Mutations in the rpoB Gene of Rifampin Resistant Myco-bacterium tuberculosis Isolated in Isfahan by PCR-SSCP

Rifampin interacts with the beta-subunit of RNA Polymerase (rpoB), thereby hindering transcription. Mutations in the rpoB locus confer conformational changes leading to defective binding of the drug to rpoB and consequently resistance. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing have been established as a rapid screening test for the detection of mutation in rpoB gene, which is responsible for rifampin resistance of Mycobacterium tuberculosis species. In this study, a total of 37 strains of M. tuberculosis, 16 sensitive and 15 resistant of Isfahan isolates, and 6 resistant strains obtained from Pasteur institute of Iran, were used. They were confirmed as M. tuberculosis by conventional methods and amplification of DR gene a region that belongs to M. tuberculosis complex group. Their sensitivity or resistance to Rifampin was initially determined by proportion method. The 193-bp region of the rpoB gene was then amplified and PCR-SSCP patterns of rifampin resistance (RIFr) and rifampin susceptible (RIFs) strains were determined by electrophoresis on 10% acrylamide gels and silver staining. Also 8 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from Rifr and one from Rifs were sequenced. By PCR-SSCP analysis, 7 PCR-SSCP distinguishable patterns were recognized in the Isfahan RIFr strains. Although 6 of these patterns were different from sensitive strain, one pattern was identical to sensitive standard strain H37Rv. Six resistant strains from Pasteur institute of Iran demonstrated two patterns that one of them was alike to pattern 4 of Isfahan RIFr but other was different. 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However, one of the sensitive isolates demonstrated different pattern. After determinations the rpoB sequences of the resistance strains, different mutations were seen in codon 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG) and 511(CTG/TTG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method. Presence of different PCR-SSCP banding patterns that were observed in this study is in accordance with the results of similar studies in other parts of the world. The two most prevalent mutations were missense mutations at the positions Ser-531 (TCG/ TTG: Ser/Leu) and His-526 (CAC/TAC: His/Tyr). This finding is comparable to the results of early studies demonstrating the rpoB mutation frequencies in isolates from other parts of the world.