Genetic characterization of rifampin resistance in Mycobacterium Tuberculosis

Abstract

Mutation in the gene encoding beta-subunit of RNA polymerase (rpoB gene) was determined for detecting resistance to rifampin and compared the results with radiometric method for susceptibility testing. Sixteen reference Mycobacterium tuberculosis strains and 108 clinical isolates were used in this study. Radiometric method detected 52 rifampin-susceptible isolates and 56 rifampin-resistant isolates. Mutation was detected by amplification of a 305-bp fragment in rpoB gene by polymerase chain reaction (PCR) and direct sequencing of the PCR product. Heteroduplex formation (HDF) analysis was also performed for detecting the mutation. The sequencing result showed that 100% of resistant isolates had mutations in this DNA region. Substitution in codon 531 showed the highest frequency (50%) followed by mutation in codon 526 (35.7%). Additional mutations were found in positions 513, 516, 519, 522, 523, and 533. No mutations were observed in rifampin-susceptible isolates. This study revealed three new types of mutation (AAC to AAA at codon 519, TCG to CAG at codon 522, and GGG to GAG at codon 523). The use of PCR-HDF technique for rapid identification of mutations in the rpoB gene was unsuccessful in testing resistant M. tuberculosis isolates, except one reference strain with insertion mutation.