2 While serving as environmental reservoir for V. cholerae infection, biofilms are also crucial for 3 intestinal colonization of the pathogen. Triterpenoids, a group of bioactive phytochemicals, have 4 been tested for antibiofilm activity against model biofilm forming bacteria in recent times. In this 5 context, glycyrrhetinic acid (GRA), ursolic acid (UA) and betulinic acid (BA), representing three 6 categorically distinct groups of pentacyclic triterpenoids, are targeted for profiling their impact 7 on Vibrio cholerae C6709 biofilms. The triterpenoids substantially affected biofilm associated 8 attributes like formation, substratum adherence and dispersion from preformed biofilms. Though 9 at variable degree, the compounds decreased cell surface hydrophobicity and composition in 10 terms of macromolecular content. Not only EPS-associated extracellular enzyme activities were 11 estimated to be reduced by triterpenoid exposure, ultra structural analysis also revealed that 12 GRA, UA and BA can affect extracellular polymeric substance (EPS) content. Albeit total 13 extracellular proteolytic activity remained unaffected by the triterpenoids, GRA treatment 14 resulted in considerable reduction of extracellular gelatinase activity. Molecular docking analysis 15 indicated potential interaction with cyclic di-GMP sensor VpsT, autoinducer-2 sensor kinase 16 LuxP-LuxQ and transcriptional activator HapR, component of complex quorum sensing 17 networks modulating biofilm formation. Comprehensive analysis of antibiotic action revealed 18 accentuation of cephalosporin antibiotics with GRA and UA while BA potentiated action of 19 fluoroquinolones, widening the scope of combinatorial therapeutic strategy.20
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