Layer-dependent modulation of S. aureus biofilms by light-induced oxidative stress

• Biofilms exhibit layer-dependent responses to light-induced oxidative stress. • Photodynamic treatment redistributes oxidative stress across biofilm layers. • Protective biofilm microenvironments associated with low penetration are disrupted. • Post-treatment biofilm recovery and metabolic activity are significantly impaired. • Repeated photodynamic exposure limits functional biofilm re-establishment. Bacterial biofilms display pronounced spatial heterogeneity, generating protective niches that contribute to antimicrobial tolerance and incomplete eradication. Photodynamic approaches based on light-induced oxidative stress have emerged as promising alternatives, yet how these effects are distributed across biofilm layers and how surviving cells respond after treatment remain poorly understood. Staphylococcus aureus biofilms (S biofilm) were exposed to light-induced oxidative stress, and post-treatment responses were evaluated through growth kinetics, biofilm reformation capacity, and layer-dependent photosensitizer penetration. S biofilms with different maturation times and structural complexities were analyzed to assess how biofilm architecture modulates photosensitizer distribution across superficial and deeper regions. 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. 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.