Principal Investigator Dr. Nerges Mistry
Project team Dr. Anirvan Chatterjee, Dr. Kayzad Nilgiriwala
Funder Department of Science and Technology (DST)
Duration August 2014- July2017
Budget Rs.54.96 Lakhs
Globally Mycobacterium tuberculosis (M.tb) is estimated to result in 8 million new cases of tuberculosis (TB) and 2 million deaths annually. The emergence of multidrug resistance (MDR), extensive drug resistance (XDR), and the recently reported total drug resistant tuberculosis (TDR) poses a threat to the TB control programme patterned on directly observed therapy short course (DOTS) strategy. Our study in DOTS compliant patients showed that 32% drug susceptible (DS) isolates at onset developed MDR at the end of 5th month of therapy.While the molecular basis of drug resistant M.tb has been attributed to point mutations in identified drug target genes, the mechanisms for rapid accumulation of drug resistance involving multiple gene loci are yet to be elucidated. The resistance conferred by such mechanisms may act through switching on/off genes, as well as through differential expression of genes in the presence of drugs and/or other stress factors. Our initial study provided evidence of a significant association of deregulation of several genes with rapid acquisition of drug resistance. However, their role as biomarkers for detecting strains predisposed to acquiring MDR can be determined through confirmation and validation of the deregulated genes using qRT-PCR. While mutation in several genes have been identified and associated with drug resistance, their role in rapid acquisition of MDR has not been reported in longitudinal clinical DS and MDR isolates.
Transcriptional analysis of genes associated with rapid acquisition of multi drug resistance in Mycobacterium tuberculosis
The previously collected longitudinal clinical isolates showing rapid amplification of drug resistance will be revived. A new set of drug susceptible (DS) clinical isolates of M.tb from patients with pulmonary tuberculosiswill be collected. A follow up of patients to detect failures and poor drug responderswill be done. Serial isolates from patients who were DS pre-treatment, but failed DOTS (20 patients)will be collected (MDR isolates). Drug sensitivity tests (DST)will be conducted using HAINS (for genotype) and MGIT (for phenotype).Genotyping (24-loci MIRU-VNTR and Spoligotyping) analysis of all isolates will be conducted after sequencing the bacterial genomes. RNA will be extractedfrom selected single colonies and standardization of qRT-PCR of 25 genes plus 2 housekeeping genes from individual colonies of each isolate will be conducted, and the results will be analyzed. Artificial evolution of H37Rv for drug tolerance/resistance will be conducted by using suboptimal concentrations of Isoniazid (H), Rifampicin (R), Ethambutol (E), and Pyrazinamide (Z) antibiotics.DST and fingerprinting of MDR H37Rv single colonies will be done. RNA extracted from H37Rv and MDR H37Rv will be quantified for expression of the selected genes and the results will be analyzed to determine the potential of usingappropriate genes as biomarker candidates for predicting MDR. qRT-PCR of selected biomarkers in 50 MDR M.tb isolates from new cases which failed treatment will be performed in order to check the validity of the selected genetic biomarkers. The results so obtained will be published and disseminated.
This project will help to identify and confirm the genes most significantly upregulated/downregulated in isolates showing rapid accumulation of MDR as determined by quantitative real-time polymerase chain reaction (qRT-PCR). This study will also test the feasibility to develop cost effective qRT-PCR methodology for a selected and limited set of genes associated with rapid acquisition of drug resistance in DOTS compliant patients and will also help to determine potential novel targets in therapyfor tuberculosis.
Andrews JR, Gandhi NR, Moodley P, Shah NS, Bohlken L, et al. (2008) Exogenous reinfection as a cause of multidrug-resistant and extensively drug-resistant tuberculosis in rural South Africa. J Infect Dis 198: 1582-1589.
Warner DF, Mizrahi V (2013) Complex genetics of drug resistance in Mycobacterium tuberculosis. Nat Genet 45: 1107-1108.
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