Full Length Research Paper

Multiple mutations in the rpoB gene of Mycobacterium tuberculosis isolates correlate with high level of resistance to rifampicin in patients with active pulmonary tuberculosis in Afghanistan border of Iran

Saeed Zaker Bostanabad^{1, 3}*, Mohammad Noghanian³, A. Edward Graviss², Ahmad Reza Bahrmand¹, P. Leonid Titov³ and Seyed Ali Nojoumi¹

¹Molecular Genetic laboratory of Mycobacteria, Department of Mycobacteriology, Pasteur Institute of Iran, Pasture Ave, Tehran, 13164, Iran.

²Pathology Department, Baylor College of Medicine, Houston, Texas.

³Belarusian Research of Epidemiology and Microbiology, Clinical Microbiology, Belarus.

*Corresponding author. E-mail: Saeedzaker20@yahoo.com.

Accepted 18 March, 2008

The aim of this study was to investigate the significance of multiple mutations in the rpoB gene, predominant nucleotide changes and their correlation with high levels of resistance to rifampicin in Mycobacterium tuberculosis isolates that were randomly collected from sputa of 50 patients with primary and secondary active pulmonary tuberculosis from the southern TB endemic border of Iran. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-nine (78%) isolates were found to have multiple mutations (composed of two to five mutations) in the rpoB (β-subunit) gene. Furthermore, it was demonstrated that the combination of mutations with predominant nucleotide changes were observed in codons 510, 523, 526 and 531, and indicated higher frequencies of mutations among patients with secondary infection. In this study, multiple mutated isolates 76% (n = 38) were found to have mutation combinations involving nucleotide changes in codons 523 (GGG→GCG), 531 (TCG→TTG, TTC), 526 (CAC→CGC, TTC, TAC, AAC, CAA), and demonstrated predominant mutations in the last nine codons of the β-subunit (523 - 531) were associated with higher levels of resistance to rifampicin (MIC = ≥ 100 µg/ml).

Key word: Multiple mutations, rpoB gene, M. tuberculosis, high level resistance, Afghanistan border.