Layer-dependent modulation of

Background: Bacterial biofilms display pronounced spatial heterogeneity, generating, yet how these effects are distributed across biofilm layers and how surviving cells respond after treatment remain poorly understood. Methods: Results: Light-induced oxidative stress altered post-treatment growth dynamics and impaired the ability of surviving cells to re-establish structured biofilms. Photosensitizer penetration was strongly layer-dependent and influenced by S biofilm maturation and complexity, resulting in spatially heterogeneous photodynamic effects. These effects differentially impacted cells located in superficial versus deeper biofilm regions. Conclusion: These findings demonstrate that photodynamic treatment modulates S. aureus biofilm behavior in a layer-dependent manner, weakening protective niches without requiring complete eradication. By disrupting biofilm microenvironments associated with tolerance and persistence, light-induced oxidative stress limits biofilm recovery and provides a mechanistic basis for photodynamic strategies aimed at controlling biofilm re-establishment.