Isolation,Identification And Biological Characteristics Analysis Of Avian Pathogenic Escherichia Coli Bacteriophage

Avian pathogenic Escherichia coli(APEC)can cause avian colibacillosis and seriously endanger the poultry industry.APEC has numerous serotypes with poor cross-protection.At present,avian colibacillosis is mainly controlled by antibiotics.However,due to the unreasonable use of antibiotics,bacterial multi-drug resistance is caused,and drug residue problems seriously threaten food safety.Due to the slow development of new antibacterial drugs,bacteriophages(phages)and their lytic enzymes,as alternatives to antibiotics,have received extensive attention from scholars at home and abroad.In this study,a phage was isolated and purified with a multidrug-resistant APEC as the host bacteria,studied its biological characteristics and whole-genome characteristics,determined its therapeutic effect on mice infected with APEC preliminarily,and further cloned and expressed the phage lysozyme to determine the antibacterial activity,which enriched the E.coli phage library,so as to provide reference and basis for the use of phage and lytic enzyme to prevent and treat APEC infection.In order to provide reference and basis for the control of avian colibacillosis by phages,this study collected fecal water samples from chicken farms,and 14 APEC phages were identified through isolation and six rounds of purification.A virulent phage with a wide lysis spectrum was screened and named v B＿Eco S＿AH50.The morphology,optimal multiplicity of infection(MOI),one-step growth curve,thermal stability,room temperature stability,p H stability,in vitro antibacterial ability and biofilm formation inhibition ability of the phage were determined.The mice were challenged with APEC by intraperitoneal injection,and the phage was injected intraperitoneally at 12 h post-infection to initially determine the therapeutic effect of phage.The results showed that phage v B＿Eco S＿AH50 belonged to the family Siphoviridae and the order Caudovirales,which can lyse O1 and O2 serotypes of E.coli.The optimal MOI of this phage to the host bacteria AH50 was 1,the latent period and the burst period were both 20 min,and the burst size was about 76 plaque-forming units(PFUs)/cell.It can maintain strong lysis ability in the range of 4～50℃ and p H 3～11,and can survive stably for 4 weeks at room temperature.At MOIs of 0.01,0.1 and 1,phage v B＿Eco S＿AH50 could efficiently inhibit bacterial planktonic cells growth and biofilm formation.In the phage treatment test,the survival rate of the infected group was 16.7%,and the survival rate of the treatment group was 50%.These results showed that phage v B＿Eco S＿AH50 can specifically lyse E.coli,with strong lysis ability,good tolerance and certain therapeutic effect for APEC infection,suggesting that it has potential application value in the prevention and treatment of avian colibacillosis.In order to understand the whole-genome characteristics of phage,the genome of phage v B＿Eco S＿AH50 was extracted,the nucleic acid type of the genome was analyzed by enzyme digestion,and then the whole genome was sequenced by Illumina Nova Seq sequencing platform.The whole genome was annotated using Gene Mark S software(v4.32)and NCBI database.The t RNA genes of the genome were identified by the t RNAscan-SE software.The virulence factor database VFDB and the antibiotic resistance gene database CARD were used to analyze the virulence genes and drug resistance genes of the phage genome.The whole genome of the phage was compared using NCBI and Easyfig software(v2.2.5).Based on the amino acid sequence of phage terminase large subunit,a neighbor-joining phylogenetic tree was constructed by MEGA software(v5.05)to analyze the evolutionary relationship.The results showed that the phage v B＿Eco S＿AH50 genome is double-stranded DNA,composed of 37268 bp,with a GC content of 54.62%.A total of 47 ORFs were predicted,21 of which had known functions,and ORF7 encoded lysozyme.There was no t RNA gene in the genome.And no lysogen-related genes,virulence genes and antibiotic resistance genes were detected in the genome,indicating that the phage is safe and has the application prospect as biological antibacterial agents.Blast analysis showed that this phage had the highest homology with E.coli phage PC2(ON184124.1),with 94.56% sequence similarity.Easyfig compared the genomes and showed that most functional regions of v B＿Eco S＿AH50 and PC2 had high homology(>82%).The phylogenetic tree based on the amino acid sequences of terminase large subunits showed that phage v B＿Eco S＿AH50 was most closely related to E.coli phage v B＿Eco S＿011D5(genus Dhillonvirus).Phage v B＿Eco S＿AH50 is a new member of the genus Dhillonvirus of the family Siphoviridae.In order to further explore the antibacterial activity of the phage lysozyme Lys AH50,the lysozyme sequence was first analyzed.The homologous proteins of the lysozyme were analyzed by NCBI alignment,and the signal peptide and transmembrane domain were predicted by Signal P(v4.1)and TMHMM(v2.0).The conserved domain was predicted using the CDD database in NCBI.The physicochemical properties of lysozyme were predicted through the online tool Ex PASy-Prot Param.Then,the recombinant expression strain BL21(DE3)Plys S-p ET28a-Lys AH50 was constructed,the protein was expressed and purified,and its lysis spectrum and the antibacterial effect of the combination with EDTA were determined.The results showed that lysozyme Lys AH50 was composed of 163 amino acids and had the highest similarity of 99.39% with the lysozyme sequence of Shigella phage EP23(NC＿016566.1).Lysozyme Lys AH50 has no signal peptide and transmembrane domain,and contains a conserved Lyz＿endolysin＿autolysin structual domain of the Lyz-like superfamily between 7-148 amino acids,acting on the β-1,4 glycosidic bond of peptidoglycan.Prot Param tool predicted that Lys AH50 has a molecular mass of 18.1 k Da,which is hydrophilic and stable.The BL21(DE3)Plys S-p ET28a-Lys AH50 strain was constructed,and the soluble recombinant lysozyme protein Lys AH50 was successfully expressed and purified,with a molecular weight of about 21 k Da.Lysozyme Lys AH50 showed lytic activity against 61 inactivated strains of avain E.coli and Salmonella,but could not lyse Staphylococcus aureus.When combined with EDTA,Lys AH50 showed strong antibacterial activity,reducing the number of viable bacteria by 2.67 orders of magnitude.Lysozyme Lys AH50 has a wide lysis spectrum and strong lysis ability,which shows its good application prospect and lays the foundation for developing new antibacterial agents for APEC.