Isolation And Identification Of A Toxic Bacteriophage PA-YS35 From Pseudomonas Aeruginosa And Analysis Of Its Complete Genome Sequence

Pseudomonas aeruginosa is a gram-negative non-fermentative aerobic bacterium commonly distributed in soil,domestic water and hospital or industrial wastewater.As one of the main pathogenic bacteria causing wound purulent infection,when the body's immune function is reduced,it will cause a variety of diseases,such as wound infection after surgery,meanwhile also can cause abscesses,acne,suppurative otitis media and other diseases.With the multi-drug resistant strains constantly updating and generating new biofilms,the difficulty of clinical treatment is further increased.As a biological agent,bacteriophage re-enters people's sights.A Bacteriophage is a virus that spreads bacteria throughout the natural world.With the accelerating rate of mutation of drug-resistant bacteria,Phage Therapy has attracted the interest of scientific researchers and the attention of some scientific research institutions.In this experiment,the host bacteria were isolated from the clinic,and the traditional double-layer AGAR plaque method was used to isolate the toxic bacteriophage from the waste water of the hospital of Ji Lin university,and named it PA-YS35.Electron microscopy showed that bacteriophage PA-YS35 belonged to the phages of Caudovirales Order and Myoviridae family.The study of biological characteristics revealed that :(1)PA-YS35's head is stereo symmetry,with a diameter of about 45 nm,and the tail and head length is similar,about 45-50 nm.(2)PA-YS35 genome is double-stranded DNA.The restrictive endonuclease map analysis showed that the genome contains several restriction sites such as Eco R I,Eco R V,Hind III,Nde I and Xba I;(3)PA-YS35 has broad-spectrum lysis,and 30 Pseudomonas aeruginosa clinical isolates can be lysed,and 21 of them can be lysed effectively.(4)The phage PA-YS35 had the best multiplicity of infection(MOI)of 0.01.(5)The one-step growth curve showed that PA-YS35 had a 3 minute incubation period,and the eruption lasted 21 minutes with an outbreak of 380 PFU/cell.(6)Phage stability experiments revealed that PA-YS35 phage still had high activity and lysis rate in the range of 50?-70? and p H 4-9.The results of phage genome sequencing showed that the bacteriophage PA-YS35 genome was a linear double-stranded DNA,with a size of 93,296 bp and GC content of 49.35%.11 t RNA-related genes were detected.Their average length was 77 bp and the total length was 850 bp.The total number of genes is 0.9111%.Gene annotation using RAST showed that the PA-YS35 genome contained 172 Open Reading Frame(ORF),of which 41 coding sequences predicted its function and the remaining 131 were unknown coding sequences.Phylogenetic tree mapping using RNA ligase nucleotide sequences revealed that the genome of phage PA-YS35 was in the same branch with the Pseudomonas phase PAK P1 phase and had a closer evolutionary relationship.The genome sequence of the bioteriophage PA-YS35 has been submitted to Gen Bank under the accession number MF974178.1.In summary,the bacteriophage PA-YS35 is a toxic bacteriophage that has a short incubation period,high cleavage efficiency,a broad spectrum of cleavage,and extremely high environmental adaptability(Maintains high lytic activity in the range of 50°C-70°C and p H 4-9).The lytic activity can be considered for the development of drug-resistatn bacteriophage biologies infected with Pseudomonas aeruginosa.