Immunomodulation and Intestinal Morpho-Functional Aspects of a Novel Gram-Negative Bacterium Rouxiella badensis subsp. acadiensis

Abstract

A novel bacterium (Rouxiella badensis subsp. acadiensis) isolated from the microbiota of wild blueberry fruit was investigated for its immunomodulation capabilities and intestinal morpho-functional aspects. The whole-genome shotgun sequencing of this bacterium led to its new taxonomy and showed absence of pathogenicity genes. Although the bacterium was used for blueberry-fermentation and enhancing its anti-inflammatory effects on neurodegeneration, diabetes, and cancer, no study has assessed the effect of the bacterium on health. In this study, we used several in vitro and in vivo assays to evaluate the interaction of R. badensis subsp. acadiensis with the intestinal mucosa and its impact on the localized immune response. The strain antibiotic susceptibility has been investigated as well as its tolerance to gastric and intestinal environment and ability to attach to human intestinal epithelial cells (Caco-2 and HT-29). In addition, Balb/c mice were used to explore the immune-modulatory characteristics of the live bacterium at the intestinal level and its impact on the morpho-functional aspects of the intestinal mucosa. In vitro assays revealed the ability of R. badensis subsp. acadiensis to survive the gastric and intestinal simulated conditions and to satisfactorily adhere to the human intestinal epithelial cells. The bacterium was shown to be sensitive to an array of antibiotics. Immuno-modulation studies with mice orally administered with R. badensis subsp. acadiensis showed a higher number of IgA positive cells in the small intestine, a higher concentration of the anti-inflammatory cytokine IL-10 in the intestinal mucosa, as well as an increase in the number of goblet cells. The anti-inflammatory cytokine miR146a was found to be increased in the ileum and brain. Furthermore, it increases the number of goblet cells which contribute to intestinal barrier integrity. Taken together, our findings reflect the ability of the tested bacterium to modulates the intestinal homeostasis and immune response. Detailed safety unpublished studies and genome data support our finding. The strain Rouxiella badensis subsp. acadiensis has been filed in a provisional patent; a U.S. Provisional Application No. 62/916,921 entitled "Probiotics Composition and Methods." Future studies are still needed to validate the potential utilization of this strain as functional food and its potential probiotic effect.

Keywords: gastro-intestinal tolerance; immuno-modulation; micoRNAs (miRNAs); mucosal immunity; probiotic.

FIGURE 1

Effect of simulated gastric juices SGF [pH 2 (A) pH 3.0 (B), pH 4.0 (C)] and simulated intestinal juice SIF [pH7 (D), pH 8 (E)] on the viability of R. badensis subsp. acadiensis that was exposed to SGF for 60, 90,and 180 min and to SIF for 60, 90,180, and 240 min. In panel (F) R. badensis subsp. acadiensis was first exposed to SGF for 180 min then to SIF for 240 min. Data represents mean log10CFU/ml ± SEM (n = 3). Significant difference exists if *p < 0.05, **p < 0.01.

FIGURE 2

Data represents percentage of adhesion of R. badensis subsp. acadiensis to HT29 and Caco2 cells. Results shown are means of three independent experiments ± SEM.

FIGURE 3

Mean ± SEM of the number of IgA positive cells populations in 10 fields of objective 100X in the ileum of mice fed 1% sucrose (CTR) or R. badensis subsp. acadiensis fed 10⁸CFU/mouse/day for 7 days. Significant difference exists if *p < 0.05, representative tissue photos are taken with 10×, 20×, and 40 × magnifications.

FIGURE 4

Mean ± SEM of the number of IgG positive cells populations in 10 fields of objective 100X in the ileum of mice fed 1% sucrose (CTR) or R. badensis subsp. acadiensis fed 10⁸CFU/mouse/day for 7 days. Significant difference exists if *p < 0.05, representative tissue photos are taken with 10×, 20×, and 40 × magnifications.

FIGURE 5

Mean ± SEM of the number of IL10 positive cells populations in 10 fields of objective 100X in the ileum of mice fed 1% sucrose (CTR) or R. badensis subsp. acadiensis fed 10⁸CFU/mouse/day for 7 days. Significant difference exists if *p < 0.05- p is < 0.001 representative tissue photos of IL10 positive cells in the lamina propria are taken with 10×, 20×, and 40 × magnifications.

FIGURE 6

Mean ± SEM of the number of goblet cells stained with PAS (Periodic Acid Schiff) in 10 fields of objective 100× in the small intestine of mice fed 1% sucrose (CTR) or R. badensis subsp. acadiensis fed 10⁸CFU/mouse/day for 7 days. Significant difference exists if *p < 0.05-p = 0.035 Representative tissue photos are taken with objective 10×, 20×, and 40× magnifications of the PAS-stained tissue.

FIGURE 7

Mean ± SEM of the number of goblet cells stained with AB (Alcian Blue) at pH 2.5 in 10 fields of objective 100× in the small intestine of mice fed 1% sucrose (CTR) or R. badensis subsp. acadiensis fed 10⁸CFU/mouse/day for 7 days. Significant difference exists if *p < 0.05-Representative tissue photos are taken with objective 10×, 20×, and 40× magnifications of the PAS-stained tissue. AB stains in blue acid glycoconjugates.

FIGURE 8

Data represents mean ± SEM of concentration of different (A) anti-inflammatory cytokines IL-10, (B) pro-inflammatory cytokine-IL-6 as well a (C) IgA in the intestinal fluid of (CTR) control mice fed 1% sucrose or R. badensis subsp. acadiensis treated group 10⁸CFU/mouse/day of R. badensis subsp. acadiensis for 7 days. Number of animals per group is n = 10. Difference is considered significant between groups if *p < 0.05 ns = non-significant difference when p > 0.05.

FIGURE 9

Mean ± SEM of relative expression of miR145 and miR146a in the brain and ileum of (CTR) control mice fed 1% sucrose or R. badensis subsp. acadiensis treated group 10⁸CFU/mouse/day of Serratia vaccinia for 7 days. Significant difference between mice exists if *p < 0.05.