Mutations in theembC-embAIntergenic Region Contribute to Mycobacterium tuberculosis Resistance to Ethambutol

Abstract

ABSTRACTThe rapid increase inMycobacterium tuberculosisresistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in theembC-embAintergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767M. tuberculosisclinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and theembC-embAIGRs of these strains were sequenced. The transcriptional activity of theembC-embAIGR mutations was examined by reporter gene assays in recombinantMycobacterium smegmatisstrains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator ofembAB, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that theembC-embAIGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level ofembAandembBmRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed intoM. smegmatisstrains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of theembC-embAIGR enhance the binding of EmbR to the promoter region ofembABand increase the expression ofembAB, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST.