Isolation and Characterization of Lytic Bacteriophages Capable of Infecting Diverse Multidrug-Resistant Strains of Pseudomonas aeruginosa: PaCCP1 and PaCCP2

Abstract

Background/objectives: Antimicrobial resistance (AMR) is a major public health threat, which is exacerbated by the lack of new antibiotics and the emergence of multidrug-resistant (MDR) superbugs. Comprehensive efforts and alternative strategies to combat AMR are urgently needed to prevent social, medical, and economic consequences. Pseudomonas aeruginosa is a pathogen responsible for a wide range of infections, from soft tissue infections to life-threatening conditions such as bacteremia and pneumonia. Bacteriophages have been considered as a potential therapeutic option to treat bacterial infections. Our aim was to isolate phages able to infect MDR P. aeruginosa strains.

Methods: We isolated two lytic phages, using the conventional double layer agar technique (DLA), from samples obtained from the influent of a wastewater treatment plant in Concepción, Chile. The phages, designated as PaCCP1 and PaCCP2, were observed by electron microscopy and their host range was determined against multiple P. aeruginosa strains using DLA. Moreover, their genomes were sequenced and analyzed.

Results: Phage PaCCP1 is a member of the Septimatrevirus genus and phage PaCCP2 is a member of the Pbunavirus genus. Both phages are tailed and contain dsDNA. The genome of PaCCP1 is 43,176 bp in length with a GC content of 54.4%, encoding 59 ORFs, one of them being a tRNA gene. The genome of PaCCP2 is 66,333 bp in length with a GC content of 55.6%, encoding 102 non-tRNA ORFs. PaCCP1 is capable of infecting five strains of P. aeruginosa, whereas phage PaCCP2 is capable of infecting three strains of P. aeruginosa. Both phages do not contain bacterial virulence or AMR genes and contain three and six putative Anti-CRISPR proteins.

Conclusions: Phages PaCCP1 and PaCCP2 show promise as effective treatments for MDR P. aeruginosa strains, offering a potential strategy for controlling this clinically important pathogen through phage therapy.

Keywords: P. aeruginosa; antimicrobial resistance; bacteria; bacteriophages; virus; wastewater.

Figure 1

Transmission electron micrographs of phages PaCCP1 (A) and PaCCP2 (B). Images show the head and tail of both phages.

Figure 2

Genome maps of phages PaCCP1 (A) and PaCCP2 (B) obtained using Proksee. ORFs with identified functions are shown. Moreover, GC content and GC skew are indicated.

Figure 3

ViPTree analysis of Pseudomonas phages PaCCP1 and PaCCP2. Phages are identified according to their official ICTV classification, with the outer and inner rings indicating their host group and virus family, respectively.

Figure 4

Heatmap showing the intergenomic similarities of phage PaCCP1 and Septimatrevirus phages.

Figure 5

Phylogenetic analysis of phage PaCCP1 and Septimatrevirus phages. The scale bar indicates the number of substitutions per site. Each species is indicated by a unique color.

Figure 6

Comparison of phage PaCCP1 and its closest relative, phage Guyu, using alignment of all annotated proteins. The arrow’s colors represent the gene clusters encoding similar proteins. The lines linking the arrows show gene-encoding proteins that share more than 80% sequence identity.

Figure 7

Heatmap of the intergenomic similarities of phage PaCCP2 with Pbunavirus phages.

Figure 8

Phylogenetic analysis of phage PaCCP2 and Pbunavirus phages. The scale bar indicates the number of substitutions per site. Each species is indicated by a unique color.

Figure 9

Comparison of phage PaCCP2 and its closest relative, phage LS1, using alignment of all annotated proteins. The arrow’s colors represent the gene clusters encoding similar proteins. The lines linking the arrows show gene-encoding proteins that share more than 80% sequence identity.