Fibrin barriers limit vancomycin penetration into staphylococcal communities and impact S. aureus transcriptional responses

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 staphylococcal community (SC) formation and is compatible with timelapse microscopy and bulk transcriptional profiling. Using this system, we show that the fibrin barrier protects SCs from vancomycin by restricting drug penetration. Furthermore, we show that the fibrin barrier impacts transcriptional responses, leading to both shared and distinct transcriptional profiles between WT and fibrin barrier-deficient SCs. Together, these findings expand our understanding of SC fibrin barriers beyond immune evasion to include modulation of antibiotic interactions and bacterial physiological state.