Characteristics of Staphylococcus haemolyticus Isolated from Humans and Animals

Staphylococcus haemolyticus is an opportunistic coagulase-negative staphylococcus increasingly recognized as an important reservoir of antimicrobial resistance within the One Health framework. However, comparative studies simultaneously evaluating isolates from humans and companion animals remain limited. This study characterized 113 S. haemolyticus isolates recovered from clinically healthy and affected humans and cats and from clinically healthy dogs using phenotypic, molecular, proteomic, and genomic approaches. Multidrug resistance was identified in 45.13% (51/113) of isolates. Whole-genome sequencing of 35 selected isolates revealed substantial genomic diversity, with no clear clustering according to host species or clinical status within the analysed subset. The VFDB-associated loci tufA, clpC, and clpP were detected in all sequenced isolates; however, their presence does not imply expression or a direct contribution to virulence-related phenotypes. Acquired antimicrobial resistance determinants showed variable distributions across the analysed genomes. Within the WGS subset, mecA showed a host-associated difference that remained significant after false discovery rate correction in a one-isolate-per-host sensitivity analysis, whereas the initially observed association for blaZ/PC1-family determinants was not retained after this sensitivity analysis. No acquired resistance determinant was significantly associated with clinical status. Among the full collection of 113 isolates, blaZ (53.98%) and tetL (33.63%) were the most frequently detected resistance genes by PCR. Genotype–phenotype concordance varied according to the antimicrobial agent and phenotypic testing method, indicating that resistance-gene detection alone was not uniformly predictive of phenotypic susceptibility. Biofilm formation, assessed in all 113 isolates, increased markedly at 39 °C despite heterogeneous, strain-dependent changes in planktonic growth. Eight representative isolates evaluated in the Galleria mellonella model showed clear inoculum-dependent and strain-variable larval mortality; however, the assay was not designed to formally assess host-species- or clinical status-associated differences. Overall, the findings demonstrate substantial genomic and phenotypic overlap among human and companion-animal S. haemolyticus isolates, together with selected host-associated differences and marked strain-level variability, supporting the value of integrated One Health surveillance.