Characterization of Bacillus velezensis DY201: Antimicrobial Mechanisms and Intestinal Health Benefits in Broilers

A novel Bacillus velezensis strain DY201, isolated from broiler feces, was characterized to assess its probiotic potential as an antibiotic alternative in poultry production. The strain demonstrated robust environmental tolerance with optimal growth at 42 °C and 51.32% survival following sequential exposure to simulated gastric and intestinal fluids. DY201 exhibited broad-spectrum antimicrobial activity against enterotoxigenic Escherichia coli K88, Staphylococcus aureus, Salmonella pullorum, and Clostridium perfringens, with activity remaining stable across pH 5.0–8.0 and retaining over 92.65% efficacy after 85 °C treatment. Scanning electron microscopy revealed metabolite-induced membrane perforation in target pathogens. Although whole-genome sequencing identified 14 biosynthetic gene clusters for lipopeptides including surfactin and fengycin, integrated proteomic and metabolomic analyses detected small-molecule metabolites—Withaferin A, 2′-hydroxy-2-methoxychalcone, and platycodigenin—as the primary antimicrobial effectors. In a preliminary broiler trial, dietary DY201 supplementation significantly increased the relative abundance of Bacillus in the ileum from 0.30% to 10.30% (p = 0.0434) and in the jejunum from 0.77% to 5.56% (p = 0.0453), enriched the generally beneficial genus Lactobacillus in the jejunum from 73.05% to 80.11% (p = 0.0323), and reduced Candidatus Arthromitus in the ileum from 13.38% to 0.59% (p = 0.0105). These findings support B. velezensis DY201 as a promising probiotic candidate for intestinal microbiota modulation in broilers, although functional intestinal health benefits require further validation through growth performance, barrier function, immune response, and pathogen challenge studies.