Citrus flavonoids diosmin, myricetin and neohesperidin as inhibitors of Pseudomonas aeruginosa: Evidence from antibiofilm, gene expression and in vivo analysis.

Citrus flavonoids are group of bioactive polyphenols. Here, we investigated the potential of diosmin, myricetin and neohesperidin as possible inhibitors of Pseudomonas aeruginosa. This bacterium is a major clinical challenge due to its propensity to form resistant biofilm. The aims of this study were to examine flavonoids antibacterial activity using the microdilution method, assays intended to determine several antibiofilm mechanisms (crystal violet, congo red binding, extracellular DNA (eDNA) test and confocal laser scanning microscopy (CLSM) live/dead cell imaging), followed by virulence genes RT-qPCR analysis. Furthermore, we aimed to examine in vivo toxicity of the compounds as well as their efficacy in P. aeruginosa zebrafish embryo infection model. Minimal inhibitory concentrations of tested flavonoids towards P. aeruginosa were in range 0.05 - 0.4 mg/mL. A high potential of the compounds to disturb both the formation of the bacterial biofilm and its eradication was recorded, including significant reduction in biofilm biomass, exopolysaccharide and eDNA production. Biofilm treatment with diosmin resulted in the lowest percentage of live microbial cells as observed in the CLSM live/dead cell imaging. The lasI, pvdS, and rhlC genes were found to be downregulated in the presence of diosmin and myricetin. Only diosmin stood out as non-embryotoxic. Consequently, in vivo analysis using a zebrafish model of P. aeruginosa infection showed an antivirulence effect of diosmin. Our findings suggest that diosmin could be potential candidate for the development of new agent that target P. aeruginosa infections by reducing its virulence mechanisms.