Isolation And Characterization Of PaP6,A Newly Isolated Pseudomonas Aeruginosa Bacteriophage Containing Three DsRNA Genomic Segments

Antibiotic resistance has been one of the major worldwide public health concerns in recent years,which due mainly to the abuse of antibiotics in clinical setting and in animal husbandry and the limited discovery of new antibiotics.Infections associated with multi-drug resistant bacteria that resist lots of antibiotics are intractable and even lethal.Pseudomonas aeruginosa is a Gram-negative bacterium that distributed in diverse environments such as water,soil and living host organisms.As an opportunistic pathogen,P.aeruginosa is associated with various infections and causes serious morbidity and mortality among compromised individuals due to its notable multi-drug resistance.Thus,it is necessary to develop an alternative therapeutic strategy other than antibiotics to combat this versatile opportunistic pathogen.Bacteriophages(phages)are bacterial viruses that are considered as the most ubiquitous and diversified organisms on Earth.Phage has a small genome and is easy to be isolated.The application of phage as antimicrobial agents to treat bacterial infections began subsequently since it was first discovered by Frederick W.T.in 1915.In this study,we report the first isolation and identification of a P.aeruginosa dsRNA phage,named PaP6.Its general biological characteristics were identified,including morphology,optional multiplicity of infection and one step growth curve.Structural proteins of PaP6 were analyzed by SDS-PAGE.Chloroform sensitivity and host range were also assessed for PaP6.The main contents and results of this study are as follows:1.Characterization of biological characteristics of the novel P.aeruginosa phage PaP6.Plaque of PaP6 obtained from double-layer agar culture method exhibits transparent and clear edge with a size of 2-4 mm in diameter.Phage particles were examined by transmission electron microscope after cesium chloride density gradient centrifugation,showing an isometric head approximately 45 nm in diameter.The PaP6 genome contains three dsRNA segments and can be digested by RNase instead of DNase.2.PaP6 Phage titer reaches the peak at the MOI of 0.1 and one step growth curve shows that its incubation period is about 10 min and the outbreak period is about 50 min,so it concluds that the burst size of PaP6 infected host bacterium PAO38 is about 10.3.The host range of PaP6 was tested with 112 P.aeruginosa clinical isolate strains,and PaP6 can infect 40.1% of these strains,suggesting the broad host range of PaP6 and implying its potential in phage therapy.To our knowledge,this study presents the first Cystoviridae phage that could infect P.aeruginosa.