Supplementary Material for: Topoisomerase Mutations That Are Associated with High-Level Resistance to Earlier Fluoroquinolones in <i>Staphylococcus aureus</i> Have Less Effect on the Antibacterial Activity of Besifloxacin

<i>Background:</i> The impact of mutations in DNA gyrase and topoisomerase IV on minimum inhibitory concentrations (MICs) was investigated to better understand why besifloxacin has a higher potency against <i>Staphylococcus aureus</i> when compared to other fluoroquinolones, which was especially pronounced against ciprofloxacin-resistant isolates. <i>Methods:</i> MICs were determined for 52 clinical isolates against besifloxacin, moxifloxacin, gatifloxacin, ciprofloxacin, and levofloxacin. The genes encoding GyrA, GyrB, ParC, and ParE were sequenced and the potential impact of mutations assessed in light of recent structural data. <i>Results:</i> For all fluoroquinolones tested, the MICs increased with the number of mutations in the quinolone resistance-determining regions. However, this increase was the smallest for besifloxacin and the largest for ciprofloxacin and levofloxacin. In addition to the commonly observed mutations in ParC and GyrA, more unusual mutations in ParE, such as Asp-432→His or Pro-585→Ser, were also detected. <i>Conclusions:</i> Compared to earlier fluoroquinolones, the higher potency of besifloxacin suggests that the drug’s unique combination of a 7-azepinyl ring and an 8-chloro-substituent results in unique interactions with DNA gyrase and topoisomerase IV.