Detection of embB Gene Mutations in EMB-Resistant Mycobacterium tuberculosis Isolates From Isfahan Province by PCR-SSCP and Direct Sequencing

Abstract

Background: Ethambutol (EMB) is the first-line drug used for the treatment of tuberculosis. Recent reports on the EMB-resistant isolates of Mycobacterium tuberculosis in different geographical regions of the world have revealed the EMB resistance patterns of M. tuberculosis and mutations in the embB gene. Objectives: In this study, we determined the emb locus in sensitive and resistant Iranian M. tuberculosis isolates using two effective methods for the detection of point mutation, i.e., single-strand conformational polymorphism (SSCP) and direct sequencing. Methods: Thirty-two M. tuberculosis isolates from the Isfahan tuberculosis center were characterized by conventional methods and specific amplification of the regions of difference (rd) gene and internal transcribed spacer (its) gene. Observing standard operational procedures, EMB susceptibility tests were performed on the LJ medium using the proportion method with 2, 5, and 10 μg/mL concentrations. PCR-SSCP and direct sequencing were used to detect different kinds of mutation in the embB gene with precision. Results: In a total of 32 isolates, two isolates (6.25%) were found to be resistant to EMB in 2, 5, and 10 μg/mL concentrations. Single-strand conformational polymorphism showed altered mobility with triple bands in the resistant isolates and double bands in the sensitive isolates. In the two EMB-resistant cases, mutation was found to occur codons 309and 299. Conclusions: We concluded from the results that the frequency of EMB-resistant M. tuberculosis cases in Iran is lower than that of many other regions. The PCR-SSCP technique can separate resistant isolates from sensitive isolates. The sequencing results of this study showed mutation in codons 309 and 299 of the embB gene. In none of the resistant isolates, mutation was observed in codon 306. Further studies are required to determine other point mutations and analyze other genetic loci associated with EMB resistance in M. tuberculosis isolates in Iran.