Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Abstract

Mycobacterium avium, a member of the M. avium complex (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) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that 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 IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- 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 the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.