Leluo Guan

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.

View record

Shiga toxin (Stx)-producing Escherichia coli (STEC) are major zoonotic pathogens, with cattle as their primary reservoir. The O157:H7 strains containing the stx2a gene are especially concerning due to their strong association with severe human disease. Phage-based interventions are a promising strategy for reducing STEC burden in livestock; however, the efficacy of this approach is challenged by the presence of bacterial antiviral defence systems (ADSs) that block phage replication, and the risk of prophage induction leading to Stx release. Non-coding RNAs (ncRNAs) are key regulators of stress response gene expression, mobile elements, and phage–host interactions, yet their potential role in modulating ADSs in bovine STEC remains unknown. This thesis characterized the landscape of ncRNAs and ADSs in 76 bovine-derived STEC genomes, including stx2a-positive (n=45) and stx2a-negative (n=21) O157:H7, and non-O157 (n=10) strains. A bioinformatic pipeline was developed to integrate covariance model-based ncRNA detection (Infernal + Rfam), homology searches (BLAST + RNAcentral), and ADS annotation (DefenseFinder). Predicted ncRNAs were filtered and classified by family, structure, and frequency of occurrence in the genomes. Potential regulatory associations between ncRNAs and predicted antiviral gene sequences were inferred based on BLAST-based homology. The analysis revealed distinct ncRNA and ADS profiles across bovine STEC genomes. The stx2a-positive O157:H7 strains were predicted to harbour the most diverse ADSs and prevalent ncRNAs associated with phage regulation and stress adaptation compared to stx2a-negative O157:H7, and non-O157 strains. Several ncRNAs were predicted to co-occur with genes encoding restriction enzymes, signalling domains, and toxin-antitoxin elements, suggesting potential ncRNA-mediated regulation of bacterial immune activity. The findings from this study provides the first comprehensive assessment of ncRNA and ADS profiles in bovine-derived STEC. This work offers a foundation for developing phage-based control strategies that bypass or exploit host regulatory mechanisms by predicting candidate ncRNAs that may suppress STEC antiviral defence pathways. These insights may help reduce prophage activation and Stx release, providing knowledge on future intervention strategies in reducing STEC shedding in cattle on farm.

View record