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Author ORCID Identifier

https://orcid.org/0000-0002-6348-1281

Date Available

2-12-2027

Year of Publication

2026

Document Type

Thesis

Degree Name

Master of Science (MS)

College

Agriculture, Food and Environment

Department/School/Program

Veterinary Science

Faculty

Yosra A. Helmy

Theodore S. Kalbfleisch

Abstract

Non-typhoidal Salmonella is a leading cause of foodborne illness globally, responsible for an estimated 93.8 million cases and 155,000 deaths annually, representing a significant One Health concern. Cattle serve as a primary reservoir, facilitating pathogen dissemination through the food chain, environmental runoff, and international trade networks. The widespread antibiotic misuse in livestock leads to the emergence of Salmonella strains that reduce the efficacy of first-line antimicrobial therapies, increase hospitalization rates, and elevate the risk of mortality in humans. Despite its epidemiological significance, a comprehensive, integrated characterization of phenotypic and genotypic aspects remains limited. The goals of this study are to: i) characterize the phenotypic antimicrobial resistance, biofilm-forming capacity, and motility profiles of S. enterica recovered from necropsied cattle, and ii) define the genomic architecture of antimicrobial resistance genes (ARGs), virulence gene complement, and mobile genetic elements (MGEs) of bovine-derived isolates, and determine their phylogenomic relationships to Salmonella lineages recovered from human clinical, food, animal, and environmental sources. To address the first goal (Chapter 2), a total of 1,008 samples were collected from necropsied cattle submitted for diagnostic evaluation in the University of Kentucky Veterinary Diagnostic Laboratory. S. enterica subspecies were identified by MALDI-TOF MS and confirmed by serotyping. Biofilm-forming capacity was quantified using a crystal violet microtiter plate assay, and motility phenotypes were assessed on semi-solid agar plates. Phenotypic antimicrobial susceptibility testing was performed against 18 antimicrobial agents across 9 drug classes, with the multiple antibiotic resistance (MAR) index calculated for each isolate. Our results showed that 27 isolates were Salmonella-positive, yielding an overall isolation rate of 2.7%. Among the confirmed samples, S. Dublin was the most prevalent serotype (25.9%). All isolates were classified as multidrug-resistant (MDR), with MAR index ranging from 0.32 to 0.74. Phenotypic resistance was highest to macrolides (100%), followed by chloramphenicol (85.2%). All isolates demonstrated biofilm-forming capacity at varying intensities, and 96.3% exhibited both swimming and swarming motility. To address the second goal (Chapter 3), whole-genome sequencing (WGS) was performed on all 27 confirmed isolates, with genomic analyses including Multi-locus Sequence Typing (MLST), ARGs, virulence genes, MGEs, and pan-genome construction with core-genome phylogenetic analysis. MLST resolved 12 distinct sequence types (STs), with ST10 predominating (25.9%). A total of 223 virulence genes were identified, including conserved invasion genes and variable toxin-encoding and immune-evasion gene complements. Genomic ARG profiling identified resistance predominantly driven by efflux pump systems (62.1%), with the aminoglycoside resistance gene aac (6′)-Ib detected in all isolates. Plasmid replicons were present in 77.8% of isolates, with 11.1% harboring integron-associated elements alongside widespread prophages and insertion sequence (IS) elements. While most resistance loci were chromosomally located (63.5%), plasmid-associated ARGs showed significantly stronger linkage to IS elements. Pangenome analysis revealed an open genome structure with a limited conserved core fraction (19.9%) and a dominant accessory genome fraction (77.6%). Core genome phylogeny II demonstrated tight clustering between cattle-derived isolates and isolates from humans, food, and environmental sources. These findings define the antimicrobial resistance (AMR) genomic architecture of clinically derived bovine Salmonella and confirm the central role of mobile genetic elements in the evolution of resistance and cross-sectoral dissemination. The open pan-genome structure and phylogenomic linkage between cattle and human food-associated lineages provide genomic evidence that cattle represent active contributors to the broader One Health Salmonella transmission network. These results have direct implications for antimicrobial stewardship policy, pre-harvest intervention design, and the development of integrated WGS- based surveillance systems spanning veterinary, food safety, and human clinical domains.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2026.427

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