Mutations in the ubiA gene are the major mechanism of ethambutol resistance in Mycobacterium avium

Mycobacterium avium-intracellulare complex (MAC) infections are the most common nontuberculous mycobacterial (NTM) 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 M. avium remain poorly understood. This study determined the minimum inhibitory concentration (MIC) on 40 M. avium 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 ubiA gene, which encodes DPPR (decaprenylphosphoryl-β-D-5-phosphoribose) synthase - an enzyme involved in cell wall biosynthesis that has been associated with high-level EMB resistance in M. tuberculosis . Complementation with the wild-type ubiA gene restored EMB susceptibility in EMB-resistant mutants and clinical strain 322 (EMB MIC=64 μg/mL) with ubiA mutation, reducing the MICs from 32 μg/mL to 4 μg/mL and from 64 μg/mL to 8 μg/mL, respectively. This study indicates that the ubiA mutation is the major mechanism of EMB resistance in M. avium , which should facilitate the development of molecular tests for rapid detection of resistance in this organism.