Mutations in <i>emb</i> B406 are associated with low-level ethambutol resistance in Canadian <i>Mycobacterium tuberculosis</i> isolates

Abstract Background In Mycobacterium tuberculosis , molecular predictions of ethambutol resistance rely primarily on the detection of mutations within emb B. However, discordance between emb B406 mutations and phenotypic drug sensitivity questions its clinical significance. This study aims to decipher the association of emb B406 mutations with ethambutol resistance in M. tuberculosis . Methods All M. tuberculosis isolates from our culture collection containing emb B406 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 μg/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). Results Two emb B406 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 μg/mL but exhibit variable resistance between 2-4 μg/mL. Of these 12 strains, a newly described frameshift mutation in regulator embR (Gln258fs) was found in 9 strains. Conclusions Mutations in emb B406 are associated with low-level ethambutol resistance currently undetectable by the critical concentration of 5 μg/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.