Abstract
Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen frequently associated with chronic and hard-to-treat infections, particularly in burn units and immunocompromised patients. Its intrinsic and acquired resistance to multiple antibiotics poses a major therapeutic challenge. While ZnO nanoparticles conjugated with thiosemicarbazone (TSC) have shown promise in general antimicrobial applications, their potential for simultaneously inhibiting biofilm formation and pyocyanin production-key virulence factors-in clinical P. aeruginosa strains remains unexplored. In this study, ZnO nanoparticles were synthesized via a hydrothermal route and conjugated with a glutamine-modified TSC ligand (ZnO@Glu-TSC) to enhance their antimicrobial efficacy. The nanoconjugate was comprehensively characterized using UV-Vis spectroscopy, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). Functional evaluations were conducted against clinical isolates of P. aeruginosa, including minimum inhibitory concentration (MIC), fractional inhibitory concentration (FIC) index, biofilm inhibition, and pyocyanin suppression assays. ZnO@Glu-TSC nanoparticles exhibited a sharp UV-Vis absorption peak at 380 nm with a band gap of 3.26 eV, and XRD confirmed a hexagonal wurtzite structure with an average crystallite size of ~ 19.8 nm. The nanoconjugate demonstrated significantly enhanced antibacterial activity with MIC values ranging from 128 to 512 µg/mL and synergistic effects in 70% of clinical isolates (FIC ≤ 0.5, p < 0.01). Biofilm inhibition assays revealed an 80% reduction in biomass (OD values approaching those of the negative control), while pyocyanin production decreased by more than 75% at 512 µg/mL (p < 0.001). These results represent the first demonstration of ZnO@Glu-TSC's dual antivirulence action against clinical P. aeruginosa strains, underscoring its therapeutic promise as a potent, multi-targeted nanoantimicrobial candidate and warranting further development for translational nanomedicine applications in combating persistent infections.