Mutations in the <i>ubiA</i> gene are the major mechanism of ethambutol resistance in <i>Mycobacterium avium</i>

<i>Mycobacterium avium-intracellulare</i> complex (MAC) infections are the most common nontuberculous mycobacterial infections, and ethambutol (EMB) is one of the main therapeutic agents used to treat MAC infections. However, the EMB resistance profile and its resistance mechanisms in <i>M. avium</i> remain poorly understood. This study determined the minimum inhibitory concentration (MIC) of 40 <i>M. avium</i> clinical strains, revealing that 97.5% (39/40) of the strains were intermediate (MIC = 4 µg/mL) or resistant (MIC ≥ 8 µg/mL) to EMB. One susceptible clinical isolate strain 245 (MIC = 2 µg/mL) was picked for EMB-resistant mutant isolation, and a total of 121 resistant mutants were isolated and subjected to whole-genome sequencing or Sanger sequencing. Integrated analysis revealed that 94.21% (114/121) of the mutants carried mutations in the <i>ubiA</i> gene, which encodes decaprenylphosphoryl-β-D-5-phosphoribose (DPPR) synthase-an enzyme involved in cell wall biosynthesis that has been associated with high-level EMB resistance in <i>Mycobacterium tuberculosis</i>. Complementation with the wild-type <i>ubiA</i> gene restored EMB susceptibility in EMB-resistant mutants and clinical strain 322 (EMB MIC = 64 µg/mL) with <i>ubiA</i> mutation, reducing the MICs from 32 to 4 μg/mL and from 64 to 8 μg/mL, respectively. This study indicates that the <i>ubiA</i> mutation is the major mechanism of EMB resistance in <i>M. avium</i>, which should facilitate the development of molecular tests for rapid detection of resistance in this organism.IMPORTANCEThis study identified <i>ubiA</i> as a key gene associated with ethambutol (EMB) resistance in <i>Mycobacterium avium</i>, a finding which has not previously been reported. Furthermore, although <i>ubiA</i> has been linked to EMB resistance in <i>Mycobacterium tuberculosis</i> (MTB), mutations in this gene account for only a small proportion of EMB-resistant MTB isolates. In contrast, our study showed that <i>ubiA</i> played a major role in EMB resistance in <i>M. avium</i>. Our findings contribute to the development of molecular assays for rapid detection of EMB resistance in <i>M. avium</i> and highlight the distinct main resistance mechanisms across different bacterial species to the same antibiotic.