Genomic diversity, farm-level persistence, and food safety risk traits of Staphylococcus aureus in small-ruminant raw milk dairy chains in central Italy.

Staphylococcus aureus is a major food safety and One Health concern in raw milk dairy chains. This study investigated its occurrence, contamination levels, antimicrobial resistance (AMR), enterotoxigenic potential, surface-adhesion phenotype, and genomic relatedness across 15 small-scale ovine-caprine dairy farms in the Umbria and Marche regions of Central Italy. Overall, 425 food, environmental, and operator hand swab samples were analysed. S. aureus was detected in 208/425 samples (48.9%), with the highest occurrence in food matrices (62.1%) and marked farm-level heterogeneity. One finished cheese product tested positive for staphylococcal enterotoxins (SEs). A total of 1662 colonies from 208 positive samples were confirmed as S. aureus, and a representative subset, including the MRSA isolates, underwent phenotypic characterization and whole-genome sequencing (WGS). No isolate carried the mupirocin resistance gene mupA. WGS revealed diverse sequence types, mainly ST133, ST700, and ST522. Phenotypic resistance was observed mainly to sulfisoxazole, tetracycline, and penicillin, whereas the most frequent AMR genes were blaZ, tet(M), and tet(K). Classical SE genes were detected in a subset of sequenced isolates, and all sequenced isolates carried at least one SE or SE-like gene. All tested isolates adhered to polystyrene, mostly moderately or strongly, and all sequenced isolates carried the biofilm-associated icaA, icaB, icaC, icaD, and icaR genes. Phylogenetic analyses indicated farm-specific clustering and persistence or recurrence of genetically related isolates across matrices and seasons. These findings support farm-specific hygiene interventions and integrated phenotypic-genomic surveillance to control AMR and potentially toxigenic S. aureus in artisanal small-ruminant raw milk dairy production.