Comprehensive analysis of Staphylococcus aureus supernatants in different culture media to mimic chronic wound conditions in vitro

Staphylococcus aureus is a common pathogen found in chronic wounds and it produces virulence factors that inhibit wound healing. There is no standard infection model, so culture supernatants based on different media are used to study the properties of this pathogen. To develop an in vitro infection model, we therefore compared the influence of different culture media on the virulence factors of S. aureus supernatants and their toxicity toward keratinocytes. Supernatants were prepared in brain heart infusion (BHI) broth, lysogeny broth (LB), nutrient broth (NB), and tryptic soy broth (TSB). We assessed bacterial growth, the protein content of the supernatants, and a range of biological activities. SDS-PAGE revealed distinct protein bands consistent with S. aureus hyaluronidase and hemolytic toxins. Hyaluronidase was present in all supernatants and the total activity was similar in BHI, LB, and TSB, but significantly lower in NB. The analysis of hemolytic activity on blood agar and sheep erythrocytes revealed that TSB supernatants showed the highest hemolytic activity closely followed by BHI. Finally, cell viability assays using HaCaT keratinocytes revealed concentration-dependent toxicity for all supernatants, particularly TSB. Our work therefore shows that the culture medium can affect the virulence factors in S. aureus supernatants and also influences their cytotoxicity. These aspects should be considered when using culture supernatants for in vitro studies, and the quantification of virulence factors may improve the relevance and reproducibility of such studies. • Reproducible S. aureus supernatants in brain heart infusion broth, lysogenic broth, nutrient broth and tryptic soy broth. • Culture supernatant protein profiles in SDS-PAGE surprisingly similar regardless of culture medium. • S. aureus produces significantly less hyaluronidase in nutrient broth. • Tryptic soy broth leads to supernatants with the strongest hemolytic and keratinocytic toxicity. • Culture media significantly influences S. aureus virulence factor production and cytotoxicity, affecting in vitro studies.