Synthetic synovial fluid drives biofilm and virulence expression in Staphylococcus aureus

Staphylococcus aureus is a leading cause of implant-associated infections, including periprosthetic joint infections (PJI). Its high virulence and ability to form biofilms make these infections particularly difficult to treat. Traditional in vitro studies rely on conventional growth media that lack key components of the infectious microenvironment, limiting their physiological relevance. To this end, a synthetic synovial fluid (SSF2) model was developed incorporating essential host compounds such as amino acids, hyaluronic acid, albumin and fibrinogen. However, how accurately this model reflects S. aureus behavior during PJI remains unclear. To investigate this, RNA sequencing was performed on a PJI isolate of S. aureus grown in SSF2 and Luria-Bertani (LB) medium, followed by differential gene expression analysis. Functional classification using Clusters of Orthologous Groups (COG) and gene ontology (GO) enrichment was applied, and known and putative virulence and resistance genes were evaluated. To assess how closely SSF2 mimics in vivo conditions, transcriptomic profiles were compared with previously published in vivo gene expression data for the same strain. Approximately half of the protein-coding genes were differentially expressed in SSF2 compared to LB (56.8%). Consistent with transcriptomic findings, growth in SSF2 resulted in reduced growth rates and enhanced biofilm formation. Furthermore, SSF2 induced a large fraction of virulence-associated genes (69.5%), including those encoding toxins, iron acquisition systems, adhesins and efflux pumps. These findings are in line with previously published in vivo data of the same strain. These results highlight the potential of the SSF2 medium to approximate in vivo conditions encountered by S. aureus during PJI. Nonetheless, more comprehensive in vivo transcriptomic datasets are needed to fully validate this model. Overall design: 4 biological replicates of clinical S. aureus isolate SAU060112 grown in LB and in SSF2 (total 8 samples)