Isolation and characterization of a "phiKMV-like" bacteriophage and its therapeutic effect on mink hemorrhagic pneumonia

Abstract

The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink both in vitro and in vivo. Five Pseudomonas aeruginosa (P. aeruginosa) strains were isolated from lungs of mink with suspected hemorrhagic pneumonia and their identity was confirmed by morphological observation and 16S rDNA sequence analysis. Compared to P. aeruginosa strains isolated from mink with hemorrhagic pneumonia in 2002, these isolates were more resistant to antibiotics selected. A lytic phage vB_PaeP_PPA-ABTNL (PPA-ABTNL) of the Podoviridae family was isolated from hospital sewage using a P. aeruginosa isolate as host, showing broad host range against P. aeruginosa. A one-step growth curve analysis of PPA-ABTNL revealed eclipse and latent periods of 20 and 35 min, respectively, with a burst size of about 110 PFU per infected cell. Phage PPA-ABTNL significantly reduced the growth of P. aeruginosa isolates in vitro. The genome of PPA-ABTNL was 43,227 bp (62.4% G+C) containing 54 open reading frames and lacked regions encoding known virulence factors, integration-related proteins and antibiotic resistance determinants. Genome architecture analysis showed that PPA-ABTNL belonged to the "phiKMV-like Viruses" group. A repeated dose inhalational toxicity study using PPA-ABTNL crude preparation was conducted in mice and no significantly abnormal histological changes, morbidity or mortality were observed. There was no indication of any potential risk associated with using PPA-ABTNL as a therapeutic agent. The results of a curative treatment experiment demonstrated that atomization by ultrasonic treatment could efficiently deliver phage to the lungs of mink and a dose of 10 multiplicity of infection was optimal for treating mink hemorrhagic pneumonia. Our work demonstrated the potential for phage to fight P. aeruginosa involved in mink lung infections when administered by means of ultrasonic nebulization.

Figure 1. Transmission electron micrograph of phage PPA-ABTNL negatively stained with uranyl acetate.

Scale bar represents 50 nm.

Figure 2. One-step growth curve of phage PPA-ABTNL.

PPA-ABTNL phage was co-incubated with culture of PA5-1-1 to absorb for 5 min at 37°C. The mixture was centrifuged to remove non-absorbed phage. The re-suspended pellets were incubated at 37°C and sampled at 10 min interval over a period of 2 h. Phage titer was measured. Data represent mean±SD from three independent experiments. n = 3.

Figure 3. Antibacterial curve of phage PPA-ABTNL at different multiplication of infection in vitro.

PA5-1-1 strain was lysed by the phage PPA-ABTNL in LB medium at 37°C. OD600 development of uninfected control culture and parallel cultures infected with phage at different multiplicities of infection (MOI) was measured. n = 3.

Figure 4. Overview of the PPA-ABTNL genome sequence analysis.

Predicted open reading frames (ORFs) are numbered. ORFs with significant homology to known genes and to the identified proteins are in dark blue. Putative genome organization. Putaitve promoters (↳), terminators (౐) are indicated. Predicted and presumed proteins and corresponding ORF number are marked.

Figure 5. Phage PPA-ABTNL cures mink hemorrhagic pneumonia caused by a clinical P. aeruginosa strain.

(A) Effect of atomization on the titer of phage. (B) Effect of different multiplicities of infection (MOIs) on the amount of bacteria in the lung of mink. (C) Effect of different MOIs on the amount of phage in the lung of mink. (D) Survival curves of mink infected with PA5-1-1 strain and treated with phage PPA-ABTNL. n = 5. Means with different superscripts differ significantly from each other (P<0.05).