Preliminary Research Of Biological Characteristics Of Escherichia Coli Phage And Phage Therapy In Mice Infection With Escherichia Coli

Escherichia coli, gram-negative bacteria, commensally colonize the lower intestinal tractof animals and humans. In healthy individuals, E. coli is a member of normal flora in intestinaltract, however, these organisms may have adverse effect on the host when they get entrance intoother parts, such as blood, of the body. Common disease caused by E. coli infections includeurinary tract infections, respiratory tract infections, digest tract infection,wound infection,abdominal abscesses, cholangitis, bacteremia and meningitis. E. coli is the highest rate ofisolation at clinic, but also it is one of the main pathogenic bacteria at the community andhospital-acquried bacterial infections. In recent years, E. coli infections have increased year byyear. Since ESBL-producing E. coli poses formidable challenge in the management of criticallypatients with bacterial infecions.Application of antibiotics is usually thought to be an effective approach for bacterialinfection. A consequence of the unjustifiable and non-efficacious overuse of antibiotics is theselection and propagation of resistant bacterial pathogens. The emergence of mutidrug-resistantbacteria has become a global cirsis.The situation is getting worse because of the lack ofinvolvement of pharmaceutical companies in developing new antimicrobial agents and alsobecause of additional restrictions on the administration of antibiotics. This calls for a search fornovel therapeutic strategies. Bacteriophages, one of the most numerous life forms on earth, areviruses infecting bacteria; they are obligate intracellular parasites and lack their ownmetabolism. Bacteriophages are characterized by their specific ability to selectively infectbacterial cells belonging to a single strain or antigenically homologous strains within a bacterialgenus or species. They have been isolated from soil, water, and human and animal feces.Bacteriophages are divided into lytic or lysogenic depending of the kind of life cycle. The lytic phase leads to rapid lysis of bacteria, whereas in the lysogenic phase the phage's geneticmaterial is integrated into host cell's nucleic acids, forming a prophage or existing as a plasmid.The lysogenic phase of phages is linked with the risk of phage conversion, i.e. the bacteriumacquiring phage genes encoding antibiotic resistance, exotoxin, enzymes and factorsresponsible for changing its antigenic properties. Therefore, preparations containing exclusivelylytic phages are used in therapy.In this study, nine strains of phage specific for E. coli were isolated and characterized fromswage, and their biological properties were investigated. Therapeutic effect of bacterio- phagesagainst E. coli infection in mice was carried out.For further exploration of phages being as an agents against bacterial infection,the goal ofthis research was to select twenty strains of E. coli as host cells to isolate phages specific for E.coli and characterize them with respect to morphology, genome size and restrictionendonuclease digestion pattern, bacterial range, activity at various pH value,sensitivity totemperatures, growth curve, optimal multiplicity of infection and therapeutic effect of phages onE. coli infection. Nine phages (EcP25, EcP26, EcP65, EcP66, EcP75, EcP90, EcP152, EcP154,and EcP161) were isolated from swage. All the phages produced similar plaques that were clearand sharpness of border. EcP25 has a narrow host spectrum, only sensitive to E. coli 25 . ButEcP65 has a broad host spectrum, sensitive to many E. coli. The two of nine phages wereexamined by electron microscopy of negatively stained preparations. The appearance of thesephages by electron microscopy showed that they has a polyhechon-dimensional symmetricheads, and a long tails, with tail fibres. The genome sizes of EcP65 were estimated, based on thesizes of fragments obtained by electrophoresis of restriction enzyme digests of phage DNAusing EcoRI and HindIII. The sizes of genomes of these phages appeared to be 35kb. Themultiplicity infection of EcP65 was 0.01. After infecting host cells, growth curve of EcP65showed that the latent period is about 23 min, the rise period is about 38 min, the burst size isabout 93. The biolytic activity of EcP65 demonstrated that EcP65 lyesd host cells for arelatively long time. Phages specific for E. coli can be kept at 4oC for two months more or less.Resistance of phage EcP65 shows the survival of the phages over a range of pH 3-13. Theoptimal pH value for the lytic activity of EcP65 is about 8-10. EcP65 was inactivated by uviolize 12min. The mutation frequency of E. coli resistant to EcP65 is about 10-8~10-9determined by end- point- titer method.Toxcity experiment was conducted to examine the security of EcP65. It showed that EcP65was safe for mice. To determine the minimum lethal dose (MLD), serial dilution of E. coli wereinjected intraperitoneally (i.p.) into mice in 500-μl aliquots. This experiments established that3×108 CFU of strain E. coli is the MLD for ICR mice, with all mice that received the MLD dosedying within 48h. This MLD was used in all the phage therapy experiments described in thisstudy. In the dose-range experiment, seven groups of animals were challenged by i. p. injectionof the MLD of E. coli. Each of these groups was treated with a single injection of EcP65 at 0.01,0.1, 1, 10, 100, 1 000 and 10 000 MOI dose. The results demonstrated that approximately 50%of the animals are rescued at the moderate dose (MOI＝1) of phage i. p. injection,and as thephage increased (MOI＝103), the survival rates of animals reached 100%. In same time, theMLD of E. coli was injected i. p. to induce death. At various intervals thereafter, ranging from 0to 60 min (0, 10, 20, 30, 40, 50 and 60 min), the mice received a single i. p. injection of the highdose (MOI＝104) of EcP65. The results showed that if treatment is delayed at the time point 40min, a single injection of phage can rescue 100% of the mice. The heat-inactivated phageinjection has no effect of anti-infection.