Prophages are bacteriophages integrated into the bacterial host’s chromosome. This research aims to analyze and characterize the existing prophages within a collection of 53 Pseudomonas aeruginosa strains from intensive care units (ICUs) in Portugal and Spain. A total of 113 prophages were localized in the collection, with 18 of them being present in more than one strain simultaneously. After annotation, five of them were discarded as incomplete, and the 13 remaining prophages were characterized. Of 13, 10 belonged to the siphovirus tail morphology group, 2 to the podovirus tail morphology group, and 1 to the myovirus tail morphology group. All prophages had a length ranging from 20,199 to 63,401 bp and a GC% between 56.2% and 63.6%. The number of open reading frames (ORFs) oscillated between 32 and 88, and in 3/13 prophages, more than 50% of the ORFs had an unknown function. With our findings, we show that prophages are present in the majority of the P. aeruginosa strains isolated from Portuguese and Spanish critically ill patients, many of them found in more than one circulating strain at the same time and following a similar clonal distribution pattern. Although a great sum of ORFs had an unknown function, number of proteins in relation to viral defense (anti-CRISPR proteins, toxin/antitoxin modules, proteins against restriction-modification systems) as well as to prophage interference into their host’s quorum sensing system and regulatory cascades were found. This supports the idea that prophages have an influence in bacterial pathogenesis and anti-phage defense.

Despite being known for decades, prophages remain understudied when compared to the lytic phages employed in phage therapy. This research aims to shed some light into the nature, composition, and role of prophages found within a set of circulating strains of Pseudomas aeruginosa, with special attention to high-risk clones. Given the fact that prophages can effectively influence bacterial pathogenesis, prophage basic research constitutes a topic of growing interest. Furthermore, the abundance of viral defense and regulatory proteins within prophage genomes detected in this study evidences the importance of characterizing the most frequent prophages in circulating clinical strains and in high-risk clones if phage therapy is to be used.