ABSTRACTMycobacterium avium, a member of theM. aviumcomplex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) ofgyrAofM. aviumis not yet clearly understood, as many FQ-resistant clinicalM. aviumisolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR ofM. aviumGyrA in the development of FQ resistance. We found four clinicalM. aviumsubsp.hominissuisisolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations ingyrAthat were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC5015- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC5012.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in thegyrAofM. aviumcontribute to FQ resistance.IMPORTANCEThe emergence of fluoroquinolone (FQ) resistance has further compounded the control of emergingMycobacterium avium-associated nontuberculous mycobacteria infections worldwide. ForM. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) ofgyrAis not yet clearly understood. Here, we report that four clinicalM. aviumisolates with a mutation in the QRDR ofgyrAwere highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performingin vitroFQ-inhibited DNA supercoiling assays. These results confirmed that, like inMycobacterium tuberculosis, mutations in the QRDR ofgyrAalso strongly contribute to FQ resistance inM. avium. Since many FQ-resistantM. aviumisolates do have these mutations, the detailed molecular mechanism of FQ resistance inM. aviumneeds further exploration.