B-244. A Bile-Induced Exopolysaccharide Required for Salmonella Biofilm Formation on Gallstone Surfaces

R. W. Crawford¹, D. L. Gibson², J. S. Gunn¹;
¹The Ohio State Univ., Columbus, OH, ²Univ. of British Columbia, Vancouver, BC, CANADA.

Salmonellae interact with bile in the intestine and gallbladder, and bile affects expression of Salmonella genes important for virulence phenotypes that likely enhance colonization and persistence in both organs. We have previously demonstrated that salmonellae form biofilms on the surface of human gallstones, which may contribute to the development of the carrier state. To eliminate reliance on human gallstones, an in vitro assay of biofilm formation on cholesterol gallstones was developed. We show that cholesterol-coated Eppendorf tubes support Salmonella biofilm formation (TBA, tube biofilm assay), suggesting that salmonellae bind to cholesterol on human gallstones. The TBA, like assays using human gallstones, has shown biofilm formation to be enhanced by bile. This enhancement was shown to be due to the effect of bile on Salmonella and its biofilm forming capacity. Although cellulose and colanic acid are the primary extracellular matrix products of Salmonella biofilms on various surfaces, a double mutant lacking these factors formed mature biofilms on gallstones and in the TBA, suggesting that a novel exopolysaccharide (EPS) was involved in gallstone biofilms. The recently identified O-antigen (O-ag) capsule was shown to be enhanced by bile. The presence of O-ag in the TBA and on gallstone biofilms was examined by ELISA and immunofluorescent microscopy, showing it to be highly expressed within cholesterol biofilms. The O-ag encoding operons (yihU-yshA and yihVW operons) were identified and shown to be transcriptionally activated in the presence of bile. O-ag mutants in S. typhimurium, S. typhi, and S. enteritidis failed to form a biofilm in the TBA, suggesting that O-ag capsule is required for biofilm formation on gallstones and perhaps colonization of the gallbladder in Salmonella carriers. A better understanding of Salmonella gallstone biofilms promises to better characterize the asymptomatic carrier state and lead to novel drug therapies.