Isolation，Identification And Complete Genomic Sequence Of The Escherichia Coli Phage VB_EcoM_ECOO78 And Mechanism Of Its Depolymerase Dpo42

Escherichia coli(E.coli)is one of the most common conditioned pathogens in hospital that can cause a variety of local and systemic infectious diseases.Moreover,E.coli is one of the bacteria that tends to form biofilms.Biofilm formation is an important cause of E.coli resistance.With the wide application of the biomedical materials in clinical practice,such as tracheal intubation,artificial heart valves,artificial joints,arteriovenous catheters,catheters,etc.,the incidence of related infections increased.It was reported that about 65% of human infections were related to the formation of biofilm,90% of patients with mechanical ventilation in the tracheal intubation at the end of bacterial colonization infection,catheter-related urinary tract infection rate was 9.23%.Furthermore,the resistance of the bacteria is more than 1000 times that of the planktonic bacteria.Therefor,treatment of these infections has become ever more difficult.Bacteriophage is a kind of virus that can specifically infect its host bacteria.Host bacteria can be bacteria,fungi,actinomycetes or spirochetes and other microorganisms.Bacteriophages use the replication of the host bacteria to synthesize their own ingredients and assembly,and release the progeny phage by destroying the host bacteria.Some phages can encode depolymerases to hydrolyze the bacterial biofilms,so phages have an important potential in dealing with bacterial infectious diseases.In this study,E.coli phage and its depolymerase(Dpo42)were used as the object to explore the potential of phage-derived depolymerase as a therapeutic agent and its mechanism against biofilm.First,E.coli O78-3 was used as host bacteria to isolate,screen and purify one phage,named v B_Eco M_ECOO78.We observed its ability to lyse five out of 34 tested E.coli clinical isolates.The highest phage titer was observed at a multiplicity of infection(MOI)of 10-5and a burst size of approximately 74 PFU/infection.Electron micrographs showed that the phage had an isometrically hexagonal head 47± 2 nm in diameter.The head was separated from the tail sheath by a collar.The tail was contractile and approximately 149 ± 3 nm long.According to the InternationalCommittee on Taxonomy of viruses(ICTV),v B_Eco M_ECOO78 belongs to the Myoviridae family.The presence of increasing halo surrounding the lysis plaques formed by v B_Eco M_ECOO78 indicated that this phage may encode a depolymerase.Based on sequencing analysis,the complete genome of v B_Eco M_ECOO78 is 41,289 bp,with a GC content of 53.07%.Additionally,v B_Eco M_ECOO78 has 56 predicted ORFs,51(91.07%)of which are assumed to be functional.A total of 51 CDSs were predicted to encode proteins ranging from 6.28 k Da(ORF16)to 86.39 k Da(ORF10).Approximately 90.2% of ORFs(46 ORFs)started with an ATG codon,7.8%(4 ORFs)used GTG,and only one ORF started with TTG.TGA and TAA were stop codons for27(52.9%)and 22(43.1%)ORFs,respectively.Only ORF48 and ORF53 had TAG as a stop codon.Based on a comparative analysis of the complete genome,five phages show similarity to v B_Eco M_ECOO78,specifically v B_Eco M-ep3,v B_Eco M_ECO1230-10,Enterobacter phage Arya,Pseudomonas phage PPp W-3 and v B_Eco M_CBA120.However,there are some genes show no identity with the five phages above in the whole genome of v B_Eco M_ECOO78,such as the fragments of487-745 bp,8044-8331 bp,15921-16054 bp,16295-16630 bp,16725-16898 bp,16937-17288 bp,18514-18826 bp,and 32937-33581 bp.Thus,we guess that the above sequences which v B_Eco M_ECOO78 have are the main cause of the difference from the five phages.Moreover,BLAST nucleotide analysis also indicated the presence of genetic drift and recombination between phages and bacteria.Synthesizing the above general biological characteristics and sequencing results we can know v B_Eco M_ECOO78 is a lytic phage.The phage v B_Eco M_ECOO78can form clear phage plaques.In the one-step growth curve,we can know the phage v B_Eco M_ECOO78 lyses the host cell and releases progeny phages.After the whole genome sequenced and blasted with the other bacteriophage genomes in the database,there is no integrase gene or immunosuppressor genes.Further sequence BLAST analysis indicated that ORF42 of v B_Eco M_ECOO78(Dpo42)shares low identity with other reported phage-associated depolymerases.Dpo42 was expressed and purified as a soluble protein using E.coli BL21.Dpo42 contained 747 amino acids with a molecular weight of 78.58 KDa and a theoretical p I of 4.77.The secondary structure of Dpo42 was included nine alpha-helices,55beta-strands.As shown in the protein-level phylogenetic tree,Dpo42 represents adifferent evolutionary offshoot branch from 10 other similar phage depolymerases.Purified Dpo42 can prevent the biofilm formation of E.coli O78-3,and progressive semi-clear spot formation was observed on the plate.Spot size constantly increased after prolonged incubation.As the concentration of Dpo42 increased,the sizes of the semi-clear circles on the plate also increased.Therefor,it indicated that Dpo42 has a dose-dependent activity to prevent the biofilm formation of E.coli.Biofilm formation ability of E.coli isolates and the antibiofilms activity of Dpo42 were tested by performing spot assays and a 96-well micro-titre plate method.Dpo42 can prevent the formation of the two strong biofilms.Maneval staining assays indicated that Dpo42 can degradate the capsular polysaccharides surrounding E.coli.Based on these results,Dpo42 is a novel phage-derived depolymerase representing a potential new strategy to prevent E.coli biofilms formation.