Long-term spatiotemporal biological and mechanobiological dynamics of Staphylococcus aureus biofilms.

In the context of implanted medical devices, biofilm formation transforms otherwise inert biomaterials into persistent sources of infection that are highly resistant to antimicrobial therapy and immune clearance. This persistent problem demands a critical re-examination of how biofilms are conceptualized, studied, and modeled. Using tissue-mimicking gelatin methacryloyl hydrogels, we show that Staphylococcus aureus spatiotemporal biofilm dynamics are strongly regulated by matrix mechanics and nutrient availability. Nutrient-rich conditions promote hyperproliferative, eDNA-rich microcolonies that actively degrade polymer networks, whereas serum-supplemented conditions produce compact aggregates enriched with vesicle-like structures and serum-derived matrix components. Notably, 3D confinement revealed a previously unrecognized 'budding' dispersal mechanism unique to embedded biofilms. Integrating 3D-printed polycaprolactone implants shows that while implant surfaces promote bacterial adhesion, the surrounding microenvironment exerts primary regulatory control. Longitudinal tracking over 3 weeks recapitulates key biofilm hallmarks and challenges surface-centric paradigms of implant infections, providing a framework for dissecting infection mechanisms and guiding therapeutic strategies.