Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates.
Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.
Negi
Sanjay Singh
SS
Microbiology Division, National Institute of Communicable Diseases, Delhi 110 054, India.
Anand
Ranjana
R
Pasha
S T
ST
Gupta
Sunil
S
Basir
Seemi Farhat
SF
Khare
Shashi
S
Lal
Shiv
S
D000293:Adolescent; D000328:Adult; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D002374:Catalase / Q000235:genetics*; D003062:Codon; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008875:Middle Aged; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012319:RNA Polymerase II / Q000235:genetics*; D012293:Rifampin / Q000494:pharmacology; D014376:Tuberculosis / Q000382:microbiology
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003062:Codon; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D012319:RNA Polymerase II; D007538:Isoniazid; D012293:Rifampin