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.

Phage therapy of pulmonary infections

“…For instance, with regard to the latter point: All but one of the animal studies involved only a single phage dosing and most 23,[54][55][56][57][58][59]62 involved topical phage application into the lungs. Exceptional were the mouse studies by Chhibber et al, 60 Semler et al, 62 and Singla et al 61 which also tested intraperitoneal phage application.…”

“…25 Thus, for example, one typically will see intranasal instillation of phages, [54][55][56][57][58][59] or intraperitoneal application, 54,60,61 both of which might less efficiently deliver phages into the lungs versus, for example, the use of nebulizers. 12,59,62 Below I provide overviews of these studies. Note that most of these treatments consist of only a single phage dose vs., e.g., multiple dosing over the course of weeks for typical human treatments of pulmonary infections (above).…”

“…Cao et al 59 isolated a phage from hospital sewage against a P. aeruginosa isolate associated with hemorrhagic pneumonia in minks. A toxicity trial was performed involving the application intranasally of 60 ml of "crude phage preparation" containing 10 12 plaque-forming units (PFU) per ml once a day for 3 d to mice.…”

Therapeutic effect of the YH6 phage in a murine hemorrhagic pneumonia model

“…3 Isolated from Dingtai mink breeding base, Dalian, Liaoning province. 4 Purchased from National Center for Medical Culture Collections (CMCC). 5 Purchased from American Type Culture Collection (ATCC).…”

“…Phage therapy has also been used for the control and treatment of multidrug-resistant P. aeruginosa lung infections in mice and in cystic fibrosis airway lung cells [1]. Cao et al evaluated the potential for phage against a mink hemorrhagic pneumonia model when administered by means of ultrasonic nebulization [4].…”