Molecular Study on Gene mutations associated with Antibiotic Resistance of Mycobacterium tuberculosis

Abstract

In response to the huge number of people who die yearly due tuberculosis and the emergence of multidrug resistant (MDR) M.tuberculosis, accurate and rapid detection of this resistance can improve the situation. The aim of this work is to identify the mutation conferring resistance of M. tuberculosis bacteria to INH and RIF, compare conventional methods (DST and MIC) and molecular methods (DNA sequencing and genotyping) for detection of resistant genes and evaluate automated DNA sequencing and a genotyping method by MTBDR plus assay for predation of TB resistance. Sputum specimens were collected from 155 patients with chest symptoms and examined by Acid Fast Stain (AFB) staining, digested using sputasol-sodium hydroxide, then decontaminated sediment was inoculated onto the LJ medium for each specimen and incubated at 37°C. Cultures were regarded as negative only if there was no growth after 8 weeks of incubation. The niacin and nitrate reduction tests were used for the primary identification of M. tuberculosis. Antibiotic susceptibility and determination of minimum inhibitory concentration (MIC) were performed. Relapsed patients represented significant percentages among rifampicin and isoniazid resistant isolates compared to other risk factors. Two molecular techniques, Genotype MTBDRplus assay and specific gene sequencing, were used to detect associated mutations in TB drug resistant isolates. The genotypic profile of Multi-drug resistant (MDR) isolates showed missing of katG wild type 1 (WT1) band. 80% of isoniazid mono-resistant isolates, showed katG MUT1, 20% showed katG MUT1 and inhA MUT1, 20% showed only inhA MUT1. The molecular techniques partly predicted the level of antibiotic resistance associated with katG and/or inhA gene mutations (for isoniazid) and rpoB gene mutation (for rifampicin). MTBDRplus could clearly detect rifampicin resistance among 66.7% of MDR isolates that showed mutation band rpoB MUT3 while 33.3% of them were considered as unknown, while 100% of mono-isoniazid resistant strains were detected.