Isolation, Identification And Preliminary Application Of Escherichia Coli O157 Bacteriophage

Escherichia coli O157 infection is a leading cause of foodborne illness. Infection with Escherichia coli O157 often leads to severe bloody diarrhea and abdominal cramps. In some persons the infection can also cause the life-threatening hemolytic uremic syndrome (HUS). Escherichia coli O157 was first recognized as a cause of illness in 1982 during an outbreak of severe bloody diarrhea in USA. Now it spreads in many country including British, Canada, Japan, and so on. An outbreak of Escherichia coli O157 infection in Jiangsu province in 1999 was reported. It indicated that Escherichia coli O157 becomes threaten of public health in China. Escherichia coli O157 has been found in the intestines of healthy cattle, deer, goats, and sheep. Ruminants are principal reservoir. Because the organism lives in the intestines of healthy cattle, it would be difficult to eradicate this pathogenic bacterium thoroughly. Antibiotic therapy could increase releasing of Shiga Toxin and lead to severe consequence.In order to explore the feasibility of phage applied to biocontrol, a new phage using Escherichia coli O157 as host cell was isolated from the sewage samples. Phages were isolated, identified and purified by double-layer agar plate method with Escherichia coli O157 as host cell. Bacteriophage enumeration was conducted by soft agar overlay procedures. Fifty bateriophages of Escherichia coli O157 were separated from ten sewage samples. According to the lysis spectrum, three of them were named DLSF12,DLSF29 and DLSF48 respectively. The concentration of the phages were all ove 109pfu/mL. The bacteriophage plaques were about diameter 7mm.The plaques of the phage DLSF48 were clear and with clear edgea, its phage titer were 9.6×10~9pfu/mL. DLSF48 was a stronglysis bacteriophage.The biological characteristics of DLSF48 indicated that : the optimum pH of the phage was pH 8; the optimum temperature of the phage infecting to Escherichia coli O157 was 37℃. DLSF48 still remained high lisis activities after incubated at 70℃for 20min or in pH 4-12 for 2h; The phage was resistent to aether and chloroform; The MOI of DLSF48 was 0.1-0.01; The latent period and rise period were 20min and 75min respectively. The average burst size was about 99.Virion buoyant density in CsCl of DLSF48 is 1.45-1.50g/mL.Electronic microscope showed that the phage had icosahedral heads of diameter 50nm , extremely flexible tails of 120×4nm, and no envelope. SDS-PAGE profiles indicated DLSF48 contained six capsid proteins , 3 main capsid proteins ,and molecular weights estimated at 32KD,28KD and 17KD respectively. The nucletic acid of the phage were prepared for agrose eletro phoresie.The rusult showed that DNA of the phage could be degradation by DNaseⅠ, and not by RnaseA and Mung Bean Nuclease. It explained that the nucletic acid of the phage were linear duplex DNA. The genome of the DLSF48 was about 30kb which restricted by enzyme EcoRⅠ,BamHⅠ,XbaⅠ. Base on the eighth bulletin of international classify commission of virus , the phage belong to T1-like virus . According to the results of genome sequencing and homology analysis, the phage was likely to be a new phage.Toxcity experiment was conducted to examine the security of DLSF48. It showed that the phage was safe for mice. we established a mice model for Escherichia coli O157 infection. The weaned mice (weight 10-12 gram) were intragastrically inoculated with a dose of 2×10~9 CFU Escherichia coli O157.DLSF48 were used in the therapy experiment for the infection of Escherichia coli O157. At the same time through intraperitoneal inoculation, the phage(2×10~9pfu) could rescue entire of mice model; Not mice surived even when the therapy was delayed for 6h, also when the treat was ahead of 6h. At the same time through oral inoculation, the phage(dose of more than 2×10~6pfu) could rescue entire of mice model; About entire of mice model surved even when the therapy was delayed for 3h by using the phage(dose of more than 2×10~7pfu) , also when the therapy was delayed for 6h by using the phage(dose of more than 2×10~8pfu). Oral inoculation is obviously better than intraperitoneal inoculation.In the simulation of lake water with Escherichia coli O157 ,there was a decrease in the number of Escherichia coli O157 when the phage was poured into the water.