Fig. 6

Prophylactic and therapeutic effects of EcN on AIEC infection

(A) Fish were added to 400 mL water containing 5 × 10⁶ CFU/mL of only AIEC or a 1:1 combination of AIEC and EcN cells for 6 h and then moved to fresh water for 18 h, or fish were added to 400 mL water containing 5 × 10⁶ CFU/mL of EcN for 6 h followed by 5 × 10⁶ CFU/mL of AIEC (pre-EcN, prophylactic) for 6 h and vice versa, i.e., AIEC first, then EcN (post-EcN, therapeutic), and then incubated in fresh water for 12 h. AIEC colonization in zebrafish intestine is shown on the y axis and is compiled from multiple experiments. Data are represented as mean +/– SEM from multiple experiments. Each dot represents the data from one fish. Total colonization per intestine was calculated after plating serial dilutions of intestinal homogenates 24 h postinfection. Statistical significance indicated above the data was determined by Student’s t test. ∗∗∗p < 0.0005 compared with only AIEC infection.

(B) Zebrafish were infected with 5 × 10⁶ CFU/mL of only AIEC, pre-EcN then AIEC (prophylactic), or AIEC then post-EcN (therapeutic) as mentioned and were sacrificed 24 hpi. mRNA was isolated from intestinal tissue. Relative gene expression levels of TNFα, s100a-10b, IFNγ, and IL-1β were determined through qRT-PCR. Gene expression was normalized against β-actin and expressed as fold change. Error bars indicate standard deviation. Data are represented as mean +/– SEM from three experiments. ∗∗p < 0.005 and ∗p < 0.05 as compared with only AIEC infection.

(C) S100A-10b protein levels were determined in intestinal homogenates by calprotectin ELISA. Error bars indicate standard deviation. TNBS, a positive stimulator of inflammatory markers, was used as a positive control. Data are represented as mean +/− SEM from three experiments. ∗∗p < 0.005 compared with the only AIEC infection.