Abstract This study evaluated whether growth medium or exposure conditions influence the production of capsular polysaccharides (CPS) by Escherichia coli O157:H7, and whether changes in CPS impact plant defense responses, consequently affecting survival on plants. E. coli O157:H7 grown in Luria–Bertani (LB) broth supplemented with manure extracts showed an approximately 58% increase in CPS production compared to cells grown in LB medium alone. Levels of CPS were significantly higher for E. coli O157:H7 cells exposed to soil or manure extracts as compared to the non-exposed LB cultured control. Arabidopsis thaliana plants expressing β-glucuronidase (GUS) under the control of the β-1,3-glucanase (BGL2) promoter were used to investigate whether E. coli O157:H7 induces defense-related gene expression. Plants inoculated with E. coli O157:H7 grown in LB containing manure extracts or cells exposed to manure extracts exhibited 3-fold and 2-fold lower GUS activity, respectively, suggesting a limited plant defense response compared to plants inoculated with cells grown in LB. On day 5 post inoculation the population of E. coli O157:H7 grown in LB supplemented with manure on plants was significantly greater than the population of E. coli O157:H7 grown in LB medium alone. E. coli O157:H7 cells exposed to soil or manure exhibited greater survival on plants compared to LB-grown E. coli O157:H7. The results of this study underscore the need to consider medium composition and cultural conditions when conducting crop challenge studies.