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

Abstract

b Ethambutol (EMB) is an essential first-line drug for tuberculosis (TB) treatment. Nucleotide substitutions at embB codon 306 (embB306) have been proposed to be a potential marker for EMB resistance and a predictor of broad drug resistance in clinical Mycobacterium tuberculosis isolates. However, discordant findings about the association between embB306 mutations and EMB resistance were reported. Hebei Province is located in the Beijing-Tianjin-Hebei integration region in China; however, little information about the genetic diversity of the embB locus in this area is available. In this study, we sequenced the region surrounding embB306 (codons 207 to 445) in 62 ethambutol-resistant (EMB r ) isolates, 214 ethambutol-susceptible isolates resistant to other first-line drugs (EMB s isolates), and 100 pan-sensitive isolates. Our data indicated that none of the pan-sensitive isolates showed mutations at embB306 and 63 drug-resistant isolates harbored embB306 substitutions, with these substitutions being found in 56.5% (35/62) of EMB r isolates and 13.1% (28/214) of EMB s isolates. A significant association between the embB306 mutation and resistance to isoniazid, rifampin, EMB, and multiple drugs was observed, and the rate of mutation of embB306 increased with increasing numbers of first-line drugs to which the isolates were resistant. The embB306 mutation is not the sole causative factor for EMB resistance, and the poor sensitivity limits its utility as a marker for drug-resistant TB. However, it may be a potential marker for broad drug resistance, especially for multidrug resistance. The mycobacterial interspersed repetitive unit-variable-number tandem-repeat profiles may serve as markers for predicting the embB306 substitutions that may occur in drug-resistant M. tuberculosis isolates under antimicrobial selection pressure.T uberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. According to estimates of the World Health Organization (WHO), in 2014 there were 9.6 million new TB cases and 1.5 million people died from this infectious disease (1). The widespread occurrence of drug-resistant (DR) TB (DR-TB), especially multidrug-resistant (MDR) TB (MDR-TB), has become a major global public health concern. In 2014, an estimated 3.3% of new TB cases and 20% of previously treated cases had MDR-TB and approximately 190,000 people died of MDR-TB worldwide. Treatment for MDR-TB is longer and requires more expensive and more toxic second-line drugs.Ethambutol (EMB) is an essential first-line drug for TB treatment and plays an important role in the chemotherapy of DR-TB (2). EMB acts against M. tuberculosis through inhibiting the membrane-associated arabinosyl transferases that are encoded by the embCAB operon (which included three contiguous genes, embC, embA, and embB) and that participate in the synthesis of arabinogalactan and lipoarabinomannan of the mycobacterial cell wall (3, 4). Mutations in the embCAB operon have been identified to confer resistance to EMB (3), with embB codon 306 (embB306) b...

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…68 Although the causal relationship between embB and ubiA missense mutations with EMB resistance has been established, missense mutations at embB can only explain 40-68% of the global EMB resistance occurrence. 12,69 Moreover, the presence of EMBsusceptible clinical isolates with embB M306V or M306I missense mutations has been reported in multiple studies. [70][71][72] Such discrepancies might be due to inappropriate critical concentrations (CC; i.e.…”

“…Whereas the occurrence of embB mutations among EMB‐resistant clinical isolates was consistent throughout the globe, occurrence of ubiA mutations in EMB‐resistant strains can vary according to the geographical region considered . Although the causal relationship between embB and ubiA missense mutations with EMB resistance has been established, missense mutations at embB can only explain 40–68% of the global EMB resistance occurrence . Moreover, the presence of EMB‐susceptible clinical isolates with embB M306V or M306I missense mutations has been reported in multiple studies .…”

Detecting Ethambutol Resistance in Mycobacterium tuberculosis Isolates in China: A Comparison Between Phenotypic Drug Susceptibility Testing Methods and DNA Sequencing of embAB

“…Furthermore, certain mutations within the upstream region (UR) of embA are associated with EMB resistance (Cui et al, 2014;Brossier et al, 2015;Zhao et al, 2015). However, the presence of these mutations in embAB was also observed in EMBsusceptible isolates (Shi et al, 2011;Zhao et al, 2015;Li et al, 2016;Sun et al, 2018). Besides, there are no mutations within the particular genes responsible for EMB resistance in some EMB-resistant isolates, implying the involvement of other resistance mechanisms (Sharma and Bisht, 2017;Sharma et al, 2018).…”

Mutations within embCAB Are Associated with Variable Level of Ethambutol Resistance in Mycobacterium tuberculosis Isolates from China

“…Mutations in the embCAB operon (mainly embB) were most commonly associated with EMB resistance, with variations of codon embB 306, 406, and 497 as the hot spots for mutations (6)(7)(8)(9). However, information on the correlation between EMB resistance and embCAB mutations was scarce until now in China (9), with most studies investigating mutations only in embB (10)(11)(12)(13). Furthermore, studies often reported aggregate test performance containing sensitivity and specificity (8,9,11,12,14) and infrequently report on EMB MIC measurements.…”

“…However, information on the correlation between EMB resistance and embCAB mutations was scarce until now in China (9), with most studies investigating mutations only in embB (10)(11)(12)(13). Furthermore, studies often reported aggregate test performance containing sensitivity and specificity (8,9,11,12,14) and infrequently report on EMB MIC measurements. To improve the accuracy of the molecular diagnostic, a detailed analysis of the differential effects of the embCAB mutations on EMB resistance is necessary.…”