Evolution of high-level ethambutol-resistant tuberculosis through interacting mutations in decaprenylphosphoryl-β-D-arabinose biosynthetic and utilization pathway genes

Abstract

To study the evolution of drug resistance, we genetically and biochemically characterized Mycobacterium tuberculosis strains selected in vitro for ethambutol resistance. Mutations in decaprenylphosphoryl-β-d-arabinose (DPA) biosynthetic and utilization pathway genes Rv3806c, Rv3792, embB and embC accumulated to produce a wide range of ethambutol minimal inhibitory concentrations (MICs) that depended on mutation type and number. Rv3806c mutations increased DPA synthesis, causing MICs to double from 2 to 4 µg/ml in a wild-type background and to increase from 16 to 32 µg/ml in an embB codon 306 mutant background. Synonymous mutations in Rv3792 increased the expression of downstream embC, an ethambutol target, resulting in MICs of 8 µg/ml. Multistep selection was required for high-level resistance. Mutations in embC or very high embC expression were observed at the highest resistance level. In clinical isolates, Rv3806c mutations were associated with high-level resistance and had multiplicative effects with embB mutations on MICs. Ethambutol resistance is acquired through the acquisition of mutations that interact in complex ways to produce a range of MICs, from those falling below breakpoint values to ones representing high-level resistance.

Substantial molecular evolution and mutation rates in prolonged latent Mycobacterium tuberculosis infection in humans

“…We did not observe mutations in any of the secondary-or tertiary isolates that could be linked to established drug resistance phenotypes. We note, however, that genes Rv3805c (aftB) and Rv3130c (tgs1), which both contained putative deleterious mutations in isolate R98-3208 (Link 2), have been previously implicated in processes that may influence the development of antibiotic resistance and tolerance (Daniel et al, 2004;Safi et al, 2013). However, the putatively deleterious tgs1 mutation in R98-3208 is particularly intriguing in a purely metabolic context as it is essential for accumulation of triacylglycerol (TAG) in response to hypoxia and other stresses (Daniel et al, 2004).…”

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

“…EMB resistance is acquired through the acquisition of mutations that interact in complex ways to produce a range of MICs, from those falling below breakpoint values to ones representing high-level resistance (23). Earlier studies showed that the embB306 substitutions were associated with an 8-to 16-fold increase in the MICs of EMB (19,24,25).…”

A Microbiological Revolution Meets an Ancient Disease: Improving the Management of Tuberculosis with Genomics

“…In the case of ethambutol resistance, a study showed that a low-level resistance that would be undetected by current phenotypic antibiotic susceptibility testing may accumulate within the genome of M. tuberculosis (41). In contrast, another study reported that phenotypic testing indicated isoniazid resistance, but WGS failed to identify the mutation responsible (45).…”

“…However, accurate sequence-based typing of XDR isolates was more challenging, with only 40.0% sensitivity and 99.3% specificity (38). Recent studies used WGS on hundreds of clinical isolates to identify known and novel antibiotic resistance-conferring mutations (39)(40)(41). The largest of these, a study of 1,000 Russian isolates, 48% of which were MDR, identified 238 single nucleotide polymorphisms (SNPs) associated with drug resistance (42).…”