Reverse-engineering an expired patent: Characterization of a previously undefined probiotic consortium suggests possible modes of action in broiler chickens

Abstract

In this study, we characterized the taxonomic composition of a previously patented but uncharacterized mucosal competitive exclusion (MCE) product to identify previously unrecognized bacterial strains and genes that may have contributed to the efficacy of this product. By comparing the composition of the MCE product to cecal communities from typical commercial broiler flocks, we were able to identify specific bacterial taxa and metabolic pathways that were significantly enriched within the MCE consortium that therefore might provide novel insights into this historical product's mode of action. We used high-throughput 16S rRNA gene sequencing to describe the taxonomic composition of archived MCE cultures and compared these data to recent surveys of commercial poultry flocks. A total of 111 amplicon sequence variants (ASVs) were observed across all samples, dominated by the phyla Firmicutes and Bacteroidota. The 20 most abundant ASVs in the MCE products were most closely related to the genera Clostridium, Bacteroides, Veillonella, Parabacteroides, Megamonas, Anaerotignum and Enterococcus . Using a taxonomy-based genomic inference approach, we identified six main metabolic pathways that are potentially enriched in the MCE product compared to samples from commercial broiler flocks: (1) phenylpropanoids biosynthesis, (2) flavones and flavonol biosynthesis, (3) glycosphingolipids (GSL) biosynthesis, (4) adipocytokine signaling, (5) carbohydrate digestion and absorption, and (6) biosynthesis of secondary bile acids. Finally, to identify specific strains with genes involved in these metabolic pathways, we queried previously-sequenced genomes of bacteria most closely-related to those found in the MCE culture. Several Bacteroides genomes including B. ovatus, B. xylanisolvens , and B. fragilis were highly enriched in several pathways such as GSL biosynthesis that may interfere with pathogen binding to GI epithelial cells. By applying modern high-throughput sequencing to a historical product previously shown to be effective against Salmonella and Campylobacter colonization, our results may help identify specific beneficial microbes and their modes of action that could reduce pathogen colonization in broiler chickens.