Ziyuglycoside II potentiates the antibacterial activity of tetracycline against multidrug-resistant Staphylococcus aureus

Multidrug-resistant Staphylococcus aureus (MDR-SA) poses a critical challenge in both clinical and livestock settings, where conventional antibiotics are increasingly ineffective. To explore alternative strategies, we screened 223 compounds and identified ziyuglycoside II as a promising candidate. Its antibacterial and synergistic effects were evaluated using minimum inhibitory concentration (MIC) and checkerboard assays, biofilm inhibition and clearance tests, and mechanistic analyses of membrane integrity, reactive oxygen species (ROS) generation, membrane potential, and ATP synthesis. Biosafety was assessed through hemolysis and mammalian cytotoxicity assays, and therapeutic potential was examined in Galleria mellonella and mouse endometritis models. Ziyuglycoside II exhibited moderate activity (MIC =32 µg/mL) but showed strong synergy with tetracycline (fractional inhibitory concentration index 0.25-0.375), effectively suppressing resistant and susceptible strains. Time-kill assays revealed a reduction of up to 5 log₁₀ in bacterial counts with the combination. The pairing also inhibited biofilm formation, eradicated mature biofilms, and produced markedly thinner, fragmented biofilm structures under confocal microscopy. Mechanistic studies have demonstrated that ziyuglycoside II potentiates tetracycline by disrupting membrane integrity, inducing ROS accumulation, depolarizing the membrane potential, and impairing ATP synthesis. Importantly, no hemolytic or cytotoxic effects were observed at active concentrations. In vivo, the combination outperformed monotherapy by improving host survival, reducing uterine bacterial burden, and alleviating inflammatory responses. Together, these findings establish ziyuglycoside II as a safe and effective tetracycline potentiator, providing a promising strategy to combat MDR S. aureus infections.