Characterization And Complete Genome Sequence Analysis Of A Novel Enterococcus Faecalis Phage IME-EF3

Objectives The bacteriophage IME-EF3is isolated and its genome is sequenced usinghigh-throughput sequencing which provides basic data for the potential phage therapyagainst antibiotics resistant Enterococcus feacalis infections.Methods The bacteriophage IME-EF3was isolated from the hospital sewage by usingclinically isolated Enterococcus faecalis as host. A double-layer agar plate method wasused to determine the shape of the plaque and the titer of the phage preparations， aswell as the best multiplicity of Infection（MOI）, one-step curve. Heat and ultravioletirradiation stability of IME-EF3was analyzed by incubating the phage preparations underdifferent temperatures and with different radiation durations respectively. The phagegenome extracted using the method of SDS/protease K was characterized by nucleasedigestion and agarose gel analysis. Genomic DNA was sequenced using Ion Shearchemistry along with the Ion One-Touch200template kit v2DL（Life Technologies）and the Fast DNA Library Prep Set for Ion Torrent （New England Biolabs）,according to the manufacturer’s instructions. Sequencing was carried out on an IonTorrent personal genome machine platform using the Ion Sequencing300kit （LifeTechnologies）. Raw sequences were assembled using the Newbler2.8assembler. Theprediction of open reading frames （ORFs） was performed using the RASTannotation server and Kodon（Applied Maths） ORF annotation was performed usingthe BLASTP and InterProScan programs. Evolutionary phylogenetic analysis of phageIME-EF3was conducted in MEGA5using the neighbor-joining method. The predictionof classifying the lifestyle of IME-EF3was performed using PHACTS.Results Bacteriophage IME-EF3was isolated successfully from hospital sewage. Theplaque of IME-EF3was1.7mm in diameter and the best MOI was1.0. IME-EF3wasrelative heat-stable，while it was sensitive to the ultraviolet irradiation. Digestion withrestriction endonucleases indicated that IME-EF3genome was dsDNA. Genomic analysisand electron microscopy suggested that IME-EF3was a member of the familySiphoviridae. The phage has an isometric head and a long, non-contractile tail, with a41-kb linear double-stranded DNA genome. The genome encodes69putative proteins, with33annotated functionally, including proteins related to phage structure, packaging,transcription, replication, and a lysis module. Interestingly, a metallo-beta-lactamase generesponsible for multi-drug resistance was found in the genome of IME-EF3. The metallo- beta-lactamase gene of Enteroroccus phage IME-EF3was the most similar toEnterococcus phages EfaCPT1, the percentage of the different amino acid sequnences is14%. The large terminase subunits,lysin and host specificity protein were compared byClustalW multiple alignment which showed to be most closely to Enterococcus phageEFAP-1and Enterococcus phage EFRM31. Reads sequence frequency analysis showedthat IME-EF3had both specific right and left end sequences, which suggests that IME-EF3contains a linear genome with redundant termini.Conclusions A bacteriophage IME-EF3lytic to antibiotics resistant Enterococcusfaecalis is isolated successfully from hospital sewage. Biological characterization ofIME-EF3provids useful information to apply IME-EF3as an alternative therapeutics totreat antibiotics-resistant Enterococcus faecalis infection. The possibility of horizontalgene transfer of the metallo-beta-lactamase gene suggests that phage IME-EF3, althoughlytic, might not be suitable for phage therapy unless one would devise a way to delete themetallo-beta-lactamase gene. Hence, whole genome sequencing should always be aprerequisite for identifying a phage therapy candidate.