Molecular Genetic Analysis of Nucleotide Polymorphisms Associated with Ethambutol Resistance in Human Isolates of Mycobacterium tuberculosis

Abstract

Ethambutol (EMB) is a central component of drug regimens used worldwide for the treatment of tuberculosis. To gain insight into the molecular genetic basis of EMB resistance, approximately 2 Mb of five chromosomal regions with 12 genes in 75 epidemiologically unassociated EMB-resistant and 33 EMB-susceptibleMycobacterium tuberculosis strains isolated from human patients were sequenced. Seventy-six percent of EMBresistant organisms had an amino acid replacement or other molecular change not found in EMB-susceptible strains. Thirty-eight (51%) EMB-resistant isolates had a resistance-associated mutation in only 1 of the 12 genes sequenced. Nineteen EMB-resistant isolates had resistance-associated nucleotide changes that conferred amino acid replacements or upstream potential regulatory region mutations in two or more genes. Most isolates (68%) with resistance-associated mutations in a single gene had nucleotide changes in embB, a gene encoding an arabinosyltransferase involved in cell wall biosynthesis. The majority of these mutations resulted in amino acid replacements at position 306 or 406 of EmbB. Resistance-associated mutations were also identified in several genes recently shown to be upregulated in response to exposure of M. tuberculosis to EMB in vitro, including genes in the iniA operon. Approximately one-fourth of the organisms studied lacked mutations inferred to participate in EMB resistance, a result indicating that one or more genes that mediate resistance to this drug remain to be discovered. Taken together, the results indicate that there are multiple molecular pathways to the EMB resistance phenotype. Ethambutol [EMB; (S, S)-2,2Ј-(ethylenediimino)di-1-buta-nol] is used worldwide as one of the primary antituberculosis agents. The mechanism of action and the molecular genetic basis of resistance to EMB are not fully understood. Only the dextro isomer of EMB is biologically active, an observation consistent with the idea that the drug binds to a specific cellular target (7, 37). Several studies have implicated membrane-associated arabinosyltransferases as targets for EMB (1,5,20,22). These enzymes are well conserved in mycobacteria and are involved in the biosynthesis of arabinan, a component of arabinogalactan present in cell walls (6,8,17,33,34,39). Inhibition of arabinan synthesis leads to accumulation of mycolic acids and eventually to cell death.Three contiguous genes encoding arabinosyltransferases and designated embC, embA, and embB have been identified in Mycobacterium tuberculosis (35). The proteins encoded by these genes are about 65% identical to each other. Previous studies based on limited sequencing of the 10-kb region containing the embCAB genes have identified mutations that result in replacement of amino acid residues and are found only in EMB-resistant organisms cultured from humans. The most commonly affected amino acid was Met306 of EmbB. For example, Sreevatsan et al. (31) identified five distinct mutant codons that resulted in replacement of wild-type Met306 with Ile, Leu, or Va...

Tuberculosis ethambutol resistance: Concordance between phenotypic and genotypic test results

“…Statistical analysis revealed a significant association between EMB resistance and embB306 mutations in univariate analysis (p < 0.0001), with embB306 mutations the only significant factor in multivariate analysis (p < 0.0001). Thus, our study confirms previous investigations that describe a clear correlation between mutations in emb306 and EMB resistance, 11,21 without any indication of an influence on the resistance to other antituberculous drugs as suggested by other authors. 13,15,16 This high concordance between both assays was, however, determined only after repeating conventional drug susceptibility testing, pointing to problems with conventional diagnostics of EMB resistance.…”

“…For example, Ramaswamy et al described in their work mutations, which were associated with EMB resistance, in 21 variant codons of the embCAB operon. 21 Further investigations of the EMB resistant isolates from Usbekistan, which had no mutation in embB306, are planned for future studies.…”

Molecular characterization and drug susceptibility profile of a Mycobacterium avium subspecies avium isolate from a dog with disseminated infection

“…Resistance to this drug is mainly ascribed to mutations in the embAB genes that encode an arabinosyltransferase involved in cell wall arabinan biosynthesis (Belanger et al, 1996). For ethambutol-resistant M. tuberculosis, several mutations of the embB gene have been described (Alcaide et al, 1997;Ramaswamy et al, 2000;Sreevatsan et al, 1997). Data on ethambutol-resistant MAC are limited, however.…”

Polymorphism in the RD1 locus and its effect on downstream genes among South Indian clinical isolates of Mycobacterium tuberculosis

“…Sreevatsan et al (1997) demonstrated a polymorphism in the 105 bp oxyR-ahpC intergenic region resulting in altered expression of KatG and correlated with resistance to isoniazid (Sreevatsan et al, 1997). Ramaswamy et al (2000) illustrated a polymorphic intergenic region between the embC and embA genes involved in ethambutol resistance (Ramaswamy et al, 2000).…”