Genome Analysis Of IME196 And IME199 Infecting Enterococcus And Phage Receptor

With the widespread use of antibiotics,increased number of clinical multidrug-resistant Enterococci,The use of natural antimicrobial phage in the treatment of multiple drug-resistant pathogens began to be concerned again.Phage can also be used in combination with antibiotics to treat infections caused by multiple drug-resistant pathogenic bacteria,which is currently one of the best ways to solve the problem of multiple drug resistance of pathogens.Phage host specificity makes the phage’ host spectrum limited,and it is necessary to analyze the reason.In this study,two strains of Enterococci were isolated from hospital sewage.Biological characterisitcs and genomics analysis of these two phages were carried out to provide the basis for phage theray.Through the comparative genomics analysis of phage-sensitive and resistant bacteria,the reasons for determining the specificity of phage host were identified initially to provide a basis for phage therapy.Isolation and its genomic analysis of phage vB_E.faecalis_IME196(IME196)A phage was isolated from the hospital sewage with Enterococcus faecalis strain 2007 as an indicator,named vB_E.faecalis_IME196.Transmission electron microscopy analysis indicated that IME196 displays morphology resembling Siphoviridae family.Biological characterization analysis showed IME196 have sensitivity to high temperature and ultraviolet,respectivly broad spectrum,has a good cleavage activity in the range of pH 7.0-9.0.High throughput sequencing results showed the genome of IME196 is a 38 895 bp in length,linear,double-stranded DNA has a G+C content of 33.9%.No toxin gene.The complete genome of IME196 was Blasted on database of NCBI,92% homology with phage EfaCPT1.The results of the gene annotation showed that there were 57 open reading frames(ORFs).Of these 57 ORFs,30 of them were hypothetical protein sequences,and the function of the remaining 27 ORFs was known.No bacterial virulence and antibiotics resistant gene.Isolation and its genomic analysis of phage vB_EfaP_IME199(IME199)A phage was isolated from the hospital sewage with Enterococcus faeciumstrain 1007 as an indicator,named vB_EfaP_IME199.Transmission electron microscopy analysis indicated that the isolated bacteriophage IME199 displays morphology resembling Podoviridaefamily.Biological characterization analysis showed IME199 have sensitivity to high temperature and ultraviolet,respectivly narrow spectrum,has a good cleavage activity in the range of pH 7.0-9.0.High throughput sequencing results showed the genome of IME199 was 18 838 bp circular dsDNA,and has a G+ C content of 34.6%.The complete genome of phage IME199 was Blasted,and only 4% coverage with IME-EFm5.The genome had very low homology to all other known bacteriophage sequences in the GenBank database.The results of the gene annotation showed that there were 22 open reading frames(ORFs).Of these 22 ORFs,10 of them were hypothetical protein sequences,and the function of the remaining 12 ORFs was known.This phage genome has a short termini reverse repeats.No bacterial virulence and antibiotics resistant gene.The mechanism analysis of E.coli BL21 that resistant to phage vB_EcoS_IME253 This study screened phage IME253 resistant Escherichia coli BL21.By sequencing genomes of phage sensitive and resistant bacteria strain,FepAgene was predicted to be related with phage resistant.CRISPR knockout technique validates the mechanism of phage receptor To prove that the fepA gene was the cause of phage resistance,the fepA gene was cut by using CRISPR/Cas9,and the Red homologous recombination system was used to knock out fepA gene seamlessly.The results demonstrated thatfepA gene knockout can lead to bacterial BL21 resistance to phage IME253.This experiment used comparative genomic analysis to establish an analytical method of host resistance of bacteriophages,and used CRIPSR technology to phage receptor related genes,which provide a supports for phage therapy.In this study,two lytic phages,IME196 and IME199,were isolated from multi-resistant Enterococci.Biological analysis and genomics analysis provided information for further study of phage.The results showed that the two phages were candidates for phage treatment.Since the phage has host specificity,which makes the phage lytic spectrum limited.In this study,we used a comparative genomics analysis platform to establish a host resistant phage mechanism analysis method to study its resistant-related genes,CRISPR seamless knockout technique validates the mechanism of phage receptorof host,analyze host-specific causes,and provide phage therapy basis.