Molecular characterization of INH-resistant Mycobacterium tuberculosis isolates by PCR-RFLP and multiplex-PCR in North India.
In the present study, among 327 Mycobacterium tuberculosis (MTB) isolates collected from patients attending three different centres of North India, we attempted to find out the most common mutations occurring both at the Ser315 codon of katG and at the regulatory region of the mabA-inhA operon to evaluate their role for INH drug resistance in India. Out of 121 phenotypically INH-resistant MTB isolates, 88 (72.7%) were resistant to INH by genotypic methods viz., PCR-RFLP with MspI and SatI digestion and multiplex-PCR. PCR-RFLP results showed that 67 (55.4%) isolates had mutation in codon 315 of katG by SatI endonuclease. Among these, eight isolates that were found resistant by SatI PCR-RFLP were found to be sensitive by MspI PCR-RFLP. By multiplex-PCR we found 49 (40.5%), 21 (17.4%) and 10 (8.3%) isolates having AGC-->ACC substitution in katG only, mutation in inhA(C-15T) only and mutation in both respectively. Simultaneous use of both PCR-RFLP and multiplex-PCR can improve the detection rate of INH-resistant strains and may have an advantage over the liquid culture system of detecting drug resistance. These findings also enhanced our understanding about potential of resistance-related mutations in M. tuberculosis clinical isolates in India and could help in development and designing of molecular methods for revealing the drug susceptibility profiles of M. tuberculosis clinical isolates.
Mathuria
Jitendra Prasad
JP
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India. jitesh141@gmail.com
Nath
Gopal
G
Samaria
Jai Kumar
JK
Anupurba
Shampa
S
D000995:Antitubercular Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002374:Catalase / Q000032:analysis / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D006801:Humans; D007194:India; D007538:Isoniazid / Q000494:pharmacology* / Q000627:therapeutic use; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000032:analysis / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology*
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D007538:Isoniazid