show Abstracthide AbstractSerial passaging was used to generate S. aureus isolates exhibiting an increased IMP-1700 minimum inhibitory concentration (MIC). Whole-genome sequencing (WGS) revealed that these isolates had a single mutation in GyrA. Subsequently, in silico docking predicted that IMP-1700 interacts with a second target in the GyrB subunit. Because zoliflodicin targets GyrB, S. aureus was passaged with zoliflodicin to generate isolates with GyrB substitutions, which were confirmed by WGS.