Functional Characterization and Application of Lacticaseibacillus and Lactobacillus Strains to Hatching Eggs for Control of Salmonella Enteritidis in Layer Hatchlings

With growing emphasis on antibiotic-free poultry production, functional probiotics represent a promising strategy to improve gut health and reduce pathogen transmission. This study characterized three lactic acid bacteria (LAB) strains Lactobacillus delbrueckii subsp. bulgaricus NRRL-B-548 (LD), Lacticaseibacillus paracasei DUP-13076 (LP), and Lacticaseibacillus rhamnosus NRRL-B-442 (LR) for their probiotic potential and evaluated their efficacy against Salmonella enterica in poultry. The LAB strains were assessed for acid and bile tolerance, lysozyme resistance, cholesterol assimilation, antimicrobial activity, surface hydrophobicity, epithelial adherence, hemolysis, and antibiotic susceptibility. Genomic analysis was performed to identify genes associated with probiotic functionality. The protective potential of LR and LP was further validated in hatchlings using a hatchery spray model challenged with Salmonella Enteritidis. All strains survived simulated gastric and intestinal conditions, exhibited strong adhesion to epithelial cells, and demonstrated high hydrophobicity, indicating robust colonization capacity. The LAB significantly inhibited Salmonella Enteritidis, S. Typhimurium, and S. Heidelberg growth in vitro and remained sensitive to clinically relevant antibiotics. In vivo application of LR and LP to hatching eggs markedly reduced S. Enteritidis colonization in the liver, spleen, and ceca of hatchlings. Further, genomic profiling of the LAB strains revealed genes for bacteriocin production, exopolysaccharide synthesis, and carbohydrate metabolism supporting probiotic function. In summary, the evaluated LAB strains exhibit multiple probiotic attributes and strong anti-Salmonella activity, confirming their potential as safe, hatchery-applied probiotics for improving gut health and biosecurity in poultry production systems.