## 3 Discussion
Taken together, our findings in this study suggest that diosgenin reduces persister cell formation through multiple mechanisms involving metabolic suppression, stringent response inhibition, and membrane modulation: The substantial reductions in _relP_ and _relQ_ expression, coupled with ATP depletion, indicate that diosgenin primarily targets the metabolic pathways essential for persister cell survival. The observed decrease in membrane fluidity, without concomitant increases in membrane permeability or significant changes in membrane potential, suggests that diosgenin may selectively affect membrane-associated signaling proteins involved in stress response pathways. This may contribute to its anti-persister activity by altering the functional dynamics of membrane-bound receptors or enzymes. However, the temporal limitation of diosgenin's anti-persister effects, specifically its reduced efficacy 6 h after exposure, suggests that its inhibitory activity may be most effective during the early phase. This observation highlights the need for further studies to determine whether combining diosgenin with other metabolic inhibitors or membrane-targeting agents can prolong its anti-persister effects for extended periods. Although our findings were derived from in vitro experiments, they provide a mechanistic foundation for future in vivo evaluations. Assessing diosgenin's bioavailability, toxicity, and therapeutic efficacy in animal models of persistent infection will be essential to determine its translational potential. Several factors may contribute to the diminished efficacy of diosgenin after 6 h of exposure. First, diosgenin may undergo chemical degradation or reduced bioavailability in the culture medium over time, which could weaken its biological activity. Second, bacterial adaptation mechanisms such as the upregulation of efflux pumps or changes in membrane composition may reduce cellular sensitivity to diosgenin during prolonged exposure. Third, once _S. aureus_ enters the stationary phase, the established persister cells may possess robust metabolic and structural defenses that render them intrinsically less susceptible to interventions targeting earlier phases of dormancy initiation. These possibilities merit further investigation and underscore the need for kinetic studies of diogenin stability and bacterial adaptive responses over extended treatment periods. Despite this study's promising findings, it has limitations that warrant further investigation. First, all our experiments were conducted in vitro, and the potential effects of diosgenin in vivo should be explored. Additionally, though we identified reductions in (p)ppGpp synthase gene expression and ATP levels, the precise molecular interactions between diosgenin and metabolic enzymes remain unclear. Future studies should focus on elucidating the molecular targets of diosgenin within the stringent response and metabolic pathways, as well as assessing its effects in infection models. Moreover, exploring potential synergies between diosgenin and conventional antibiotics may provide valuable insights into its clinical applicability as an anti-persister agent in chronic and recurrent infections.