Berberine-ZnO loaded chitosan nanoparticles inhibits biofilm formation in Pseudomonas aeruginosa PAO1 through targeting extracellular polymeric substances

Biofilm formation is an important strategy for coping with antimicrobial agents and worsening bacterial infections. In this investigation, we synthesized berberine-zinc oxide-loaded chitosan nanoparticles (CS-ZnO-Ber NPs) and explored their potential as anti-biofilm agents against Pseudomonas aeruginosa PAO1. CS-ZnO-Ber NPs exhibited an amorphous structure with particle sizes ranging from 34.7 to 407.7 nm and a zeta potential of − 38.5 mV. The biofilm inhibitory activity of the fabricated NPs was validated using a crystal violet staining assay. Based on the SEM imaging, cells treated with NPs displayed thin biofilms with low aggregation, whereas the untreated group exhibited robust biofilms with dense architecture. Moreover, at sub-inhibitory concentration of CS-ZnO-Ber NPs, the production levels of extracellular matrix components, including exopolysaccharides, pellicle, and alginate, decreased by 92.81 ± 0.84%, 81.45 ± 3.1%, and 31.74 ± 2.91%, respectively. Additionally, exposure to CS-ZnO-Ber NPs resulted in a significant reduction in pyocyanin production and attenuation of bacterial motility, including swimming, swarming, and twitching. CS-ZnO NPs and CS-ZnO-Ber NPs also decreased the EtBr MIC four-fold and eight-fold, respectively, indicating their efflux pump–inhibitory activity. Furthermore, molecular docking simulations revealed a favorable binding orientation of berberine with biofilm-associated proteins (AlgD, PelD, and PslG), as well as with quorum-sensing regulators LasI and LasR. Overall, CS-ZnO-Ber NPs substantially diminished extracellular matrix components, leading to the formation of weak and unstable biofilms. Pending further investigation, these findings suggest that CS-ZnO-Ber NPs hold promise as effective agents for controlling biofilm-related infections caused by P. aeruginosa.