, 1998; Barkocy-Gallagher et al., 2004). Infected cattle are capable of shedding 102–105 CFU of E. coli O157:H7 per gram of feces (Wang et al., 1996; Campbell et al., 2001), and it can persist in manure and slurry (Kudva et al., 1998; Bolton et al., 1999; Lau & Ingham, 2001; Avery et al., 2005) and in soil, water, sediment, and animal carcasses

for extended periods of time (Mead & Griffin, 1998). Thus, contamination of the soil and surface water with E. coli O157:H7 in the vicinity of infected cattle herds occurs at high frequency, making it the main source of contamination of nonmeat food products (McGee et al., 2002). While E. coli O157:H7 is not thought of as an intracellular pathogen, it has been shown to survive within human macrophages for at least 24 h (Poirier et al., 2008) and in the soil protozoan Everolimus clinical trial Acanthamoeba polyphaga for at least 45 days (Barker et al.,

1999). This bacterial–protozoal interaction has certain implications as protozoa are widely acknowledged as reservoirs for bacterial pathogens such as Legionella, Listeria, Campylobacter, Pseudomonas, Helicobacter, Mycobacterium, Coxellia, Salmonella, Staphylococcus, and the harboring of these pathogens Idasanutlin within protozoa has been associated with increased survival and persistence in environment (King et al., 1988), increased virulence (Cirillo et al., 1994; Rasmussen et al., 2005), and increased resistance to antibiotics (Barker et al., 1995; Miltner & Bermudez, 2000). With this in mind, protozoa may serve as a vehicle for E. coli O157:H7 environmental persistence and transmission Tolmetin as well as preparing E. coli O157:H7 for enhanced survival during its journey through the rumen of cattle. We sought to characterize the transcriptome of E.

coli O157:H7 after exposure to the protozoan Acanthamoeba castellanii environment as a model for environmental and rumen exposure using microarrays to measure the transcriptional changes that occur in E. coli O157:H7 following uptake compared with standard planktonic growth conditions. Our results demonstrate that a significant portion of the E. coli O157:H7 genome, including many virulence-related genes, are differentially expressed as a result of the A. castellanii intracellular environment. Escherichia coli O157:H7 EDL933 (ATCC 43895) was grown in Luria–Bertani (LB) broth at 37 °C. Following overnight incubation, these cultures were diluted 1 : 100 in LB broth and incubated with shaking for 2 h before use in the Acanthamoeba assay. Acanthamoeba castellanii (ATCC 30010) was grown in ATCC PYG712 broth at 30 °C. An estimate of A. castellanii cell numbers was obtained using a Coulter particle counter. Acanthamoeba castellanii cultures were centrifuged at 100 g for 5 min, resuspended in fresh PYG712 broth to a density of 2 × 106 cells mL−1. Wells within six-well cell culture plates were seeded with 1 mL of this suspension. After 2 h of incubation, E.