Mutations in embB406 are associated with low-level ethambutol resistance in Canadian Mycobacterium tuberculosis isolates

BackgroundIn Mycobacterium tuberculosis, molecular predictions of ethambutol resistance rely primarily on the detection of mutations within embB. However, discordance between embB406 mutations and phenotypic drug sensitivity questions its clinical significance. This study aims to decipher the association of embB406 mutations with ethambutol resistance in M. tuberculosis. MethodsAll M. tuberculosis isolates from our culture collection containing embB406 mutations (n=16) and pan-sensitive control isolates (n=10) were selected for this study. Phenotypic drug susceptibility testing for ethambutol was performed in duplicate on the BACTEC MGIT 960 at concentrations of 2, 3, 4, and 5 g/mL with strain H37Rv as assay control. Whole genome sequencing was performed on Illumina Miseq for drug resistance predictions (MyKrobe Predictor v.0.7.0), phylogenomics (SNVPhyl v.1.2.3) and single nucleotide polymorphism analysis (Snippy). ResultsTwo embB406 mutation subtypes were found among 16 strains: Gly406Asp and Gly406Ala. MyKrobe predicted all strains of either subtype to be ethambutol resistant. However, 12 of 16 strains appear phenotypically sensitive at 5 g/mL but exhibit variable resistance between 2-4 g/mL. Of these 12 strains, a newly described frameshift mutation in regulator embR (Gln258fs) was found in 9 strains. ConclusionsMutations in embB406 are associated with low-level ethambutol resistance currently undetectable by the critical concentration of 5 g/mL for ethambutol. Novel mutations are predicted to exacerbate variability in ethambutol resistance. We suggest amendment to molecular and phenotypic drug susceptibility testing to improve ethambutol DST sensitivity and specificity as well as concordance between rapid and gold standard methods.