Pathogen Challenge and Dietary Shift Alter Microbiota Composition and Activity in a Mucin-Associated in vitro Model of the Piglet Colon (MPigut-IVM) Simulating Weaning Transition

Abstract

Enterotoxigenic Escherichia coli (ETEC) is the principal pathogen responsible for post-weaning diarrhea in newly weaned piglets. Expansion of ETEC at weaning is thought to be the consequence of various stress factors such as transient anorexia, dietary change or increase in intestinal inflammation and permeability, but the exact mechanisms remain to be elucidated. As the use of animal experiments raise more and more ethical concerns, we used a recently developed in vitro model of piglet colonic microbiome and mucobiome, the MPigut-IVM, to evaluate the effects of a simulated weaning transition and pathogen challenge at weaning. Our data suggested that the tested factors impacted the composition and functionality of the MPigut-IVM microbiota. The simulation of weaning transition led to an increase in relative abundance of the Prevotellaceae family which was further promoted by the presence of the ETEC strain. In contrast, several beneficial families such as Bacteroidiaceae or Ruminococcaceae and gut health related short chain fatty acids like butyrate or acetate were reduced upon simulated weaning. Moreover, the incubation of MPigut-IVM filtrated effluents with porcine intestinal cell cultures showed that ETEC challenge in the in vitro model led to an increased expression of pro-inflammatory genes by the porcine cells. This study provides insights about the etiology of a dysbiotic microbiota in post-weaning piglets.

Keywords: ETEC; gene expression; in vitro model of colonic microbiota; intestinal cells; piglet; weaning.

FIGURE 1

Experimental design of the in vitro fermentations and denomination of the conditions studied in the MPigut-IVM.

FIGURE 2

Short chain fatty acids (SCFA) relative abundances (A) and Evolution of the total concentration of SCFAs (B) produced by fermentation activity of the microbiota inhabiting the MPigut-IVM in the CTRL and ETEC conditions for the runs #1, 2, and 3. Groups (days) associated with different letters are significantly different (P < 0.05).

FIGURE 3

NMR-based metabolomics analysis of mucin bead medium. Left panel: Individual plot of PLS-DA. Right panel: Heatmap representing the relative concentrations of all identified metabolites (rows) in individual samples (columns). The color represents the Z-scores (row-scaled relative concentration) from low (blue) to high values (red). The days sharing the same letters are not significantly different from each other (p > 0.05) whatever the conditions.

FIGURE 4

Q -PCR quantification of total bacteria, Escherichia/Shigella and methanogenic archaea populations in the bioreactor medium (A) and the mucin beads (B) of the MPigut-IVM for the runs #1, 2, and 3 in the CTRL and ETEC conditions. The days sharing the same letters are not significantly different from each other (p > 0.05) whatever the conditions.

FIGURE 5

Relative abundances of the main bacterial families in the bioreactor medium (A) and the mucin beads (B) in MPigut-IVM during the runs #1, 2, and 3 which were subjected to a simulated weaning transition and challenged or not with the ETEC Ec105 strain.

FIGURE 6

Mean relative abundance of the 30 more abundant bacterial genera in the bioreactor medium (A) and mucin beads (B) of MPigut-IVM in the CTRL and ETEC conditions (n = 3).

FIGURE 7

Relative abundance of the archaeal genera in the bioreactor medium (A) and mucin beads (B) of the MPigut-IVM for the runs #1, 2, and 3, in the CTRL and ETEC conditions. Day 7 and 9 of run #1 were removed due to their low number of sequences.

FIGURE 8

Quantification of the ETEC LT gene in the MPigut-IVM by qPCR for the runs #1, 2, and 3 in the CTRL and ETEC conditions (n = 3).

FIGURE 9

Differentially abundant genera between the samples of the recovery period containing the ETEC Ec105 strain or not in the bioreactor medium (A) and mucin beads (B) of the MPigut-IVM. Only significant Log2 fold changes are represented on the figure. Positive Log2 fold changes indicate genera that were significantly more abundant in the presence of the pathogen. P values were corrected for multiple testing.

FIGURE 10

Log2 fold changes of gene expression of IPI-2I cells incubated with bead medium supernatants of the MPigut-IVM collected at days 11, 13, and 15 and challenged or not with the ETEC Ec105 strain. Values were normalized with basal gene expression profiles for the targeted genes from IPI-2I cells incubated with their usual glutamine and FCS complemented DMEM medium (n = 3).