## 2 Results
### Effect of Diosgenin on Persister Cell Formation  
The effect of diosgenin on _S. aureus_ persister cell formation was evaluated under antibiotic stress induced by oxacillin, ciprofloxacin, and gentamicin at 10 \(\times\) minimum inhibitory concentration (MIC) (Figure 2). Persister cell survival was quantified as colony  
Figure 1: Effect of diosgenin on _Staphylococcus aureus_ growth: growth curves of _S. aureus_ cultures treated with ethanol (\(\blacksquare\); control) or 80 uM (\(\blacklozenge\)) or 160 uM (\(\blacktriangle\)) diosgenin over a 12 h period. Bacterial growth was monitored by measuring absorbance at 600 nm (Abs\({}_{600}\)). Data are presented as means \(\pm\) the standard deviation of three independent experiments. forming units (CFU), evaluated as the percentage of surviving cells relative to the initial CFU count, with antibiotics administered 0, 1, 3, or 6 h after the diosgenin pretreatment. In the control group, pretreated with ethanol instead of diosgenin only, the number of persister cells remained relatively stable when antibiotics were administered during the first 3 h after the ethanol treatment, regardless of the antibiotic used. However, a substantial increase in persister cell formation was observed when antibiotics were administered after 6 h, with persister cell counts increasing 21-, 13-, and 7-fold between the treatments at hours 3 and 6 for oxacillin, ciprofloxacin, and gentamicin, respectively. This surge in persister cell populations coincides with the transition of _S. aureus_ cultures into the stationary phase (Figure 1), indicating that persister cell formation may be associated with metabolic downregulation during growth arrest. Pretreatment with 80 uM and 160 uM diosgenin significantly reduced persister cell formation at the 3 h time point across all antibiotic treatments. Under oxacillin stress, the persister cell fraction in the control group was 0.0919%, but pretreatment with 80 uM and 160 uM diosgenin reduced the persister cell fraction to 0.015% and 0.0104%, representing 83% and 89% reductions, respectively. A similar inhibitory effect was observed under ciprofloxacin treatment. In the control group, the persister cell fraction was 0.095%, while pretreatment with 80 uM diosgenin decreased it to 0.0169%, and pretreatment with 160 uM diosgenin further reduced it to 0.0128%, corresponding to reductions of 82% and 87%, respectively. In cultures treated with gentamicin, persister cell formation was also markedly reduced by diosgenin pretreatment. The control group exhibited a persister cell fraction of 0.187%, which was reduced to 0.0271% and 0.0108% in the 80 uM and 160 uM diosgenin-treated cultures, respectively. These reductions correspond to decreases of 85% and 94%, respectively, indicating that diosgenin effectively suppresses persister cell formation 3 h after it is applied across different antibiotic treatments. At the 6 h time point, persister cell fractions in the diosgenin-treated groups began to increase, aligning with the trend observed in the control group. This increase coin  
Figure 2: Effects of diosgenin on persister cell survival in _Staphylococcus aureus_ under antibiotic stress. The survival of persister cells was assessed in _Staphylococcus aureus_ cultures pretreated with ethanol (�❝E; control) or 80 uM (•) or 160 uM (•) diosgenin dissolved in ethanol after exposure to oxacillin (**A**), ciprofloxacin (**B**), or gentamicin (**C**) for 24 h, with antibiotic treatments administered immediately (0 h) or 1, 3, or 6 h after diosgenin exposure (x-axis).