Combination of genetically diverse Pseudomonas phages enhances the cocktail efficiency against bacteria

Abstract

Phage treatment has been used as an alternative to antibiotics since the early 1900s. However, bacteria may acquire phage resistance quickly, limiting the use of phage treatment. The combination of genetically diverse phages displaying distinct replication machinery in phage cocktails has therefore become a novel strategy to improve therapeutic outcomes. Here, we isolated and studied lytic phages (SPA01 and SPA05) that infect a wide range of clinical Pseudomonas aeruginosa isolates. These relatively small myophages have around 93 kbp genomes with no undesirable genes, have a 30-min latent period, and reproduce a relatively high number of progenies, ranging from 218 to 240 PFU per infected cell. Even though both phages lyse their hosts within 4 h, phage-resistant bacteria emerge during the treatment. Considering SPA01-resistant bacteria cross-resist phage SPA05 and vice versa, combining SPA01 and SPA05 for a cocktail would be ineffective. According to the decreased adsorption rate of the phages in the resistant isolates, one of the anti-phage mechanisms may occur through modification of phage receptors on the target cells. All resistant isolates, however, are susceptible to nucleus-forming jumbophages (PhiKZ and PhiPA3), which are genetically distinct from phages SPA01 and SPA05, suggesting that the jumbophages recognize a different receptor during phage entry. The combination of these phages with the jumbophage PhiKZ outperforms other tested combinations in terms of bactericidal activity and effectively suppresses the emergence of phage resistance. This finding reveals the effectiveness of the diverse phage-composed cocktail for reducing bacterial growth and prolonging the evolution of phage resistance.

Figure 1

Morphological, biological, and genomic characteristics of phages SPA01 and SPA05. Plaque morphology of phages SPA01 (A) and SPA05 (E). Transmission electron micrographs of phages SPA01 (B) and SPA05 (F). Scale bar equals to 100 nm. Adsorption assays within 25 min of phages SPA01 (C) and SPA05 (G) with P. aeruginosa strain PAO1. One-step growth curve of phages SPA01 (D) and SPA05 (H) in P. aeruginosa strain PAO1 during a window of 70 min. Schematic whole genome maps of phages SPA01 (I) and SPA05 (J). The innermost circles colored in green and purple indicate the positive and negative GC skew, respectively. The open reading frames (ORFs) are indicated in blue color with arrows indicating the ORF direction. The functional annotation of these ORFs in phages SPA01 and SPA05 is shown in Table S1. The data shown in (C,D,G,H) represent the mean ± standard deviation of at least triplicates.

Figure 2

Potential of phages SPA01 and SPA05 in P. aeruginosa suppression and formation of phage-resistant bacteria. Killing profiles of phages SPA01 (A) and SPA05 (B) against P. aeruginosa strain PAO1 in vitro at MOIs of 0.01, 0.1, 1, 10, and 100. Survival P. aeruginosa PAO1 (CFU/ml) in the presence of SPA01 (C) and SPA05 (D) for 12 and 24 h, showing the increasing emergence of phage resistance through time. Quantitation of adsorption of phages SPA01 and SPA05 among phage resistant strains, suggesting cross resistance to phages through mutations of bacterial receptors (E). Unabsorbed phage titers were measured after incubation with each phage-resistant strain: SPA01-resistant isolates (R1 to R10-SPA01) and SPA05-resistant isolates (R11 to R20-SPA05). The data shown in (A–D) represent the mean ± standard deviation of at least triplicates.

Figure 3

Phages SPA01 and SPA05 are genetically and mechanistically diverse from the nucleus-forming jumbophages. Comparative genome analysis of small phage genomes (SPA01 and SPA05) with giant phage genomes (PhiKZ and PhiPA3) (A). The green arrows represent the coding sequence directions and locations, and gray shaded lines reflect the degree of homology between them. Phylogenetic tree based on whole-genome sequence comparisons of selected phages, generated with Geneious version 2022.2.2 using the neighbour-joining method and visualized with iTOL (B). The single-cell level assay reveals the bacterial morphological changes triggered during infections of small (SPA01 and SPA05) and giant (PhiKZ and PhiPA3) phages at MOI 5. P. aeruginosa cells at OD₆₀₀ ~ 0.4 were infected with phages at MOI 5 and fixed at 20 min post infection (mpi), followed by staining cell membrane with FM4–64 (red) and nucleoid with DAPI (blue). Scale bar equals to 1 micron (C). The cytological profile was performed by Uniform Manifold Approximation and Projection (UMAP), showing cell clusters of uninfected cells (gray) and cells infected with small phages (SPA01; light blue, and SPA05; blue) and jumbophages (PhiKZ; orange, and PhiPA3; red).

Figure 4

Giant phages PhiKZ and PhiPA3 efficiently suppress the growth of SPA01 and SPA05-resistant bacteria and improve the efficacy of the phage cocktail. Killing profiles of phages PhiKZ (A) and PhiPA3 (B) in all SPA01 and SPA05-resistant strains (R1 to R10-SPA01 and R11 to R20-SPA05) at a MOI of 1, indicating the susceptibility of small phage-resistant strains to giant phages. EOP of phages PhiKZ and PhiPA3 in all SPA01 and SPA05-resistant strains compared to P. aeruginosa PAO1, suggesting medium to high production of giant phages in the resistant isolates (C). Survival P. aeruginosa PAO1 (CFU/ml) at 24 and 48 h in six different formulas of phage cocktails: (1) SPA01-SPA05, (2) SPA01-PhiKZ, (3) SPA01-PhiPA3, (4) SPA05-PhiKZ, (5) SPA05-PhiPA3, and (6) PhiKZ-PhiPA3 (D). The revival frequency of P. aeruginosa PAO1 at 24 and 48 h in six different formulas of phage cocktails (E). The data shown in (A-E) represent the mean ± standard deviation of at least triplicates. Statistical significance in (D-E) was calculated by two-way ANOVA. Different letters above bars show values that are significantly different (p < 0.05).