Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS 6110 Clustering Rather than Ethambutol Resistance

Abstract

Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P ‫؍‬ 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P ‫؍‬ 0.008) and katG315 (odds ratio, 1.99; P ‫؍‬ 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.The antibiotic ethambutol (EMB) appears to inhibit the growth of both Mycobacterium tuberculosis and Mycobacterium smegmatis by blocking the synthesis of arabinogalactan. Arabinogalactan biosynthesis is dependent on the activity of the embABC gene cluster, which encodes the arabinotransferases that mediate the polymerization of arabinose into arabinan. Several lines of evidence suggest that EMB exerts its toxic effect on mycobacteria by inhibiting the embABC-encoded proteins (26,27,34,35,46), and mutations in embABC also appear to play a key role in the development of EMB resistance in both M. tuberculosis and M. smegmatis (34). Associations between EMB resistance and mutations in embA, embB, and embC have been reported in clinical strains of M. tuberculosis (41), and mutations in codon 306 of the embB gene (embB306) in M. tuberculosis are seen in approximately 50% of all EMB-* Corresponding author. Mailing address:

Drug-Resistance inMycobacterium Tuberculosis:Molecular Basis and Genotypic Detection

“…however all eMBsusceptible MDR isolates in our study had embB306 wild type allele (57). Accordingly, it appears that a hypothesis of hazbon et al (16) about embB306 as a marker of multidrug resistance is only partly supported by our data.…”

“…our own results on embB306 variation in Bulgarian population of M. tuberculosis (57) are in line with earlier findings (45,46) and German results (43) which correlated mutations in embB306 with eMB resistance. they are in contradiction with more recently reported discrepancies between genotypic and phenotypic eMB resistance (16,34, and references therein). A number of explanations of these contradictory findings have been proposed.…”

“…Mokrousov et al (34) hypothesized an unknown mechanism in MDR M. tuberculosis strains that leads to susceptibility to eMB. hazbon et al (16) suggested that the clear association between mutations in embB306 and eMB resistance found in several earlier studies might be due to the use of pansusceptible strains as control groups. Regarding this latter point, our own study in the Bulgarian setting found embB306 mutation in 7 isolates of which 6 isolates were resistant to more than one drug.…”

“…the prevalence of resistance to eMB, initial and acquired, is not generally high and varies in different countries from 1.3% in canada (47) to 23.3% in pakistan (22), with an overall average of 4-9% for initial resistance (67,68), and is predominant among patients with MDR TB. For the first time, Mokrousov et al (34) highlighted a presence of embB306 mutations in eMB-susceptible strains and hazbon et al (16) suggested an association of embB306 mutations with broad drug resistance and clustering rather than eMB resistance. our own results on embB306 variation in Bulgarian population of M. tuberculosis (57) are in line with earlier findings (45,46) and German results (43) which correlated mutations in embB306 with eMB resistance.…”

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

“…The prevalence in our work is significantly lower than that reported in Latvia, Thailand, Mozambique, and Uganda by WHO (3) and the study by Plinke et al (24) that obtained a resistant rate of 63.5%. The frequency of resistance was reported as 18.8% by Hazbon et al (25).…”