Isolation,identification And Application Of Carbapenem-resistant Klebsiella Pneumoniae Phage VB＿KonP1

Objective: Klebsiella pneumoniae is a conditionally pathogenic bacterium widely present in the environment and susceptible to infecting immunocompromised patients.With the long-term irrational use of antibiotics,bacterial resistance is a growing problem.According to the Clinical and Laboratory Standards Institute(CLSI),carbapenem-resistant Klebsiella pneumonia(CRKP)refers to Klebsiella pneumoniae that are resistant to any of the carbapenem antibiotics,such as imipenem,meropenem or ertapenem.In this study,CRKP from sputum culture of a patient with severe pneumonia in the intensive care medicine department of our hospital was used as the host bacterium.A new virulent phage strain vB＿KpnP1 was isolated from the unsterilized effluent in our hospital’s wastewater treatment station,and its biological properties,genome-wide information,in vitro bacterial inhibitory ability and in vivo efficacy assessment in mice were investigated to provide a basis for the development of novel antibacterial agents.Methods:1.A virulent phage strain was isolated and purified from the untreated sewage of our hospital using a double-layer agar plate method and named vB＿KpnP1.The phage particles were concentrated,placed under transmission electron microscopy and observed for its morphological.2.23 strains of carbapenem-resistant Klebsiella pneumoniae(CRKP1-CRKP23),1 strain of Escherichia coli(Eco01),1 strain of Serratia(Serratia01),1 strain of Enterobacter cloacae(Ecl01),and 1 strain of Enterobacter aerogenes(Eae01)were collected from patients in our hospital,and the phage was determined using a combined spot test and double-layer agar plate method lysis spectrum of vB＿KpnP1.3.The biological properties of phage vB＿KpnP1 were obtained by measuring the chloroform,temperature and p H stability of the phage.4.The optimal multiplicity of infection,one-step growth curve and adsorption experiment of phage vB＿KpnP1 were performed;thus,it’s optimal multiplicity of infection,the incubation period,the lysis period,and the amount of lysis and adsorption rate were obtained.5.The phage genome was first extracted using a Proteinase K/SDS method,and then the phage was sequenced using the Illumina platform.The sequenced phage genomes were annotated with RAST and compared with the online BLAST tool to predict the genome function,and the whole genome was maped using CG View Server software.The phylogenetic evolutionary tree of the whole genome,terminal enzyme large subunits and DNA polymerase genes was constructed by maximum likelihood method using MEGA 11.0 software.Easyfig 2.2.3 software was used for comparative gene analysis.6.In vitro bacterial inhibition assay of phage vB＿KpnP1 was performed using a fully automated growth curve analyzer.7.The safety of the phage was assessed in mice by intraperitoneal injection of highly potent phage;then a mouse sepsis model was established by intraperitoneal injection of a minimal lethal dose of logarithmic growth phase CRKP1 bacteriophage,which was treated with phage vB＿KpnP1 of different multiplicity of infection 1 hour later.Results:1.A virulent phage strain,named vB＿KpnP1,was isolated from the sewage of our hospital,which formed a 2-3 mm transparent circular phage spot surrounded by a distinct halo;Electron microscopy showed that the head size of the phage was about 68.2×64.3 nm,and the tail length was about 11 nm;The head of the phage was a regular icosahedron with a non-contractile short tail,which was thus identified as a member of the Autographiviridae family and the Caudovirales order.2.The spot experiment suggested that phage vB＿KpnP1 lysis rate of CRKP was 59.09%(13/22),and the double-layer AGAR plate method showed that phage lysis rate of CRKP was 54.55%(12/22),but it could not lyse Escherichia coli,Serratia marcescens,Enterobacter cloacae and Enterobacter aerogenes.3.The phage vB＿KpnP1 was not sensitive to chloroform,and the potency of phage was more stable under the conditions of temperature 10-50 ℃ and p H 4-12.4.The optimal multiplicity of infection was 0.0001,the incubation period was 10 min,the lysis period was 30 min,and the amount of lysis was 26PFU(plaque forming unit)/cell.the adsorption curve showed that when it was mixed with the host bacteria and incubated for 4 min about 60% of the phage adsorbed to the host bacteria.5.The genomic analysis showed that the phage vB＿KpnP1 was a double-stranded DNA(ds DNA)with full genome length of 40,352 bp,GC content of 53.09% and 50 CDS.no t RNA structure was predicted,and no known drug resistance genes or virulence factors were found.The homology evolutionary tree indicated that the phage belonged to the family Autographiviridae,subfamily Studiervirinae,genus Przondovirus,unclassified species of Przondovirus.6.In vitro inhibition experiments showed that phage vB＿KpnP1 added at different infection complex ratios could inhibit the growth of host bacteria,but after 5 h,the absorbance increased rapidly presumably for the production of phage resistant strains.7.In vivo tests showed that phage vB＿KpnP1 of different infection complexes reduced mortality in mice;the phage improved structural and inflammatory lesions in lung,liver,spleen and kidney tissues of septic mice and promoted slow healing,indicating its ability to treat sepsis caused by CRKP infection.In addition,safety assessment experiments in mice did not reveal any significant adverse effects of phage on mice.Conclusion: A virulent Klebsiella pneumoniae phage vB＿KpnP1 was successfully isolated,and its biological properties,genome-wide information,in vitro bacterial inhibitory ability and in vivo efficacy assessment in mice were studied to support the subsequent treatment of patients with carbapenem-resistant Klebsiella pneumoniae infection.