<abstract> <p>Antibiotic-resistant strains of <italic>Pseudomonas aeruginosa (P. aeruginosa</italic>) pose a major threat for healthcare-associated and community-acquired infections. <italic>P. aeruginosa</italic> is recognized as an opportunistic pathogen using quorum sensing (QS) system to regulate the expression of virulence factors and biofilm development. Thus, meddling with the QS system would give alternate methods of controlling the pathogenicity. This study aimed to assess the inhibitory impact of chitosan nanoparticles (CS-NPs) on <italic>P. aeruginosa</italic> virulence factors regulated by QS (e.g., motility and biofilm formation) and <italic>LasI</italic> and <italic>RhlI</italic> gene expression. Minimum inhibitory concentration (MIC) of CS-NPs against 30 isolates of <italic>P. aeruginosa</italic> was determined. The CS-NPs at sub-MIC were utilized to assess their inhibitory effect on motility, biofilm formation, and the expression levels of <italic>LasI</italic> and <italic>RhlI</italic> genes. CS-NPs remarkably inhibited the tested virulence factors as compared to the controls grown without the nanoparticles. The mean (±SD) diameter of swimming motility was decreased from 3.93 (±1.5) to 1.63 (±1.02) cm, and the mean of the swarming motility was reduced from 3.5 (±1.6) to 1.9 (±1.07) cm. All isolates became non-biofilm producers, and the mean percentage rate of biofilm inhibition was 84.95% (±6.18). Quantitative real-time PCR affirmed the opposition of QS activity by lowering the expression levels of <italic>LasI</italic> and <italic>RhlI</italic> genes; the expression level was decreased by 90- and 100-folds, respectively. In conclusion, the application of CS-NPs reduces the virulence factors significantly at both genotypic and phenotypic levels. These promising results can breathe hope in the fight against resistant <italic>P. aeruginosa</italic> by repressing its QS-regulated virulence factors.</p> </abstract>
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