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Abstract

The emerging cattle-adapted pathogen Salmonella enterica serotype Dublin threatens the dairy industry by causing lethal infection in calves and reducing productivity in cows. Given the lack of effective interventions, novel mitigation strategies are needed. Probiotics offer a promising strategy to inhibit enteric pathogens. This study isolated fourteen novel Lactobacilli strains (L1– L14) from healthy 24-h-old dairy calves and obtained their genome sequences using Illumina MiSeq (300 paired-end). Comparative genomic analyses of strains L1–L14 revealed enrichment of genes encoding urease (ureC), S-ribosylhomocysteine lyase (luxS), ethanolamine utilization (eutJ), and diol dehydratase (pduCDE) in Limosilactobacillus reuteri (strains L8, L11, and L12). Phylogenomic analysis was extended to include 376 reported Lm. reuteri genomes, suggesting that L8, L11, and L12 are bovine-adapted and revealing host- and lineage-specific enrichment of ureC, eutJ, pduCDE, and glutamate decarboxylase (gad). Culture-based experiments assessed whether strains L1–L14 or their metabolites modulate S. Dublin growth or virulence gene expression: sigma S factor of RNA polymerase (rpoS), invasion protein (invA), Salmonella outer protein E2 (sopE2), and secretion system regulator (ssrA). Metabolites were quantified using gas and high-performance liquid chromatography, growth by visible spectrophotometry and colony counts, and gene expression with RT-qPCR. Data were analyzed using parametric or nonparametric tests with appropriate post hoc analyses. All Lactobacilli produced lactate, with four strains showing elevated acetate after 24 h. Only Lm. reuteri grew on 1,2-propanediol, producing propionate and propanol. Lm. reuteri L8, L11, and L12 supplemented with 1,2-propanediol or glycerol generated significantly more acetate (P < 0.0001 and P = 0.002) and reached higher cell densities (P < 0.05). Coculture with Lm. reuteri L8, L11, or L12 reduced S. Dublin below detection limits, likely due to acidification. While most pH-adjusted metabolites did not alter S. Dublin growth, high-dose metabolites (5:1 to 100:1) from Ligilactobacillus agilis inhibited S. Dublin growth rate (P < 0.001). Lm. mucosae L1 metabolites reduced expression of rpoS, invA, sopE2, and ssrA, whereas Lg. agilis L6 and Lactobacillus amylovorus L7 increased expression. Collectively, these findings demonstrate that calf-derived Lactobacilli employ diverse metabolic strategies that influence S. Dublin growth and virulence, providing a foundation for developing targeted probiotic interventions.