Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen that forms biofilms and produces virulence factors via quorum sensing (QS). Blocking the QS system in P. aeruginosa is an excellent strategy to reduce biofilm formation and the production of virulence factors. RhlR plays an essential role in the QS system of P. aeruginosa. We synthesized 55 analogues based on the chemical structure of 4-gingerol and evaluated their RhlR inhibitory activities using the cell-based reporter strain assay. Comprehensive structure–activity relationship studies identified the alkynyl ketone 30 as the most potent RhlR antagonist. This compound displayed selective RhlR antagonism over LasR and PqsR, strong inhibition of biofilm formation, and reduced production of virulence factors in P. aeruginosa. Furthermore, the survival rate of Tenebrio molitor larvae treated with 30 in vivo greatly improved. Therefore, compound 30, a pure RhlR antagonist, can be utilized for developing QS-modulating molecules in the control of P. aeruginosa infections.

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor-mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis-2-decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.

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