Inhibitory effects of zinc oxide nanoparticles and morin on virulence-associated gene expression in Staphylococcus aureus

Objective: The emergence of multidrug-resistant Staphylococcus aureus has prompted interest in antivirulence strategies that attenuate pathogenicity without exerting bactericidal pressure. This study evaluated the inhibitory effects of zinc oxide nanoparticles (ZnO-NPs) and the flavonoid morin, individually and in combination, on transcriptional regulation of key S. aureus virulence genes. Methods: ZnO-NPs were synthesised and characterised using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The reference strain S. aureus ATCC 25923 and multidrug-resistant clinical isolates were employed. Minimum inhibitory concentrations (MICs) were determined by broth microdilution following CLSI guidelines. Sub-MIC concentrations of ZnO-NPs, morin, and their combination were used to evaluate transcriptional changes in eta, seb, agrA, sarA, and RNAIII by RT–qPCR, with 16S rRNA as a housekeeping control. Results: ZnO-NPs demonstrated high crystallinity and nanoscale morphology (70–92 nm). At sub-MIC concentrations, ZnO-NPs significantly suppressed seb (0.12-fold) and eta (0.42-fold). Morin strongly inhibited seb (0.05-fold) but paradoxically induced eta (6.82-fold). Ciprofloxacin also upregulated eta (2.97-fold). The ZnO-NPs + morin combination neutralised morin-induced eta upregulation (3.01-fold) and produced complete suppression of agrA transcripts, with marked inhibition of sarA and RNAIII. Conclusions: ZnO-NPs and morin act synergistically to repress S. aureus virulence gene expression, particularly through suppression of global regulators and toxin genes. Unlike conventional antibiotics, this combination reduces pathogenicity without applying bactericidal selection pressure. These findings highlight ZnO-NP–flavonoid combinations as promising antivirulence therapeutics, warranting further in vivo and translational studies.