Characterization, Genomic Analysis and Application of Five Lytic Phages Against Carbapenem-Resistant Pseudomonas aeruginosa

The high pathogenicity rate of carbapenem-resistant <i>Pseudomonas aeruginosa</i> (CRPA) has resulted in substantial economic losses for humans and the breeding industry. Consequently, there is an urgent need to develop new alternatives to mitigate antibiotic use. Phage therapy has demonstrated promising results in numerous studies. In this study, lytic phages targeting CRPA were isolated from feces and river water samples in Shandong, China. A total of 94 phage strains with CRPA as hosts were obtained, exhibiting lysis rates that ranged from 29% to 76% for <i>P. aeruginosa</i> derived from humans and different types of animals (n = 246). We further examined five representative phages, the host bacteria of which were CRPA from clinical patients and poultry, and these phages included two myoviruses and three podoviruses. Their optimal multiplicities of infection (MOIs) ranged from 10^-3 to 10^-5, with latent periods of less than 5 to 15 min and burst durations of 140 to 175 min, resulting in burst sizes of 133 to 352 PFU/cell. All five phages exhibited the ability to survive at temperatures up to 60 °C and within pH levels of 3 to 11. Whole-genome sequencing revealed that these five phages were all double-stranded DNA phages and did not possess resistance genes or virulence factors. The two myoviruses, sharing similar sequences, were classified into the genus <i>Pakpunavirus</i>, with a size of 92,509 bp and 92,293 bp, 149 to 152 ORFs and 20 to 22 tRNAs. In contrast, the three similar podoviruses belong to the genus <i>Phikmvvirus</i> and all contained a perforin-lyase system, with a size of 43.35 kb, a GC content of 62%, 49 to 50 ORFs and 16 to 20 tRNAs. A spray disinfection experiment demonstrated that the phage cocktail exhibited a high sterilization effect after spraying and showed good efficacy against cement and metal surfaces. This study provides foundational information for further research into the elimination of CRPA in the environment.