The global spread of antimicrobial-resistant bacteria, including high-risk clones, poses a significant threat to human health, causing an estimated 1.27 million deaths annually. Low-income countries, like Zimbabwe, are particularly vulnerable due to high AMR prevalence and limited healthcare resources. Antibiotic overuse in clinical and agricultural settings is a primary driver of AMR emergence. Antibiotics used in livestock production can lead to the emergence of resistant bacteria that spread between animals, farms, and into the food chain, representing a major pathway for AMR pathogen transmission to humans. Non-typhoidal *Salmonella* (NTS) serotypes are a significant public health concern globally. While often causing self-limiting gastroenteritis, multidrug-resistant (MDR) strains are associated with increased morbidity and mortality. Invasive NTS (iNTS) disease can be severe, requiring antibiotic treatment, and the emergence of strains resistant to fluoroquinolones and extended-spectrum cephalosporins has limited treatment options. *S. Kentucky* infections are frequently linked to contaminated poultry and readily acquire resistance. A globally dispersed clone of sequence type (ST)198, initially identified in Egypt, has spread widely, exhibiting multidrug resistance encoded on *Salmonella* genomic island 1 (SGI1). This study aimed to investigate the molecular epidemiology and extent of ESBL-producing *S. Kentucky* in Zimbabwe by analyzing isolates from human clinical infections, poultry farms, and related sources using whole-genome sequencing (WGS), and to investigate the distribution and genetic flux of AMR determinants.

Existing literature highlights the global threat of antimicrobial resistance (AMR), particularly in low-income countries where healthcare resources are limited. Studies have established a strong link between antibiotic use in livestock and the emergence of MDR bacteria in the food chain. *Salmonella* species are significant human pathogens, and the emergence of MDR strains, especially those resistant to fluoroquinolones and extended-spectrum cephalosporins, has complicated treatment strategies. The *S. Kentucky* ST198 clone has been recognized as a high-risk global MDR clone due to its rapid spread and acquisition of resistance genes, primarily through the acquisition of SGI1 variants. Previous research has detailed the genomic characteristics of this clone, including the presence of AMR genes on SGI1 and chromosomal mutations conferring resistance to fluoroquinolones. However, data on the epidemiology of ESBL-producing *S. Kentucky* in Sub-Saharan Africa, particularly Zimbabwe, remained limited before this study.

A total of 245 non-typhoidal *Salmonella* (NTS) strains, isolated between 2016 and 2020 from human clinical infections (n=162), chicken farms (n=82), crocodile meat (n=1), and a school dining table (n=1) in Zimbabwe, underwent whole-genome sequencing (WGS). *In silico* serotyping identified 44 distinct serotypes. Thirty-seven isolates were identified as *S. Kentucky*, with 11 from human clinical infections and 26 from chicken farms. For the 37 *S. Kentucky* isolates, sequence type (ST) was determined, and a maximum likelihood phylogenetic tree was constructed based on core genome sequence variation. Antimicrobial resistance (AMR) genes and plasmid replicons were identified using bioinformatic tools. For a subset of strains, long-read sequencing was performed to obtain complete plasmid sequences. Phylogenetic analysis of the *S. Kentucky* ST198 isolates from Zimbabwe was conducted in the context of a global collection of ST198 strains. The presence of AMR genes in the global collection was compared to determine the extent of AMR gene diversity.

Of the 37 *S. Kentucky* isolates, 36 belonged to ST198 and one to ST152. All ST198 isolates possessed multiple AMR genes (6-15), with 92% having at least ten. SGI1-KIV, along with mutations in *gyrA* and *parC* genes, was found in all ST198 isolates. The *blaCTX-M-14.1* and *fosA3* genes, conferring resistance to extended-spectrum cephalosporins and fosfomycin, respectively, were present in 92% of ST198 isolates, located on an IncH12 plasmid. Phylogenetic analysis revealed that Zimbabwean *S. Kentucky* ST198 isolates formed a closely related clade, emerging from a previously reported global epidemic population. A significant finding was the close genetic relationship between human clinical and poultry isolates, supporting the hypothesis that poultry is a crucial source of highly resistant *S. Kentucky* in Zimbabwe. The IncH12 plasmid, designated pGTZIM1, carrying multiple AMR genes including *blaCTX-M-14.1* and *fosA3*, was identified through long-read sequencing. Another plasmid, pGTZIM2, carrying *blaCMY-2*, and a ColpVC plasmid, pGTZIM3, were also identified. Comparison with a global collection of *S. Kentucky* ST198 isolates showed that the Zimbabwean strains exhibited a greater number of AMR genes, reflecting recent acquisition of resistance determinants, particularly on pGTZIM1. This highlights the significant increase in AMR gene content in Zimbabwean ST198 strains compared to isolates from other regions.

This study provides strong evidence supporting the hypothesis that poultry represent a critical reservoir for the highly drug-resistant *S. Kentucky* ST198 clone in Zimbabwe. The close genetic relatedness and shared AMR gene profiles between human clinical and poultry isolates highlight potential transmission pathways. The identification of pGTZIM1, carrying multiple AMR genes including *blaCTX-M-14.1*, explains the concerning high level of resistance observed in these strains. The emergence of this novel plasmid in Zimbabwe emphasizes the need for heightened surveillance and intervention strategies to curtail the further spread of this MDR clone. Comparison with global ST198 isolates suggests a recent acquisition of the IncH12 plasmid, potentially within Zimbabwe. The high level of resistance observed necessitates a reconsideration of treatment strategies for *S. Kentucky* infections. The findings underscore the need for a One Health approach to address AMR, integrating surveillance and intervention strategies across human and animal health sectors.

This study demonstrates the emergence and spread of a highly drug-resistant *Salmonella* Kentucky ST198 clone in Zimbabwe, with strong evidence implicating poultry as a significant source. The identification of a novel IncH12 plasmid (pGTZIM1) carrying multiple AMR genes including the *blaCTX-M-14.1* gene highlights a concerning rise in resistance, emphasizing the urgent need for enhanced surveillance and control measures. Future research should focus on detailed investigation of transmission dynamics within the poultry industry and the wider community to inform effective intervention strategies. Furthermore, exploring the wider ecological niches that the ST198 clone inhabits in Zimbabwe is crucial to fully understanding its spread and potential for further AMR gene acquisition.

The relatively small sample size (37 *S. Kentucky* isolates) might limit the generalizability of the findings. The study primarily focused on poultry and human isolates, and data on other potential sources of *Salmonella* transmission, such as other animal species or environmental factors, were limited. Furthermore, the study's reliance on bioinformatic predictions for AMR gene identification requires experimental validation to confirm the phenotypic resistance profiles.