Prevalence, trends and associations of antimicrobial resistance and virulence factors in Salmonella enterica, including the genomic characterization (WGS) of monophasic variant 4,[5],12:i:- isolated along the food chain from Marche Region, Italy (2005-2022)

Salmonellosis is reported as the second most frequent foodborne zoonotic disease in the European Union (EU). The evolution of antimicrobial resistance (AMR) and virulence in Salmonella spp. over time is inevitable. The acquisition and recombination events of mobile genetic elements (MGEs), including transposons, genomic islands, plasmids, and integrons, act as primary drivers of evolution. This study investigates the prevalence, trends, and associations of antimicrobial resistance (AMR) and virulence genes in a wide Salmonella enterica strain panel isolated from the food chain of the Marche region of Italy (2005–2022). Inclusivity of invA and ttrC-based PCR assays was also analyzed for genus-specific Salmonella detection. A monophasic variant of S. enterica serovar Typhimurium (MVST) 4,[5],12:i:-, has emerged as a major public health threat worldwide, including Italy. The genomic characterization of MVST isolates was carried out to analyze in depth the AMR, plasmid, and virulence profiles, along with the fljBA operon region variability and the genetic relatedness of MVST isolates through cgMLST analysis. The ttrC and the invA-based real-time PCR assays showed 100% inclusivity and specificity performance. Human isolates showed the highest resistance to sulfisoxazole (61%), tetracycline (56%), and ampicillin (54%); food and veterinary (food-vet) to pefloxacin (68%), tetracycline (62%), and nalidixic acid (59%); surface water to sulfisoxazole (56%), streptomycin/tetracycline (52%). Higher MDR levels were observed in humans (61%), followed by food-vet (57%), and surface water (54%). Increasing trends of resistance were shown by humans and food-vet isolates (not towards all classes) across all three periods (2005-10, 2011-16, 2017-22). The presence of the invA gene was ubiquitous in the tested isolates. Human isolates always carried sopB, sipA, sipB, and tolC (100%), with sifA (86%), followed by flgK (85.1%), flgL (81.6%), ssaR (75.4%), sipD (73.7%), sopD 51.8% and spvB (32.5%) the least prevalent. Food-vet isolates showed the highest prevalence of sopB (80.6%), tolC (79%), sipA (77.4%), and sipB (74.7%), while spvB had the lowest (3.8%). Surface water isolates had tolC (100%), sipA (97%), sipB (95%), flgL (73%), and flgK (70%), the highest, while the least prevalent were spvB (27%) and sipD (17%). Statistically significant associations (p < 0.05) for some virulence genes and serovars, the strongest association for virulence genes and resistance profiles in food-vet, and highly significant associations between virulence genes and origins were detected. S. Enteritidis (humans), S. Infantis (food-vet), and S. Typhimurium (surface water) carried the widest range of virulence genes. Variable temporal trends for virulence gene prevalence were found among S. Enterica strains. The presence of different ASSuT variants (ASSuT, ASSuT-plus, and incomplete-ASSuT) was detected in MVST isolates. Overall, 29 resistance genes belonging to eleven different antimicrobial classes were found. Genes conferring resistance to ASSuT phenotype were more frequent; aph(6)-Id, aph(3'')-Ib/sul2 (100%), and blaTEM-1 (96%), tet(B) (86%). The analysis of the genetic region surrounding the fljBA operon revealed an extensive genetic variability, shaped by transposon insertions, AMR, and heavy metal resistance genes (HMRGs). Two main regional clusters and two outliers were found in the cgMLST analysis, suggesting a potential for transmission along the food chain. The observed prevalence and rising trends of AMR highlight the need for improved antibiotic stewardship and surveillance. Virulence gene prevalence and variable trends observed over the time periods represented the serovar-specific pathogenic potential of Salmonella in different origins. These results emphasize the need for integrated genomic surveillance along with One Health monitoring to track and control the spread of this pathogen along the food chain of Central Italy.