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INTRODUCTION: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was employed for rapid detection of ethambutol (EMB) resistant clinical isolates of Mycobacterium tuberculosis. MATERIALS AND METHODS: From 182 clinical isolates of M tuberculosis collected from different regions, 103 strains were entered in the investigation. DNA was extracted by Chelex 100 method and PCR was performed using specific primers for embB gene. Polymerase chain reaction products were digested with HaeIII and NlaII restriction endonucleases and the patterns of restriction fragments were analysed. Some randomly selected samples were sequenced. RESULTS: Out of 103 studied strains, 52 were resistant to EMB. The cases of secondary tuberculosis were 53 (51.50 ± 1.77%), and primary cases 50 (48.50 ± 1.77%; p > 0.05). From 63 extensively drug-resistant (XDR), pre-XDR and multidrug-resistant (MDR) isolates, 27 (87%), 18 (81.8%) and 7 (70%) strains were resistant to EMB, respectively. Results of PCR-RFLP method showed that from 27R EMB XDR isolates, 13 (sensitivity 48% with CI: 0.307, 0.66 and specificity 100%), from 18R EMB pre-XDR strains, 4 (sensitivity 22% with CI: 0.09, 0.45 and specificity 100%) and of 7R EMB MDR, 2 (sensitivity 28% with CI: 0.082, 0.64 and specificity 100%) had mutation in ATG-Met codon 306. Results of sequencing were concordant with RFLP method. Overall, sensitivity of the molecular method was 36.5% (CI: 0.09, 0.45) and specificity 100%. None of the 40 pansusceptible strains was embB306 mutants. Extensively drug-resistant strains had a higher proportion of embB306 mutants (43%) than pre-XDR and MDR isolates (odds ratio 6.78; p < 0.001). CONCLUSION: Fast detection of susceptibility to EMB drug is possible by PCR-RFLP. The embB306 locus is a candidate marker for rapid prediction of high resistance consisting of MDR and XDR forms to anti-tuberculosis drugs using this method.
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Resistance phenomenon in M tuberculosis is mainly based on decreased permeability of the bacterial envelope and function of effluent pumps. The regulatory gene of the whiB7 transcription determines drug resistance in these bacteria. Increases in WhiB7 protein activity induce transcription of resistance genes leading to intrinsic multidrug resistance. The aim of this work was to evaluate the whiB7 gene sequence in susceptible, MDR and XDR clinical isolates of M tuberculosis in order to further design an inhibitor. Thirty-three clinical isolates of MTB identified as susceptible, MDR and XDR-TB were investigated by PCR for sequencing of the entire promoter (429 bp), structural gene (279 bp) and the end of the upstream gene uvrD (265 bp). No differences were detected in the sequences of the structural gene in susceptible and MDR with XDR isolates and all of them terminated at TGA as stop codon. Examination of sequence profiles of the promoter part of whiB7 by several sets of primers proved that there were no differences between sequence of susceptible, MDR and XDR isolates by type strain (H37Rr). Furthermore, the structure of WhiB7 protein was studied in achieved sequences from clinical isolates. We found that the promoter and structural gene of whiB7 are highly conservative in clinical susceptible and resistant isolates. It is a key finding that would assist in the design of an inhibitor for the WhiB7 protein in all clinical forms in further studies.