## 3 Discussion  
The present study provides new insights into the mechanisms by which diosgenin modulates persister cell formation in _S. aureus_. By systematically analyzing its effects on bacterial growth, persister cell formation, (p)ppGpp synthase gene expression, intracellular ATP levels, and membrane integrity, we have identified multiple pathways through which diosgenin exerts anti-persister activity. Diosgenin exhibited a dose-dependent inhibitory effect on bacterial growth, with 80 uM and 160 uM treatments reducing cell density by 9% and 12%, respectively (Figure 1). Figure 5: Effect of diosgenin on membrane permeability and fluidity in _Staphylococcus aureus_ cells. (**A**) Membrane permeability of _S. aureus_ cells treated with ethanol (; control) or 80 uM () or 160 uM () diosgenin, as measured by fluorescence intensity (arbitrary unit, AU). (**B**) Membrane fluidity of _S. aureus_ cultures treated with 80 uM or 160 uM diosgenin and untreated control cultures (none). Benzyl alcohol was used as a positive control for membrane fluidization. Data are presented as means \(\pm\) the standard deviation of three independent experiments. Statistically significant differences at the 99% confidence level (\(p<0.01\)) between the treatment means and the control mean are indicated by asterisks (***). Although the reductions were modest, they were statistically significant and indicate that diosgenin exerts a mild bacteriostatic effect rather than bactericidal action. This observation is consistent with previous reports identifying diosgenin as a metabolic modulator without direct bactericidal properties. The more pronounced effect of diosgenin was observed in its reduction in persister cell populations under antibiotic stress (Figure 2). Pretreatment with 80 uM and 160 uM diosgenin significantly decreased persister cell fractions across oxacillin, ciprofloxacin, and gentamicin treatments, with reductions ranging from 82% to 94% 3 h after diosgenin application. This reduction aligns with a previous study showing that metabolic disruption can effectively inhibit persister cell formation by preventing the metabolic shift to dormancy [27]. Interestingly, we found that persister cell inhibition was most pronounced during a relatively short period after diosgenin exposure (1-3 h), with the effect diminishing after 6 h, as cultures transitioned to the stationary phase. This trend suggests that diosgenin may prevent the initial establishment of persister cells but is less effective once persister cells are fully established, highlighting a temporal limitation in its anti-persister activity. The stringent response, mediated by (p)ppGpp, plays a pivotal role in persister cell formation by downregulating metabolic activity under stress conditions [28]. In this study, diosgenin treatments significantly reduced the expression of _relP_ and _relQ_, the two key synthases involved in (p)ppGpp synthesis (Figure 3). When cultured in the 80 uM diosgenin medium, _relP_ expression in _S. aureus_ cells decreased by 27%, while the 160 uM diosgenin treatment reduced _relP_ expression by 46%. The dose-dependent suppression of _relP_ is particularly relevant given its established role in (p)ppGpp synthesis. In contrast, _relQ_ expression exhibited a more substantial reduction in 80 uM diosgenin-treated cells (60%), with no further reduction when the concentration increased to 160 uM (55%). This suggests that _relQ_ expression is more sensitive to diosgenin and that a suppression threshold exists at or below 80 uM, beyond which increasing the diosgenin concentration does not significantly enhance inhibition. Such a plateau in _relQ_ suppression may indicate that other regulatory factors limit the extent of diosgenin-induced inhibition at higher concentrations. The plateau in _relQ_ suppression at 80 uM diosgenin, despite further increases in diosgenin concentration, may result from several regulatory dynamics.