Assessing host-adaptation of new sequence types of livestock-associated methicillin-resistant Staphylococcus aureus...

The importance of animals as reservoirs of human pathogens is widely recognised, with livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) being consistently reported. This study presents a phenotypic and genotypic characterization of five MRSA isolates from raw bovine milk in northern Portugal, using culturomics and whole genome sequencing (WGS), to assess their potential risk for human infection. Resistance to beta-lactams was confirmed by the presence of the mecA resistance gene as part of the SCCmec type V element (5C2&5), complemented by a resistance profile to other antibiotics characteristic of adaptation to animal hosts. Several virulence factors were identified, including some with potential human tropism, such clumping factors, gamma-hemolysins and staphylococcal complement inhibitors. All MRSA isolates gave rise to new sequence types (ST), newly added to the MLST database as ST8475, ST8894 and ST8895, which show a variation of 1 or 2 alleles compared to the recognised LA-MRSA ST398/CC398 lineage. A phylogenetic comparison of these MRSA isolates with other S. aureus isolates confirmed a close genetic relationship with the ST398/CC398 lineage. This finding further supports the inclusion of these new STs in this clonal complex. A pathogenicity in vivo test using the Galleria mellonella model, which has an innate immune system similar to humans, showed a high pathogenicity of the MRSA isolates, and an assessment of biofilm production showed a similar capacity for biomass formation among all isolates. These findings demonstrate a typical adaptation of the isolates to animal hosts, while retaining or acquiring traits associated with the ancestral human host of S. aureus. Such dual adaptation emphasizes their zoonotic potential and strengthens the relevance of LA-MRSA as a critical public health concern.