PhD Thesis defense by Clara Samper Cativiela at the VISAVET Centre of the Complutense University of Madrid

June 20^th, 2025

Non-typhoidal Salmonella (NTS) serovars cause salmonellosis, a globally relevant foodborne zoonosis primarily characterized by gastrointestinal manifestations. Since the mid-20th century, surveillance and control measures have been implemented along the food chain to mitigate its health and economic impact. However, human incidence and prevalence of NTS infection in animal reservoirs remain high. This is due to the complexity of its epidemiology, involving multiple animal species that can act as asymptomatic carriers, and the increasing emergence of antimicrobial-resistant strains, a phenomenon that complicates control efforts and limits the effectiveness of available treatment. Additionally, anthropogenic and environmental factors contribute to the spread of this pathogen, underscoring the need of considering these aspects to maximize the success of control measures.

In this context, the present thesis addresses the issue of NTS through a cross-disciplinary approach using epidemiological and microbiological analyses based on data and biological material obtained from surveillance programs in Spain. Specifically, data from the National Control Plans (NCPs) in laying hens—considered one of the primary sources of infection— were analysed along with microbiological and epidemiological data from isolates of serovars of public health importance obtained from clinical cases, livestock, food, and wildlife. Analytical methods based on Bayesian statistics (Study I) and whole-genome sequencing (WGS) (Studies II and III) were applied.

Despite the ubiquity of NTS, the consumption of poultry products, particularly eggs, is the primary source of human infection. Since 2008, NCPs have been implemented in poultry production in Spain and other European Union countries, generating valuable data on the prevalence of NTS and, in particular, of target serovars (Enteritidis, Typhimurium, and 4,[5],12:-.). Study I analysed the association between factors at the farm, flock, and sampling levels and the probability of detecting NTS in laying hen farms using a hierarchical Bayesian model. Data from the NPCs collected between 2015 and 2020 were used. The study found that the geographic location of farms (measured at the regional level) was associated with significant variations in the odds of NTS detection, suggesting the influence of environmental and management factors. Additionally, caged systems were linked to a higher probability of detection compared to alternative systems, likely due to their higher animal density and challenges ensuring their proper disinfection. A seasonal effect was also observed, with higher detection probabilities in autumn/winter, along with an impact of the agent carrying out the sampling: samples taken by competent authorities had between 5.63- and 7.75-times higher odds of detection compared to those collected by food business operators. Although the study is observational and may be subjected to certain biases (e.g., spatial and temporal dependencies in sampling results), its findings underscore the need for a continuous review of surveillance and control strategies. This is particularly relevant in the context of emerging NTS clones of public health concern, such as S. Kentucky ST198 and S. Enteritidis ST11.

In Studies II and III, WGS was applied to a representative panel of isolates of these two serovars, characterizing their genomes and antimicrobial resistance profiles. The serovar Kentucky has raised concerns due to the emergence of multidrug-resistant (MDR) lineages, particularly ST198, widely associated with global poultry production. Study II analysed 66 S. Kentucky isolates obtained through the implementation of the NCPs between 2011 and 2017 to determine whether the observed increase in resistance in poultry S. Kentucky isolates was linked to the MDR ST198 lineage, and to characterize its distribution and resistance profiles. Genomic results confirmed that all isolates belonged to the ST198 clone, and were grouped into three main clades with a different spatiotemporal distribution. These strains harbored an extensive set of resistance genes, surpassing what has been reported in other ST198 strains worldwide, and included genes encoded on IncHI2/IncI1 plasmids. The repeated detection of similar plasmids over several years in certain laying hen farms suggests the existence of persistent sources of infection and/or failures in cleaning and disinfection protocols. These findings highlight the widespread distribution of the MDR ST198 clone in Spanish poultry production and underscore the need to strengthen surveillance and control strategies to mitigate its impact on public health.

The serovar Enteritidis, historically one of the leading causes of foodborne outbreaks associated with poultry products, was the focus of Study III. A total of 298 isolates obtained between 2002 and 2021 from clinical cases, food, animals, and the environment in Spain were analysed to characterize their genomes and establish relationships based on their origin. Two main ST11 clades exhibiting clear genomic and phenotypic differences were identified. Clade I, genetically more heterogeneous and predominantly pansusceptible, included 57% of the human cases. It was associated with the "Atlantic Clade" or MGT14-CC13, a lineage historically linked to modern poultry farming and widely distributed in Europe and North America. Clade II, associated with the "Global epidemic clade," was in contrast mainly composed of isolates from animal, food, and environmental origins (77.2%). It showed greater genomic homogeneity and a higher frequency of mutations in quinolone resistance-determining regions (QRDRs) and ?-lactam resistance genes located on IncX1 plasmids. While resistance was more frequent in animal isolates, the most concerning resistance phenotypes (e.g., ESBL and pAmpC) were only found in human isolates.

The cross-disciplinary approach adopted in this thesis has allowed the identification of factors associated with NTS detection in laying hen farms and clarified the epidemiology of two emerging NTS clones (S. Kentucky ST198 and S. Enteritidis ST11) in food animals in Spain. These findings indicate the need for continuous evaluation and optimization of surveillance and control strategies, considering both host and pathogen factors, to reduce NTS incidence and mitigate the impact of antimicrobial resistance on public health.