Impact of Heat and Acidic Stress on the BiochemicalComposition of Pseudomonas aeruginosa BacteriophagesA Nanoscale AFM-IR Study

Antimicrobial resistance (AMR) poses a significant global health threat, highlighting the need for effective bacteriophage (phage) therapy formulations. Understanding phenotypic and genotypic changes is essential for optimizing phage formulation and delivery. Here, we examine the chemical composition changes of Pseudomonas aeruginosa-specific phages subjected to external stresses, including heat and acidification, using atomic force microscopy-infrared spectroscopy (AFM-IR), with results confirmed by transmission electron microscopy (TEM) and biological assays. We assessed two structurally distinct phageslong-tailed myovirus PEV20 and short-tailed podovirus PEV31to evaluate their differential resilience. We observed variations in signal intensity and peak positions in the amide I and II regions, indicating protein degradation on phage capsids, as well as changes in the nucleic acid content related to capsid stability and integrity. TEM analysis confirmed structural alterations such as ruptured capsids and tail detachment, which were more pronounced in PEV31, correlating with a greater loss of viability observed in biological assays. These findings enhance our understanding of phage resilience to stress and support the development of stable phage-based therapies for combating multidrug-resistant bacterial infections.