Naringenin as a Potent Natural Biofilm Inhibitor of Pseudomonas aeruginosa in Diabetic Foot Ulcers Through lasR Competitive Inhibition

Chronic non-healing foot ulcers are a major complication in diabetic patients, contributing to significant morbidity and mortality. Microorganisms in these wounds form biofilms, conferring greater virulence and enhanced protection from antibiotics. Hence, we examined naringenin, and other natural compounds like chlorofuranone, 4-nitropyridine N-oxide, and quercetin as a positive control against the major pathogenic organism that forms biofilm in foot ulcers. Here, we focused on Pseudomonas aeruginosa, which produces high levels of biofilm in diabetic foot ulcers. Naringenin (47.10 µg/ml for PA21; 124.7 µg/ml for PA333) and other natural compounds were tested for their ability to inhibit biofilm formation and virulence in vitro, and their effect on biofilm-associated gene expression was studied. The biofilm inhibitory mechanism of naringenin was elucidated using in silico analysis and in vitro reporter gene assay. In vitro biofilm assays, confocal and scanning electron microscopy showed that natural compounds effectively inhibited biofilm, without causing cell death. Treatment with these compounds significantly altered the expression of genes associated with quorum sensing in P. aeruginosa, such as lasR, pslA, algA, gacS, and pelA. Naringenin decreased the production of major virulence factors in P. aeruginosa. Molecular docking showed that naringenin exhibited the strongest binding affinity to LasR, and the same was validated by reporter gene assay using plasmid pSB1142 indicating its role as a competitive inhibitor in the las quorum sensing system in P. aeruginosa. The findings of this study could be extrapolated to in vivo diabetic wound infection models to help optimize the use of naringenin in effective biofilm control for better wound management in diabetic patients.