Isolation of a bacteriophage targeting Pseudomonas aeruginosa and exhibits a promising in vivo efficacy

Abstract

Pseudomonas aeruginosa is an important pathogen that causes serious infections. Bacterial biofilms are highly resistant and render bacterial treatment very difficult, therefore necessitates alternative antibacterial strategies. Phage therapy has been recently regarded as a potential therapeutic option for treatment of bacterial infections. In the current study, a novel podovirus vB_PaeP_PS28 has been isolated from sewage with higher lytic activity against P. aeruginosa. Isolated phage exhibits a short latent period, large burst size and higher stability over a wide range of temperatures and pH. The genome of vB_PaeP_PS28 consists of 72,283 bp circular double-stranded DNA, with G + C content of 54.75%. The phage genome contains 94 open reading frames (ORFs); 32 for known functional proteins and 62 for hypothetical proteins and no tRNA genes. The phage vB_PaeP_PS28 effectively inhibited the growth of P. aeruginosa planktonic cells and displayed a higher biofilm degrading capability. Moreover, therapeutic efficacy of isolated phage was evaluated in vivo using mice infection model. Interestingly, survival of mice infected with P. aeruginosa was significantly enhanced upon treatment with vB_PaeP_PS28. Furthermore, the bacterial load in liver and kidney isolated from mice infected with P. aeruginosa and treated with phage markedly decreased as compared with phage-untreated P. aeruginosa-infected mice. These findings support the efficacy of isolated phage vB_PaeP_PS28 in reducing P. aeruginosa colonization and pathogenesis in host. Importantly, the isolated phage vB_PaeP_PS28 could be applied alone or as combination therapy with other lytic phages as phage cocktail therapy or with antibiotics to limit infections caused by P. aeruginosa.

Keywords: Antibiotic resistance; Bacteriophage; Biofilm; Genomic analysis; Phage therapy; Pseudomonas aeruginosa.

Fig. 1

Isolation of bacteriophage. a Clear lytic zone on bacterial lawn by spot assay of phage lysate from sewage. b Plaque morphology of isolated phage double layer agar plate. c Transmission electron microscope (TEM) images of vB_PaeP_PS28. Phage particles were negatively stained by 2% phosphotungstic acid. Scale bar = 100 nm

Fig. 2

Physical properties of vB_PaeP_PS28. a Thermal stability; b pH stability. Error bars represent mean ± SE for three replicates

Fig. 3

One-step growth curve of vB_PaeP_PS28. Phage was incubated with exponential culture of PS28 for 10 min, centrifuged and pellet was resuspended in TS broth. Titer of free phages was determined by double layer agar technique. Three biological replicates were performed and data were presented as mean ± SE

Fig. 4

Bacteriolytic activity of phage vB_PaeP_PS28 against the host strain P. aeruginosa PS28 (a) and PAO1 b. Early exponential bacterial cultures were incubated with and without isolated phage suspension at MOI of (0.1, 1 and 10) at 37 °C for 24 h. Bacterial growth was determined and measured spectrophotometrically at OD₆₀₀. The results were expressed as means ± SE of three independent experiments

Fig. 5

The antibiofilm activity of vB_PaeP_PS28 against various P. aeruginosa isolates. Biofilms were formed in 96-well plates for 24 h and treated with phage at different MOIs (0.1, 1 and 10) for 24 h. Formed biofilms were stained by 1% crystal violet and measured spectrophotometrically at OD₆₀₀. The experiment was carried out at three independent replicates and data was expressed as means ± SE with P < 0.05 was considered significant

Fig. 6

Genomic characterization of vB_PaeP_PS28. a Circular genomic map of vB_PaeP_PS28; from inside to outside, the first to third circles represent the scale, GC Skew, and GC content respectively; the fourth represents the position of ORFs. The prediction and direction of ORFs are indicated by arrow heads. The genomic map was generated and visualized using CGView b Phylogenetic analysis of vB_PaeP_PS28 and other closely related sequences. c Phylogenetic tree analysis based on the amino acid sequence of terminase large subunit. d Phylogenetic tree analysis based on the amino acid sequence of RNA polymerase large subunit. Phylogenetic trees were constructed using BLAST (Basic Local Alignment Search Tool) using neighbor-joining method

Fig. 6

Genomic characterization of vB_PaeP_PS28. a Circular genomic map of vB_PaeP_PS28; from inside to outside, the first to third circles represent the scale, GC Skew, and GC content respectively; the fourth represents the position of ORFs. The prediction and direction of ORFs are indicated by arrow heads. The genomic map was generated and visualized using CGView b Phylogenetic analysis of vB_PaeP_PS28 and other closely related sequences. c Phylogenetic tree analysis based on the amino acid sequence of terminase large subunit. d Phylogenetic tree analysis based on the amino acid sequence of RNA polymerase large subunit. Phylogenetic trees were constructed using BLAST (Basic Local Alignment Search Tool) using neighbor-joining method

Fig. 7

Comparative genomic analysis between phage vB_PaeP_PS28 and related sequences. Pseudomonas phage vB_PaeP_FBPa1 (GenBank Acc. No. ON857943.1), Pseudomonas phage VB_PaeS_VL1 (GenBank Acc. No. OK665488.1), Pseudomonas phage YH6 (GenBank Acc. No. KM974184.1) and Pseudomonas phage PA26 (GenBank Acc. No. NC_041907.1). Sequence similarity is represented by the gray scale bar. The coding sequences are represented by directional arrows. Predicted ORFs in vB_PaeP_PS28 genome are listed below. Comparative analysis was performed using Easyfig

Fig. 8

VIRIDIC heatmap of vB_PaeP_PS28 phage and its closest homologues. Intergenomic similarities between the genomic nucleotide sequences of vB_PaeP_PS28 and related bacteriophages infecting P. aeruginosa; Pseudomonas phage vB_PaeP_FBPa1 (GenBank Acc. No. ON857943.1), Pseudomonas phage YH6 (GenBank Acc. No. KM974184.1), Pseudomonas phage PA26 (GenBank Acc. No. NC_041907.1) and Pseudomonas phage VB_PaeS_VL1 (GenBank Acc. No. OK665488.1). Color coding scales are represented above the matrix with intensity of color corresponding to level of similarity

Fig. 9

In vivo characterization of the influence of phage vB_PaeP_PS28 on P. aeruginosa pathogenesis in mice infection model. a Survival curves of mice infected with P. aeruginosa and treated with isolated phage. Mice in first group were infected with P. aeruginosa (2.5 × 10⁷ CFU/mL), mice in second group were injected with vB_PaeP_PS28 (2.5 × 10⁹ PFU/mL) and mice in third group were infected with P. aeruginosa and treated with the phage vB_PaeP_PS28. Uninfected and PBS-injected mice served as negative controls. Mice survival was monitored in each group daily for 4 days and plotted using Kaplan–Meier survival curve. Bacterial burden and phage titers were determined in liver (b) and spleen (c) of infected mice. Mice were anesthetized, liver and spleen were obtained and homogenized for enumeration of CFU and PFU at 24, 48, 72 h post infection. Of note that, bacterial load in P. aeruginosa infected mice was determined at 24 h post infection only as all mice in this group died after 24 h. Bacterial and phage load were represented on left and right y axis, respectively. Data are expressed as means ± SE of three independent experiments