Figure 4.

Effects of differently encapsulated B. breve ATCC15700 on their protection offered to intestinal epithelial layers against a 2 h E. coli Hu734 challenge in the absence or presence of tetracycline. Cell layers were co-cultured for 4 h with B. breve that were first exposed for 30 min to SGF, followed by a 2 h E. coli challenge and continued co-culturing for another 2 h. Finally, growth was pursued in the modified medium without or with tetracycline for another 22 h with tetracycline exposure for the first 3 h (10 μg/mL). (A) Number of B. breve CFUs adhering to intestinal epithelial layers that survived an E. coli challenge in the absence of tetracycline exposure. Absence of data indicates CFU/cm² below detection. (B) Same as panel (A), now for B. breve surviving an E. coli challenge in the presence of tetracycline exposure. Absence of data indicates CFU/cm² below detection. (C) The number of E. coli CFUs adhering to intestinal epithelial layers that were killed by differently encapsulated B. breve in the absence of tetracycline exposure. (D) Same as panel (C), now for E. coli killed by adhering differently encapsulated B. breve in the presence of tetracycline exposure. (E) TEER values of intestinal epithelial cell layers with adhering B. breve and challenged by E. coli in the absence of tetracycline exposure. The horizontal band represents the TEER values of intestinal cell layers with or without adhering B. breve in the absence of an E. coli challenge. Note that these TEER values were not affected by the presence of adhering unencapsulated or differently encapsulated B. breve (Figure S6A). (F) Same as panel (E), now for the TEER of intestinal epithelial cell layers with adhering B. breve and challenged by E. coli in the presence of tetracycline exposure. (G) Surface coverage of the transwell membrane by intestinal epithelial cell layers with adhering B. breve and challenged by E. coli in the absence of tetracycline exposure. The horizontal band represents the surface coverages of intestinal cell layers with or without adhering B. breve in the absence of an E. coli challenge (Figure S6B). Surface coverages were calculated from fluorescence images, as presented in Figure S6C. (H) Same as panel (G), but now representing surface coverage by intestinal epithelial cells of the transwell membrane in the presence of tetracycline exposure. The horizontal band represents the membrane surface coverage by intestinal cell layers without adhering B. breve in the absence of an E. coli challenge. Surface coverages were calculated from fluorescence images, as presented in Figure S6D. Error bars represent the SEM over three experiments with separately grown cells and bacteria. * indicates statistically significant differences (one-way ANOVA) between unencapsulated and encapsulated B. breve, while # indicates the difference between cellular layers with E. coli challenges in the absence of colonizing B. breve and in the presence of unencapsulated and encapsulated B. breve. Significance was accepted at p < 0.05. The absence of significance is indicated as “ns”, while seemingly missing data are too close to the axes to be visible.