Mutations in embB406 Are Associated with Low-Level Ethambutol Resistance in Canadian Mycobacterium tuberculosis Isolates

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 (TM) MGIT (TM) 960 at ethambutol concentrations of 2-5 mu g/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 mu g/mL ethambutol with variable resistance between 2-4 mu g/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 mu g/mL). These novel mutations may exacerbate variability in ethambutol resistance.