Mutations in embB 406 Are Associated with Low-Level Ethambutol Resistance in Canadian Mycobacterium tuberculosis Isolates

Abstract

In Mycobacterium tuberculosis, molecular predictions of ethambutol resistance rely primarily on the detection of mutations within embB. However, discordance between embB406 mutations and gold standard phenotypic drug sensitivity testing (DST) questions the significance of embB406 mutations used in molecular DST. This study tabulates embB mutations found in Canadian M. tuberculosis isolates and evaluates the impact of specific mutations on ethambutol resistance. The National Reference Centre for Mycobacteriology culture collection (n = 2796) was screened for isolates with embB mutations. Phenotypic DST was performed on the BACTEC™ MGIT™ 960 at ethambutol concentrations of 2-5 μg/mL. Whole genome sequencing was used for drug resistance predictions, phylogenomics and single nucleotide polymorphism analysis. Detection of resistance-associated embB mutations corresponded to a positive predictive value of 64.3%, negative predictive value of 99.2%, 98.7% specificity, and 73.3% sensitivity compared to phenotypic DST. Two embB406 mutation subtypes (Gly406Asp, Gly406Ala) were found among 16 isolates, of which 12 were sensitive at 5 µg/mL ethambutol with variable resistance between 2-4 µg/mL. A novel frameshift mutation in regulator embR (Gln258fs) was found in nine isolates. Mutations in embB406 were associated with low-level ethambutol resistance undetectable at the recommended critical concentration (5 μg/mL). These novel mutations may exacerbate variability in ethambutol resistance.

Keywords: Mycobacterium tuberculosis; embB; ethambutol; resistance.

Figure 1

Ethambutol Susceptibility Predictions among M. tuberculosis Isolates Harbouring embB406 Mutations. Phylogenomic analysis was performed for M. tuberculosis study isolates 1–26 using SNVPhyl (v1.2.3) with reference M. tuberculosis H37Rv (NC_000962.3) and included 94.27% of all genomic positions in the core genome. The adjacent H37Rv was cultured and underwent whole genome sequencing in-house. Molecular susceptibility predictions were determined by MyKrobe Predictor (v0.10.0). Ethambutol phenotype is depicted at the critical concentration, 5 μg/mL, and a lower concentration, 2 μg/mL.

Figure 2

Mutations and Codon Changes Found in the embCAB Operon of M. tuberculosis Isolates. Linear gene (green) and protein domain (key) map of single nucleotide polymorphisms in the embCAB operon. Identity and position of base pair (bp) and amino acid (aa) substitutions are shown. n indicates the total number of ethambutol-resistant isolates harbouring a given mutation. The black arrows represent mutations found in embB406 mutants only. The white arrows indicate mutations found in both embB406 mutants and pan-susceptible control isolates.