after MDR tuberculosis treatment initiation from a patient with previous documented tuberculosis due to M. tuberculosis resistant to at least rifampicin and isoniazid. The primary outcome was defined as good (ie, cure or treatment completion) or poor (ie, treatment failure, treatment default, or death). A secondary outcome was time to culture conversion. Cox proportional hazard models were used to determine the association between phenotypic DST heterogeneity and outcomes.ResultsPhenotypic DST heterogeneity was identified in 33 of 475 patients (7%) with MDR tuberculosis. Poor outcome occurred in 126 patients (28%). Overall, patients with MDR tuberculosis who had phenotypic DST heterogeneity were at greater risk of poor outcome than those with MDR tuberculosis but no phenotypic DST heterogeneity (adjusted hazard ratio [aHR], 2.1; 95% confidence interval [CI], 1.2-3.6). Among HIV-infected patients with MDR tuberculosis, the adjusted hazard for a poor outcome for those with phenotypic DST heterogeneity was 2.4 (95% CI, 1.3-4.2) times that for those without phenotypic DST heterogeneity, whereas among HIV-negative patients with MDR tuberculosis, the adjusted hazard for those with phenotypic DST heterogeneity was 1.5 (95% CI, .5-4.3) times that for those without phenotypic DST heterogeneity. HIV-infected patients with MDR tuberculosis with phenotypic DST heterogeneity also had a longer time to culture conversion than with HIV-infected patients with MDR tuberculosis without phenotypic DST heterogeneity (aHR, 2.9; 95% CI, 1.4-6.0).ConclusionsPhenotypic DST heterogeneity among persons with HIV infection who are being treated for MDR tuberculosis is associated with poor outcomes and longer times to culture conversion.

Dataset Information

Whole Genome Sequencing of Mycobacterium tuberculosis Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility.

Publications

Whole Genome Sequencing of <i>Mycobacterium tuberculosis</i> Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility.

Similar Datasets

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