Preliminary Research On Isolation And Biological Characteristics Of A Lytic Bacteriophage Against Enterohemorrhagic Escherichia Coli O157: H7

The emergence of enterohemorrhagic Escherichia coli O157: H7, which causesinfectious diarrhea, remains a continuous public health thread worldwide. Infectionwith enterohemorrhagic Escherichia coli O157: H7may result in asymptomaticcarrier, diarrhea, hemorrhagic colitis, hemolytic uremic syndrome andthrombocytopenia. Bacteriophages (phages) are viruses infecting bacteria anddistribute widely in environment. Both specifically efficiency of lysing bacteria byphages and phage therapy, a method using phages for the treatment of bacterialinfectious diseases (particularly caused by drug-resistant bacteria) lead to publicattention. Therefore, further investigation on phages against enterohemorrhagicEscherichia coli O157: H7has an important impact on both realistic significance andpotential utilization.In studies addressing isolation of lytic Escherichia coli O157: H7phages insamples taken from sewage, a lytic phage specific against Escherichia coli O157: H7was identified using double-agar plaque method. Electron microscope demonstratedthat the morphology of head of phages is cubic symmetry,40nm in diameter andtheses phages had a long tail (approximately100nm long). Based on morphologicalfeatures,lytic Escherichia coli O157: H7phages were classified into order Caudovirales,family Siphoviridae. Purified phages were subjected to CsCl gradient centrifugationand ultrafitration for phage enrichment, a high titer of phages was obtained. Theelectrophoresis of phage genomes digested with restriction enzymes showed that thesize of phage genomes is proximately30kb, harboring EcoR I and EcoR Vrestriction sites. SDS-PAGE analysis suggested that13bands (capsid structural andnon-structural proteins) with molecular weight ranging from16～220kD.Multiplicity of infection is one of biological features closely associated with relationship between phage infection and its releasing amount from host. Aftermixing phages and host cells in different portions, a bactericidal efficiency byphages was carried out at different times. This study revealed that when the ratio ofhost cells (Escherichia coli O157) to phages is1:100and the incubation period is180min, the number of bacterial cells declined dramatically (decreased from10~8CFU/ml to10~4CFU/ml). One-step growth curve showed that the latent time is10min, which indicated a period from phages attaching host cells to progeny phagesliberating. With continuous lysis of host cells and releasing of progeny phages, thenumber of progeny phages reached highest level, therefore, the period from latenttime to highest level of phages is referred to burst time. In our experiments, the bursttime was30min and the lysis amount is130PFU/cell. The adsorption rate indicatesa period from early stage of phage attachment to host cell to late stage of all phagespenetrating host cells. Our data demonstrated that upon infection of Escherichia coliO157in the early-exponential phase with phages, approximately100%of the phageparticles adsorbed to host cells within10min. The host range experimental resultsindicated that these isolated phages are lytic and Escherichia coli O157specific.Three methods of conservation for isolated phages were investigated, chloroform:SM buffer is an appropriate mean for conserving isolated phage particles at4℃forat least8months.Comprehensive understandings of biological characteristics of phage enable us tostudy and to develop phage agents against bacterial infection. On the basis ofisolation of Escherichia coli O157phages, researches on biological characteristics ofphages lay the foundation for the further study and animal experiments.