Single-Cell Membrane-Permeabilization Kinetics Reveal Species-Specific Responses to Antimicrobial Photodynamic Treatment

Abstract Bacterial populations that appear uniform can hide striking differences in how individual cells respond to antimicrobial stress. This hidden heterogeneity is especially important for antimicrobial photodynamic treatment (aPDT), where population-averaged assays report whether bacteria recover but cannot reveal when individual cells become damaged or why some cells respond later than others. Here, we developed an agarose-pad single-cell imaging platform to track membrane permeabilization during aPDT mediated by Br2B, a brominated boron dipyrromethene (BODIPY) dye. Using extraintestinal pathogenic Escherichia coli UMN026 and Staphylococcus aureus SA113 as model pathogens, time-resolved propidium iodide (PI) fluorescence trajectories were fitted for individual bacteria to extract the onset of PI entry (ti), final PI-positive transition time (tf), and transition duration (Δt = tf – ti). This approach revealed distinct species-specific response architectures that were not apparent from bulk measurements alone. In E. coli, photodynamic response heterogeneity was distributed across both delayed PI-entry onset and prolonged transition duration, particularly under nutrient-rich LB conditions and in mixed-founder populations. In contrast, S. aureus displayed a compressed onset phase, with heterogeneity emerging mainly after PI entry had begun. Br2B uptake was consistently higher in S. aureus than in E. coli, supporting a model in which envelope architecture and photosensitizer access determine where heterogeneity appears during photodynamic damage. Post-aPDT regrowth assays further showed that recovery kinetics were species- and population-dependent, linking single-cell membrane-permeabilization dynamics with later population-level outgrowth. Finally, mutation-rate estimates indicated that colony-derived inocula are not genetically identical, but that mutation-derived diversity is unlikely to be the dominant source of the observed timing patterns. Together, these results demonstrate that aPDT response is not a single synchronized event but a structured, species-dependent process shaped by photosensitizer access, nutrient context, founder-lineage history, and physiological heterogeneity. This work establishes single-cell PI-entry kinetics as a powerful framework for uncovering hidden antimicrobial response architectures during photodynamic treatment.