Characterization Of Lytic Phages Of Klebsiella Pneumoniae And Its Application In Foods

Klebsiella pneumoniae(K.pneumoniae)is an important foodborne pathogen,considered to be the most common hospital pathogen,which can cause human pneumonia,serious infection,sepsis and other diseases.Due to the overuse of antibiotics,a large number of multidrug resistant Klebsiella pneumoniae have emerged,which makes it extremely difficult to prevent and control it in medical treatment.At present,Klebsiella pneumoniae is often detected in fruits,vegetables,dairy products,chicken,seafood and other foods.The multi-drug resistant Klebsiella pneumoniae is causing more and more harm.It is urgent to develop a new product to replace antibiotics.Phages have been shown as antibacterial drugs,which can be used to control antibiotic resistant strains in food.In order to explore the solution to multidrug resistant K.pneumoniae,this study isolated and purified the phage of K.pneumoniae from the untreated sewage of Shanghai Sixth People’s Hospital,measured the temperature,p H tolerance,adsorption rate and one-step growth curve of the phage,and observed the biological morphological characteristics of the phage through transmission electron microscopy.Then the genome analysis and phylogenetic analysis of bacteriophages were carried out,and the functional annotation of the structural proteins of bacteriophages was carried out to clarify the classification status of bacteriophages.Finally,the bacteriostatic ability of bacteriophage in vitro,the clearance of biofilm in the environment and milk,and the therapeutic effect of bacteriophage on milk and chicken infection models were determined.The main results are as follows:1.Analysis of biological characteristics of bacteriophageThe phage HS37 belongs to the order of tailed bacteriophages and the family of short-tailed bacteriophages;The bacteriophage HS106 belongs to the order of tailed bacteriophages and the family of long-tailed bacteriophages;Phage HS23 belongs to the order Tailed Phage and the family Short-tailed Phage.The three bacteriophages have a wide range of temperature tolerance.Under 60 ℃,the activity of bacteriophages remains unchanged.At 70 ℃,the activity of bacteriophages decreases.At 80 ℃,the three bacteriophages are completely inactivated.At the same time,it also has a wide range of acid-base tolerance.At p H 4-10,the activity of bacteriophage remains stable.The optimal MOI of HS37 is 0.01,HS106 is 0.001,HS23 is 0.1,and the optimal MOI of three phages is less than 1.The incubation period of HS37 is 10 min,the fragmentation period is 40 min,and the explosive amount is about 83PFU/Cel.The incubation period of HS106 is 10 min,the fragmentation period is 40 min,and the explosive amount is 183PFU/Cell.The incubation period of HS23 is 10 min,the cleavage period is 50 min,and the outbreak amount is 97PFU/Cell.The incubation period of the three phages is relatively short,the outbreak amount of HS106 is high,and the outbreak amount of HS23 and HS37 is average.2.Whole genome analysis of bacteriophageThe phage HS37 genome consists of a 74084 bp linear double-stranded DNA with an average G+C content of 45.13%.Genome annotation predicts that there are121 open reading frames(ORFs)in the phage HS37 genome,accounting for 80.37%of the genome(59544 bp/74084 bp).96 ORFs are in the positive chain,and only 25 ORFs are in the negative chain.Thirty ORFs(24.8%)are known functional protein-coding genes,while the other 68 ORFs(75.2%)are putative proteins.These30 ORFs were predicted to encode functional proteins related to phage DNA replication,packaging,structural proteins and host cell lysis.v B＿Kp P＿HS37 is a new species of the genus Sugarlandvirus of Demerecvridae.The phage Hs106 genome consists of a 76430 bp linear double-stranded DNA with an average G+C content of 44.0%.The genome annotation predicted that there were 95 ORFs in the phage Hs106 genome,accounting for 92.85% of the genome(70968bp/76430bp).73 ORFs are on the positive chain,and only 22 are on the negative chain.These 27 ORFs(28.4%)are protein coding genes with known functions,while the other 68 ORFs(71.6%)encode hypothetical proteins.These 27 ORFs were predicted to encode functional proteins related to phage DNA replication,packaging,structural proteins and host cell lysis.Phage v B＿Kp P＿Hs106 is a new species of the genus Efbeekayvirus of Schitoviridae family.Both strains of bacteriophages encode bacteriophage holin and endolysin protein,and do not contain any lysogen factor,indicating that they are strictly lytic bacteriophages,and do not carry genes encoding virulence factors,toxins or other antibiotic resistance.It provides data support for subsequent applications in food.3.Application of bacteriophageThe phages HS37 and HS106 of K.pneumoniae have good in vitro lysis ability to host bacteria under different MOI.Within 24 hours,HS106 and HS37 can inhibit the growth of corresponding host bacteria,and the OD value is always close to 0.Phages HS37 and HS106 can play a very good role in removing the biofilm formed in the medium and milk.The removal rate of biofilm formed in the medium reaches more than 30%.The biofilm formed in milk can reduce the number of bacteria in the membrane by more than 1 log.Application of phage HS106 in milk: at 25 ℃,compared with the control group,the phage HS106 treatment group decreased by about 1.6 log10 CFU/m L at 6 h.When used in chicken: the bacteriophage HS106 treatment group reduced about 2 log CFU/m L compared with the control group at 6h.The above results showed that the bacteriophage HS106 could be used in food for antibacterial treatment.Application of phage HS37 in milk: Compared with the control group,the phage HS37 treatment group decreased about 2.3log10 CFU/m L at6 h.At 25 ℃,the phage HS37 treatment group reduced about 3 log CFU/m L compared with the control group at 12 hours,and the effect was good.The results showed that the bacteriophage HS106 could be used in food for antibacterial treatment.In summary,the bacteriophages isolated from the untreated sewage of the hospital in this paper have good temperature and p H tolerance,wide spectrum of lysis,short incubation period and large amount of lysis.The bacteriophages can clear the biofilm in the environment and food,have therapeutic effect on the food infected by Klebsiella pneumoniae,and have the prospect of being used as a new antibacterial agent in food.