UBC Theses and Dissertations
Influence of vitamin exposure on Escherichia coli O157:H7 attachment, stress response and virulence Cancarevic, Ana
Fresh produce is a natural source of vitamins in our diet. Additionally, our enteric flora produces several vitamins, including biotin, cobalamin, folate, menaquinone, pantothenate, and riboflavin. The aim of this study was to determine whether enterically-produced or food-related vitamins may increase attachment of Escherichia coli O157:H7 to leafy green produce, and trigger expression of key stress and virulence genes, thereby enabling its gastrointestinal survival. Late logarithmic phase E. coli O157:H7 grown in M9 minimal medium was exposed to α-tocopherol, ascorbate, biotin, cobalamin, folate, menaquinone, pantothenate, or riboflavin. Following 1.5 and 3 h exposure, HeLa cell assays were performed to assess adherence, while the impact of ascorbic acid, cobalamin and pantothenate on Shiga toxin (Stx) production was quantified by Stx ELISA. Expression of stress response genes (dnaK, osmC, rpoS) was monitored using lux-promoter fusions. Expression of relevant stress response and virulence genes was examined by a quantitative real-time polymerase chain reaction. Lastly, to determine attachment behavior, treated E. coli O157:H7 cells were spotted onto spinach leaves. Treatments with α-tocopherol, biotin, cobalamin, and pantothenate significantly increased adherence to HeLa cells (p<0.05), though only pantothenate (50 mg/mL) produced a >1-log10 increase in adherence. Cobalamin treatment resulted in significantly increased Stx1 and 2 levels (p<0.001), while ascorbate and pantothenate led to lower levels (p<0.001) than the control. In general, exposure to vitamins affected expression of selected virulence and stress response genes. Lastly, vitamin-treated cells attached to the surface of spinach leaves similarly to controls, with the exception of ascorbate and pantothenate treatments where fewer cells attached over a period of 24 h. Increases in E. coli O157:H7 cell concentration on leaves were observed up to 24 h in all treatments, except ascorbate (10 mg/mL) which caused cell numbers to decrease. This study suggests vitamins present in the enteric environment or food have the potential to influence stress response and virulence of E. coli O157:H7, though high concentrations above those found in food or enteric environments are required to elicit these. Ascorbic acid warrants further study as a potentially effective antimicrobial agent for decreasing E. coli O157:H7 contamination on the surface of spinach.
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