Revised Title - Quercetin Combined with Ciprofloxacin and Gentamicin Inhibits Biofilm Formation and Virulence in Staphylococcus aureus

Biofilm formation, extracellular substance synthesis, and virulence factor production all have a major impact on drug tolerance and infection propagation caused by Staphylococcus aureus. Flavonoid compounds have been explored as potential solutions to enhance antibiotic efficacy against the biofilm formation of pathogenic microbes. Quercetin (QER) has previously demonstrated antibacterial and antibiofilm properties. This study examines the potential of QER on enhancing the antibacterial, antibiofilm, and antivirulent potential of conventional antibiotics gentamicin (GEN), and ciprofloxacin (CIP) and aims to decipher the underlying mechanisms of action. Our research demonstrates that combining QER with GEN or CIP enhances their antibacterial activity, disrupts S. aureus cell membrane integrity, and increases reactive oxygen species production, leading to enhanced bacterial cell lysis. Furthermore, the combinatorial effect of QER with sub-MIC of GEN and CIP markedly inhibits biofilm formation, reduces viable cell counts, and diminishes the extracellular matrix components. The inhibition of biofilm after combinatorial treatment is confirmed through fluorescence microscopy and scanning electron microscopy. The study also found that QER-antibiotics combinations strongly reduce virulence characteristics in S. aureus, (spreading ability, protease, and hemolysin production) controlled by global key regulatory factors AgrA and SarA.Gene expression analysis revealed down regulation of key regulatory genes (sarA and agrA) and the virulence gene (hla). Molecular docking experiments have revealed the interaction between QER and the quorum sensing regulatory proteins SarA and AgrA, predicting another possible mechanism by which QER improves the anti-biofilm and antivirulence efficacy of GEN and CIP. Collectively, our findings indicate that QER enhances the efficacy of GEN and CIP antibiotics in reducing the antibiofilm and virulent characteristics of S. aureus, highlighting its potential as a broad-spectrum strategy for controlling S. aureus pathogenicity.