A novel virulent Litunavirus phage possesses therapeutic value against multidrug resistant Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a notable nosocomial pathogen that can cause severe infections in humans and animals. The emergence of multidrug resistant (MDR) P. aeruginosa has motivated the development of phages to treat the infections. In this study, a novel Pseudomonas phage, vB_PaeS_VL1 (VL1), was isolated from urban sewage. Phylogenetic analyses revealed that VL1 is a novel species in the genus Litunavirus of subfamily Migulavirinae. The VL1 is a virulent phage as no genes encoding lysogeny, toxins or antibiotic resistance were identified. The therapeutic potential of phage VL1 was investigated and revealed that approximately 56% (34/60 strains) of MDR P. aeruginosa strains, isolated from companion animal diseases, could be lysed by VL1. In contrast, VL1 did not lyse other Gram-negative and Gram-positive bacteria suggesting its specificity of infection. Phage VL1 demonstrated high efficiency to reduce bacterial load (~ 6 log cell number reduction) and ~ 75% reduction of biofilm in pre-formed biofilms of MDR P. aeruginosa. The result of two of the three MDR P. aeruginosa infected Galleria mellonella larvae showed that VL1 could significantly increase the survival rate of infected larvae. Taken together, phage VL1 has genetic and biological properties that make it a potential candidate for phage therapy against P. aeruginosa infections.

Figure 1

Plaque and morphology of the phage vB_PaeS_VL1. (a) Plaque morphology of phage VL1 formed in double-layer agar plate with P. aeruginosa ATCC27853 as host strain. (b) Transmission electron micrographs of phage VL1 negatively stained with 2% (w/v) uranyl acetate. The scale bar represents 200 nm.

Figure 2

Phage vB_PaeS_VL1 whole genome analysis. (a) Map of genome organization of phage VL1. The outer ring shows a total of 92 open reading frames (ORFs) with their transcription direction and highlighted with different colors according to their functions. This circular map was generated using Artemis version 17.0.1. (b) Comparison of the whole genome sequences of the phage VL1 with similar phages (Pseudomonas YH6 and PA26). The colored arrows indicated ORFs according to their predicted function. The homologous regions between phages are indicated by gray shading. (Scale = base pairs).

Figure 3

Neighbor-Joining phylogenetic tree based on (a) DNA polymerase gene, (b) complete genome sequences, or (c) terminase large subunit gene of phage vB_PaeS_VL1 and related phages in Litunarviruses. Numbers are shown next to the branches indicated by the percentage of replicate trees of the bootstrap test (1000 replicates). The evolutionary distances were processed using the Maximum Composite Likelihood method. The isolation source and country of each species are presented with a different colored rectangle.

Figure 4

In vitro characterization of the phage vB_PaeS_VL1. (a) Kinetic adsorption of the phage to the host strain (P. aeruginosa ATCC27853) at an MOI of 0.01. (b) One-step growth curve of phage VL1 at a MOI of 0.001. (c) Bacterial killing activity of phage VL1 against host cells at different MOI (0.1, 1, and 10) in TSB medium. (d) Phage VL1 stability under different pH conditions. (e) Stability of Phage VL1 under different temperature conditions. Data are presented as mean ± SD of three independent experiments. The asterisks indicate significant differences (*P < 0.05 and ****P < 0.0001, one-way ANOVA followed by Dunnett's post hoc test) between the experimental and control groups.

Figure 5

Efficiency of the lytic activity of the phage vB_PaeS_VL1 against three different clinical isolates of MDR P. aeruginosa. (a) The P. aeruginosa strains MDR PA897, (b) MDR PA269 or (c) MDR PA294 were cocultured with the phage vB_PaeS_VL1 at different MOIs (i.e., MOI of 1, 10 and 100) and incubated at 37 °C. Every 1 h for 6 h, bacterial growth was measured by optical density measurement. Bacterial culture without phages was included as a control. Data are presented as mean ± SD of three independent experiments. The asterisks indicate significant differences (*P < 0.05, one-way ANOVA followed by Dunnett's post hoc test) between the experimental and control groups.

Figure 6

Biofilm eradication activity of the phage vB_PaeS_VL1 against clinical isolates of MDR P. aeruginosa. The viable bacterial cell counts in 48 h-old biofilms of P. aeruginosa strains (a) MDR PA897, (b) MDR PA269 or (c) MDR PA294 after treatment with phage VL1 for 6, 12 and 24 h were measured using the standard plate count. The total biofilm mass of 48 h-old biofilms of P. aeruginosa strains (d) MDR PA897, (e) MDR PA269 or (f) MDR PA294 after treatment with phage VL1 for 6, 12 and 24 h. The absorbance of each well was measured at 595 nm after a crystal violet stain. Data are presented as mean ± SD of three independent experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01 or *P < 0.05, Students t test, indicates statistically significant differences between experimental and control groups.

Figure 7

Percent survival of MDR P. aeruginosa infected G. mellonella larvae after phage vB_PaeS_VL1 treatment. Groups of 10 larvae were inoculated with either MDR P. aeruginosa strains (a) PA897, (b) PA294 or (c) PA269 follow by treatment with phage VL1 at MOI of 1,000 or 10,000 at 1 h p.i.. The numbers of dead larvae were determined every 12 h for 72 h p.i. GraphPad Prism software was used to graph and analyze the data using a Log-rank (Mantel-Cox) test. Asterisks indicated significant differences (*P < 0.05 or **P < 0.01) between bacterial infection groups, and the phage treatment groups. Data is representative of that obtained in three independent experiments (n = 3 biological replicates).