The comparative superior in vitro activity of DW-224a was supported by the downward MIC distribution due to the weakened influence of alterations within target enzymes in ciprofloxacin-resistant Staphylococcus aureus. The MI$C_{50}$ for DW-224a was 4 $mu$g/mL, similar to that of gemifloxacin, 8-fold less than that of sparfloxacin and 16-over-fold less than that of ciprofloxacin. We constructed combinations of amino acid changes, located at codon 80, 83 or 84 within GrlA and 84, 85 or 88 within GyrA, which were associated with MIC increase. The amino acid changes were less influential to the MIC of DW-224a compared to those of other fluoroquinolones, and it was verified from the requirement of a total of two GrlA- and two GyrA-alterations to reach the MIC of DW-224a over 32 $mu$g/mL.