CHARACTERISTICS OF CLINICAL NONTYPHOIDAL SALMONELLA ISOLATES FROM A HISTORICAL BIOBANK WITH A FOCUS ON THE INVASIVE SEROTYPE CHOLERAESUIS, LOMBARDY REGION (2001-2016)

Introduction. Salmonella enterica is a leading cause of foodborne infections worldwide, with clinical presentations ranging from self-limiting gastroenteritis to invasive systemic disease. While typhoidal serotypes are well recognized as agents of enteric fever, a subset of nontyphoidal serotypes also displays an unusually high invasiveness index, posing a major challenge for surveillance and control. In this context, analyses of historical biobanks of clinical Salmonella strains represent an invaluable tool to improve knowledge of the biological, clinical, and epidemiological characteristics of the pathogen, as well as to investigate pathogenicity and antimicrobial resistance mechanisms. Aim. The aim of this study was to enhance and expand the potential of the historical Salmonella enterica preserved at the EpiSoMI laboratory (Center of Coordinate Research – “Epidemiologia e Sorveglianza Molecolare delle Infezioni”) and established during the operational period of the Centro Enterobatteri Patogeni per l’Italia Settentrionale (CEPIS). Clinical S. enterica strains isolated in Lombardy between 2001 and 2016 and archived in our laboratory biobank were analyzed. Each isolate was associated with epidemiological, clinical, and demographic data corresponding to the related salmonellosis case. Furthermore, this thesis sought to better characterize the burden of invasive Salmonella serotypes in Lombardy, with a specific focus on S. Choleraesuis, a swine-adapted yet highly invasive human pathogen. Materials and Methods. Datasheets from 6,624 laboratory-confirmed salmonellosis cases (2001–2016) were collected and analyzed to assess trends in incidence, demographics, clinical presentation, and hospitalization rates. Invasive serotypes were identified and characterized. Archived isolates underwent phenotypic assays to assess fitness and survival under acidic conditions. Particular attention was devoted to the S. Choleraesuis serotype due to its invasive properties. Genomic analyses included whole-genome sequencing (WGS), core-genome multilocus sequence typing (cgMLST), and in silico investigation of antimicrobial resistance determinants. Results. A total of 172 nontyphoidal serotypes (NTS) were identified. The most prevalent were S. Typhimurium (41.8%, n = 2,772), S. Enteritidis (18.5%, n = 1,223), and S. 1,4,[5],12:i:- (9.8%, n = 649), which were primarily associated with gastroenteritis. In addition, a subset of invasive serotypes linked to systemic infections and higher hospitalization rates emerged. Bloodstream infections were identified in 244 patients, with S. Choleraesuis, S. Typhimurium, and S. Napoli being the most common serotypes (24.6%, n = 60; 22.5%, n = 55; 15.8%, n = 38, respectively). S. Choleraesuis displayed one of the highest invasive indexes (76.9%), disproportionately affecting older adults and frequently leading to severe extraintestinal disease. Phenotypic assays revealed comparable growth fitness across serotypes, but S. Choleraesuis exhibited enhanced acid tolerance, consistent with its ability to overcome the gastric barrier and disseminate systemically. Genomic analyses demonstrated that isolates of this serotype formed a highly clonal population, frequently harboring multidrug resistance genes, including determinants conferring resistance to aminoglycosides, sulfonamides, tetracyclines, and β-lactams. Discussion. Although our biobank data consists of Salmonella enterica isolates collected over several years without any clear information as to how these samples and data were collected, it provides a realistic snapshot of the serotype’s circulation. These findings highlight the dual nature of salmonellosis: a substantial burden of self-limiting gastroenteritis caused by common serotypes, alongside a smaller but clinically significant fraction of invasive infections associated with specific serotypes characterized by host adaptation and multidrug resistance. The persistence of clonal invasive lineages, such as S. Choleraesuis, underscores the zoonotic interface between swine and humans and illustrates the risks posed by antimicrobial resistance. Conclusion. This study demonstrates the value of combining long-term biobank resources with clinical and demographic data to capture shifts in serovar distribution, antimicrobial resistance, and pathogenic potential over time, providing a comprehensive view of invasive Salmonella infections in Lombardy. The results emphasize the need for continuous One Health-based surveillance, targeting both common and invasive serotypes, to mitigate the impact of salmonellosis on human health.