Investigating the mode of action of Bacillus subtilis C-3102 probiotic.

Bacillus probiotics have been utilized to improve body weight gain or feed conversion ratio in poultry production since 1970s, however, the mechanisms of action of Bacillus probiotics are not well delineated. Two major pathways were hypothesized; a direct pathway mediated by probiotic organism and an indirect pathway mediated via modified host intestinal microbiota. Two new gnotobiotic experimental systems were developed to differentiate these pathways. Firstly, the direct hypothesis was accessed by combination of in ovo inoculation and sterilized individual HEPA-filtered containers. Bacillus subtilis C-3102 spores (BS) were delivered to amniotic fluid at E17 by in ovo inoculation. Vegetative growth and mono-association by BS was observed at hatch. Multiple direct effects were observed including modified body and organ weight as well inflammation, barrier function, and nutrient digestion gene expression. The response to mono-association with other bacteria, including Enterococcus faecalis (ENT03), Lactobacillus salivarius (LCT01), Escherichia coli (ECL01 and ECL02), Bacteroides fragilis (BCT06), and Clostridium butyricum (CLS01), representing taxonomic families dominant in chicken were also investigated. Live but not heat-killed E.coli inoculation were lethal before E20. Other strains demonstrated variation in colonization density, and intestinal gene expression of inflammation and barrier function. Mixture of 5 bacterial species, Simplified Microbiota (SM) inoculant, were administered to 1-day-old germ-free birds in HEPA-filtered isolators. In 2 of 4 isolators, irradiated feed was supplemented with BS. At 14 day of age, culture of intestinal contents demonstrated colonization with all SM species without contamination. Vegetative BS was observed in BS supplemented SM birds associated with reduced abundance of ECL01 and ENT01. BS reduced relative organ weight, increased digestion and nutrient transport gene expression. Observations suggest probiotic actions of B. subtilis in broilers result from both direct and indirect mechanisms. Direct mechanisms increasing digestive and absorptive capacity are supported by observations in ovo and in SM model. Colonization patterns observed in SM model suggest competitive reduction of E.faecalis and E.coli, may be indirect mechanisms. The in ovo and SM models developed here to simplify the intestinal microbial environment were demonstrated to aid in the differentiation of direct and indirect mechanisms of action of gut modifiers such as probiotic bacteria