Biofilm inhibition by silver nanoparticles produced from Stachys spectabilis

Green synthesis of silver nanoparticles (AgNPs) has emerged as an environmentally friendly approach for developing novel antimicrobial materials. Although several Stachys species have been investigated for their pharmacological properties, the antibacterial and antibiofilm activities of AgNPs synthesized from Stachys spectabilis have not previously been evaluated. Therefore, this study aimed to investigate the antimicrobial and antibiofilm potential of Stachys spectabilis-derived AgNPs against clinically relevant biofilm-forming bacterial pathogens.

AgNPs were fabricated through an aqueous green synthesis approach and characterized using standard analytical techniques. Antibacterial activity was assessed via liquid microdilution to determine minimum inhibitory concentrations (MICs) against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Antibiofilm activity was quantified using the crystal violet assay in 96-well microplates.

Disc diffusion results revealed measurable inhibition zones only in E. coli and P. aeruginosa (6 mm), corresponding largely to the disc diameter and indicating limited nanoparticle diffusion in agar. In contrast, MIC and MBC analyses demonstrated a clear concentration-dependent antibacterial effect: S. aureus showed the highest susceptibility with a MIC of 1024 µg/mL, while the remaining strains exhibited MICs of 2048 µg/mL. Consistently higher MBC values confirmed the inhibitory-to-bactericidal threshold. AgNPs also displayed substantial antibiofilm activity, achieving biomass reductions of 59.7% in P. aeruginosa, 57.0% in E. coli, 54.9% in S. aureus, and 40.1% in K. pneumoniae. Overall, the results demonstrate that although AgNPs exhibit limited diffusion in solid media, they possess concentration-dependent antibacterial and antibiofilm activity in liquid media.

The synthesized silver nanoparticles demonstrated substantial antibacterial and antibiofilm efficacy despite their limited diffusion in solid media. While disc diffusion assays produced minimal inhibition zones, MIC and MBC analyses revealed concentration-dependent inhibitory and bactericidal effects, with S. aureus showing the greatest susceptibility. Additionally, significant reductions in biofilm biomass across all tested species indicate that AgNPs not only inhibit planktonic bacterial growth but also effectively disrupt established biofilms. These findings suggest that AgNPs hold considerable potential as alternative antimicrobial agents, particularly in applications where both planktonic and biofilm-associated bacterial forms must be controlled. To the best of our knowledge, this is the first study to demonstrate the antibacterial and antibiofilm activities of Stachys spectabilis-derived AgNPs, highlighting their potential as sustainable antimicrobial agents for the control of biofilm-associated bacterial infections.