Flavonoids interfere with biofilm formation by targeting diguanylate cyclases in multidrug resistant Vibrio cholerae

Rise of antibiotic resistance has led to increased treatment failures in severe cholera cases across the globe. The etiological agent Vibrio cholerae is known for its ability to persist in human gut and environmental settings through biofilm formation. Consequently, the targeting of biofilms is suggested as a viable alternative approach to address the pathogen alongside the rising challenge of drug resistance. Anti-biofilm efficacies of two flavonoids baicalein and fisetin were explored. Minimum biofilm inhibitory concentrations (MBIC) were 40 and 30 µg/mL and minimum biofilm eradication concentrations (MBEC) were 70 and 50 µg/mL for baicalein and fisetin respectively against studied multidrug resistant high biofilm-forming V. cholerae strains. Additionally, both the flavonoids were able to reduce accessory adhesion factors like auto-aggregation abilities, cell surface hydrophobicity of V. cholerae. Disintegration of biofilm moieties post treatment with flavonoids were visualized by scanning electron and atomic force microscope. Fisetin displayed greater efficacy than baicalein in both the studied strains. Both the flavonoids could reduce gene expressions levels of biofilm regulatory DGCs viz., cdgA, cdgH, cdgK, cdgL, cdgM and vpvC and three important biofilm regulators vpsR, vpsT and aphA that correspond to intercellular cyclic-di-GMP levels in V. cholerae. Besides, molecular docking and dynamic simulation studies also demonstrated superiority of fisetin over baicalein in targeting the DGC active site, potentially explaining its enhanced biofilm suppression. Presence of hydroxyl groups at C3, C4, C7, and C3' positions for fisetin could be the underlying reason as the therapeutic efficacy of flavonoids is significantly contributed by the arrangement of hydroxyl groups. The present study gains its prominence by documenting, maiden insights on the capacity of studied flavonoids to target DGCs and interfere c-di-GMP signalling pathways, to exert their anti-biofilm properties.