Background/Objective: Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide and presents a significant One Health concern due to zoonotic transmission. Although antibiotic therapy remains a standard approach for treating salmonellosis in severe cases in animals, the widespread misuse of antibiotics has contributed to the emergence of multidrug-resistant (MDR) Salmonella strains. This study provides insights into the genotypic and phenotypic characteristics among Salmonella isolates from necropsied cattle. Methods: A total of 1008 samples were collected from necropsied cattle. Salmonella enterica subspecies were identified by MALDI-TOF MS and subsequently confirmed by serotyping. The biofilm-forming ability of the isolated bacteria was assessed using a crystal violet assay. The motility of the isolates was assessed on soft agar plates. Additionally, the antimicrobial resistance genes (ARGs) and virulence genes were investigated. Antimicrobial resistance patterns were investigated against 19 antibiotics representing 9 different classes. Results:Salmonella species were isolated and identified in 27 necropsied cattle. Salmonella Dublin was the most prevalent serotype (29.6%). Additionally, all the isolates were biofilm producers at different levels of intensity, and 96.3% of the isolates exhibited both swarming and swimming motility. Furthermore, virulence genes, including invA, hilA, fimA, and csgA, were detected in all the isolates. The highest resistance was observed to macrolides (azithromycin and clindamycin) (100%), followed by imipenem (92.6%), and chloramphenicol (85.2%). All isolates were multidrug-resistant, with a multiple antibiotic resistance (MAR) index ranging between 0.32 and 0.74. The aminoglycoside resistance gene aac(6′)-Ib was detected in all the isolates (100%), whereas the distribution of other antimicrobial resistance genes (ARGs) varied among the isolates. Conclusions: The increasing prevalence of MDR Salmonella poses a significant public health risk. These resistant strains can reduce the effectiveness of standard treatments and elevate outbreak risks. Strengthening surveillance and regulating antibiotic use in livestock are essential to mitigating these threats.

Non-typhoidal Salmonella remains a leading global cause of foodborne disease and an important contributor to the global burden of antimicrobial resistance (AMR) within the One Health framework. Cattle serve as an important reservoir, facilitating the dissemination of antimicrobial-resistant Salmonella through the food chain and the environment. In this study, we analyzed the genomes of 27 Salmonella enterica isolates recovered from necropsied cattle in central Kentucky to characterize their genomic AMR, virulence, and mobile genetic element profiles. Multilocus sequence typing (MLST) identified 12 sequence types (STs), with ST10 ( S . Dublin) representing the dominant lineage (25.9%). Genomic screening revealed 4,618 virulence gene occurrences, including conserved factors associated with secretion and adhesion, as well as variable toxin and immune evasion genes. Antimicrobial resistance genes (ARGs) were detected across the isolate collection, with resistance primarily associated with efflux systems (62.1%) and genes conferring resistance to aminoglycosides, tetracyclines, sulfonamides, phenicols, and β-lactams. Plasmids were detected in 77.8% of isolates, integrons were identified in 11.1% of isolates, and prophages and insertion sequence (IS) elements were widely distributed. While most resistance loci were chromosomal (63.5%), plasmid-associated ARGs (11.7%) demonstrated significantly greater proximity to IS elements, suggesting an increased potential for mobilization. Pan-genome analysis revealed an open pan-genome structure with a limited core (19.9%) and a dominant accessory genome (77.6%). Core-genome phylogenetic analysis incorporating 97 publicly available genomes demonstrated close clustering of cattle-associated isolates with isolates from food, human, and environmental sources, suggesting the presence of shared or closely related lineages across interconnected One Health compartments. These findings characterize the genomic architecture of AMR in cattle-associated Salmonella and highlight the potential contribution of mobile genetic elements to shaping AMR evolution and dissemination across interconnected One Health systems. IMPORTANCE: This study reveals that multidrug-resistant Salmonella enterica in cattle is sustained by a dual genomic architecture in which resistance determinants are both chromosomally embedded and mobilized through plasmids, prophages, and insertion sequence (IS) elements. The presence of plasmid-borne resistance genes and IS elements suggests their potential involvement in transposition-mediated dissemination. The predominance of chromosomal resistance may indicate greater persistence of resistance determinants within bacterial lineages. The dominance of the ST10 ( S. Dublin) lineage and its close genomic relatedness to food-associated isolates, together with an open pan-genome structure, suggest ongoing circulation and genomic diversification of high-risk lineages across interconnected animal, food, and environmental systems.