Isolation and identification a novel Pseudomonas aeruginosa phage PaP4

Abstract:

[Objective] To isolate and identify a novel Pseudomonas aeruginosa phage named PaP4 via its biological properties. [Methods] We used the double-layer agar culture method to make a single plaque. Phage particles of PaP4 were purified with CsCl2 gradient centrifugation and the ultrastructure of PaP4 was examined by electron microscopy. We extracted the genome of PaP4 and identified the type of nucleic acid with restriction enzyme analysis. The host bacteria were infected with PaP4 at six different MOI. Three hours and thirty minutes later, the phage titers were determined, respectively. For one step growth experiments, phages and its host with an MOI of 10 were used with a 15 min adsorption. Following centrifugation, the pellet was washed twice and resuspended with LB broth and incubated. Samples were taken at 15 min-intervals (up to 120 min) and immediately tittered by the double layer agar plate method. [Results] The plaques of PaP4 were transparent and their sizes were about 3 mm?5 mm in diameter. PaP4 had an isometric head approximately 50 nm in diameter and a long tail with 30 nm in length. 0.001 MOI-infected host bacteria gave the highest phage offsprings. One-step growth curve was determined according to the results of one-step growth experiments. [Conclusion] PaP4 is a virulent phage and belongs to Podoviridae. The whole genome can be digested by the restriction enzyme, indicating that PaP4 is a dsDNA virus. The optimal MOI of PaP4 is 0.001 and one-step growth experiment shows latent and burst periods are 25 min and 20 min, respectively, burst size is 150.