Systematic evaluation of phenotypic variations induced by prophages in a clinical isolate of <i>Pseudomonas aeruginosa</i>

With the rise of antibiotic resistance, nosocomial infections caused by <i>Pseudomonas aeruginosa</i> present a significant challenge to healthcare systems. Prophages integrated into bacterial chromosomes play a key role in generating phenotypic and genotypic diversity, influencing bacterial pathogenicity and complicating antimicrobial treatment strategies. Recent advances in high-throughput sequencing have facilitated the precise localization of prophages within bacterial genomes. However, the mechanisms by which prophages influence host phenotypes remain poorly understood, particularly in clinical polylysogens. In this study, we conducted a comprehensive investigation of prophages predicted in the clinical <i>P. aeruginosa</i> isolate ZS-PA-05 in the absence of an external trigger, focusing on their contribution to bacterial phenotype through the use of prophage deletion mutants. Our findings indicate that ZS-PA-05 harbors both active and cryptic prophages, each exhibiting unique spontaneous induction rates. Notably, the absence of certain prophages significantly affects bacterial growth, motility, biofilm formation, antibiotic susceptibility, and phage proliferation, as well as virulence. Intriguingly, the deletion of prophage Y significantly increased pyocyanin production, enhancing interspecies competition and cell line survival, despite impaired bacterial growth. This underscores the complexity of prophage-host interactions and emphasizes the role of prophages in bacterial adaptation, with far-reaching implications for antimicrobial therapies and beyond.IMPORTANCEUpon infecting a bacterial host, phages can follow one of two developmental pathways: the lytic or lysogenic cycle. In the lysogenic state, prophages remain dormant, integrating into the bacterial genome and being vertically transmitted through binary fission. These prophages profoundly influence bacterial phenotypic and genetic diversity and contribute to the structuring of microbial communities. Here, we systematically assess the beneficial and detrimental impacts of prophage carriage in the clinical multilysogen <i>Pseudomonas aeruginosa</i> strain ZS-PA-05. Our results reveal marked variation in spontaneous induction frequencies among co-resident prophages and demonstrate prophage-driven phenotypic heterogeneity. By uncovering key aspects of prophage-host interactions, this study highlights the critical role of prophages in shaping the behavior of clinical isolates, particularly in the context of antimicrobial interventions such as antibiotic and phage therapies.