Isolation,Biochemical Characterization And Bioinformatics Analysis Of Escherichia Coli Phage From Dairy Farm

The abuse of antibiotics has led to the emergence of bacterial resistance,drug residues and other dairy safety issues.Phage has the unique bactericidal mechanism,high host specificity and safety,is expected to become a new means of treating bacterial mastitis in dairy cows.In this study,6 strains of E.coli(K2,Hm4,7140,7525,4525,4818)were used as host bacteria,and mixed with the pretreated dairy farm sewage sample to filter and obtained bacteriophage solution.Firstly,the obtained bacteriophage solution was screened and verified by double-layer plates and topical application.Secondly,the bacteriophage was purified,enriched and concentrated,and its morphological structure was observed by transmission electron microscopy.Identification of the biochemical properties of phage isolated through multiplicity of infection,one-step growth curve and optimum growth temperature,optimal growth of potential hydrogen,SDS-PAGE protein components,genomic nucleic acid types,etc.Finally,the whole genome of bacteriophage isolated was sequenced by high-throughput sequencing technology and the bioinformatics analysis was applied include sequence assembly,evaluation,Functional annotation and phylogenetic tree analysis,etc.The results are as follows:Bacteriophage isolates:Escherichia coli Hm4 and 7140 were used as host strains,and two Escherichia coli phage were successfully isolated and named as Hm4 phage and 7140 phage;The results of transmission electron microscopy showed that the heads of the two phages were positive icosahedrons with contractile tail sheath and tail silk,belonging to the phage of the Myoviridae;Biochemical characteristics:The optimal multiplicity of infection of the two phages was 10-3.the optimum temperature was 40～50℃,and the optimum pH was 5～10;the incubation period of Hm4 phage and 7140 phage was 10 min,5 min,respectively;the burst period of Hm4 phage and 7140 phage was 60 min and 45 min.respectively;the lysis amount of Hm4 phage and 7140 phage was 24 PFU/mL and 12 PFU/mL,respectively.Bioinformatics analysis:The nucleic acid of the two phage is ds DNA;the Hm4 phage genome is 168,370 bp in length,the GC content is 31.6%,and there are 10 t RNA and 11 restriction sites,After annotation,277 CDS,524 ORFs were obtained;7140 phage genome is 169,642 bp in length and has a GC content of 37.58%,It has two kinds of tRNA and 11 restriction sites,After annotation,270 CDS and 704 ORFs were obtained.No resistance genes and virulence genes were found in the two phages.