Biological characterization, and whole genome sequencing of bacteriophage against Pseudomonas aeruginosa isolated from forest musk deer (Moschus berezovskii)

Abstract:

[Background] Pseudomonas aeruginosa (PA) is a common opportunistic pathogen. The septic diseases caused by PA account for more than 50% of the deaths in captive forest musk deer populations. Phages have become popular substitutes of antibiotics due to the increased resistance of bacteria. [Objective] To isolate a bacteriophage using PA isolated from the lungs of dead forest musk deer as the host bacteria, and to conduct biological characterization, whole genome sequencing, and in vivo bacteriostasis test on it. [Methods] Double-layer agar culture method was used to isolated a phage against PA from the daily drinking water of forest musk deer in Sichuan Institute of Musk Deer Breeding. The same method was used to determine the host range, optimal multiplicity of infection (MOI), one-step growth, thermostability, and pH range of this phage. Phage morphology was observed by transmission electron microscopy (TEM). The whole genome of the phage was sequenced and analyzed, and in vivo bacteriostasis test was carried out in mice. [Results] A phage strain against PA was isolated and named as vB_PaeM_PAMD02. It produced a transparent plaque with clear edge and no halo ring and had a narrow host range. The phage strain had high thermostability and was stable in weak basic environment, with the optimal MOI of 0.1 and the incubation period of 40 min. The whole genome sequencing showed that the genome of this phage had a length of 66 264 bp and the GC content of 55.59%. The annotation results showed that the phage had 92 open reading frames and carried no virulence gene or resistance gene. The results indicated that the phage belonged to the Myoviridae family. Animal experiments showed that the phage effectively inhibited the pathogenicity of bacteria in mice. [Conclusion] The isolated phage vB_PaeM_PAMD02 has high lysis efficiency and good tolerance to adverse environments. It has great potential to be applied in clinical prevention and treatment of PA infection.

Key words:bacteriophage;Pseudomonas aeruginosa;biological characterization;whole genome sequencing;forest musk deer