Isolation,Identification And Genomic Analysis Of K.pneumoniae Lytic Phage Kp-8329

Klebsiella pneumoniae belongs to the family Enterobacteriaceae,Klebsiella,which is found in the respiratory tract and normal intestinal flora of healthy people.It is generally not pathogenic,its pathogenesis is related the defensive function defect,dysbacteriosis and a variety of predisposing factors of the host.Klebsiella pneumoniae is more likely to cause pneumonia,but also can cause meningitis,liver abscess,urinary tract infection,wound infection.In recent years,with the abuse of broad-spectrum antibiotics,Klebsiella pneumoniae has developed resistance to a variety of antibiotics,leading to failure of clinical antibiotic treatment and prolonged disease progression.Phage has become one of the effective methods for treating infection due to its biological characteristics,and has been widely used in many fields.This experiment collected pathogens from the Second Hospital of Jilin University,use the isolated pathogen as the host strain,and isolated the lytic phage from the hospital sewage by double-layer agar plate method.The phage was named Kp-8329,and a series of experiments were used to identify the phage of the pathogen:(1)Transmission electron microscopy showed that the phage Kp-8329 is a phage of the Cardioviral,a long-tailed phage,with a head diameter of about 45-50 nm and a tail with a diameter of about 130-140 nm.(2)The Kp-8329 genome is double-stranded DNA,and the genome contains EcoR I,EcoRV,Hind III,Nde I and other cleavage sites;(3)The optimal multiplicity of infection of the phage Kp-8329(MOI)is 0.01;(4)The one-step growth curve shows that Kp-8329 has a 15-minute incubation period,the burst period lasts 15 minutes,and the burst volume is 147 PFU/cell;(5)Kp-8329 still has a high activity and cleavage rate in the range of pH 4-12 and below 60 °C;(6)Kp-8329 has higher cleavage rate,12 of 19 clinical isolates of Klebsiella pneumoniae could be cleavaged efficiently;(7)The genomic sequencing and genome-wide sequence analysis showed that the Kp-8329 genome was 46,907 bp in length,Contains 116 functional ORFs and among them,32 have high similarity to the functional proteins that have been published in the database.Based on the phylogenetic tree of DNA polymerase,it was found that the bacteriophage had high homology with Escherichia phage.We can infer that the evolutionary relationships are similar.The whole genome sequence of bacteriophage Kp-8329 has been submitted to GenBank,its login number is MK327140.1.In summary,through this study,we screened a phage with high cleavage rate and adaptability,and it does not carry virulence genes and drug resistance genes.All of these provide a new way of thinking that research the bacteriophage biological preparation for treatment of clinical multiple drug resistant bacterial infections.