Aws Nasser, Khalid H Alobaidi, Jameel R Al-Obaidi
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated mortality, with multidrug resistance and biofilm formation limiting treatment options. Anti-virulence strategies targeting adhesin gene expression offer a promising alternative, yet the ability of green-synthesised ZnO-NPs to suppress MRSA virulence remains underexplored. In this study, ZnO-NPs were synthesised using Dodonaea viscosa leaf extract, with D-Fructose-3-O-methyl identified as the dominant phytochemical. Nanoparticles were characterised by UV-Visible spectroscopy, FTIR, SEM, EDS, and AFM. Antibacterial and anti-biofilm activities were evaluated against a clinically isolated MDR MRSA strain (VITEK 2 confirmed; resistant to 15 antibiotic agents) using MIC determination and crystal violet microplate biofilm assay. Virulence gene expression (fnbA, cna) was quantified by RT-qPCR using the 2⁻ΔΔCt method with 16 S rRNA as a reference gene. ZnO-NPs exhibited a characteristic UV absorption at 321 nm and an AFM mean diameter of 34.95 nm. EDS confirmed Zn (28.94%) and O (22.54%) as principal elements, with residual C and Cl attributable to phytochemical surface capping and precursor, respectively. The MIC of ZnO-NPs against MRSA was 4.312 mg/mL - a 7.5-fold improvement over crude D. viscosa extract (32.25 mg/mL). Anti-biofilm inhibition reached 41% and 58% for two clinical isolates at sub-MIC concentrations. RT-qPCR revealed significant downregulation of fnbA (fold change 0.37; 63.4% reduction) and cna (fold change 0.46; 54.0% reduction) relative to untreated controls. These findings demonstrate that D. viscosa-mediated ZnO-NPs combine direct antibacterial activity with transcriptional suppression of MRSA adhesin-encoding virulence genes at sub-lethal concentrations, offering a promising anti-virulence platform with reduced potential for resistance selection compared with conventional bactericidal agents.