Characterization And Preliminary Application Of The Lytic Bacteriophage VB_KpnP_Bp5 Of Klebsiella Pneumoniae

Klebsiella Pneumoniae is a common zoonotic pathogen that may be the cause of many diseases in clinic and economic losses in breeding industry.At present,multidrug resistant K.pneumoniae has aroused people's attention.Bacteriophages are viruses that specifically attack and kill their host bacteria.They are the most promising antibiotic substitutes and biological disinfectants because of their natural bactericidal properties.In this study,we screened a bacteriophage that can lyse klebsiella pneumoniae with high viscosity phenotype and multiple drug resistance.In addition,we analyzed its biological characteristics and clinical application effect in environment and in vivo.We first isolated a highly pathogenic strain of multidrug-resistant klebsiella pneumoniae with high viscosity phenotype from a pig farm and named it GXKP-J05.Next,we screened a lytic K.pneumoniae phage and named it vB_KpnP_Bp5.It's host bacteria was GXKP-J05.Then we analyzed the biological characteristics and genome of phage vB_KpnP_Bp5.Our results showed that it has clear and transparent plaque and no halo around it.Thus,the phage was a typical lytic phage.The phage was classified as a member of tail bacteriophages,short tail virus family based on electron microscopy analysis.The phage has an icosahedron head with a diagonal length of about 60nm and a short tail about 15nm long.One step growth curve of the phage revealed that the latent period was 5 min and the outbreak period was 40 min.The burst size was24 PFU/cell.The optimal number of infections was 0.001.The phage could withstand 50?temperature.The optimal p H value was 4.0 to 10.0.Whole genome sequencing revealed that the ds DNA length of phage was 43872 bp.BLASTn analysis showed that it shared 94.06%identity(94%genome coverage)with K.pneumoniae phage v B?Kpn P?SU552A of complete genome idefix.RAST genome analysis showed that the bacteriophage genome was small,with only 52ORFs,and the number of functional proteins was relatively consistent with other bacteriophages.In addition,we detected the number of Klebsiella pneumoniae in the surface treated with phage 10~9PFU/m~2and PBS of equal volume respectively.The results showed that the count of Klebsiella pneumoniae was 0 CFU/ml after3 h in phage group,indicating that phage vB_KpnP_Bp5 had bactericidal effect in the external environment.In vivo,we evaluated the effect of intraperitoneal injections of phage on mice(each with 2×10~8PFU)by testing survival rate and pathological change.The experiment was divided into three groups(5 mice of each group):no bacterial infection,bacterial infection and longer duration of bacterial infection.The results showed that phage injection could significantly improve the survival rate of mice.When all the mice in the positive control group died,the survival rate of mice in the pre-infection injection group and the synchronous injection group was 100%,while the survival rate of mice in the post-infection injection group was 60%.Pathological sections of lungs in each group showed that phage treatment could effectively protect the structural integrity of lungs and reduce exudate.The earlier the phage injection,the better the effect on mice.In summary,the phage vB_KpnP_Bp5 provided a new insight to purify the environment for farms and has great potential to develop environmental biological disinfectant.It possessed good application prospect in prevention,control and treatment of Klebsiella pneumoniae infection.