Isolation Of Bacteriophage From Domestic Sewage And Genome Research

Urban sewage contains a variety of potential pathogens.Once it enters the animals and plants,it will cause various diseases.The treated sewage may still contain a high number of microorganisms.Phages are viruses that infect microorganisms such as bacteria,and have strong host specificity and diversity.People began to use the characteristics of bacteriophage to conduct pathogen control research in various fields.The samples needed for the experiment came from Qinghe Reclaimed Water Plant of Beijing Drainage Group.After enrichment and isolation,52bacterial strains were obtained and using them as hosts,3 bacteriophages were specifically isolated.The biological characteristics of these bacteriophages such as bacteriophage spectrum,potency,infection complex number and growth curve were analyzed,and the structure of bacteriophage genome was preliminarily explored using transmission electron microscopy,whole genome sequencing,splicing and functional annotation.A total of 52 strains of bacteria were isolated in this experiment.After 16S rRNA sequencing of the isolated and purified bacteria,the type of bacteria was determined according to the sequence comparison results of the NCBI database.The 52 strains of bacteria were used as potential hosts to isolate the phage.Finally,3 strains of LW03,EK06,and NM19 were isolated by Raoultella ornithinolytica,Enterobacter kobei,and Citrobacter freundii.The results showed that the three bacteriophages LW03,EK06,and NM19 could form transparent plaques with clear edges;under the electron microscope,the three bacteriophages had regular regular polyhedron heads and obvious tails.LW03 belongs to the Siphoviridae;EK06 and NM19 belong to the Myoviridae.All three bacteriophages have host specificity within the range of bacteria isolated in this study.The basic biological analysis of bacteriophage showed that the titer of the suspension of EK06 can reach 1.03×10¹⁰ PFU/mL,the titer of the suspension of NM19 can reach 2.37×10¹⁰ PFU/mL;the optimal multiplicity of infection of EK06 is0.1,The optimal multiplicity of infection for NM19 is 1.By measuring the one-step growth curve of phage,we found that the incubation period of EK06 is about 50min and the outbreak period is about 50-70min;the incubation period of NM19 is about30min and the outbreak period is about 30-50min.?3?Through genome sequencing and analysis,it was found that the length of LW03 genome is 109430bp,which contains a total of 93 open reading frames?ORF?and 24 tRNA,of which 47 ORF functions are known,and 46 ORFs are hypothetical protein expression sequences.The EK06 genome is 178140 bp in length and contains a total of 270 ORFs and 1 tRNA,110 of which have known ORF functions,and 160 ORFs are hypothetical protein expression sequences.The NM19 genome is 89089 bp in length and contains a total of132 ORFs and 26 tRNAs.Among them,32 ORFs have known functions,and 100ORFs are hypothetical protein expression sequences.The protein coding sequence?CDS?that has been given functions can be divided into five categories:structural protein modules,DNA packaging modules,cleavage modules,DNA recombination and replication modules,and hypothetical protein modules;genome-wide alignment found that LW03 is homologous to Bacteriophage Eos Sexuality is the highest?83.42%?and may belong to a genus in the Siphoviridae.EK06 has the highest homology?98.28%?with Cronobacter phage vB_CsaM_GAP161,and may belong to the genus T4-like bacteriophage of the Myoviridae.NM19 has the highest homology?88.84%?with Escherichia phage fjerdesal,so NM19 may belong to the genus?H-like bacteriophage of the Myoviridae.In this study,multiple bacterial strains were isolated from domestic sewage.Based on this,3 bacteriophages were obtained,and systematic biological identification and genomic analysis were carried out on them.To a certain extent,the microorganism strain resources were accumulated and enriched.The research results can also provide a certain data reference and theoretical basis for the elimination and prevention of environmental microbial pollution by phages and their lyases.