Synergistic tissue destruction by Staphylococcus aureus and Staphylococcus epidermidis in a 3D human skin biofilm equivalent.

Staphylococci are common skin commensals that can transition into opportunistic pathogens, particularly in biofilm-associated and polymicrobial infections. However, how interspecies interactions modulate virulence remains poorly understood, partly due to a lack of human-relevant models. We adapted a human cell-based three-dimensional skin equivalent (3DSE) into a biofilm infection model using monospecies biofilms of Staphylococcus aureus or Staphylococcus epidermidis, and a dualspecies co-culture. Biofilm architecture and spatial distribution were analysed by histology and fluorescence in situ hybridisation, while bacterial dominance was assessed by colony-forming unit counts. Host responses were evaluated using a composite biofilm destruction score, lactate dehydrogenase release, apoptosis and tight junction integrity, and cytokine profiling. The 3DSE supported robust, species-specific biofilm formation. Notably, despite reduced biofilm mass, dualspecies biofilms caused the most severe tissue damage, cytotoxicity and epithelial disruption. Although S. aureus dominated in co-culture, pathogenicity was not dependent on bacterial load. These findings demonstrate synergistic host modulation in polymicrobial staphylococcal biofilms and establish the 3DSE as a physiologically relevant platform for studying skin biofilm infections.