| Enterococci are important pathogenic bacterium causing nosocomial infection,Enterococcus faecalis take the second place in nosocomial infections induced by gram possitive bacteria,which occupied about 80-90% enterococcal infections involved multiple organs and tissue.Contrasting to rapid increasing of multidtrug resistant strain detection,slow exploration of new antibiotics resulted treatment of enterococcus in a dilemma.Bacteriophages are the viruses of bacteria,actinomycetes,spirochetes,unicellular algae and fungi.Rapid and efficient multiplication of lytic bacteriophage induce lysis of specific host cell and production of large number of progeny phages.Lytic phages with wide host range and high liysis efficiency are becoming the new hot spot of multi-drug resistance enterococcus infection.In this research,the clinical isolated enterococci were identified by 16 SrDNA sequencing and random amplified polymorphic DNA.After the drug resistance testing,multidrug resistantce strains were used as the hosts to isolate and idetified of lytic pahge from hospital sewege.Series of biological properties of the isolated phages,such as the morphology,one-step growth curve,optimal multiplicity of infection,temperature and pH stability,were tested and analyzed.And the genome of two lytic phages were sequenced and annotated.In this study,30 strains of Enterococcus faecalis were obtained,and all of them were identified as multi-drug resistant strains.Two strains of lytic phage were obtained,named as LY0322 and LY0323,respectively.Both of them belong to Caudovirales order,Siphoviridae family.The optimal multiplicity of infection(MOI)of LY0322 was 0.1,and optimal MOI of LY0323 was 0.001.One-step growth curve showed that LY0322 had an latent period of 20 min,the burst period was 20 min,and the burst size was 40(PFU/cell).LY0323 had an latent period of 25 min,the burst period was 55 min,and the burst size was 257(PFU/cell).Both phages were able to maintain high activity at temperatures below 50℃ and pH of 6 and 7.LY0322 can lyse 8 strains of MDR Enterococcus faecalis.LY0323 can lyse 7 strains of Enterococcus faecalis,two strains of Enterococcus faecium.All sensetive hosts of LY0323 are also multi-drug resistant bacteria.SDS-PAGE analysis showed that the stucutural proteins of LY0322 distribute in a molecular mass range of 35 KD.And the structural proteins of LY0323 distributed in molecular mass range of 15-35 KD.The genome of LY0322 is a linear double-stranded DNA and consisted of 40934 bp,contained 64 ORFs,with a G+C content of 34.84%.The LY0322 genome occupied highly homologous to Enterococcus phage vB_EfaS_AL3.The genome of LY0323 is also a linear double-stranded DNA and consisted of 40876 bp,contained 60 ORFs,with a G+C content of 34.67%.The genome of LY0323 is highly homologous to that of Enterococcus bacteriophage IME-EF4,vB_EfaS_AL2 and Ec-ZZ2.Both phage genomes do not contain virulence genes and drug resistance genes.The study obtained the following conclusions.1.30 strains of Enterococcus faecalis were obtained,and 30 sequences of 16 SrDNA were uploaded to GenBank.2.30 strains of multi-drugresistant Enterococcus faecalis were identified.3.Two lytic phages belong to Siphoviridae were abtained,and their biological characteristics were analyzed.4.The genome sequence of two lytic phages were determined and uploaded to GenBank with Numbers MH193369 and MH375074.Two strains of phage were newly discovered,and virulence genes or drug resistance genes were not found.To sum up,Enterococcus faecalis phages in this study have strong adaptability and safty,which provided important basic data for future practical applications. |
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