Nano-Phyto Synergy: Zinc Oxide and Silver Nanocomposites Combined with Regional Phytometabolites Combat Multispecies Biofilms

Abstract

Multispecies biofilms represent a clinically and environmentally significant challenge owing to their heightened resistance to conventional antimicrobials and the synergistic interplay among constituent microbial species. The present study investigated the combinatorial antibiofilm potential of nanostructured surface-active synthetic surfactants — specifically sodium dodecyl sulfate-functionalized zinc oxide nanoparticles (SDS-ZnO NPs) and cetyltrimethylammonium bromide-coated silver nanoparticles (CTAB-AgNPs) — along with bioactive plant-derived secondary metabolites extracted from Azadirachta indica (neem), Ocimum sanctum (tulsi), and Terminalia chebula (harad), all collected from environmentally distinct sampling sites across Raipur, Chhattisgarh, India. Biofilm-forming organisms including Staphylococcus aureus (MTCC 96), Pseudomonas aeruginosa (MTCC 1688), Candida albicans (MTCC 227), and Escherichia coli (MTCC 443) were isolated, characterised, and subjected to quantitative biofilm assays using the 96-well microtiter crystal violet method. Nanoparticles were synthesised and characterised by UV-visible spectrophotometry, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and dynamic light scattering (DLS). Plant extracts were profiled by gas chromatography-mass spectrometry (GC-MS) and evaluated for total phenolic content (TPC) and total flavonoid content (TFC). Minimum biofilm inhibitory concentration (MBIC₅₀) values ranged from 3.91 to 62.5 μg/mL for SDS-ZnO NPs, 1.95 to 31.25 μg/mL for CTAB-AgNPs, and 62.5 to 500 μg/mL for plant extracts. Synergistic combinations of CTAB-AgNPs with T. chebula extract demonstrated fractional inhibitory concentration index (FICI) values ≤ 0.5, indicating potent synergism against all tested species. Confocal laser scanning microscopy (CLSM) confirmed architectural disruption of mature biofilms. Cytotoxicity screening via MTT assay demonstrated acceptable safety profiles at sub-inhibitory concentrations. These findings establish a mechanistic basis for deploying nanostructured surface-active molecules combined with phytochemicals as next-generation, broad-spectrum antibiofilm agents for clinical, industrial, and environmental applications.