Green-synthesized ZnO nanoparticles from endophytic fungi inhibit virulence exoenzymes and biofilm formation in Staphylococcus aureus

Abstract

The rapid emergence of multidrug-resistant Staphylococcus aureus (MRSA), specifically, methicillin-resistant strains, has become a global health concern. Biofilm-forming ability, enhanced virulence and resistance to multiple antibiotics have challenged treatment strategies. Therefore, immediate attention is necessary to design alternative therapeutic strategies including nanotechnology-based solutions, novel drug targets, and anti-virulence strategies, to combat this global public health threat. Findings of this study elucidates the binding interaction and inhibition potential of zinc oxide nanoparticles (ZnONPs) biosynthesized using an endophytic isolate obtained from Hibiscus rosa-sinensis and identified as Penicillium citrinum using morphology and ITS rRNA gene sequencing. The comprehensive characterization of biosynthesized ZnONPs was done using UV- visible spectrophotometry, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential analysis. XRD and TEM analyses revealed the ZnONPs with spherical to elliptical shape with mean size of ~58 nm. The FTIR analysis confirmed the functional groups responsible for stabilizing and capping of ZnONPs. The biosynthesized ZnONPs displayed colloidal stability with a mean hydrodynamic diameter of 240.4 nm with a ζ-potential of −13 mV, as revealed in Dynamic light scattering (DLS) studies. The anti-staphylococcal studies of ZnONPs revealed an excellent antibacterial activity (22 ± 1.42) with a minimum inhibitory concentration (MIC) of 1000 µg/mL and demonstrated time-dependent bactericidal effects as revealed in time-kill assay. In addition, ZnONPs inhibited biofilm formation in a concentration-dependent manner, with highest reduction (68.7% ± 1.12%) in biofilm biomass, as revealed in a dye-based assay, accompanied by suppression of metabolic activity in biofilm-embedded cells. Importantly, ZnONPs displayed excellent inhibitory activities against the key virulence-associated exoenzymes, including lipase (IC₅₀ ≈ 380 ± 1.12) and hyaluronidase (IC₅₀ ≈ 330 ± 1.12 µg/mL). The cytotoxicity assessment using human red blood cells (hRBCs) revealed negligible haemolytic activity (< 2%) at biologically effective concentrations, confirming their safety and hemocompatibility. Considering the green synthesis, biocompatible nature and outstanding anti-staphylococcal potential, the endophyte derived ZnONPs showcases their potential as a sustainable nanotechnology-based alternative to conventional antimicrobial strategies against biofilm-associated infections of S. aureus.