PMC

Overview of the experimental setup of the intracystic survival assay.

TEM micrographs of A. castellanii cysts with internalized bacteria (A to E) and an amoebal monoculture control (F). (A) S. enterica serovar Enteritidis at d0. (B) L. monocytogenes 1/2a at d14. (C) E. coli O:26 at d0. (D) Y. enterocolitica 2/O:9 at d0. (E) C. jejuni highly invasive toward Caco2-cells at d0. b, bacteria; ec, ectocyst; en, endocyst; EDG, electron-dense granule; n, condensed nucleus; v, vacuole.

Entry efficiencies of different strains belonging to five foodborne bacterial pathogen species in A. castellanii trophozoites. Shown are the percentages of internalized viable, cultivable bacteria related to the initial inoculum after 30 min of cocultivation with A. castellanii in HS buffer at 25°C. Bars represent the mean ± standard error from four replicate experiments. *, no viable bacteria could be recovered (detection limit of 1 CFU/ml). inv., invasiveness towards Caco2 cells.

(A) Encystment process of A. castellanii; (B) Yersinia enterocolitica strain 2/O:9 inhibits encystment of A. castellanii. (A) A. castellanii trophozoite (panel 1), immature cyst (panel 2), and mature cyst (panel 3). Note the presence of pseudopodia (p) and vacuoles (v) in panel A1 and the thick double-layered cell wall (c) in panel A3. (B) Percentages of A. castellanii trophozoites (gray) and (im)mature cysts (black) in the total amoebal count when cultivated in the presence (coculture) or absence (monoculture) of Y. enterocolitica at 25°C in HS buffer. Bars represent the mean ± standard error from four replicate experiments. *, significant difference (P < 0.05) between the means of coculture and the corresponding monoculture condition.

Survival (S. enterica, L. monocytogenes, Y. enterocolitica, and E. coli) or presence (C. jejuni) of foodborne pathogen strains inside A. castellanii cysts. Ten strains belonging to five foodborne pathogenic bacterial species were cocultivated with A. castellanii trophozoites (MOI of 1:100 in HS buffer at 25°C). After 6 days, cysts were treated with HCl and gentamicin and stored in HS buffer at 25°C (t = 0). To detect the intracellular survival of bacteria, excystment was induced at different time points (d0, d1, d3, d7, d14, and d21) by replacing HS buffer with nutrient-rich medium. Viability of bacteria was confirmed by colony counting. Bars represent the presence of viable, cultivable bacteria after excystment (n ≥ 3). For each species, two strains were tested, as indicated by the black and gray bars. *, presence of bacteria inside cysts (as determined by transmission electron microscopy), which were not cultivable after excystment.