Analysis of embCAB Mutations Associated with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates from China

Abstract

e Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(؊16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306 -497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.M ultidrug-resistant tuberculosis (MDR-TB) is attributed to an estimated 3.7% new cases and 20.2% previously treated cases of TB annually worldwide and is becoming a major threat to global public health (1). In China, the significantly high prevalence (5.7% new cases and 25.6% previously treated cases) of MDR-TB makes TB control especially challenging (2). Ethambutol (EMB) is an important first-line anti-TB drug routinely recommended for therapy of drug-resistant TB, including MDR-TB. Disturbingly, in some regions of China, substantial proportions (51.3% to 66.7%) of MDR-TB isolates demonstrated EMB resistance (3-5). Development of new rapid and reliable molecular methods for detecting drug resistance is essential to optimize treatment regimens, prevent treatment failure, and thus reduce the further spread of drug-resistant isolates. However, these molecular assays require precise knowledge of the genetic mutations associated with drug resistance. Prior studies indicated that the characteristics of resistance-associated mutations vary in different regions (6, 7). EMB acts against TB by inhibiting membrane-associated arabinosyl transferases encoded by the embCAB operon (including embC, embA, and embB), which are involved in the synthesis of cell wall arabinogalactan (8,9). Approximately 50% to 70% of EMB-resistant TB isolates harbor mutations in a relatively short fragment (codons 306 -497) in embB genes, with mutations occurring most frequently at codon 306 in embB (embB306), embB406, and embB497 (5,8,(10)(11)(12)(13). Sequence analysis of this fragment has been a tool for the rapid detection of EMB resistance. However, approximately one third of EMB-resistant isolates do not carry changes in this region and therefore are not detectable by using DNA sequencing (12,14). Although other mutations in the embCAB...

Molecular Analysis of the embCAB Locus and embR Gene Involved in Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis in France

“…In these studies, the percentage of ETB-R isolates with mutations in the embCAB locus ranged from 65% (16) to 96% (46). In our study, 70/71 (98%) ETB-R isolates harbored mutations in the embCAB locus that likely contribute to ETB resistance.…”

“…Few publications have reported sequencing of the entire embCAB locus (16,20,46); most previous studies investigated only part of the embCAB locus (5,26), part of embC-embB and embR almost entirely (23), or the embC-embA intergenic region entirely and part of embB (15). In these studies, the percentage of ETB-R isolates with mutations in the embCAB locus ranged from 65% (16) to 96% (46).…”

“…Some authors (38,(50)(51)(52) have argued that mutations at codon 306 in EmbB do not cause ETB resistance but predispose M. tuberculosis isolates to the development of drug resistance and increase the capacity of resistant isolates to be transmitted (38,(50)(51)(52). Accordingly, several reports have described isolates with mutations at codon 306 of EmbB that remain susceptible to ETB (2,4,8,13,17,25,29,30,34,36,46,51,52). There are two explanations for the presence of a mutation at codon 306 in EmbB in an ETB-S isolate: the ETB concentration recommended for drug susceptibility testing varies from 2 to 7.5 mg/liter depending on the phenotypic method used for susceptibility testing (39), and the ETB MICs induced by mutations in codon 306 could be close to the breakpoint defining ETB resistance (37,50).…”

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

“…Notably, the high HIV prevalence combined with inadequate infection control have undoubtedly contributed to the spread of XDR tuberculosis in South Africa. 84 Molecular analysis of MDR tuberculosis strains worldwide shows a strong association between MDR tubercu losis and resistance to ethambutol 121,122 or pyrazinamide. 123,124 This association probably reflects firstline treatment of undiagnosed MDR tuberculosis resulting in acquisition of resistance to ethambutol and pyrazinamide, 125 followed by transmission.…”

Association between embB Codon 306 Mutations, Phenotypic Resistance Profiles, and Genotypic Characterization in Clinical Mycobacterium tuberculosis Isolates from Hebei, China

“…Overall, approximately 50% to 60% of EMB-resistant isolates carry a mutation in embB306 (5-7). However, discrepancies between an embB306 mutation and phenotypic EMB resistance have limited the utility of molecular assays based on the detection of an embB306 mutation for the prediction of EMB resistance (6,8,9). Prior studies have demonstrated a strong association between embB306 mutations and resistance to isoniazid (INH) or rifampin (RIF) or MDR (8, 10, 11).…”