Isolation And Identification Of STEC Bacteriophages And Its Therapeutic Effect On Infected Mice

Shiga toxin-producing Escherichia coli(STEC)is a type of pathogen that can cause a series of diseases in humans and animals,such as watery diarrhea,hemorrhagic colitis,and uremia syndrome.Shiga toxin is the main virulence factor of STEC.Till now,antibiotics are the main method for treating STEC infections in China.However,the excessive use of antibiotics over the years has caused more and more antibiotic resistance in bacteria.Finding effective treatments that can replace antibiotics has become an urgent problem.Bacteriophage is a virus that can lyse bacteria and is one of the most widely distributed populations in the viruses.The phage is highly host-specific,and its genetic material is enveloped by a protein shell.Compared to antibiotics,bacteriophages have many advantages:easy isolation,host-specific,and safety.Over the years,phages have been used as a powerful weapon against bacteria.Phage therapy is considered to have great potential in combating bacterial diseases.The aim of this study was to isolate and identify phages of STEC,study their biological characteristics,and investigate the therapeutic effects of these phages on STEC-infected mice.1.Isolation and identification of phage and study of its biological characteristicsIn this study,52 strains of E:coli phage were isolated from 200 feaces samples in a chicken farm,and all these phages were purified and stored.Using 97 E.coli strainsstored in the laboratory as host bacteria,the lysis rates of 10 phages(NJ-6,NJ-9,NJ-10,NJ-14,NJ-22,NJ-33,NJ-34,NJ-36,(NJ-37,NJ-38)were determined,the results showed that the lysis rates of of the 10 phages against the 97 E.coli strains were 20.78%,27.28%,55.84%,28.57%,19.48%,11.69%,11.69%,22.08%,22.08%,22.08%,respevtively.Further studies were performed on the biological characteristics of one of the phages,NJ-38,including morphology,optimal multiplicity of infection(MOI),thermal stability,pH stability,and one-step growth curve.The results showed that the optimal growth temperature of NJ-38 is 40℃ and the optimal multiplicity of infection(MOI)is 0.1;NJ-38 can tolerate in a pH range of 3-1l,with the optimal pH of 7.0.The one step growth curve showed that NJ-38 had a latent period of 10 min,a burst phase time of 40 min and a burst size of 26.2.Therapeutic effect of phage NJ-10,NJ-20,NJ-38 on infected STEC miceThe minimum lethal dose of STEC strain CVCC193 was determined in mice,the results showed that the minimum lethal dose of CVCC193 was 4 X 107CFU/mice.Subsequently,the therapeutic effects of three phages NJ-10,NJ-20,and NJ-38 were tested.80 mice were randomly divided into 8 groups.The first,second and thirdgroup of mice was intraperitoneally injected with 4 X 107 CFU/mice of CVCC193,followed by 1 X 107 PFU/mice phage NJ-10,NJ-20,and NJ-38 3h later respectively;the fourth group of mice was injected intraperitoneally with 4 X 107 CFU/mice of CVCC193,followed by SM buffer 3h later;the fifth,sixth and seventh group of mice was injected intraperitoneally with 1 X 107 PFU/mice of phage NJ-10,NJ-20,and NJ-38 respectively,followed by 4 X 107CFU/mice of CVCC193 24 h later;the eighth group of mice was injected with SM solution intraperitoneally,followed by 4×107CFU/mice of CVCC193 24 h later.The death of the mice was observed for 7 days.The results showed that the survival rate of mice injected with phage 3h after being challenged with CVCC193 was 40%-50%;the survival rate of mice injected with phage 24 h before being challenged with CVCC193 reached 80%-100%,and the group challenged with CVCC193 3h after or 24 h before being injected with SM buffer were all died.In summary,NJ-10,NJ-20,and NJ-38 have a certain therapeutic effect on mice infected with CVCC193.Compared with post-infection treatment,preventive treatment is better.Theclinical symptoms,pathological anatomy,bacterial load in different organs,and the expression level of inflammatory factors of the infected mice were observed in each period of the experiment.The results showed that the mice injected with phage 3h after or 24 h before being challenged CVCC193 were significantly decreased in the degree of organ lesions,bacteria load,and expression level of inflammatory factors.It was further confirmed that NJ-10,NJ-20,and NJ-38 had a potential therapeutic effect on mice infected with STEC.