IN VIVO TRANSCRIPTIONAL DYNAMICS OF VIRULENCE FACTORS IN CLINICAL STAPHYLOCOCCUS AUREUS ISOLATES DURING FRACTURE-RELATED INFECTION

Aim Fracture-related infections (FRI) remain a major challenge in trauma surgery and are most commonly caused by Staphylococcus aureus. Clinical manifestations range from acute systemic infections to chronic, persistent disease, indicating that bacterial virulence is a key determinant of infection dynamics. However, in vivo regulation of virulence factors in implant-associated environments remains poorly understood. This study aimed to characterize the in vivo transcriptional dynamics of virulence-associated genes in clinical S. aureus isolates and to assess their impact on pathogenicity and host immune response in implant-associated infection. Methods Two clinical S. aureus isolates with distinct virulence profiles (EDCC 5458: high virulence; EDCC 5464: low virulence) were investigated. Expression of key virulence genes (hlgA, hlgB, hlgC, scn, sak, aur, and sarA) was quantified using RT-qPCR under in vitro conditions (planktonic growth and biofilm formation) and in vivo using a Galleria mellonella infection model. Implant-associated infection was simulated by inserting titanium K-wires colonized with a bacterial biofilm. Bacterial burden was quantified by CFU analysis, while larval survival and host immune response markers were assessed to evaluate infection severity and host–pathogen interactions. Results Under planktonic in vitro conditions, the high-virulent strain EDCC 5458 showed marked upregulation of γ-hemolysin genes, whereas EDCC 5464 exhibited no relevant transcriptional changes. In the hematogenous infection model, no significant differences in larval survival were observed between strains. In contrast, biofilm-associated infection resulted in significantly reduced survival in the EDCC 5458 group (13.3%) compared to EDCC 5464 (46.6%; p ≤ 0.01). During biofilm formation, both strains demonstrated a general downregulation of virulence-associated genes. However, following in vivo biofilm maturation, EDCC 5458 exhibited a ≥2.5-fold increase in γ-hemolysin gene expression, accompanied by significantly higher bacterial load on implants (0.5–2.0 log 10 CFU/implant) and in surrounding tissue (0.3–1.0 log 10 CFU/larva; p < 0.05). This was associated with a markedly enhanced host immune response compared to EDCC 5464. Conclusions These findings demonstrate a dynamic and environment-dependent regulation of virulence expression in Staphylococcus aureus during implant-associated infection. Differences between high- and low-virulent strains are particularly reflected in the activation of cytotoxic factors and the magnitude of the host immune response. The Galleria mellonella model enables standardized in vivo transcriptional analysis and provides valuable insights into host–pathogen interactions in fracture-related infection. In the long term, this approach may contribute to the development of improved diagnostic strategies and targeted therapeutic concepts in musculoskeletal infections.