Identification and Genetic Diversity of Etambutol Resistant Strains of Mycobacterium Tuberculosis by Allelic-Specific PCR and Spologiotyping

Ethambutol is one of the four main drugs in treatment of tuberculosis. The most common mutation associated with this drug resistance usually occurs in codon 306 of embB. The aim of this study was to detect ethambutol resistance using Allele-Specific PCR and Spoligotyping in various subtypes of Mycobacterium tuberculosis.

140 sputum specimens were collected from suspected TB patients. They were digested and decontaminated using Pettrof method before culturing them on LJ medium. Drug susceptibility testing was performed on 106 culture positive specimens using proportional method. DNA was extracted from the isolated organisms and subsequently subjected to Allele-Specific PCR to detect any mutationin embB306. Spoligotyping was then used to determine the subtypes.

Out of 106 cultures positive samples، 36 samples (33. 9%) showed resistance to ethambutol using proportional method. Allele-Specific PCR assay identified 93 as sensitive and 13 (27. 6%) as resistant strains. The results of PCR were in agreement with result of proportional method. The PCR method revealed that 61. 5% of mutation occurred in the first and 38. 5% in third nucleotides. Spoligotyping differentiated Mycobacterium tuberculosis strains into Beijing (10; 9. 4%)، Bovis (2; 1. 8%)، CAS (24; 22. 6%)، EAI (1; 0. 9%)، Haarlem (27; 25. 4%)، LAM (5; 4. 7%)، Manu (5; 4. 7%)، T (27; 25. 4%) and U (2; 1،8%) families. The high frequency of mutation in embB gene was belonged to Haarlem، CAS and T subfamilies.

Based on results current study، mutations in the genes other than embB might have occurred in the resistant strains that gave negative result in Allele-Specific PCR assay. Therefore other mechanisms of resistance to this antibiotic should be investigated.