Interactions between Escherichia coli O157:H7 and environmental protozoa: The role of protozoa in seasonal variation of E. coli O157:H7 on cattle farms and effects of Shiga toxin-encoding bacteriophages on protozoan grazing

Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. Clinical cases of E. coli O157:H7 occur more frequently in summer months, paralleling seasonal variation in colonization of the principal animal reservoir, cattle. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs, such as water troughs, may also be important sources of infection for both cattle and humans, and similar seasonal variation of E. coli O157:H7-contaminated water troughs on cattle farms has been documented. The main objectives of these studies were to determine the relationship between water trough protozoa (common aquatic microfauna) and seasonal variation of E. coli O157:H7 in the northwestern U.S. In addition, we wanted to determine the role of bacteriophage-encoded Shiga toxins (Stx) carried by E. coli O157:H7 in relation to grazing environmental protozoa, as there have been suggestions in the literature that these prophages may provide a selective advantage for survival of E. coli O157:H7 in the environment, possibly through their toxic effects on grazing protozoa.;With regards to seasonal variation of protozoa within cattle water troughs, predominant genera of protozoa were similar across seasons; however, the density of free-living protozoa in water troughs in the winter was higher than in the summer. In addition, protozoa tended to graze E. coli O157:H7 at higher rates in the winter than in the summer. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon isolated from cattle water troughs, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1 - 3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933) and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon). Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli.;Overall, the abundant grazing of Shiga-toxigenic E. coli by environmental protozoa with increased grazing rates in the winter along with the increased wintertime density of protozoa may help to explain the reduced incidence of E. coli O157:H7-contaminated water troughs in the winter. Subsequently the decrease in cattle fecal shedding may be explained by less exposure of cattle to E. coli O157:H7 during these winter months. Manipulating water troughs to promote bacterial grazing by protozoa or adding highly bactivorous protozoa such as Paramecium to water troughs in the summer may help to reduce E. coli O157:H7 loads during the peak season.