Honey-biosynthesized silver nanoparticles against vancomycin-resistant enterococci: integrated antibacterial, antibiofilm, mechanistic and preliminary safety evaluation

Vancomycin-resistant enterococci (VRE), including Enterococcus faecalis and Enterococcus faecium , are multidrug-resistant pathogens with limited treatment options. This study evaluated honey-biosynthesized silver nanoparticles (AgNPs) against VRE using integrated antibacterial, antibiofilm, mechanistic, and preliminary safety assessments. Methods: AgNPs were synthesized using natural honey as a reducing and capping agent and characterized by UV-visible spectroscopy, dynamic light scattering, zeta potential analysis, SEM-EDS, FTIR, and stability assays. Antibacterial activity was assessed using MIC, MBC, agar well diffusion, and time-kill assays. Antibiofilm activity was evaluated by crystal violet assay. Mechanistic responses were examined by molecular docking, RT-qPCR analysis of pbp5 and srtA , PBP5 Bocillin-FL binding, and Sortase A activity assays. Preliminary safety was assessed using HEK293 and HepG2 cell viability, LDH release, and erythrocyte hemolysis assays. Results: AgNPs showed MICs of 16 µg/mL against E. faecalis and 32 µg/mL against E. faecium , with MBCs of 32–64 µg/mL and inhibition zones of 14–18 mm. Time-kill assays showed > 5 log10 CFU/mL reduction for E. faecalis and approximately 4 log10 CFU/mL reduction for E. faecium after 24 h. AgNPs had a hydrodynamic diameter of 18 ± 3 nm, PDI of 0.24 ± 0.03, and zeta potential of -32.5 ± 2.1 mV. Biofilm inhibition ranged from 61.4 to 86.5%. AgNP exposure downregulated pbp5 and srtA , reduced PBP5 labelling, and decreased Sortase A activity. Cell viability remained ≥ 81%, and hemolysis was < 3%. Conclusion: Honey-biosynthesized AgNPs demonstrated bactericidal and antibiofilm activity against VRE with mechanism-linked effects and acceptable preliminary in vitro tolerance at antibacterial concentration ranges.