Isolation And Biological Characteristics Of Chicken-derived Enterococcus Faecalis Phage

Enterococcus faecalis infection leads to the death of chicks and joint lesions of chickens,and the decrease of egg production rate,which brings potential risks to the livestock breeding and human health.Antibiotic are still the first choice to against bacterial infections.The application of antibiotics induces the formation of drug resistance in E.faecalis,especially the emergence of vancomycin-resistant E.faecalis,which has aroused widespread concern in the field of global public health.Bacteriophages are the main viruses that infect bacteria and have the advantages of wide distribution,easy isolation,strong specificity to the host,strong fecundity,and high stability.Therefore,it is of great significance to isolate and analyze the biological characteristics of E.faecalis bacteriophage for replacing antibiotics in the prevention and treatment of E.faecalis.We isolated,cultured,and purified the phage that lysis chicken-derived E.faecalis by double-layer agar plate method.The phage titer and optimal multiple of infection were detected,and the one-step growth curve was drew.The host spectrum was determined by dot blot.The effects of ambient temperature,ultraviolet radiation,and p H on the activity of the phage were examined.In the study,a lytic bacteriophage of chicken-derived Enterococcus faecalis was isolated and purified from the sewage,named EFC1.After Double-layer plate culture,the plaques with clear boundary and transparent were obtained.The titer of the bacteriophage was 6×10~8,and the optimal multiplicity of infection was 1.The incubation period was 90 min,the outbreak period was 20 min,and the lysis amount was 9 PFU.Bacteriophage EFC1 specifically lyses multiple strains of chicken-derived Enterococcus faecalis with good thermal stability,intolerance to environmental temperature higher than 50°C,sensitive to ultraviolet radiation and wide p H adaptation range(4-12).The biological characteristics of bacteriophage EFC1 provide experimental basis for clinical application.To explore the genomic characteristics of bacteriophage EFC1,the phage was purified and condensed,and its nucleic acid was extracted.The whole genome sequence was analyzed by high-throughput sequencing assembly,and its coding genes were predicted and annotated.Bacteriophages were stained by negative staining and observed by electron microscope.The morphological characteristics of the bacteriophage belonged to the family Siphoviridae of the order Caudovirales.The genome was a circular double-stranded DNA with a total length of 56099 bp and a G+C ratio of 39.96%.It had the highest homology with the Enterococcus phage v B＿Efa S＿IME198,which was 96.66%.The whole genome was predicted to have 89 coding genes and 2 t RNA coding fragments,which were compared and annotated by NCBI Blastp.Among them,27 coding proteins were known functions,including phage intron,structure,transcription,packaging,replication,modification,and lysis,while the rest were hypothetical proteins.They were not found to carry genes associated with bacterial virulence and antibiotic resistance.Phage EFC1 is a newly discovered lytic phage.These results provide a basis for further research by preliminarily annotating E.faecalis phage gene functions.To verify the potential of bacteriophage against Enterococcus faecalis infection,the lethality test of chicken embryos was used to determine the median lethal dose of Enterococcus faecalis.Sixty 9-day-old chicken embryos were randomly divided into six groups(10 in each group).The prevention group(inoculated with bacteriophage before Enterococcus faecalis),the killing group(inoculated with Enterococcus faecalis and bacteriophage),the treatment group(inoculated with Enterococcus faecalis before bacteriophage),the positive control group(inoculated with Enterococcus faecalis),the negative control group(inoculated with BHI broth medium),and the bacteriophage group(inoculated with bacteriophage).The mortality rate of chicken embryos in each group was determined 7 days after inoculation.The results showed that the median lethal dose of chicken embryos was 9.063 CFU/ml.In the anti-E.faecalis infection test,the killing group,the prevention group and the treatment group could significantly reduce the mortality of chicken embryos,with the mortality rates of 40%,50%and 33%,respectively,while the mortality of the positive control group was 87.5%,which was significantly different from that of the killing group,the prevention group and the treatment group(P<0.05).There was no embryo death in the negative control and phage groups.This suggested that bacteriophage EFC1 had no obvious toxic and side effects on chicken embryos,and had obvious anti-E.faecalis infection effect,and had a certain inhibitory effect on the growth of chicken E.faecalis,which was expected to replace antibiotics against E.faecalis infection.