Acanthamoeba castellanii cysts harbor viable foodborne pathogens on fresh and thermally processed leafy greens

Foodborne pathogens internalized by free-living amoebae pose a significant risk to the safety of fresh produce. This study investigated the role of Acanthamoeba castellanii cysts as reservoirs for four foodborne pathogens-Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus-on fresh and steamed spinach and cabbage. Coculture experiments were conducted at a multiplicity of infection (MOI) of 10, and bacterial uptake was quantified. All pathogens were efficiently internalized, with E. coli O157:H7 exhibiting a significantly higher uptake index (p ≤ 0.05) than the others. Encystment of pathogen-laden trophozoites proceeded at a rate of over 80% within 24 h, unaffected by the bacterial species. For survival assays, produce samples (n = 3 per time point) were spot-inoculated with 10⁴ cysts and stored at 4 °C (9 days for fresh, 21 days for steamed). Viability was assessed periodically by inducing excystment and detecting bacteria in the resultant trophozoites and culture medium. No excystment or bacterial release occurred on the produce during storage. However, all pathogens survived within cysts. On fresh produce, intracellular bacteria persisted for the entire 9-day storage. In steamed produce, survival was significantly longer (p ≤ 0.05), with S. Typhimurium and L. monocytogenes demonstrating the greatest resilience, persisting for up to 19-20 days in spinach and 16-18 days in cabbage. E. coli O157:H7 and S. aureus showed shorter survival times in steamed produce. These findings demonstrate that A. castellanii cysts can harbor viable foodborne pathogens on leafy greens even after steaming, and that internalized pathogens remain viable within the cysts, posing a challenge to produce safety.