Unveiling the mechanistic aspect of biogenic copper oxide nanoparticles harnessing Citrus pseudolimon for eradicating multi-drug-resistant gram-negative bacilli

Objective: Bacterial infections caused by multidrug-resistant (MDR) strains pose a serious global health threat. This study aimed to evaluate the antibacterial efficacy of green-synthesized copper nanoparticles (G–CuNPs) against MDR strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella spp., and Escherichia coli. Significance: Emerging MDR pathogens necessitate the development of novel, eco-friendly alternatives. G–CuNPs synthesized using Citrus pseudolimon peel extract may offer a biocompatible and sustainable approach to combating MDR infections. Methodology: Clinical bacterial isolates were obtained from diagnostic specimens including urine, pus, wound swabs, sputum, and blood collected from hospitalized patients at a tertiary care hospital. Bacterial identification and antimicrobial susceptibility testing were performed using the VITEK 2 automated system. Phenotypic detection of metallo-β-lactamase (MBL) and extended-spectrum β-lactamase (ESBL) production was conducted. G–CuNPs were synthesized and characterized for physicochemical properties. Antibacterial activity was assessed using a CFU-based time-kill assay. Mechanistic studies included evaluation of cell membrane integrity, reactive oxygen species (ROS) generation, and DNA degradation. Interaction with bacterial enzymes was analyzed via molecular docking. Hemolytic and cytotoxicity assays were performed to assess biocompatibility. Results: G–CuNPs (10 mg/mL) displayed potent antibacterial activity by disrupting cell membranes, inducing ROS accumulation, and degrading bacterial DNA. Molecular docking confirmed strong binding affinities to key bacterial enzymes. Compared to chemically synthesized CuNPs, G–CuNPs (Indian Patent No. 202111048797) exhibited minimal hemolytic and cytotoxic effects. Conclusion: G–CuNPs demonstrate promising antibacterial potential and biocompatibility, highlighting their applicability in biomedical domains such as implant coatings and wound care. Further in vivo studies are warranted to validate their clinical utility.