Saccharomyces Cerevisiae Var Boulardii CNCM I-1079 Reduces Expression of Genes Involved in Inflammatory Response in Porcine Cells Challenged by Enterotoxigenic E. Coli and Influences Bacterial Communities in an In Vitro Model of the Weaning Piglet Colon

Abstract

Enterotoxigenic Escherichia coli (ETEC) is the main infectious agent responsible for piglet post-weaning diarrhea with high mortality rates. Antimicrobials represent the current principal strategy for treating ETEC infections in pig farms, but the occurrence of multi-resistant bacterial strains has considerably increased in the last decades. Thus, finding non-antibiotic alternatives becomes a real emergency. In this context, we investigated the effect of a live yeast strain, Saccharomyces cerevisiae var boulardii CNCM I-1079 (SB) in an in vitro model of the weaning piglet colon implemented with a mucus phase (MPigut-IVM) inoculated with ETEC and coupled with an intestinal porcine cell line IPI-2I. We showed that SB was able to modulate the in vitro microbiota through an increase in Bacteroidiaceae and a decrease in Prevotellaceae families. Effluents collected from the SB treated bioreactors were able to mitigate the expression level of genes encoding non-gel forming mucins, tight junction proteins, innate immune pathway, and pro-inflammatory response in IPI-2I cells. Furthermore, SB exerted a significant protective effect against ETEC adhesion on porcine IPEC-J2 intestinal cells in a dose-dependent manner and showed a positive effect on ETEC-challenged IPEC-J2 by lowering expression of genes involved in pro-inflammatory immune responses. Our results showed that the strain SB CNCM I-1079 could prevent microbiota dysbiosis associated with weaning and protect porcine enterocytes from ETEC infections by reducing bacterial adhesion and modulating the inflammatory response.

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

Figure 1

Percentage of adherent ETEC cells on IPEC-J2 intestinal cells pre-treated or not with different doses of SB CNCM I-1079. The concentration of ETEC adherent cells on control IPEC-J2 cells (non-treated with SB yeasts) was normalized to 100% (n = 3). The denominations e6, e7, and e8, respectively, correspond to the doses of 10⁶, 10⁷, and 10⁸ CFU/well of SB incubated on the ETEC-challenged IPEC-J2 cells (p value codes: *** <0.000).

Figure 2

Gene expression of IPEC-J2 cells challenged with the ETEC Ec105 strain and treated or not with different doses of SB CNCM I-1079. Log₂ fold changes represent the differential gene expression between non-SB treated and SB treated ETEC-challenged IPEC-J2 cells (n = 3). Conditions with the same letters are not statistically different from each other, while different letters indicate that the conditions are statistically different from each other. The denominations e6, e7, and e8, respectively, correspond to the doses of 10⁶, 10⁷, and 10⁸ CFU/well of SB incubated with the ETEC-challenged IPEC-J2 cells (p value codes: *** <0.000; ** <0.001).

Figure 3

Quantification of the LT enterotoxin gene in the MPigut-IVM during ETEC and SBETEC conditions. Each value is an average of two to three technical replicates.

Figure 4

Total SCFA concentration in the MPigut-IVM during ETEC and SBETEC conditions. Each value is an average of two technical replicates.

Figure 5

Mean relative abundances of the 30 main bacterial genera in the bioreactor medium (A) on the mucin beads (B) of the MPigut-IVM during the ETEC and SBETEC conditions.

Figure 6

Log₂ fold changes of IPI-2I cells gene expression when incubated with bead medium supernatants collected from the MPigut-IVM under ETEC and SBETEC conditions. Fold changes are calculated compared to the control condition where IPI-2I cells were incubated with their usual glutamine and FCS complemented DMEM medium. Each value is an average of two technical replicates.

Figure 7

Experimental design of in vitro fermentation procedures and denomination of the MPigut-IVM.