Biological Characteristics And Genome Analysis Of Enterococcus Faecalis Phage VB＿EfS＿L1

Objective:In recent years,the problem of antibiotic resistance has become increasingly serious,and hospitals are often unable to provide effective drug treatment options when patients are infected with drug-resistant bacteria.Phages have received attention from researchers because of their advantages,such as the ability to specifically infect host bacteria.In this study,we isolated the Enterococcus faecalis phage vB＿EfS＿L1 using drug-resistant Enterococcus faecalis Ef-01 as the host bacterium,analyzed the biological properties of the phage,sequenced it,explored and analyzed the genomic characteristics of the phage,and provided a theoretical basis and experimental basis for using the phage to treat infections caused by Enterococcus faecalis.Methods:In this study,Enterococcus faecalis EF-01 was isolated and purified,and the morphological characteristics of the colonies were observed.The strain was identified by16S rDNA for molecular biology and 15 antibiotics were selected for drug sensitivity analysis.The lytic phage was screened in untreated sewage using Enterococcus faecalis EF-01 as the host bacterium and the potency of the phage was determined using a double layer plate method.Phage concentrates were prepared and the morphology of the phages was observed using transmission electron microscopy.The biological properties of the phage were analysed by determining the host spectrum,optimal infection complex,sensitivity to chemical reagents,temperature and pH stability,stability under UV irradiation and one-step growth curve.Extraction of phage genomic DNA and whole-genome sequencing of phage vB＿EfS＿L1.Using bioinformatics software Gene Mark S,Proksee,MEGA 11,VFDB and CARD,genome-wide analysis,functional gene annotation and virulence and resistance factor analysis were performed on vB＿EfS＿L1;using bioinformatics sites and bioinformatics websites such as TCDB,Signal 6.0,ExPASy,Port Scale and Phyre2 and The programme was used to perform primary structure analysis,physicochemical characterisation,hydrophobicity prediction,secondary structure prediction and structural domain 3D modelling of phage lyase related genes.Results:After isolation and purification,the single colony form of Enterococcus faecalis EF-01 was obtained,the colony form was round,creamy white in colour,shiny,with neat edges.The results of drug sensitivity analysis showed that Ef-01 showed sensitivity to ciprofloxacin,linezolid,ampicillin,levofloxacin,tigecycline and vancomycin,moderate sensitivity to furantadine,amoxicillin,cotrimoxazole and piperacillin,and resistance to penicillin,cefradine,gentamicin,teicoplanin and amikacin.A strain of Enterococcus faecalis phage vB＿EfS＿L1 was successfully isolated using EF-01 as the host bacterium.After multiple purifications,phage spots of uniform size and morphology could be observed on bilayer plates and tested for potency up to 10¹²（PFU/m L）.The lytic ability of vB＿EfS＿L1 was tested against 15 clinical isolates of Enterococcus faecalis and Enterococcus faecium.vB＿EfS＿L1 was found to be lytic against only three of the enterococci.vB＿EfS＿L1 had an optimal infection complex of0.01;an incubation period of 0-30 min,a lytic period of 30-90 min and a lytic amount of270 PFU/cell,a lytic phage.vB＿EfS＿L1 is active below 50℃,gradually inactivated above 70℃;stable at 4≤p H≤10.vB＿EfS＿L1 is completely inactivated after 35min exposure to UV lamp;insensitive to chemical reagents chloroform and ether,sensitive to SDS and proteinase K.vB＿EfS＿L1 is a lytic phage.Whole genome sequencing analysis revealed that vB＿EfS＿L1 is a double-stranded DNA phage with a full genome length of 41,151 bp,a GC content of 34.69%and no t RNA.The genome contains 64 genes,41 of which are located on the righteous strand and 23 on the negative-sense strand.The average length of each gene is 581bp,with the longest being 2355bp and the shortest 156bp;there are 33 genes（51.56%）with protein lengths of more than 100 amino acids.The whole genome sequence of phage vB＿EfS＿L1has been submitted to the Gen Bank database under accession number OP254195.1.The genome contains the lytic enzyme Lys-v BL1 and the perforin Holin.No virulence factors or antibiotic resistance genes were identified in phage vB＿EfS＿L1.Conclusion:In this study,the lytic phage vB＿EfS＿L1 was isolated using Enterococcus faecalis Ef-01 as the host bacterium.Through characterisation of the biological properties,genome-wide information and lytic enzyme properties of the phage vB＿EfS＿L1,it was concluded that the phage vB＿EfS＿L1 is a strain that can efficiently lyse Enterococcus faecalis Ef-01 with high host specificity and specificity,and without any virulence factors and antibiotic resistance genes,and has potential for the clinical treatment of Enterococcus faecalis infections.