Analysis of publicly available genomic data

Ethambutol (EMB) is a vital drug for treating Mycobacterium avium-intracellulare (MAI) infections; however, the genomic mutations underlying EMB resistance in MAI remain unclear. Herein, we evaluated eight sets of MAI clinical isolates, each containing at least two serial isolates collected from the same patient who received EMB in Japan. In four sets, the isolates independently increased EMB MIC by 4-fold, coinciding with mutations in the upstream region of embA or those corresponding to Mycobacterium tuberculosis (Mtb) embB Met306Val and Gln497Arg. Based on the increased EMB MIC values, we defined normal and elevated EMB MICs as [≤]8 {micro}g/mL and [≥]16 {micro}g/mL, respectively. In the other four sets, all of the isolates had elevated EMB MICs. In silico promoter prediction and expression analysis indicated that the upstream region of embA corresponds to the embA-embB promoter region, and mutations in this region increased the transcription of embA and embB, increasing EMB MICs. Furthermore, the analysis of 60 epidemiologically unrelated strains revealed that isolates with mutations in the embA-embB promoter and at embB codons 306/497 exhibited significantly higher EMB MICs compared with those without mutations. Publicly available genomic data demonstrate the worldwide occurrence of these mutations in clinical isolates. These results establish an association between elevated EMB MICs and mutations at embB codons 306/497 and the embA-embB promoter and are expected to predict EMB resistance.