An Escherichia coli phage, its phage composition and its application

Abstract

This invention discloses an *Escherichia coli* bacteriophage, specifically *Escherichia coli* bacteriophage RDP-EC-22219, with accession number CGMCC No. 45442. This invention also provides a bacteriophage composition comprising *Escherichia coli* bacteriophage RDP-EC-22219 as described above and other bacteriophages. Preferably, the bacteriophage composition comprises *Escherichia coli* bacteriophage RDP-EC-22219, *Escherichia coli* bacteriophage RDP-EC-22216, and *Escherichia coli* bacteriophage RDP-EC-22220; the accession number of *Escherichia coli* bacteriophage RDP-EC-22216 is CGMCC No. 45439; and the accession number of *Escherichia coli* bacteriophage RDP-EC-22220 is CGMCC No. 45443. This invention also provides the use of Escherichia coli bacteriophages and the phage composition thereof in the preparation of medicaments for the prevention and treatment of diseases caused by Escherichia coli. The phage composition provided by this invention has significantly improved lytic activity compared to a single Escherichia coli bacteriophage, and can be used for the prevention and treatment of diseases caused by Escherichia coli, while also having the advantages of safety and no side effects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an Escherichia coli bacteriophage, a bacteriophage composition thereof and application thereof. Background Art

[0002] Escherichia coli, also known as Escherichia coli, belongs to the Enterobacteriaceae family. Some of its bacteria may be spherical or filamentous. It can move with flagella, but its motility is weak. The antigens of Escherichia coli are mainly divided into bacterial antigens (O antigens), flagellar antigens (H antigens) and capsule antigens (K antigens). So far, 181 O antigens, 103 K antigens and 53 H antigens have been discovered. It is divided into two categories according to pathogenicity: non-pathogenic Escherichia coli, which is the normal flora of the intestine; pathogenic Escherichia coli, which can cause food poisoning. Pathogenic Escherichia coli can be divided into five types: enterotoxigenic Escherichia coli, hemorrhagic Escherichia coli, pathogenic Escherichia coli, intestinal adherent Escherichia coli and invasive Escherichia coli. Among them, diarrhea caused by enterotoxigenic Escherichia coli is the most common.

[0003] Avian colibacillosis refers to the general term for acute or chronic infectious diseases caused by pathogenic Escherichia coli, which often causes extraintestinal diseases in poultry and can cause disease throughout the year. It often causes extremely serious diarrhea and sepsis in young livestock (poultry). The incidence rate in chickens is about 11%-69%, and the mortality rate is about 3.8%-72.9%. With the development of intensive breeding, avian colibacillosis has become an important infectious disease threatening the poultry industry, posing a serious threat to the poultry industry and causing huge economic losses. At present, antibiotics are mostly used to prevent and treat avian colibacillosis, resulting in a significant increase in Escherichia coli resistance. The resistance of animal-derived Escherichia coli not only causes huge losses to the livestock industry, but also spreads to humans through the food chain and other means, thus posing a serious threat to human health and food safety.

[0004] At present, E. coli disease has caused serious economic losses to the poultry industry. It is the most commonly reported disease in poultry disease surveys and the main cause of carcass discard. Poultry of all ages can be infected with E. coli, but it often occurs in young poultry, especially in chicks, where the incidence and mortality rates are high. When the environmental hygiene and feeding management of the poultry flock are poor, the climate changes, or the chickens have infectious bursal disease and inclusion body hepatitis, it is easy to cause a large-scale outbreak. E. coli disease is often complicated by mycoplasma septicemia, infectious bronchitis, Newcastle disease, coccidiosis, blackhead disease and duck pasteurella disease. According to the symptoms and lesions, it is divided into E. coli septicemia, yolk peritonitis, salpingitis, arthritis, enteritis, panophthalmitis, E. coli granuloma, dead embryo, newborn yolk infection and omphalitis.

[0005] Bacteriophages are viruses that specifically infect bacteria and replicate inside bacterial cells. They are the most abundant and common organisms on Earth, playing an important role in microbiology, physiology, evolution, and therapy. In recent years, there have been many successful cases of using bacteriophages to treat chronic bacterial infections in humans and animals. Compared with antibiotics, bacteriophages have higher efficacy and safety. Compared with antibiotics, bacteriophages are highly specific. The advantage of this mechanism of action is that bacteriophages only lyse target strains and do not cause damage to symbiotic intestinal flora. At the same time, bacteriophages have the advantages of self-replication, small doses required for treatment, non-toxicity, short R&D cycle, and low production cost. Phage therapy as an alternative means of eliminating pathogens has a very broad and attractive prospect, which deserves in-depth exploration by researchers.

[0006] Therefore, the prior art needs further research. Summary of the invention

[0007] The present invention aims to address the deficiencies of the prior art and provide an Escherichia coli phage, a phage composition and an application thereof, in order to solve the problems of poultry in farms being infected with Escherichia coli, developing a rapid disease and being untreated in time.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] In the first aspect, the present invention provides an Escherichia coli phage, the phage is Escherichia coli phage RDP-EC-22219, and its deposit number is CGMCC No.45442. It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee on March 23, 2023, and the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the phage RDP-EC-22219 has a polyhedral three-dimensional symmetrical head, wrapped with nucleic acid, with a diameter of 100nm, and a tail about 129nm long, with a neck connecting the head and the tail. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage RDP-EC-22219 is classified as the Myotail Bacteriophage family of the Caudata order. The phage RDP-EC-22219 has a full genome size of 52963bp, containing 73 genes, with an average gene length of 668bp and a G+C% content of 46.5%. According to the prediction analysis, the whole genome size of bacteriophage RDP-EC-22219 is 52963bp, including 73 genes, with an average gene length of 668bp and a G+C% content of 46.5%. According to the prediction analysis, the genome of bacteriophage RDP-EC-22219 contains 73 open reading frames (ORFs) with an average length of 222bp, including 28 known functional proteins, which are divided into 3 parts according to protein function: 17 structural proteins, 10 functional proteins, 1 other protein, of which ORF10 is its lytic enzyme, and 45 hypothetical proteins.

[0010] In the second aspect, based on the same inventive concept, the present invention provides a phage composition, characterized in that it includes the Escherichia coli phage RDP-EC-22219 and other phages as described above. Preferably, the phage composition is Escherichia coli phage RDP-EC-22219, Escherichia coli phage RDP-EC-22216 and Escherichia coli phage RDP-EC-2222.

[0011] The Escherichia coli phage RDP-EC-22216 has a deposit number of CGMCC No. 45439, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on March 23, 2023, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the phage RDP-EC-22216 has a polyhedral three-dimensional symmetrical head, which wraps the nucleic acid with a diameter of 71nm, a tail of about 86nm, and a neck connecting the head and the tail. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage RDP-EC-22216 is classified as the Myocaudal Bacteriophage family of the order Caudata.

[0012] The Escherichia coli phage RDP-EC-22220 has a deposit number of CGMCC No. 45443, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on March 23, 2023, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the phage RDP-EC-22220 has a polyhedral three-dimensional symmetrical head, which wraps nucleic acid with a diameter of 71nm, a tail of about 71nm, and a neck connecting the head and the tail. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage RDP-EC-22220 is classified as the Myocaudal Bacteriophage family of the order Caudata.

[0013] The above-mentioned Escherichia coli phage RDP-EC-22219, Escherichia coli phage RDP-EC-22216 and Escherichia coli phage RDP-EC-22220 can all be used to prevent and treat poultry diseases caused by Escherichia coli infection, such as poultry sepsis, air sacitis, pericarditis, enteritis, peritonitis, etc.

[0014] Preferably, the Escherichia coli phage provided by the present invention is Escherichia coli phage RDP-EC-22219, whose lysis rate is 68%, which is higher than that of Escherichia coli phage RDP-EC-22216 and Escherichia coli phage RDP-EC-22220. Moreover, the titer of phage RDP-EC-22219 is the highest at pH=6, and its titer can be maintained at 10 at pH 5-9. 8 -1010 The higher the PFU / mL level, the better the acid and alkali resistance.

[0015] In the phage composition as described above, the volume ratio of the Escherichia coli phage RDP-EC-22219, the Escherichia coli phage RDP-EC-22216 and the Escherichia coli phage RDP-EC-22220 of the same titer is 1:1:1; preferably, the total content of the phage is not less than 10 8 PFU / mL. Preferably, the Escherichia coli phage RDP-EC-22219, the Escherichia coli phage RDP-EC-22216 and the Escherichia coli phage RDP-EC-22220 are fermented separately, filtered and mixed and compounded at a volume ratio of 1:1:1 with the same titer. Under this condition, the phage composition can lyse Escherichia coli with a lysis rate of 74%, which is higher than that of a single Escherichia coli phage. More preferably, the Escherichia coli phage RDP-EC-22219, the Escherichia coli phage RDP-EC-22216 and the Escherichia coli phage RDP-EC-22220 are placed in the same fermenter at a volume ratio of 1:1:1 with the same titer and fermented together. The phage composition produced by co-fermentation in the same fermentation tank can lyse Escherichia coli. The lysis rate of 300 strains of Escherichia coli isolated from breeding areas such as Weifang, Shandong, Binzhou, Shandong, Heze, Shandong, Handan, Hebei, Cangzhou, Hebei, Baoding, Hebei, Guangxi, Shenyang, Jiangsu, and Shaanxi was 87%, which is much higher than that of a single Escherichia coli phage, broadens its bactericidal range, increases its bactericidal activity, and its lysis rate is even 13% higher than that of the phage composition obtained by fermenting and filtering each of the above phages separately and then mixing and compounding.

[0016] Furthermore, the phage composition further comprises a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient comprises one or more of a dispersant, a stabilizer, a gelling agent, a filler and a solvent. Preferably, the excipient may be SM buffer, sodium alginate, chitosan, methylcellulose, sodium hydroxymethylcellulose, polyvinyl alcohol, glucose, sucrose, maltodextrin, etc.

[0017] In a third aspect, based on the same inventive concept, the present invention provides an application of the above-mentioned Escherichia coli phage or the above-mentioned phage composition in the preparation of a drug for preventing and treating diseases infected by E. coli. The application method is: adding the phage composition as a therapeutic drug to the feed of livestock and poultry suffering from diseases infected by E. coli, or orally administering the phage composition to livestock and poultry, or injecting the phage composition subcutaneously or intramuscularly, or applying the phage composition through the drinking water of livestock and poultry. Preferably, the phage composition is injected subcutaneously or intramuscularly to livestock and poultry.

[0018] The use of the above-mentioned phage composition in the preparation of a drug for preventing and treating diseases caused by E. coli can treat diseases in chicks caused by E. coli and reduce the morbidity and mortality of diseases caused by E. coli in chicks. It has been verified through experiments that in 30-day-old chicks infected with E. coli, 10 9 The phage combination of PFU / mL can reduce the mortality rate of diseased chicks from 80% to 30% and 10% within 3 days. In particular, the phage combination produced by co-fermentation in the same fermenter has the best therapeutic effect, with a survival rate of 90% and a mortality rate of 10%, which is much higher than the therapeutic effect of a single phage and also higher than the therapeutic effect of a phage combination produced by fermenting each phage separately and mixing them together.

[0019] In a fourth aspect, based on the same inventive concept, the present invention provides a phage pharmaceutical preparation, the active ingredient of which includes the Escherichia coli phage as described above or the phage composition as described above.

[0020] In a fifth aspect, based on the same inventive concept, the present invention provides a feed additive, wherein the active ingredient of the feed additive comprises the Escherichia coli phage as described above or the phage composition as described above; preferably, the content of the phage in the feed additive is at least 10 8 PFU / mL.

[0021] In a sixth aspect, based on the same inventive concept, the present invention provides a disinfectant, wherein the active ingredient of the disinfectant includes the Escherichia coli phage as described above or the phage composition as described above; preferably, the content of phage in the disinfectant is at least 10 8 PFU / mL. The application method of the disinfectant is: spraying, immersion, drinking water, etc., and the objects of disinfection include but are not limited to livestock and poultry pens, livestock and poultry secretions and excretions, feed, bedding, drinking water, equipment, vehicles entering and leaving, and other places, utensils and articles that may be contaminated, so as to effectively control the growth and activity of Escherichia coli and prevent Escherichia coli contamination in the environment.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention screened out an Escherichia coli phage RDP-EC-22219, which can be used to prevent and treat diseases caused by Escherichia coli infection.

[0024] 2. The present invention provides a phage composition, which is Escherichia coli phage RDP-EC-22219, Escherichia coli phage RDP-EC-22216 and Escherichia coli phage RDP-EC-22220. Compared with the lysis of a single Escherichia coli phage, the phage composition has a higher lysis rate and increased bactericidal activity. In particular, the phage composition obtained by co-fermentation in the same fermenter has the best therapeutic effect, and its survival rate reaches 90%, which is not only much higher than the therapeutic effect of a single phage, but also higher than the therapeutic effect of a phage composition obtained by fermenting each phage separately and mixing them together.

[0025] 3. The bacteriophages involved in the present invention are obtained from nature, have a wide range of sources, and are easy to separate, providing a source for drugs for preventing and treating diseases infected by Escherichia coli using a phage composition of Escherichia coli phage RDP-EC-22219, Escherichia coli phage RDP-EC-22216 and Escherichia coli phage RDP-EC-22220 as an active ingredient.

[0026] 4. The phage composition involved in the present invention has a wide range of applications. Experiments have shown that the phage composition provided by the present invention is safe, green, and has no toxic side effects. Therefore, it can be used in phage pharmaceutical preparations, feed additives, disinfectants, etc., for the prevention and treatment of poultry diseases caused by Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the colony image of Escherichia coli EC-22219;

[0028] Figure 2 The phage plaque images of Escherichia coli phage RDP-EC-22216, Escherichia coli phage RDP-EC-22219 and Escherichia coli phage RDP-EC-22220 (from left to right);

[0029] Figure 3 Electron microscope photos of Escherichia coli phage RDP-EC-22216, Escherichia coli phage RDP-EC-22219 and Escherichia coli phage RDP-EC-22220 (from left to right);

[0030] Figure 4 The best infection multiplicity graphs for three strains of Escherichia coli phages;

[0031] Figure 5 One-step growth curves of three Escherichia coli phages;

[0032] Figure 6 This is the growth temperature diagram of three strains of Escherichia coli phages;

[0033] Figure 7 It is the pH stability diagram of three strains of Escherichia coli phage;

[0034] Figure 8 This is a diagram of the genetic stability of three Escherichia coli phages. DETAILED DESCRIPTION

[0035] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art of the present invention.

[0037] Example 1 Phage Screening and Identification

[0038] 1. Isolation and identification of Escherichia coli:

[0039] (1) Take samples from a chicken farm in Guangxi, inoculate the samples on a chromogenic medium for E. coli, and incubate them in a 37°C constant temperature incubator for 18-24 hours. Observe the morphology and color of the colonies on the plate. Select colonies with smooth surfaces and blue pigments, such as Figure 1 shown.

[0040] (2) Streak inoculate on E. coli chromogenic medium, subculture and purify 3-5 times until uniform colonies are obtained, pick a single colony and streak inoculate on LB agar medium, pick the colony, and then identify it as E. coli through 16SrRNA gene sequencing technology and E. coli specific primer PCR; after identification, scrape the bacterial lawn and place it in 30% glycerol broth and store it at -80°C.

[0041] 2. Isolation and purification of Escherichia coli phage:

[0042] (1) Feces treatment: Feces were collected from three different farms. 5 g of chicken feces was weighed and added to 10 mL of SM buffer and soaked overnight. The extract was then centrifuged at 10,000 rpm for 5 min. The supernatant was filtered with a 0.22 μm filter and the filtrate was stored at 4 °C for later use.

[0043] (2) Phage enrichment: 0.1 mL of E. coli suspension and 1 mL of filtrate were added to 5 mL of LB broth and cultured overnight at 37°C on a shaking incubator. The culture was then centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter and the filtrate was used for later use.

[0044] (3) Phage isolation: The double plate method was used to isolate phages. 0.1 mL of the host Escherichia coli suspension was mixed with 0.6% LB soft agar and spread on an LB solid plate. After the soft agar solidified, 0.1 mL of the filtrate was spotted on the plate and allowed to stand until the mixed solution was absorbed. The plate was placed in a 37°C incubator for 6-8 h, and then transparent plaques were picked up with an inoculation loop and placed in 2 mL of SM buffer. The plate was stored at 4°C overnight to allow the phages to be completely released. Three strains of phages were obtained according to the above method and were named Escherichia coli phage RDP-EC-22216, Escherichia coli phage RDP-EC-22219 and Escherichia coli phage RDP-EC-22220, respectively. Among them, Escherichia coli phage RDP-EC-22216 came from a farm in Weifang, Shandong, Escherichia coli phage RDP-EC-22219 came from a farm in Qingdao, Shandong, and Escherichia coli phage RDP-EC-22220 came from a farm in Northeast China.

[0045] (4) Phage purification: The double plate method is used to separate phages. Take 0.1 mL of the filtrate from the mixed bacterial suspension and 0.2 mL of the host Escherichia coli suspension from 3 tubes, mix them evenly, add soft agar cooled to about 50°C and mix them evenly, then spread on two plates, place them in a 37°C incubator for incubation for 6-8 hours, pick out a single transparent spot with smooth edges and put it in 1 mL of SM buffer, and store it at 4°C overnight. Take 0.1 mL of the overnight stored extract and 0.1 mL of the bacterial suspension, mix them, add soft agar cooled to about 50°C and mix them evenly, spread on two plates, and incubate them in a 37°C incubator for 4-6 hours. Follow these steps to purify until the plaques are uniform in size and have smooth edges, and the purified phages are obtained. Figure 2 As shown, the phage RDP-EC-22216 has clear plaques without halos, regular edges, and a diameter of about 0.5 cm; the phage RDP-EC-22219 has clear plaques without halos, regular edges, and a diameter of about 0.2 cm; the phage RDP-EC-22220 has clear plaques without halos, regular edges, and a diameter of less than 0.1 cm.

[0046] (5) Preparation of phage: 0.2 mL of E. coli suspension and 1 mL of purified phage suspension were added to 50 mL of LB broth, incubated overnight at 37°C in a shaking incubator, and then centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter and the phage filtrate was used for later use.

[0047] (6) Phage preservation: Phage filtrates of three strains of Escherichia coli from different sources were added to 60% glycerol at a volume ratio of 1:1 and stored at -80°C.

[0048] 3. Electron microscopic observation of bacteriophage

[0049] Take 20 μL of liquid containing crude phage particles and drop it on the copper mesh. Let it precipitate naturally for 15 minutes, and use filter paper to absorb excess liquid from the side. Add a drop of 2% (W / V) phosphotungstic acid (PTA) on the copper mesh to stain the phage for 10 minutes, then use filter paper to absorb the staining liquid from the side. After the sample is dry, use an electron microscope to observe the phage morphology, such as Figure 3 shown.

[0050] The bacteriophage RDP-EC-22216 has a polyhedral, three-dimensionally symmetrical head that encapsulates nucleic acid, with a diameter of 71nm, a tail about 86nm long, and a neck connecting the head and the tail. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the bacteriophage RDP-EC-22216 is classified as the family Myocaudaphaga of the order Caudata.

[0051] The bacteriophage RDP-EC-22219 has a polyhedral, three-dimensionally symmetrical head that encapsulates nucleic acid, with a diameter of 100 nm, a tail about 129 nm long, and a neck connecting the head and tail. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the bacteriophage RDP-EC-22219 is classified as the family Myocaudaphaga of the order Caudata.

[0052] The bacteriophage RDP-EC-22220 has a polyhedral, three-dimensionally symmetrical head that encapsulates nucleic acid with a diameter of 71nm, a tail about 71nm long, and a neck connecting the head and the tail. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the bacteriophage RDP-EC-22220 is classified as the family Myocaudaphaga of the order Caudata.

[0053] 4. Phage whole genome sequencing and analysis:

[0054] Using Illumina TruSeq TM Nano DNA Sample Prep Kit method to construct the library; the specific steps are as follows:

[0055] ① The library was constructed with 1 μg of phage genomic DNA as the starting amount;

[0056] ②Covaris M220 ultrasonically breaks DNA into 300-500bp;

[0057] ③ Fill in the 3' end and add A, connect the index adapter (TruSeqTM Nano DNA Sample Prep Kit);

[0058] ④Library enrichment, PCR amplification for 8 cycles;

[0059] ⑤2% agarose gel to recover the target band (Certified Low Range Ultra Agarose);

[0060] ⑥TBS380 (Picogreen) quantification, mixed according to the data ratio and loaded on the machine;

[0061] ⑦ Bridge PCR amplification was performed on the cBot solid phase carrier to generate clusters;

[0062] ⑧Illumina Hiseq sequencing platform, performing 2×150bp sequencing.

[0063] Phage genome analysis:

[0064] The whole genome of bacteriophage RDP-EC-22219 is 52963bp, containing 73 genes, with an average gene length of 668bp and a G+C% content of 46.5%. According to the prediction analysis, the genome of bacteriophage RDP-EC-22219 contains 73 open reading frames (ORFs) with an average length of 222bp, containing 28 known functional proteins, which are divided into 3 parts according to protein function: 17 structural proteins, 10 functional proteins, 1 other protein, of which ORF10 is its lytic enzyme, and 45 hypothetical proteins.

[0065] The whole genome of bacteriophage RDP-EC-22216 is 52818bp, containing 72 genes, with an average gene length of 673bp and a G+C% content of 46.6%. According to the prediction analysis, the genome of bacteriophage RDP-EC-22216 contains 72 open reading frames (ORFs) with an average length of 224bp, containing 28 known functional proteins, which are divided into two parts according to protein function: 16 structural proteins, 12 functional proteins, of which ORF7 is its lytic enzyme, and 44 hypothetical proteins.

[0066] The whole genome of bacteriophage RDP-EC-22220 is 52963bp, containing 73 genes, with an average gene length of 666bp and a G+C% content of 46.5%. According to the prediction analysis, the genome of bacteriophage RDP-EC-22220 contains 73 open reading frames (ORFs), with an average length of 221bp, and contains 28 known functional proteins: 17 structural proteins, 10 functional proteins, 1 other protein, of which ORF5 is its lytic enzyme, and 45 hypothetical proteins.

[0067] Example 2 Determination of phage titer

[0068] 1. Experimental methods:

[0069] Take 100 μL of host bacterial solution cultured to the exponential phase, mix it in a 5mL centrifuge tube, shake it up and down, place it on LB semi-solid agar, and completely cover it on a 20mL LB solid agar plate (9cm diameter). After the semi-solid is dried, 100 μL of the purified phage concentrate is taken and placed on the plate. Three plates of one phage are spread, cultured at 37°C overnight, and the plaques are picked up in 20mL SM buffer and placed at 4°C. The next day, the supernatant is taken, filtered through a 0.22μm filter membrane, and 100 μL of the filtrate is diluted 10 times with sterile water. Then, 100 μL of the bacterial solution and the phage solution are mixed on double plates, and the plates are cultured in a 37°C incubator overnight. The above method was used to determine the phage titer of phage RDP-EC-22216, phage RDP-EC-22219, and phage RDP-EC-22220. Select countable plates, count phage plaques, and calculate the phage titer (PFU / mL) as the number of plaques × dilution factor / sample volume (mL).

[0070] 2. Experimental results and analysis:

[0071] The titers of bacteriophage RDP-EC-22216, RDP-EC-22219, and RDP-EC-22220 can all reach 10 10 PFU / mL.

[0072] Example 3 Determination of the optimal multiplicity of infection (MOI) of bacteriophage

[0073] 1. Experimental methods:

[0074] MOI refers to the ratio of the number of phages to the number of host bacteria at the initial infection. The concentration of the host bacteria cultured to the exponential phase was adjusted to 10 8 CFU / mL. Set the MOI to 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, respectively. Add the titered phage solution to the bacterial solution in proportion, 500 μL of bacterial solution and phage solution respectively, mix well, and culture at 37°C, 200 r / min for 5 hours. Centrifuge the mixed culture at 10000 r / min for 10 minutes, and measure the phage titer. The infection multiplicity with the highest titer is the optimal infection multiplicity. The above method was used to determine the optimal infection multiplicity of bacteriophage RDP-EC-22216, bacteriophage RDP-EC-22219, and bacteriophage RDP-EC-22220.

[0075] 2. Experimental results and analysis:

[0076] The results are as follows Figure 4 The results show that the phage titer of RDP-EC-22216 is the highest when the infection multiplicity is 0.1. Therefore, the optimal infection multiplicity of phage RDP-EC-22216 is 0.1, corresponding to the highest titer of 10 10 PFU / mL; Therefore, the optimal infection multiplicity of bacteriophage RDP-EC-22219 is 0.01, and the corresponding highest titer is 10 10 PFU / mL; The phage titer of RDP-EC-22220 is the highest when the infection multiplicity is 0.1, so the optimal infection multiplicity of phage RDP-EC-22220 is 0.1, corresponding to the highest titer of 10 10 PFU / mL.

[0077] Example 4 One-step growth curve of bacteriophage

[0078] 1. Experimental methods:

[0079] Mix 500 μL of phage liquid and host bacterial liquid at the ratio required for the optimal infection multiplicity, and incubate at 37°C for 15 minutes. Centrifuge at 10000 r / min for 1 minute, and wash the precipitate with LB liquid 3 times. Add 10 mL of LB liquid medium preheated at 37°C, mix well, and quickly place in a 37°C shaker for shaking culture. Take samples every 10 minutes, measure the titer of the phage, make 3 parallels, and take the average value of the results. Use the infection time as the horizontal axis and the logarithm of the phage titer in the infection system as the vertical axis to draw a one-step growth curve, and obtain the incubation period and outbreak period of phage RDP-EC-22216, phage RDP-EC-22219, and phage RDP-EC-22220, respectively, and calculate the outbreak volume.

[0080] The burst size = the total number of phages at the end of the burst / the total number of bacteria at the beginning of the burst.

[0081] 2. Experimental results and analysis:

[0082] The results are as follows Figure 5 As shown, the incubation periods of phage RDP-EC-22216, phage RDP-EC-22219 and phage RDP-EC-22220 were 60 min, 30 min and 50 min, respectively, and the outbreak periods were 90 min, 110 min and 90 min, respectively. The outbreak amounts of the three phages were 10 4 , 10 5 , 10 4 PFU / mL.

[0083] Example 5 Determination of the optimal growth temperature of bacteriophage

[0084] 1. Experimental methods:

[0085] The phage stock solution was diluted tenfold to a suitable concentration, 100 μL was taken and mixed with an equal amount of bacterial solution, poured into double-layer plates, and cultured at 20°C, 30°C, 37°C, 40°C and 50°C for 4 hours to detect the phage titer. The growth temperature curves of phage RDP-EC-22216, phage RDP-EC-22219 and phage RDP-EC-22220 were drawn with temperature as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0086] 2. Experimental results and analysis:

[0087] The results are as follows Figure 6 As shown, the suitable growth temperature for the three phage strains is between 30 and 40°C, and the optimal growth temperature is 37°C.

[0088] Example 6 Determination of pH stability of bacteriophage

[0089] 1. Experimental methods;

[0090] The pH value of LB liquid medium was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and 100 μL of each was mixed with an equal volume of phage stock solution, placed at 37°C for 2 hours, and the titer was determined by double-layer plate method, and 3 replicates were set for each pH. With pH as the abscissa and the logarithm of phage titer as the ordinate, the pH stability curves of phage RDP-EC-22216, phage RDP-EC-22219, and phage RDP-EC-22220 were plotted.

[0091] 2. Experimental results and analysis:

[0092] The results are as follows Figure 7 As shown in Figure 2, RDP-EC-22216 and RDP-EC-22220 have the highest titers at pH = 7; RDP-EC-22219 has the highest titer at pH = 6, and its titer can be maintained at 10 at pH 5-9. 8 -10 10 PFU / mL higher level.

[0093] Example 7 Determination of Genetic Stability of Bacteriophage

[0094] 1. Experimental methods:

[0095] The phage was subcultured 30 times, and equal amounts of phage and host bacteria were added to the culture medium for each generation. The culture time for each generation was 6 hours, and then double plates were spread. After the phage was subcultured for 29 generations, its titer was determined. The stability genetic curves of phage RDP-EC-22216, phage RDP-EC-22219 and phage RDP-EC-22220 were drawn respectively.

[0096] 2. Experimental results and analysis:

[0097] The results are as follows Figure 8 As shown, the titers of the three phage strains were generally stable during the culture process, indicating that the three phage strains had good biological genetic stability and were excellent choices for the production process.

[0098] Example 8 Lysis rate experiment of Escherichia coli phage RDP-EC-22219

[0099] 1. Experimental methods:

[0100] The double-layer plate method was used to determine the lysis effect of Escherichia coli phage RDP-EC-22219 on 300 strains of Escherichia coli from Weifang, Shandong, Binzhou, Shandong, Heze, Shandong, Handan, Hebei, Cangzhou, Hebei, Baoding, Guangxi, Shenyang, Jiangsu, Shaanxi and other breeding areas stored in the strain library. Prepare different Escherichia coli bacterial suspensions, take 100 μL of the test bacterial suspension cultured for 6 hours, add it to 0.6% LB semi-solid culture medium, mix well, pour it into a plate, and let it stand to solidify. Take 20 μL of phage suspension and drop it on the soft agar plate, culture it at 37°C for about 6-8 hours, and observe whether plaques appear.

[0101] Lysis rate = number of strains that can be lysed by phage / total number of strains × 100%

[0102] 2. Experimental results and analysis:

[0103] According to the lysis results, the Escherichia coli phage RDP-EC-22219 lysed 204 strains out of 300 strains of Escherichia coli, with a lysis rate of 68%.

[0104] Example 9 Lysis rate experiment of Escherichia coli phage RDP-EC-22216

[0105] 1. Experimental methods:

[0106] The double-layer plate method was used to determine the ability of Escherichia coli phage RDP-EC-22216 to lyse Escherichia coli stored in the strain library and the lysis effect on 300 strains of Escherichia coli. Prepare different Escherichia coli bacterial suspensions, take 100 μL of the test bacterial suspension cultured for 6 hours, add it to 0.6% LB semi-solid culture medium, mix well, pour it into a plate, and let it stand to solidify. Take 20 μL of phage suspension and drop it on the soft agar plate, culture it at 37℃ for about 6-8 hours, and observe whether plaques appear.

[0107] 2. Experimental results and analysis:

[0108] According to the lysis results, the Escherichia coli phage RDP-EC-22216 lysed 300 strains of Escherichia coli, and 171 of them were lysed, with a lysis rate of 57%.

[0109] Example 10 Lysis rate experiment of Escherichia coli phage RDP-EC-22220

[0110] 1. Experimental methods:

[0111] The double-layer plate method was used to determine the lysis effect of Escherichia coli phage RDP-EC-22220 on 300 strains of E. coli. Prepare different E. coli bacterial suspensions, take 100 μL of the test bacterial suspension cultured for 6 hours, add it to 0.6% LB semi-solid medium, mix well, pour it into a plate, and let it stand to solidify. Take 20 μL of phage suspension and drop it on the soft agar plate, culture it at 37℃ for about 6-8 hours, and observe whether plaques appear.

[0112] 2. Experimental results and analysis:

[0113] According to the lysis results, the Escherichia coli phage RDP-EC-22220 lysed 300 strains of Escherichia coli, and 186 strains were lysed, with a lysis rate of 62%.

[0114] Example 11 Lysis rate experiment of three Escherichia coli phage complexes

[0115] 1. Experimental methods:

[0116] Three strains of Escherichia coli phage were fermented separately, filtered and mixed in a volume ratio of 1:1:1 with the same titer (concentration). The double-layer plate method was used to measure the lysis effect of the three mixed phages on 300 strains of Escherichia coli stored in the strain library. Prepare different Escherichia coli bacterial liquids, take 100μL of the test bacterial liquid cultured for 6 hours, add it to 0.6% LB semi-solid culture medium, mix well, pour it into a plate, and let it stand to solidify. Take 20μL of phage suspension and drop it on the soft agar plate, culture it at 37℃ for about 6-8 hours, and observe whether plaques appear.

[0117] 2. Experimental results and analysis:

[0118] According to the lysis results, the phage mixed with 3 strains of E. coli lysed 300 strains of E. coli and 222 strains of E. coli, with a lysis rate of 74%, which is higher than the lysis rate of a single phage. This shows that the phage combination has a strong lysis ability and can make up for the limitations of the host spectrum when a single phage is used.

[0119] Example 12 Lysis rate experiment of three strains of Escherichia coli phage co-fermentation products

[0120] 1. Experimental methods:

[0121] Three strains of Escherichia coli phages were co-fermented in the same fermenter at a volume ratio of 1:1:1 with the same titer (concentration) to prepare a phage composition. After filtration, the double-layer plate method was used to determine the lysis effect of the phage composition on 300 strains of Escherichia coli stored in the strain library. Prepare different Escherichia coli bacterial liquids, take 100μL of the test bacterial liquid cultured for 6 hours, add it to 0.6% LB semi-solid culture medium, mix it, pour it into a plate, and let it stand to solidify. Take 20μL of phage suspension and drop it on the soft agar plate, culture it at 37℃ for about 6-8 hours, and observe whether plaques appear. Among them, the fermentation tank parameters are: seed tank 200-300rpm, 37℃, ventilation 0.6-1vvm, culture 6-8h. Fermentation tank: 120-150pm, 37℃, ventilation 0.6-1vvm, culture 8-10h. Culture medium ingredients: yeast extract powder 0.5%, peptone 1%, beef extract 0.1%, glycerol 0.5%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, calcium chloride 20ppm, magnesium chloride 20ppm.

[0122] 2. Experimental results and analysis:

[0123] According to the lysis results, the phage combination lysed 300 strains of E. coli and 261 strains of E. coli, with a lysis rate of 87%, which is 19%-30% higher than the lysis rate of a single phage and 13% higher than the lysis rate of a mixed phage. It is much higher than the lysis rate of a single phage, indicating that the phage combination obtained by co-fermentation has stronger lysis and broad spectrum.

[0124] In summary, the phage composition formed by co-fermentation of the three phages in the same fermenter and filtration has a better lysis effect on Escherichia coli than the phage composition formed by fermenting the three phages separately and then filtering them, or by fermenting and filtering the three phages separately and then mixing and compounding them.

[0125] Table 1 Lysis effect of bacteriophage and its combination

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] “√”: cleavable, “×”: not cleaved

[0135] Example 13 Phage treatment experiment on broiler chickens

[0136] 1. Experimental methods:

[0137] 80 healthy 30-day-old white-feathered broilers with similar body weight were selected and divided into negative control group, positive control group, challenge group, treatment group 1, treatment group 2, treatment group 3, treatment group 4, and treatment group 5, with 10 chickens in each group. The negative control group received a subcutaneous intramuscular injection of 0.1 mL of sterile saline; the positive control group received a subcutaneous intramuscular injection of 0.1 mL of the co-fermented phage composition obtained in Example 12; the challenge group received a subcutaneous intramuscular injection of 0.1 mL of 10 9 CFU / mL Escherichia coli liquid; treatment group 1, subcutaneous injection of 0.1mL10 9 CFU / mL Escherichia coli solution, 2 hours after treatment, 0.1mL 10 9 PFU / mL Escherichia coli phage RDP-EC-22219 was used for treatment; treatment group 2, 0.1mL 10 9 CFU / mL Escherichia coli solution, 2 hours after treatment, 0.1mL 10 9 PFU / mL Escherichia coli phage RDP-EC-22216 was used for treatment; treatment group 3, 0.1mL 10 9 CFU / mL Escherichia coli solution, 2 hours after treatment, 0.1mL 10 9 PFU / mL Escherichia coli phage RDP-EC-22220 was used for treatment; treatment group 4, subcutaneous injection of 0.1mL 10 9CFU / mL Escherichia coli solution, 2 hours after treatment, 0.1mL 10 9 PFU / mL Example 11 Equal amounts of compounded phage compositions (phage RDP-EC-22216: phage RDP-EC-22219; phage RDP-EC-22220 were mixed at a volume ratio of 1:1:1 with the same potency concentration) were used for treatment; treatment group 5, subcutaneous injection of 0.1 mL 10 9 CFU / mL Escherichia coli solution, 2 hours after treatment, 0.1mL 10 9 PFU / mL The co-fermented phage composition obtained in Example 12 was used for treatment. The broiler chickens were observed for 3 consecutive days and the mortality of the broiler chickens was recorded. The broiler chicken treatment groups and treatment results are shown in Table 2.

[0138] Table 2 Broiler treatment groups and treatment results

[0139]

[0140] 2. Experimental results and analysis:

[0141] The results showed that there was no death in the negative control group and the positive control group, indicating that the chicks were in good health, and the phage was non-toxic and had no side effects; the mortality rate of the attack group was 80%, the mortality rate of treatment group 1 was 40%, the mortality rate of treatment group 2 was 60%, the mortality rate of treatment group 3 was 50%, the mortality rate of treatment group 4 was 30%, and the mortality rate of treatment group 5 was 10%, which showed that treatment group 1, treatment group 2, treatment group 3, treatment group 4 and treatment group 5 had therapeutic effects compared with the attack group, among which the co-fermented phage composition in treatment group 5 had the best therapeutic effect, which was higher than the single phage group and the equal compound group. It can be concluded that the Escherichia coli phage RDP-EC-22216, phage RDP-EC-22219, phage RDP-EC-22220 and the phage composition of the three of the present invention can be used to treat avian Escherichia coli disease, among which the therapeutic effect of the phage composition co-fermented in the same fermentation tank is the best, and the phage composition of equal compounding after fermentation alone is second.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0143] The complete genome sequence of bacteriophage RDP-EC-22219 is assembled by sequentially splicing SEQ ID NO: 1 to SEQ ID NO: 6. The complete genome sequence of bacteriophage RDP-EC-22219 is as follows:

[0144]

[0145] The complete genome sequence of bacteriophage RDP-EC-22216 is assembled by sequentially splicing SEQ ID NO: 7 to SEQ ID NO: 12. The complete genome sequence of bacteriophage RDP-EC-22216 is as follows:

[0146]

[0147] The whole genome of bacteriophage RDP-EC-22220 is assembled by sequentially splicing SEQ ID NO: 13-SEQ ID NO: 18. The whole genome sequence of bacteriophage RDP-EC-22220 is as follows:

[0148]

Claims

1. A bacteriophage composition, characterized in that: The phage composition comprises Escherichia coli phage RDP-EC-22219, Escherichia coli phage RDP-EC-22216 and Escherichia coli phage RDP-EC-22220; the phage is Escherichia coli phage RDP-EC-22219, and its deposit number is CGMCC No.45442; the deposit number of Escherichia coli phage RDP-EC-22216 is CGMCC No.45439; the deposit number of Escherichia coli phage RDP-EC-22220 is CGMCC No.45443; the phage composition is prepared by placing the Escherichia coli phage RDP-EC-22219, the Escherichia coli phage RDP-EC-22216 and the Escherichia coli phage RDP-EC-22220 with the same potency in a volume ratio of 1:1:1 in the same fermenter for co-fermentation.

2. Use of the bacteriophage composition according to claim 1 in the preparation of a drug for treating diseases infected by Escherichia coli.

3. A bacteriophage pharmaceutical preparation, characterized in that: The active ingredient of the phage pharmaceutical preparation comprises the phage composition according to claim 1.

4. A feed additive, characterized in that: The active ingredient of the feed additive comprises the bacteriophage composition as claimed in claim 1; the content of bacteriophage in the feed additive is at least 10 8 PFU / mL.

5. A disinfectant, wherein the active ingredient of the disinfectant comprises the bacteriophage composition according to claim 1; the content of bacteriophage in the disinfectant is at least 10 8 PFU / mL.

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