A Rapid Labeling Platform for Real-Time Visualization of Pseudomonas aeruginosa Phage-Host Interactions

Directly visualizing phage-host interactions is critical to understanding why phage therapy often fails against Pseudomonas aeruginosa biofilms, yet imaging tools for this pathogen system are scarce and typically rely on laborious dye conjugation or genetic modifications. To address this, we developed an imaging platform by adapting a rapid, non-genetic Syto staining method and tested it on the P. aeruginosa podovirus PEV2, demonstrating the generalizability of the approach. By optimizing this technique for confocal microscopy, we can visualize the entire infection cycle in real-time at single-cell resolution with negligible impact on phage efficacy. The translocated dye serves as a direct indicator of successful infection and is compatible with other viability stains. Crucially, this platform is poised to enable direct investigation of phage dynamics within biofilms, which will permit the dissection of resistance and tolerance mechanisms and inform the rational design of more effective anti-biofilm strategies.