Staphylococcus haemolyticus (S. haemolyticus) is one of the Coagulase-negative staphylococci (CoNS) that
inhabits the skin as a commensal. It is increasingly implicated in opportunistic infections, including
diabetic foot ulcer (DFU) infections. In contrast to the abundance of information available for S. aureus
and S. epidermidis, little is known about the pathogenicity of S. haemolyticus, despite the increased
prevalence of this pathogen in hospitalized patients. We described, for the first time, the pathogenesis of
different clinical isolates of S. haemolyticus isolated from DFU on primary human skin fibroblast (PHSF)
cells. Virulence-related genes were investigated, adhesion and invasion assays were carried out using
Giemsa stain, transmission electron microscopy (TEM), MTT and flowcytometry assays. Our results
showed that most S. haemolyticus carried different sets of virulence-related genes. S. haemolyticus
adhered to the PHSF cells to variable degrees. TEM showed that the bacteria were engulfed in
a zipper-like mechanism into a vacuole inside the cell. Bacterial internalization was confirmed using
flowcytometry and achieved high intracellular levels. PHSF cells infected with S.haemolyticus suffered
from amarked decrease in viability and increased apoptosis when treated with whole bacterial suspensions or cell-free supernatants but not with heat-treated cells. After co-culture with PBMCs, S. haemolyticus induced high levels of pro-inflammatory cytokines. This study highlights the significant
development of S. haemolyticus, which was previously considered a contaminant when detected in
cultures of clinical samples. Their high ability to adhere, invade and kill the PHSF cells illustrate the severe
damage associated with DFU infections.