Fibrin barriers limit vancomycin penetration into staphylococcal abscess communities and maintain S. aureus in an unstressed, transcriptionally-responsive state

During Staphylococcus aureus infection, bacteria are frequently organized into staphylococcal abscess communities (SACs), multicellular bacterial aggregates encased within a host-derived fibrin barrier. Fibrin barriers are thought to insulate SAC-resident bacteria from host immune cells, as strains unable to form these barriers exhibit marked attenuation of virulence in animal infection models. However, the clinical consequences of these structures on antibiotic penetration, and whether the structures also impact bacterial physiology and alter antibiotic susceptibility, remains understudied. Here, we built upon prior work to develop a three-dimensional (3D) collagen gel matrix-based model, utilizing human coagulation components, that supports high throughput in vitro SAC formation and is compatible with timelapse microscopy and bulk transcriptional profiling. Using this system, we show that the fibrin barrier protects SACs from clinically relevant dosages of vancomycin by restricting drug penetration. Furthermore, we show that the fibrin barrier maintains stationary phase SACs in an unstressed and transcriptionally responsive state, in stark contrast with stationary phase planktonic S. aureus and fibrin barrier-deficient SACs. Together, these findings expand our understanding of SAC fibrin barriers beyond immune evasion to include modulation of antibiotic interactions and bacterial physiological state. AUTHOR SUMMARYOver the course of infection, many bacterial pathogens organize into clustered communities, which limits our ability to fully eliminate bacterial cells with antibiotic treatments. During Staphylococcus aureus infection, bacteria form dense clusters surrounded by a protective fibrin barrier derived from host blood clotting machinery, in structures known as staphylococcal abscess communities (SACs). SAC fibrin barriers have been shown to protect against immune cell invasion, but any effects on bacterial physiology and responses to antibiotics remain unclear. In this study, we developed an in vitro system to rapidly grow SACs in a 3D collagen gel matrix suspension, closely mimicking human tissue. Using this system, we show that fibrin barriers restrict vancomycin penetration into maturing SACs, protecting them from exposure to this clinically relevant antibiotic. We also show that bacteria within mature SACs are maintained in an unstressed and transcriptionally responsive state when the fibrin barrier is present, in contrast with fibrin barrier deficient SACs. These findings not only suggest that fibrin barriers serve as a physical barrier to drug diffusion but also show the importance of fibrin barriers in bacterial physiology, highlighting their importance in SAC biology and infection persistence.