Tuberculosis

Projects

Whole genome sequencing of longitudinal drug susceptible and resistant clinical M.tb isolates from Mumbai

Principal Investigator Dr. Nerges Mistry

Project team Dr. Anirvan Chatterjee, Dr. Purva Bhatter

Funder Corpus grants to FMR from Jamsetji Tata Trust

Duration May 2013- June 2014

Background

In Project 1, comparative global transcriptional levels of genes in three genotypically identical pairs of longitudinal clinical M.tbisolates from drug compliant patients showing drug resistance was performed. The study employed spoligotyping and MIRU-VNTR to confirm genotypic identity of the longitudinal M.tb isolates. Although the two genotyping techniques together have been shown to provide highest strain discrimination, they do not confirm genomic identity of the strains. Recent advances in whole genome sequencing provide the opportunity to compare whole genomes and analyze genomic variations associated with rapid amplification of drug resistance.

Thus we performed whole genome sequencing of the longitudinal DS and DR clinical M.tb strains from Mumbai, India, to confirm their genomic identity and further explore genomic changes in longitudinal MDR isolates as compared to the DS isolate from the same patient.

Objective

Explore whole genome sequence similarity between genotypically identical M.tb showing amplification in drug resistance.

Key Findings

(Left: A DNA gel picture of clinical isolates of M. tuberculosis)

The current study compared whole genomes of drug susceptible (DS) and multidrug resistant (MDR) Mycobacterium tuberculosis (M.tb) clinical isolates which were earlier shown to be genotypically identical. The M.tb strains were isolated from patients residing in Mumbai, a region which has reported rapid increase in advanced forms of drug resistance in the last decade. The MANU1 and CAS1_Delhi isolates were collected longitudinally from same patients and exhibited DS phenotype at initial diagnosis but exhibited multidrug resistance after 5 months of compliant DOTS. Additionally we compared whole genomes of 2 independent DS and extremely drug resistant (XDR) Beijing isolates.

Next generation sequencing (NGS) was performed using Ion Torrent PGM sequencer on the 318 chip using barcoded fragments derived from DNA library of individual M.tb isolates. The sequences were assembled using reference H37Rv and de novo assembly. A mean read length of 174 bp was achieved with average coverage depth of reference 218.6 X and a 99% mean raw accuracy at 1X.

We found the genotypically identical isolate to have 99.99% genetic identity. This observation confirmed that the MDR M.tb isolated after completion of Directly Observed Treatment Shortcourse (DOTS) had in fact evolved from the DS strain which infected the patient prior to treatment. Further analysis of the genomic variation between the strains was performed using a coverage >10 and base call quality >30, 827 variants were observed between the DS and MDR isolates. The Beijing DS and XDR isolates had the maximum number of variants (395) followed by MANU1 DS and MDR (242) and CAS1_Delhi DS and MDR (191). The genes with the variants between DS and drug resistant phenotypes were categorized into 10 functional categories. The highest proportion of variants were observed in the information pathway genes (203/827; 24.5%) followed by genes coding for cell wall and cell processes (148/827; 17.9%), intermediary metabolism and respiration (148/827; 17.9%), lipid metabolism (56/827; 6.8%), regulatory proteins (32/827; 3.9%), virulence, detoxification, adaptation (32/827; 3.9%), PE/PPE (24/827; 2.9%) and insertion sequences and phages (22/827; 2.7%). Fifty seven variants were detected in intergenic regions and 105 variants were observed in conserved hypothetical genes. We observed 10 common variants between MDR and the XDR isolates and 10 common variants were found between the MANU1 and CAS1_Delhi MDR isolates. Thus our study reports the whole genome sequence of clinical M.tb isolates including MDR and XDR M.tb isolates from India. The study indicates the complex mechanism of development of drug resistance under numerous selection stresses in addition to known drug target mutations. The results represent alternate modes of drug tolerance including lowering of cellular metabolism and alteration of cell wall transport. The highest TB burden in the world is recorded in India. Hence mega whole genome sequencing of clinical isolates from the region and will be critical for determining future diagnostics and therapeutics for drug resistant tuberculosis.

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