Molecular Characterization of Rifampicin associated mutation in Mycobacterium tuberculosis isolates

Abstract

Mycobacterium tuberculosis is one of the most lethal pathogens causing infection in 1.8 billion people annually.  Mycobacterium tuberculosis majorly cause pulmonary infection which spreads easily by the air. Rifampicin is a first line drug used for the treatment of this disease. By binding to the 30S ribosomal subunit it inhibits the protein synthesis. The misuse of this drug and incomplete treatment causes alteration at the genetic level of rpoB gene of the MTB. The aim of our study was to find the mutations in the rpoB gene from the clinical isolates of the tuberculosis patients. We collected 412 sputum samples from patients suspected of tuberculosis and cultured in the microbiology laboratory of THQ Fatehpur, Layyah. Positive sputum cultures were analyzed for drug susceptibility test. Genomic DNA was isolated by sonication method using 20 Hz frequency of ultrasonic waves. After that primers were designed using bioinformatics tools for amplification of the gene.  Additionally, the rpoB was amplified by a hemi-nested PCR technique. 91 samples were positive for sputum culture which consisted of 54 males and 37 females. Out of the 91 positive cultures, four samples showed rifampicin resistance. Two samples carried single missense mutation at position 526 and 531 in the amino acid sequence of rpoB gene whereas third and fourth sample carried a single missense mutation at position 516   in the amino acid sequence of rpoB gene. Moreover, 22% of the tested sputum samples were positive for tuberculosis. This means that in the population of tehsil Fatehpur, Layyah tuberculosis is prevalent.  Furthermore, 4.3% of this 22% were found to be rifampicin resistant. In the future researches the harmful effect of rpoB gene mutations which is associated with function of the β-subunit of RNA polymerase in 16S rRNA and its interaction with rifampicin can be estimated by performing molecular docking