| Escherichia coli is a kind of gram-negative bacteria prevalent in animal intestines,which can be divided into symbiotic type,diarrhea type and enteral pathogenic type.Diarrhea type and enteral pathogenic type are also referred to as pathogenic E.coli,which are important foodborne pathogenic bacteria.It is estimated that pathogenic E.coli causes 260,000 infections,3,600 hospitalizations and billions of dollars in related medical costs each year in the United States,and is the leading cause of diarrhea in children in our country.Antibiotic treatment and chemical disinfection are the most effective methods to control foodborne pathogens.However,the widespread use of antibiotics and chemical disinfectants has led to the emergence and spread of antibiotic and disinfectant resistant strains.Therefore,it is a necessary and urgent task to find new ways to deal with foodborne pathogenic bacteria.Phage therapy is a bactericidal mechanism that destroys host bacteria with the help of phage growth and reproduction,and is one of the best options for the prevention and control of pathogenic E.coli.However,studies have shown that E.coli phages have a narrow cleavage spectrum,and their effect on pathogenic E.coli with numerous serotypes is limited,which limits the clinical application of E.coli phages to a certain extent.The purpose of this study is to establish a screening method for broad-spectrum phages,screen the broad-spectrum phages that can lyse a variety of pathogenic E.coli,and evaluate the bacteriostasis effect in milk,chicken,pork and lettuce samples,so as to provide alternatives for the effective prevention and control of foodborne pathogenic E.coli.The established screening method can also be used to screen the broad-spectrum phages of other foodborne pathogens,which is of great significance for promoting the development of phage therapy in the field of veterinary medicine.Specific research results are as follows:1.E.coli was isolated and broad-spectrum phages were screenedIn this study,71 sewage samples were collected from livestock and poultry farms in 24 cities of 9 provinces in China,146 E.coli strains were isolated and 4 strains of broadspectrum phages were screened.Broad-spectrum phage screening methods are as follows:Two hundred E.coli isolates were infected by mixed environmental phages,16 strains that could not be infected were eliminated,and the remaining 184 strains were randomly divided into 4 groups.The isolates were ranked according to the phage titer after engineering infection.The screening process of broad-spectrum phages in group 1 was taken as an example:the phages obtained by the above engineering bacteria infected 184 isolates were mixed in equal quantities and infected with the first strain,the obtained phages infected with the second strain,and the obtained phages infected with the third strain until all the E.coli in the group were infected.In this study,a total of four broad-spectrum phages were screened:EPEC 133 phage,STEC 65 phage,STEC 60 phage and EPEC 66 phage.During the screening process,they stopped screening after they were infected with 8,12,10 and 9 E.coli strains,respectively.To verify the broad-spectrum of the 4 phages,the 4 phages were infected with 100 randomly selected E.coli isolates.The results showed EPEC 133 phage,STEC 65 phage,STEC 60 phage,EPEC 66 phage and Cocktail Phage(a mixture of 4phages)can infect 14,17,9,21,31 strains of E.coli isolates,indicating that the screened phages have broad spectrum,and the method of screening broad spectrum phages has been successfully constructed.2.Analysis of broad-spectrum phage characteristicsIn order to evaluate the stability and genomic information of the screened phages,morphological,biological and genomic analyses were carried out.Morphological analysis:Transmission electron microscopy was used to observe 4 phages,and the results showed that all 4 phages had tail phages,among which EPEC 133 phage and STEC 65 phage were myotail phages,STEC 60 phage was long tail phage,and EPEC 66 phage was short tail phage.Biological analysis:In order to determine the stability of phages,4 strains of phages were treated at different temperatures and pH for 1 h.The results showed that all 4 strains of phages could stably survive under the conditions of 4-60℃ and pH 4-10,indicating that phages had the stability of clinical application.In order to quantitatively describe the proliferation efficiency of phages,one-step growth curve was measured,and the results showed that the bursts of EPEC 133 phage,STEC 65 phage,STEC 60 phage and EPEC 66 phage were about 81,79,98 and 84 phages/cells,respectively.The results showed that the screened phage had high proliferation ability and could be produced on a large scale.In order to determine the pyrolysis characteristics of 4 strains of phages,the pyrolysis curves of 4 strains of phages were measured.The results showed that the larger the MOI was,the shorter the time for phage to completely lyse the bacteria,and when the MOI was 5,all the four bacteria could be completely lysed within 1 hour.Genomic analysis:In order to evaluate whether the 4 phages carried virulence genes,drug resistance genes and lysogenicity genes,the genomes of the phages were extracted and sent to the company for second-generation sequencing.The results showed that the screened phages did not contain virulence genes,drug resistance genes and lysogenicity genes.The results showed that the selected phages did not cause horizontal transfer of appeal genes in clinical application.3.Application of broad-spectrum phagesTo further evaluate the bacteriostatic effect of screened phages in food,the cocktail of 4 phage strains was inoculated into 29 E.coli treated milk,chicken,pork,and lettuce samples at MOI=1,000 and MOI=10,000,respectively.The titer of bacteria was counted 3 h,6 h,and 12 h post-treat,respectively.The results showed that the experimental group(MOI=1,000 and MOI=10,000)had stronger bacteriostatic effect than the negative control group(PBS)in milk samples.There was no significant difference in bacteriostatic effect between the experimental groups,but the bacteria amount increased with the extension of time in 3-12 h.In chicken,pork,and lettuce samples,the phages screened in the experimental group also showed strong bacteriostatic effect compared with the control group.There was no significant difference in bacteriostatic effect between the experimental groups,and the bacteriostatic effect continued to 12 h.The four broad-spectrum bacteriophages of E.coli(EPEC 133 phage,STEC 65 phage,STEC 60 phage,EPEC 66 phage)screened by this institute can infect 31 of the 100 randomly selected E.coli isolates,and the broad-spectrum bacteriophage screening method is successfully constructed.The 4 broad-spectrum phages have a variety of morphologies and different types,do not contain virulence genes,pathogenic genes,lysogene genes,and the cocktail phages they compose can effectively sterilize in milk,chicken,pork,and lettuce foods. |
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