Study On Antibacterial Activity Of Klebsiella Pneumoniae Phage Vb_KpnP_ZX10 And Its Lyase

Klebsiella pneumoniae(KP)is a common opportunistic pathogen of zoonosis,which exists in various environments and in tissues and organs with mucous membranes,such as respiratory tract,gastrointestinal tract and urethra.It causes a variety of diseases,including liver abscess,pneumonia and septicemia,and in severe cases leads to death.At present,many kinds of Klebsiella pneumoniae(Hypervirulent K.pneumoniae,hvKp)producing high virulence capsular polysaccharides have been identified,especially the K.pneumoniae producing K1 and K2 capsular polysaccharides is harmful to human and animals.Due to the long-term irregular use of antibiotics by health institutions and farms,the emergence of bacterial drug resistance and the widespread prevalence of multi-drug-resistant bacteria are causing concern and concern from all walks of life.Phage is a virus that infects and kills bacteria.Compared with antibiotics,phage has the characteristics of low development cost,easy separation and purification,high host specificity,high safety,environment-friendly and so on.Phage therapy has become one of the hot research directions of antibiotics.The purpose of this study is to develop a lytic phage for hvKp,analyze its biological characteristics and whole genome in vitro,and verify its safety and therapeutic efficacy in mouse model in vivo.On this basis,its lyase gene was cloned and expressed to verify its bacteriostatic activity in vitro.To provide theoretical basis and technical support for the development of new drugs for hvKp.1.Isolation,identification and biological characteristics of phage of HvKpUsing Klebsiella pneumoniae producing K1 capsular serum as indicator bacteria,a phage named vB_KpnP_ZX10 was isolated and purified from the sewage and feces of a duck farm in Henan Province.Through the isolation and purification of the plaque morphology,it was preliminarily judged to be a lytic phage.According to its short tail and icosahedral head shown by TEM characterization,it is considered to be a member of Autographiviridae family.The determination of cleavage spectrum shows that it has high host specificity.The best MOI determination and one-step growth curve show that the best MOI is 0.001,the incubation period is about 30 min,the explosive amount is 201 PFU/mL,which indicated its good cleavage ability in vitro.The phage stability test showed that it had high stability under the condition of 40℃ and 60℃ and pH of 2-13.and retained 21.37%activity after being stored at room temperature for one month.The results of UV and chloroform sensitivity test showed that it was sensitive to UV and insensitive to chloroform.The bacteriostatic curve in vitro further proved that it was a lytic phage,which could significantly reduce the bacterial load in the environment in a short time and inhibit bacterial growth continuously and efficiently within 4 hours.Finally,the therapeutic effect and safety of it on hvKp infected mice were verified by animal experiments.In a word,experiments in vivo and biological characteristics show that vB_KpnP_ZX10 has good antibacterial activity and safety in vivo and in vitro,and has the potential to be used in clinical treatment.2.Sequencing and Analysis of the whole Genome of PhageOn the basis of phage concentration by NaCI-PEG8000 method,phage particles were further purified and concentrated by CsCl density gradient centrifugation.Phage DNA was extracted by virus genome extraction kit.After passing the quality inspection,the whole genome was sequenced on the Illumina NovaSeq PE150 platform,and the data were filtered and assembled.The whole phage genome sequence was analyzed by NCBI Blastn(https://blast.ncbi.nlm.nih.gov),the base content and GC content of phage genome were analyzed by DNA Star7.0 Megalign,and the possible tRNA coding genes were predicted by tRNAscan-SE(http://lowelab.ucsc.edu/tRNAscan-SE).The phage genome was compared in VFDB(http://www.mgc.ac.cn/VFs/main.htm)and CARD database(https://card.mcmaster.ca)to predict whether it contains virulence factors and antibiotic resistance genes.BLAST Ring Image Generator(BRIG)was used to visualize the phage genome,and multiple sequence alignment was realized by Easyfig 2.2.5.The phage terminase large subunits and major capsid proteins were compared and phylogenetic analyzed by MEGA-X to further determine their taxonomic status,and OAT was used to determine the phage species by improved orthogonal ANI algorithm.The results showed that the total genome length of phage vB_KpnP_ZX10 was 39301 bp and the GC content was 53.14%.42 open reading frames(ORFs)were predicted,of which 28 had known functions.No integrase gene was found in the phage genome,which further confirms that it is a lytic phage at the genetic level.The function of the encoded protein belongs to DNA metabolism,bacteriophage cleavage,phage structure and DNA packaging respectively.No tRNA coding genes,virulence factors and drug resistance genes were found,which proved its safety at the genetic level.ANI analysis and phylogenetic tree construction further confirmed its biological taxonomic status(Autographiviridae family,Studiervirinae subfamily,Przondovirus genus).3.Preparation and bacteriostatic activity evaluation of phage lyaseThe lyase gene was expressed and purified in E.coli competent cell BL21(DE3)by prokaryotic expression.The expressed lyase was purified and concentrated by Ni-NTA column and 10kDa ultrafiltration tube,and the solution was replaced.The in vitro antibacterial activity and spectrum of LysinZX10 were tested by plate lysis test and turbidimetric comparison test,and the optimum reaction temperature and pH were determined,as well as the effect of combined use with EDTA.The results showed that the phage lyase genome was successfully cloned and ligated to the vector pET-32a,and the recombinant plasmid was successfully transformed into the prokaryotic expression system.After a large number of expression and purification,the protein concentration was determined to be 0.51 g/mL by BCA method.The cleavage spectrum of phage lyase was wider than that of the prophage,and it had good bactericidal ability when combined with EDTA in vitro.The range of action conditions of lyase LysinZX10 is wide.In a word,lyase LysinZX10 has good bacteriostatic ability in vitro and has clinical value in the prevention and control of Klebsiella pneumoniae infection.