Wound infection continues to pose a significant challenge in the field of wound healing. Multi-drug resistant bacteria (MDR) make it even more complicated. Therefore, there is a pressing need to fabricate wound dressings with enhanced antimicrobial activity, particularly against MDR strains. In this study, we aimed to incorporate three antimicrobial nanoparticles into a chitosan-based nanofibrous mat (CS) and assess their antimicrobial activity against MDR bacteria. We fabricated CS mats incorporated with silver nanoparticles (CS-nAg), CuO nanoparticles (CS-nCuO), ZnO nanoparticles (CS-nZnO), a combination of Ag and CuO nanoparticles (CS-nAC), and a combination of Ag, CuO, and ZnO nanoparticles (CS-nACZ). The antimicrobial activity of these mats against MDR clinical isolates of four common wound bacteria including Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Acinetobacter baumannii were investigated. Each strain was tested using 10 isolates. According to in-vitro study, mats incorporated with a single type of nanoparticle demonstrated antimicrobial activity against certain isolates of P. aeruginosa, S. aureus, and E. coli. However, the antimicrobial activity was enhanced when multiple types of nanoparticles were combined (CS-nAC and CS-nACZ). Notably, CS-nACZ was the only mat that demonstrated antimicrobial activity against A. baumannii. In addition to its superior antimicrobial activity, CS-nACZ did not exhibit cytotoxicity towards fibroblasts. In an animal study using an infected wound rat model, the CS-nACZ mat demonstrated superior antimicrobial activity and excellent wound regeneration ability. These results suggest that the use of multiple nanoparticles in CS based wound dressing can lead to enhanced antimicrobial effects and effective wound regeneration.

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