Molecular epidemiology and virulence determinants of emerging Staphylococcus aureus clones in farmed rabbits

Staphylococcus aureus is a major pathogen in commercial rabbit farming, causing significant economic damage and animal welfare concerns. Staphylococcosis have been associated with a limited number of clones, particularly the highly virulent ST121 and the low-virulence ST96. However, in recent years, veterinarians and rabbit producers have reported increasingly virulent and persistent outbreaks of S. aureus in rabbit farms across Portugal and Spain. These outbreaks may reflect the evolution of novel clones with distinct epidemiological and pathogenic profiles, facilitated by the species’ inherent genomic versatility. This study aimed to characterise S. aureus isolates through genotyping, geographical mapping, whole-genome sequencing, and in vivo virulence assessment. A total of 200 isolates were analysed, revealing the predominance of ST121 (27%), ST2855 (20.5%), and ST3764 (16%), with ST2855 and ST3764 representing emerging clones with wide regional distribution. Genomic analysis identified differences in virulence genes and mobile genetic elements, including the presence of enterotoxin clusters, bbp , and type VII secretion system components in CC121 strains, which were absent in CC96. Despite genetic changes potentially linked to increased virulence, in vivo assays showed that ST2855 and ST398 strains were unable to establish infection under experimental conditions, while ST3764 strains exhibited infectivity comparable to ST121. • Emerging clones ST2855 and ST3764 identified in Iberian rabbit farms, indicating a shift in the population structure of Staphylococcus aureus traditionally dominated by ST121 and ST96. • ST2855 shows wide geographical distribution, particularly in northern Spain and Portugal, suggesting possible local adaptation or recent expansion across multiple regions. • Genomic analysis revealed distinct virulence profiles among clonal complexes: CC121 strains harbor bbp adhesin, the egc enterotoxin cluster, and type VII secretion system genes, features absent in CC96 strains, which may explain differences in pathogenic potential. • ST2855 differs from classic CC96 strains, having lost the Sa3int-type phage and acquired mutations in the dltB gene, although these genetic changes did not translate into increased virulence in experimental infection models. • In vivo assays confirmed that ST3764 exhibits infectivity comparable to ST121, while ST2855 and ST398 were unable to establish infection under the tested conditions, highlighting the complexity of virulence beyond genomic predictions.