Real-time Imaging of Response to Host-Induced Stress in Pseudomonas aeruginosa Aggregates

Chronic infections such as those in cystic fibrosis (CF) are sustained by small, highly tolerant Pseudomonas aeruginosa aggregates that persist despite immune and therapeutic pressures. Unlike classical biofilms, these aggregates represent a distinct pathogenic unit - microscale, spatially organized communities that maintain structural integrity and physiological homeostasis under host-induced stress. However, the mechanisms that enable aggregates to remain intact under these conditions, and whether these homeostatic processes can be selectively disrupted, remain poorly defined. A key barrier to addressing this gap has been the lack of tools capable of capturing the dynamic, spatially resolved processes that link physiological stress to structural stability and collapse in real time. This study presents an integrated imaging and analytical workflow to quantify aggregate responses to host-relevant stressors. Using the voltage-sensitive dye DiBAC4(5), membrane depolarization was monitored as an early indicator of physiological disruption within aggregates formed in synthetic cystic fibrosis sputum medium (SCFM2). High-resolution time-lapse confocal microscopy enables visualization of aggregates in both stable and stress-induced states, while image segmentation and voxel-based analysis provide quantitative mapping of spatial heterogeneity in membrane integrity and aggregate disassembly at single-cell resolution. This workflow establishes a reproducible and adaptable platform for linking physiological stress to structural outcomes in multicellular bacterial aggregates. By enabling quantitative dissection of the processes that preserve - or compromise - aggregate integrity, this approach provides a critical foundation for identifying and targeting the homeostatic mechanisms that underpin aggregate resilience, advancing new strategies to disrupt this clinically significant mode of bacterial persistence.