Genome-Resolved In Silico Analysis of Poultry and Swine Lactobacillales Provides a Data- Driven Framework for Elucidating Metabolic Synergies in Multi-Strain Probiotics

Probiotics are live microorganisms that provide health benefits to the host by improving digestion, enhancing nutrient absorption, and modulating the immune system. Among them, lactic acid bacteria are known for producing vitamins and short-chain fatty acids, both essential for intestinal health. In this in silico study, we performed high-fidelity (PacBio HiFi) whole-genome sequencing and comprehensive comparative genomic analysis of five Lactobacillales strains ( Enterococcus lactis , Enterococcus mundtii , Ligilactobacillus agilis , Limosilactobacillus reuteri , Limosilactobacillus vaginalis ) isolated from the intestinal microbiota of chickens and pigs. The assembled genomes ranged from 1.8 to 2.8 Mb, with more than 98% completeness and less than 1.31% contamination. Taxonomic classification, presence of antimicrobial resistance genes, bacteriocin biosynthetic potential, carbohydrate-active enzyme repertoires and vitamin biosynthesis pathways, and capacity to degrade plant polysaccharides were investigated. Functional characterization identified 65 families of carbohydrate-active enzymes, with E. mundtii presenting the greatest diversity (43 families) and absolute number (100 terms) of enzymes. Metabolic reconstruction suggested functional specialization among strains, with xylooligosaccharide degradation exclusive to E. mundtii and pectin utilization limited to E. lactis . Genes related to the biosynthesis of B-complex vitamins, including riboflavin, folate, and menaquinone, showed heterogeneous and complementary distribution among strains. These findings suggest the potential for metabolic complementarity and cross-feeding, where metabolites produced by one strain serve as precursors for biosynthetic pathways in others. Collectively, these genome-resolved insights offer a data-driven framework for designing multi-strain probiotics aimed at improving intestinal health and feed efficiency in poultry and swine.