Background: Quorum sensing (QS) controls the virulence of P. aeruginosa. This study aims to determine the anti-QS activity of aspirin alone and in combination with chitosan to reach maximum inhibition. We tested ten virulent Pseudomonas aeruginosa (P. aeruginosa) isolates and screened for N-acyl homoserine lactone (AHL) production using Agrobacterium tumefaciens as a biosensor. P. aeruginosa isolates were treated with sub-minimum inhibitory concentrations (MICs) of aspirin and chitosan–aspirin. We used broth microdilution and checkerboard titration methods to determine the MICs and the synergistic effect of these two compounds, respectively. Real-time polymerase chain reaction (PCR) was used to estimate the anti-QS activity of the aspirin–chitosan combination on the expression of lasI and rhlI genes. Results: Aspirin decreased the motility and production of AHLs, pyocyanin, and biofilm. Chitosan potentiated the inhibitory effect of aspirin. The chitosan–aspirin combination inhibited lasI and rhlI gene expression in PAO1 (ATCC 15692) by 7.12- and 0.92-fold, respectively. In clinical isolates, the expression of lasI and rhlI was decreased by 1.76 × 102- and 1.63 × 104-fold, respectively. Molecular docking analysis revealed that aspirin could fit into the active sites of the QS synthases lasI and rhlI with a high binding affinity, causing conformational changes that resulted in their inhibition. Conclusions: The chitosan–aspirin combination provides new insights into treating virulent and resistant P. aeruginosa.
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