Escherichia coli bacteriophage PD2061, bacteriophage composition and application thereof

Abstract

The invention belongs to the technical field of bacteriophages, and discloses an Escherichia coli bacteriophage PD2061, a bacteriophage composition and application thereof, the preservation number of the Escherichia coli bacteriophage PD2061 is CGMCC NO.46163, the Escherichia coli bacteriophage PD2061 is preserved in China General Microbiological Culture Collection Center on August 16, 2024, and the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The Escherichia coli phage PD2061 not only has a high splitting rate on Escherichia coli, but also has certain splitting performance on salmonella, and has greater advantages in prevention and treatment of diseases caused by mixed infection of Escherichia coli and salmonella. The bacteriophage can be used for preparing a medicinal preparation for treating or preventing animal-derived colibacillosis and / or salmonellosis, the survival rate of poultry suffering from colibacillosis / salmonellosis or mixed infection diseases is increased, the feed utilization rate is increased, and the death and culling rate of the poultry in breeding is reduced.

Description

Technical Field

[0001] The present invention relates to the field of bacteriophage technology, in particular to an Escherichia coli phage PD2061, a bacteriophage composition and applications thereof. Background Art

[0002] Escherichia coli belongs to the Enterobacteriaceae family and is widely distributed in animal intestines, airborne particles, clothing, farm environments, rivers, and lakes. In recent years, the gradual expansion of chicken farming and the increase in stocking density, coupled with poor management, poor environmental sanitation, and a high number of stress-inducing factors in some chicken farms, have significantly increased the incidence of E. coli in chickens.

[0003] E. coli can be transmitted both vertically and horizontally, primarily causing omphalitis, incomplete yolk absorption, pericarditis, air sac inflammation, perihepatitis, diarrhea, and sepsis in poultry. Some cases can also cause arthritis, wet eyes, and subcutaneous abscesses. It can also cause animals to eat less, fail to digest feed, and experience reduced feed conversion rates and decreased production performance.

[0004] Antibiotics are commonly used to treat Escherichia coli in livestock and poultry production. However, the unscientific use of antibiotics brings about a series of problems, including the development of multidrug-resistant strains, the disruption of intestinal flora balance, the reduction of animal immune function, animal poisoning caused by excessive antibiotic dosage, environmental pollution, and antibiotic residues in animal products. Therefore, there is an urgent need to find efficient, safe, and residue-free bactericidal preparations to replace antibiotics.

[0005] Bacteriophage is a highly specific virus that infects bacteria, fungi and other microorganisms. As a natural antibacterial agent, bacteriophage has advantages that antibiotics cannot match. In addition, bacteriophage has a long history of use in clinical treatment and has achieved good results. It is an antibiotic alternative with broad prospects.

[0006] However, the lysis spectrum of existing Escherichia coli phages is limited due to their host lysis specificity, and multiple phage strains usually need to be used in combination to expand the lysis spectrum. Therefore, the existing Escherichia coli phage resources need to be further developed. Summary of the Invention

[0007] To address the above-mentioned problems, the present invention provides an Escherichia coli phage PD2061, a phage composition, and applications thereof. The Escherichia coli phage PD2061 has a broad lysis spectrum and certain lysis performance against both Escherichia coli and Salmonella. It can be used to prepare pharmaceutical preparations for treating or preventing Escherichia coli and / or Salmonellosis in chickens, thereby improving the survival rate of chickens suffering from Escherichia coli / Salmonellosis or mixed infections. The phage can also be combined with antibiotics as a combined preparation for the prevention and treatment of the above-mentioned diseases.

[0008] To solve the above problems, this application provides the following technical solutions: In the first aspect, the present application provides an Escherichia coli ( Escherichia coli bacteriophage ) phage PD2061, whose deposit number is CGMCC NO.46163. The phage was deposited in the General Microbiology Center of China Culture Collection Administration on August 16, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.46163.

[0009] Electron microscopy showed that the phage PD2061 has a polyhedral head structure and a contractile tail. The head is 60~65nm wide and 78~83nm long, and the tail is about 90~93nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage PD2061 of the present application conforms to the characteristics of the Myotailed Phage family and belongs to the Myotailed Phage.

[0010] The titer of the E. coli phage PD2061 is high, reaching 3×10 11 pfu / ml.

[0011] This bacteriophage has good high temperature resistance and can be used in high temperature and high humidity environments such as sheds.

[0012] The E. coli phage PD2061 exhibits excellent acid resistance, exhibiting minimal potency loss when exposed to gastric acid, allowing it to fully exert its effects after entering the intestines. Experiments have shown that the phage is stable at pH 4.0 to 10.0, and its potency remains detectable even after 3 hours of exposure to pH 1.0 and 13.0.

[0013] The E. coli phage PD2061 has a broad spectrum of lytic activity against E. coli, with a lysis rate of 90.00%. Furthermore, the phage also has some lytic activity against Salmonella, with a lysis rate of 20%. Therefore, E. coli phage PD2061 offers significant advantages in preventing and treating mixed infections of E. coli and Salmonella.

[0014] In this application, bacteriophage PD2061 includes mutants that undergo point, deletion, or addition mutations with greater than 98% or 99% homology and maintain substantially the same bactericidal activity. Because bacteriophages are highly susceptible to mutation during replication, such mutants are also within the scope of protection claimed in this application. The genomic sequence of bacteriophage PD2061 can be sequenced using known methods based on the biological material deposited according to the present invention. Screening for mutants with extremely similar properties based on the bacteriophage provided by the present invention does not require inventive effort for those skilled in the art.

[0015] In a second aspect, the present application also provides a phage composition, which includes the aforementioned Escherichia coli phage PD2061.

[0016] In practical applications, to further broaden the phage's lysis spectrum, fully leverage the differences in lysis spectra between different phages, and complement each other's strengths, the E. coli phage PD2061 can be used in combination with other phages, such as one or more of the E. coli phages PD328 (disclosed in Patent Publication No. CN115717126A), PD06, and PD114 (both phages disclosed in Patent Publication No. CN111349618B). This can be used to expand the bactericidal spectrum against E. coli and eliminate as many E. coli pathogens as possible in the environment, thereby preventing and treating E. coli disease. Furthermore, the E. coli phage PD2061 can also be combined with other different types of phages (to inhibit different pathogens that cause the same disease, such as existing Salmonella phages) for the prevention and treatment of salmonellosis, or for the prevention and treatment of combined E. coli and Salmonella infections.

[0017] In a third aspect, the present application further provides the use of the E. coli phage PD2061 or the aforementioned phage composition in the preparation of a medicament for preventing and treating diseases caused by E. coli and / or Salmonella infection. Such prevention and treatment includes both prophylaxis and treatment. The term "prevention" herein refers to all actions that inhibit or delay the disease by administering the composition. The term "treatment" herein refers to all actions that ameliorate or improve the disease by administering the composition.

[0018] Optionally, the diseases caused by the Escherichia coli infection include: acute sepsis, omphalitis, air sacculitis, yolk peritonitis, salpingitis, etc. caused by chicken-derived Escherichia coli.

[0019] Optionally, the diseases caused by the Salmonella infection include: pullorum, fowl typhoid, fowl paratyphoid, etc. caused by chicken-derived Escherichia coli.

[0020] The Escherichia coli phage PD2061 or the aforementioned phage composition is effective in preventing and treating diseases caused by chicken-derived Escherichia coli and / or Salmonella infection.

[0021] In a fourth aspect, the present application further provides a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned Escherichia coli phage PD2061 or the aforementioned phage composition.

[0022] Preferably, the phage pharmaceutical preparation also includes other antibacterial or bactericidal active ingredients; the pharmaceutical preparation is in the form of an oral dosage form, a topical dosage form, or a parenteral dosage form. The phage pharmaceutical preparation can be used by adding the phage or its combination as a therapeutic drug to drinking water or feed, or by gavage, subcutaneous injection, or intramuscular injection into chickens. These methods can prevent and treat E. coli disease and improve survival rates.

[0023] Optionally, the phage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant irritation to an organism and does not eliminate the biological activity and characteristics of the administered active ingredient. In order to prepare the pharmaceutical composition as a liquid preparation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, ethanol, various culture media, etc. They can be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers, and antibacterial agents, can be added as needed. When combined with a diluent, dispersant, surfactant, adhesive, and / or lubricant, the composition of the present invention can also be prepared into injection and oral dosage forms (e.g., aqueous solutions, suspensions, and emulsions, pills, capsules, granules), and other intermediate dosage forms, such as lyophilized agents.

[0024] Optionally, the phage pharmaceutical preparation is a powder, which further comprises skim milk powder and lactose as protective agents.

[0025] Optionally, the phage powder comprises the following ingredients in parts by weight: skimmed milk powder, lactose and phage suspension.

[0026] The preparation method of the bacteriophage powder comprises the following steps: mixing corresponding amounts of skimmed milk powder, lactose and bacteriophage suspension, and spray drying the mixture to obtain the bacteriophage powder.

[0027] Preferably, the spray drying conditions are: inlet temperature of 120° C., air flow velocity of 120 L / min, spray rate of 100%, nozzle cover size of 4.0 μm, and spray drying for about 1 hour.

[0028] In a fifth aspect, the present application further provides a feed additive comprising the aforementioned Escherichia coli phage PD2061 or the aforementioned phage composition. Preferably, the concentration of the phage is 10 10 PFU / mL or above.

[0029] By adding the above-mentioned drinking water additive or feed additive into water or mixing it with feed and feeding it to the chickens, the drinking water and feed in the chicken farm can be disinfected and sterilized. The acid resistance of the bacteriophage can survive in the stomach acid environment, effectively preventing and treating Escherichia coli disease.

[0030] The present application also provides a poultry drinking water additive, which includes the aforementioned Escherichia coli phage PD2061 or the aforementioned phage composition. Preferably, the concentration of the phage is 10 10 PFU / mL or above.

[0031] The drinking water additive and feed additive are in the form of liquid dosage form, powder dosage form or solid dosage form, but are not limited to the above three dosage forms.

[0032] In a sixth aspect, the present application also provides an environmental disinfectant, the active ingredient of which includes the aforementioned Escherichia coli phage PD2061 or the aforementioned phage composition.

[0033] Preferably, the concentration of phage is 10 8 Preferably, the environmental disinfectant further comprises other active ingredients for inhibiting or eliminating bacteria in the environment.

[0034] The present application also provides the use of the above-mentioned environmental disinfectant in the disinfection of a chicken farm environment. The application method of the environmental disinfectant is: disinfecting the breeding environment and feeding equipment against Escherichia coli and / or Salmonella by spraying or soaking. The breeding environment includes chicken houses, chicken cages, waterers, troughs and other feeding tools, feces and bedding.

[0035] The application method includes but is not limited to disinfecting and decontaminating the water distribution system, breeding facilities, feeding equipment or other environmental surfaces of the chicken farm by liquid immersion, spraying, and combined use with an aqueous carrier, and disinfecting and preserving feed. The environmental disinfectant can be used to replace antibiotics or traditional disinfection products to achieve a disinfection effect, and is safe to use and will not cause harm to humans and poultry.

[0036] In a seventh aspect, the present application also provides a chicken disinfectant, which includes the aforementioned Escherichia coli phage PD2061 or the aforementioned phage composition.

[0037] The chicken disinfectant can be sprayed or soaked on the surface of raw chicken meat after processing in a slaughterhouse, thereby achieving the sterilization of Escherichia coli and / or Salmonella.

[0038] In addition, studies have shown that when antibiotics and phages are used in combination, they exhibit synergistic effects (Phage–Antibiotic Synergy, PAS): Phages can enhance bacterial lysis through mechanisms such as certain antibiotics causing bacterial cell elongation or filamentation, increasing bacterial sensitivity to phage lysis, increasing plaque size, and accelerating phage amplification, thereby enhancing phage lysis. Furthermore, phages can increase bacterial sensitivity to certain antibiotics, thereby lowering the minimum inhibitory concentration (MIC) of antibiotics and reducing antibiotic usage. Therefore, the development of combined antibiotic and phage formulations has market application value.

[0039] Therefore, in the eighth aspect, the present application also provides a phage antibiotic combination preparation, the active ingredient of which is the aforementioned Escherichia coli phage PD2061 or a mixture of the aforementioned phage composition and an antibiotic; the antibiotic is any one of sulfadiazine, gentamicin sulfate, spectinomycin hydrochloride, and lincomycin hydrochloride, or a combination of two or more thereof.

[0040] Experiments have shown that the addition of bacteriophage PD2061 reduced the MIC of compound sulfadiazine suspension by 64-fold, the MIC of gentamicin sulfate powder by 32-fold, and the MIC of a mixed powder of spectinomycin hydrochloride and lincomycin hydrochloride by 8-fold. Furthermore, when treated with the same antibiotic, the mortality rate of chickens infected with E. coli was significantly reduced in the phage-treated group compared to the group without phage, demonstrating that the combination of phage PD2061 and antibiotics is more effective than using only one antibiotic.

[0041] The above results show that the use of phage-antibiotic combination preparations can not only significantly reduce the amount of antibiotics used, but also achieve better results in preventing and treating E. coli in poultry. The antibiotics can be mixed directly with the phage in different dosage forms or diluted before mixing with the phage.

[0042] Optionally, the active ingredient of the phage antibiotic combination preparation is the aforementioned Escherichia coli phage PD2061 or a mixture of the aforementioned phage composition and antibiotics. When diluted for use, the content of phage PD2061 per 1L of water is 1.0×10 7~ 10 8 PFU, the antibiotic is compound sulfadiazine or gentamicin sodium sulfate or a mixture of spectinomycin hydrochloride and lincomycin hydrochloride.

[0043] Among them, per 1L of water, the content of compound sulfadiazine is 0.08~0.18g (or 0.2~0.45ml compound sulfadiazine suspension (concentration of 0.4g / ml)), the content of sodium gentamicin sulfate is 0.1~0.15g (or 2~3g sodium gentamicin sulfate soluble powder (0.05g / g spectinomycin hydrochloride lincomycin hydrochloride soluble powder)), and the contents of spectinomycin hydrochloride and lincomycin hydrochloride are: 0.2~0.4g spectinomycin, 0.1~0.2g lincomycin (or 0.5~1g spectinomycin hydrochloride lincomycin hydrochloride soluble powder (spectinomycin 0.4g / g, lincomycin 0.2g / g)).

[0044] Preferably, when used, the amount of bacteriophage PD2061 is 1.0×10 8 PFU / L, the content of compound sulfadiazine is 0.08g / L (or 0.2ml / L of compound sulfadiazine suspension (concentration of 0.4g / ml)), the content of sodium gentamicin sulfate is 0.1g / L (or 2g / L sodium gentamicin sulfate soluble powder (0.05g / g spectinomycin hydrochloride and lincomycin hydrochloride soluble powder)), the contents of spectinomycin hydrochloride and lincomycin hydrochloride are: 0.2g / L spectinomycin, 0.1g / L lincomycin (or 0.5g spectinomycin hydrochloride and lincomycin hydrochloride soluble powder (spectinomycin 0.4g / g, lincomycin 0.2g / g)).

[0045] The present invention has the following beneficial effects: The present invention provides a broad-spectrum Escherichia coli phage PD2061, which not only has a high lysis rate against E. coli but also can lyse Salmonella, offering significant advantages in preventing and treating mixed infections of E. coli and Salmonella. This phage can be used to prepare pharmaceutical preparations for treating or preventing E. coli and / or Salmonellosis in chickens, improving the survival rate of chickens with E. coli / Salmonellosis or mixed infections, increasing feed utilization, and reducing chicken mortality rates during farming.

[0046] This bacteriophage can be used as an active ingredient in environmental disinfectants, feed additives, and chicken raw meat disinfectants. While solving pathogen infections, it avoids the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics.

[0047] 2. The bacteriophages involved in the present invention are obtained from nature and are easy to industrialize. The drugs or disinfectants prepared from the bacteriophages can not only reduce costs, but also have the advantages of being green and environmentally friendly and will not harm beneficial bacteria in the environment.

[0048] 3. The present invention also provides a phage-antibiotic combination preparation, which effectively reduces the amount of antibiotics used in actual applications, reduces the production cost, and improves the prevention and treatment effect of corresponding diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a picture of the plaque of Escherichia coli phage; Figure 2 This is an electron microscope image of Escherichia coli bacteriophage; Figure 3 The pH stability test results of Escherichia coli phage; Figure 4 is the one-step growth curve of E. coli phage; Figure 5 These are the test results of the temperature stability of Escherichia coli phage. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in this field. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0051] Example 1 Isolation and purification of Escherichia coli phage PD2061 1. Experimental methods (1) Resuscitation and cultivation of host bacteria Twenty strains of Escherichia coli (JD1-JD20) stored in our laboratory were selected, and the freezing solution was dipped into a sterile inoculum and streaked on MAC medium for recovery. The culture was incubated in a 37°C incubator for 18-24 hours to obtain a single colony. A single colony was picked and inoculated into 5 mL of NB broth, and the culture was shaken at 37°C and 170 rpm / min for 16 hours to obtain a fresh E. coli culture solution.

[0052] (2) Isolation of bacteriophage An appropriate amount of chicken feathers, feces, and other samples from Shandong Province were placed in a sample bottle, added to an appropriate amount of broth culture medium, and the 20 Escherichia coli strains obtained after proliferation were added. The mixture was placed in a shaking culture at 37°C and 170 rpm / min for 12 hours, centrifuged at 11,000 rpm for 5 minutes, and then filtered through a 0.22 μm sterile microporous filter membrane to obtain phage proliferation liquid; the phage stock solution was diluted 10-fold, and the appropriate gradient phage dilution liquid was mixed with 20 Escherichia coli strains (JD1-JD20) one by one in a 1:1 ratio. After incubation at 37°C for 5 minutes, 200 μL of the mixture was aspirated and placed on the upper agar layer (agar concentration of 0.7%). After mixing, it was quickly poured onto the lower agar layer (agar concentration of 1.5%) plate, shaken and placed flat until the culture medium solidified. After incubation in a 37°C incubator for 4-6 hours, a double-layer plate with phage plaque formation was obtained.

[0053] (3) Phage purification A single plaque was picked from the double-layer agar medium where plaques were formed and placed in 1 mL of NB broth in a shaker at 37°C, 170 rpm, and incubated for approximately 30 minutes to obtain a phage extract. The phage extract was then mixed with the corresponding plaqued E. coli (hereinafter referred to as the host strain) in a 1:1 ratio (incubated at 37°C for 5 minutes). 200 μL of the extract was then placed on the top agar plate, mixed thoroughly, and quickly poured onto the bottom agar plate. The plate was shaken and incubated flat until the medium solidified. After incubation inverted at 37°C for 4–6 hours, another double-layer plate with plaques was obtained. A single plaque was picked from the double-layer medium where plaques were formed using sterile forceps and placed in 1 mL of NB broth. The phage extract was then incubated at 37°C, 170 rpm, and incubated for approximately 30 minutes to obtain a phage extract. This procedure was repeated three times to obtain the purified phage extract.

[0054] (4) Phage proliferation and titer determination Take equal amounts of purified phage extract and host bacterial growth liquid in 5 mL of liquid NB medium, culture at 37°C, 170 rpm shaking until the liquid becomes clear, centrifuge the clear liquid at 11000 rpm for 10 minutes, take the supernatant, filter it with a 0.22 μm sterile microporous filter membrane to obtain phage growth liquid, and use the double-layer plate method to determine the titer of the newly isolated phage. Figure 1 As shown, Escherichia coli phage forms clear plaques on the double-layer agar medium plate, with no halo around and clearly visible edges.

[0055] 2. Experimental results and analysis According to the above experimental method, 12 E. coli phages were screened from 20 E. coli isolates, numbered PD2061-PD2070. These 12 E. coli phages all formed clear plaques on double-layer agar plates, without a halo around them, with clearly visible edges and a diameter of approximately 0.2 mm to 1.0 mm; their titers were between 2.8×10 9 PFU / mL-3.0×10 11 PFU / mL, the results are shown in Table 1 below.

[0056] Table 1 Titers of 12 newly isolated Escherichia coli phages (pfu / mL)

[0057] Example 2 Morphological Observation and Identification of Escherichia coli Phage PD2061 1. Experimental Method: Copper Grid Preparation and Electron Microscope Observation: 20 µL of phage sample was dropped onto a carbon-coated copper grid. The grid was allowed to settle naturally for 15 minutes. The grid was then blotted dry with filter paper and stained with 2% (W / V) phosphotungstic acid (PTA) for 1–2 minutes. The grid was blotted dry with filter paper and then observed and photographed under a transmission electron microscope.

[0058] 2. Identification results like Figure 2 As shown, electron microscopy observation showed that the phage PD2061 has a polyhedral head structure and a contractile tail. The head is 60-65 nm wide and 78-83 nm long, and the tail is about 90-93 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage PD2061 of the present application conforms to the characteristics of the Myotailed Phage family and belongs to the Myotailed Phage.

[0059] Example 3 Determination of the lysis spectrum of Escherichia coli phage PD2061 1. Experimental Materials Host bacteria: The 20 clinical isolates of Escherichia coli (JD1-JD20) in Example 1 and 60 strains of Escherichia coli from different animal sources stored in the laboratory were selected, including: chicken Escherichia coli, duck Escherichia coli, pigeon Escherichia coli, goose Escherichia coli, pig Escherichia coli, donkey Escherichia coli, etc. (see Table 2 for details).

[0060] 2. Experimental methods: 80 pathogenic strains of Escherichia coli isolated from different regions and sources and preserved in the laboratory were selected, and the lysis rates of Escherichia coli phage PD2061 against the above 80 strains of Escherichia coli were determined by the double-layer plate method.

[0061] 3. Experimental results and analysis From the results of the fragmentation spectrum experiment in Table 2, we can see that: (1) Escherichia coli phage PD2061 can lyse 72 of 80 strains of Escherichia coli from different animal species, with a lysis rate of up to 90.00%, which is a phage with a broad lysis spectrum.

[0062] (2) E. coli phage PD2061 has a high lysis rate against E. coli from different animal species. Among them, the lysis rate against E. coli from chickens was 90% (27 / 30), and the lysis rate against E. coli from other zoos was also basically 90%.

[0063] Table 2 Lysis spectrum of Escherichia coli phage PD2061 against 80 strains of Escherichia coli

[0064]

[0065]

[0066]

[0067] Example 4 Lysis test of bacteriophage PD2061 on Salmonella 1. Experimental methods In this example, 10 strains of chicken-derived Salmonella were selected, and the lysis spectrum of Escherichia coli phage PD2061 was measured according to the lysis spectrum measurement method in Example 3.

[0068] 2. Experimental results and analysis As shown in the results in Table 3, the lysis rate of E. coli phage PD2061 against these 10 Salmonella strains was 20%. This result shows that phage PD2061 can lyse some Salmonella, play a certain role in the prevention and treatment of Salmonella, and has application advantages in the prevention and treatment of mixed infections of E. coli and Salmonella.

[0069] Table 3 Lysis spectrum of E. coli phage PD2061 against 10 strains of Salmonella

[0070] Example 5 pH tolerance of Escherichia coli phage PD2061 1. Experimental methods Take sterile test tubes and add 4.5 mL of NB broth with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), three tubes each as one portion, take two portions in total, then place the test tubes in a 37°C water bath. After the temperature stabilizes, add 500 μL of 3×10 11Pfu / mL bacteriophage PD2061 proliferation solution was mixed and incubated in a 37°C water bath for 1, 2, or 3 hours. Immediately after the incubation period, an appropriate amount of 1 mol / L HCl or NaOH was added to the mixture to adjust its pH to approximately 7. The mixture was diluted 10-fold and titered using an appropriate dilution gradient. Three replicates were performed at each pH value. A phage pH stability curve was plotted with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0071] 2. Experimental results and analysis from Figure 3 The titer of phage PD2061 remained unchanged within the pH range of 4.0 to 10.0, indicating that the phage can tolerate a range of acid and alkaline environments. The titer of the phage was still detectable after 3 hours at pH 1.0 and 13.0, demonstrating its strong acid and alkaline resistance and its ability to adapt to a range of strong acid and alkaline environments over a period of time. Furthermore, the phage exhibited excellent acid resistance, exhibiting minimal potency loss when exposed to gastric acid, allowing it to fully exert its effects after entering the intestines.

[0072] Example 6 Temperature stability of bacteriophage 1. Experimental methods: The same volume of 3×10 11 Containers of phage proliferation fluid containing 100 pfu / mL were placed at 60°C, 70°C, 80°C, and 90°C, with two replicates at each temperature. After incubation for 10, 20, and 60 minutes, samples were taken after the incubation period and immediately cooled in an ice bath. The samples were then diluted 10-fold and titered using an appropriate dilution gradient. A phage thermostability curve was plotted with temperature as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0073] 2. Experimental results and analysis: like Figure 5 The results showed that the phages maintained their original activity after being exposed to 60℃ for 60 min, and the titer remained at 10 after being exposed to 70℃ for 60 min. 10 pfu / mL or above; after 60 min at 80℃, the titer remained at 10 5 pfu / mL or more; after 60 minutes at 90℃, the titer can still be detected and a certain level of activity can be maintained. This shows that the phage has high thermal stability.

[0074] Example 6 One-step growth curve of Escherichia coli phage PD2061 1. Experimental methods 1 mL of phage proliferation fluid and 1 mL of fresh host bacterial proliferation fluid with a multiplicity of infection of 0.001 were mixed thoroughly (the timer started at this point), incubated at 37°C for 5 minutes, centrifuged at 13,000 rpm for 30 seconds, and the supernatant was removed as much as possible with a micropipette. The supernatant was then washed once with 5 mL of NB broth (centrifuged at 13,000 rpm for 30 seconds), and the supernatant was discarded. The pellet was suspended in preheated NB broth (total volume 5 mL) and mixed thoroughly. The pellet was quickly placed in a shaker at 37°C with shaking at 170 rpm. 150 μL was removed at time 0 and every 10 minutes, and centrifuged at 10,000 rpm for 1 minute. After making a 10-fold dilution with NB broth, the phage titer was measured using the double-layer plate method. Three parallel experiments were performed and the results were averaged. A one-step growth curve was drawn with the infection time as the horizontal axis and the phage titer in the infection system as the vertical axis to obtain the incubation period and outbreak period of the phage and calculate the outbreak volume.

[0075] Burst size = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak 2. Experimental results and analysis from Figure 4 The results showed that after phage PD2061 infected the host bacteria, the phage lysis cycle lasted about 80 minutes, the incubation period was about 10 minutes, and the phage outbreak period was about 70 minutes. After 80 minutes, the phage number remained basically unchanged and entered a stable period. At this time, the titer could reach 3×10 11 pfu / mL, and the burst amount of bacteriophage PD2061 was 150.

[0076] Example 7 Determination of the Optimal Multiplicity of Infection (MOI) of Escherichia coli Phage PD2061 against Escherichia coli 1. Experimental methods Pick a single E. coli colony and inoculate it into 5 ml of NB broth medium. Incubate at 37°C and 170 rpm for 12-16 hours to obtain a bacterial solution. Determine the bacterial concentration by pouring the solution into the broth and adjust the concentration to 1×10 9 cfu / mL, 1×10 8 cfu / mL...1×10 5 cfu / mL. The concentration of phage isolated in Example 1 was adjusted to 1×10 5 ~1×10 7PFU / mL. Add phage and bacterial solution to NB medium according to the phage to bacterial count ratio shown in Table 4. Proliferate at 37°C, 170 rpm, and shake until the solution becomes clear. Record the proliferation time. Take an appropriate amount of the clear solution and centrifuge at 11,000 rpm for 10 minutes. Remove the supernatant and filter it through a 0.22 μm sterile microporous filter. Determine the phage titer in the filtrate using the double-plate method. The MOI (number of phages / number of bacteria) with the highest phage titer is the optimal multiplicity of infection for that phage.

[0077] 2. Experimental results The results are shown in Table 4. When the MOI was 0.001:1, the PD2061 titer reached the highest, which was 3.0×10 11 PFU / mL. This result shows that when the initial input of bacteriophage PD2061 is small during production, the highest reproduction yield can be achieved, which is conducive to its large-scale industrial production.

[0078] Table 4 Titers of Escherichia coli phage PC817 at different multiplicity of infection

[0079] Example 8 Application of Escherichia coli phage in environmental disinfection 1. Experimental methods A broiler chicken hatchery in Binzhou, Shandong Province, was found to have high levels of E. coli, and chemical disinfectants were ineffective. This study used a disinfectant prepared from E. coli phage PD2061 to spray disinfect the hatchery and determine its effectiveness.

[0080] The preparation method of the bacteriophage environmental disinfectant is as follows: take the bacteriophage PD2061 proliferation liquid and dilute it with water at a ratio of 1:1000 before use, and the potency is 1.0×10 7 PFU / mL. At the same time, benzalkonium bromide disinfectant diluted with water at a ratio of 1:25 was used as a control.

[0081] (1) Air disinfection method: Two hatchery rooms of equal size were selected to sample the air before and after the hatchery rooms using the natural sedimentation method. A total of five test points were selected, including the central point and four corner points. The sampling points were 0.3m from the ground and the four corner points were 1m from the wall. A 9cm diameter XLD agar plate was placed at each point. Before disinfection, two XLD agar plates were used at each sampling point. The culture dish lid was opened and the sample was collected for 10 minutes. The farm's own fog line was used for disinfection. Experimental and control groups were set up. The experimental group used a bacteriophage environmental disinfectant (10mL / m 3 ) were used for treatment, and the control group was disinfected with benzalkonium bromide (benzalkonium bromide diluted 1:25).

[0082] After 30 minutes of disinfection, place two XLD agar plates at each of the five sampling points, open the petri dish lid, and sample for 10 minutes. Place the sample petri dishes before and after disinfection in a 37°C constant temperature incubator for 12-24 hours, and count the cultured bacteria.

[0083] The total number of colonies was counted according to the Ostwald formula: C = 50,000 N / AT, where C is the total number of colonies per cubic meter (CFU / m3); N is the number of colonies per dish; A is the area of ​​the culture dish (cm2); and T is the sampling time (min).

[0084] 2. Experimental results and analysis The results, shown in Table 5, show that in the control group, after disinfection with benzalkonium bromide, the elimination rate of E. coli in the hatchery air was 47.47%. In the experimental group, after disinfection with the bacteriophage mixture, the elimination rate of E. coli in the hatchery air was 89.52%. This shows that the bacteriophage mixture is significantly more effective in environmental disinfection than the existing chemical disinfectant benzalkonium bromide and is safer to use.

[0085] Table 5 Escherichia coli colony counts before and after disinfection with bacteriophage disinfectant and benzalkonium bromide disinfectant

[0086] Therefore, the above results show that the phage mixture preparation has an excellent disinfecting effect on Escherichia coli in the hatchery workshop and can be promoted and applied as a new type of biological environmental disinfectant.

[0087] Example 9 Therapeutic Effect of Escherichia coli Phage on Escherichia coli Disease in Chicken Farms 1. Experimental methods A laying hen farm in northern China was selected. The mortality rate in the farm reached 2.91% in the past week, and deformed soft-shell eggs appeared. The egg production rate dropped by about 15%. After dissecting the dead chickens on site, it was found that the surface of organs such as the liver was covered with a layer of yellow cheesy substance. The liver samples were collected aseptically and taken back to the laboratory for testing. It was found that the main cause of the increased mortality rate and decreased egg production quality in this chicken farm was Escherichia coli infection. Then two chicken houses with 1,200 chickens were selected as the experimental group and the control group respectively.

[0088] (1) Treatment of the experimental group: The prepared phage preparation was added to the water line to make the total phage concentration in the water line 1×10 8 pfu / mL for five consecutive days. No phage was added to the waterline of the control group. Other immunization and medication procedures were performed according to farm procedures.

[0089] (2) At the same time, the daily mortality rate and egg production rate of the chicken houses in the experimental and control groups were counted, and the dead chickens in the experimental and control groups were autopsied to isolate and identify the bacterial strains in the livers of the dead chickens.

[0090] 2. Experimental results and analysis (1) The results are shown in Table 6. The experimental group using Escherichia coli phage PD2061 can effectively reduce the number of chicken deaths, reduce the mortality rate of E. coli, gradually increase the egg production rate, and has a good therapeutic effect on E. coli infection.

[0091] Table 6 Statistics of therapeutic effects of phage preparations

[0092] Example 10 Preventive Effect of Phage Preparation on Chicken Escherichia coli Disease 1. Experimental methods One-day-old chicks were selected from a broiler farm in southern China. The experimental group included 100 chicks and the control group included 100 chicks. The experiment was conducted according to the following method: (1) Preparation of phage spray-dried powder: The phage preparation used in this example is in the form of a freeze-dried powder. The preparation method is as follows: skim milk powder and lactose are selected as protective agents, and phage preparation is added, wherein the specific ratio of skim milk powder, lactose, and phage is 1:1:8. The powder is dried using a Swiss BUCHIB-90 nano spray dryer. After spray drying for about 1 hour, the spray-dried particles are collected to finally obtain a phage powder with a total phage concentration of 1×10 10 PFU / g.

[0093] The process parameters of the spray drying are as follows: inlet temperature of 120° C., air flow velocity of 120 L / min, spray rate of 100%, and nozzle cover size of 4.0 μm.

[0094] (2) Experimental group treatment: 500 g / ton of the prepared phage powder was added to the feed. Control group treatment: No phage powder was added to the feed. Other immunization and medication procedures were carried out according to the farm procedures.

[0095] (3) Detect the total mortality rate after the first and second weeks, detect the bacterial strains in the livers of the culled chickens, and calculate the total body weight growth rate.

[0096] 2. Experimental results and analysis The results are shown in Table 7. The overall mortality rate of the phage group was lower than that of the control group without phage addition. The detection rate of E. coli in the dead chickens in the phage group was much lower than that in the control group. The weight growth rate of the phage group (1.25%) was higher than that of the control group (100%), and the isolation rate of E. coli (12.5%) was much lower than that of the control group (69.23%). These results indicate that the phage preparation of the present invention has an excellent effect in preventing E. coli, and can improve feed utilization and increase the weight gain of chicks.

[0097] Table 7 Statistics of the preventive effect of phage mixture preparations

[0098] Example 11 In vitro synergistic effect of phage preparations and antibiotics 1. Experimental Methods (1) Select a strain of Escherichia coli DC1 isolated from a chicken farm that is sensitive to bacteriophage PD2061, and adjust the bacterial concentration to 1×10 7 cfu / ml.

[0099] (2) Dissolve the antibiotic compound sulfadiazine suspension, gentamicin sulfate powder, spectinomycin hydrochloride and lincomycin hydrochloride soluble powder in sterile water at a ratio of 1:9, and then place in a shaker at 170 rpm and 37°C and mix thoroughly for 30 minutes to obtain various antibiotic solutions.

[0100] (3) The minimum inhibitory concentration (MIC) of the above antibiotics was determined using a 96-well plate method (MIC value is expressed as the dissolution multiple of the powder / suspension), and then the dilution multiple of the above antibiotic MIC was diluted 2-fold, diluted 10 times, and 50ul of all the dilutions were added to the 96-well plate, and then 50ul of phage PD2061 was added to each well (the titer of the added phage was 1.0×10 7 PFU / ml), then take 100ul NB broth as culture medium and add it to the wells, put it into the microbial growth curve analyzer, set the parameters at 37℃800rpm for 12h, and observe and compare the statistical results the next day.

[0101] 2. Experimental Results (1) The results are shown in Table 8. It can be seen from the results that when no phage is added, the MIC value of the antibiotic sulfadiazine suspension against Escherichia coli DC1 is 0.025 mg / ml. After the addition of phage PD2061, the sulfadiazine suspension still has an antibacterial effect when diluted to 0.00039 mg / ml, and the MIC value is reduced by 64 times.

[0102] The MIC value of the antibiotic sodium sulfate gentamicin powder against Escherichia coli DC1 is 0.0015 mg / ml. After the addition of bacteriophage PD2061, the gentamicin sulfate powder still has an antibacterial effect when diluted to 0.000048 mg / ml, and the MIC value is reduced by 32 times.

[0103] The MIC value of the antibiotic spectinomycin hydrochloride against Escherichia coli DC1 is 0.2 mg / ml. After adding bacteriophage PD2061, spectinomycin hydrochloride and lincomycin hydrochloride (this is a finished mixed antibiotic) still have an antibacterial effect when diluted to 0.025, and the MIC value is reduced by 8 times.

[0104] (2) After adding bacteriophage PD2061 to various antibiotics, the minimum inhibitory concentration of the antibiotics was significantly reduced. By using bacteriophages and antibiotics together, the amount of antibiotics used can be effectively reduced, the cost of antibiotic use can be reduced, and antibiotic residues in the environment can be reduced.

[0105] Table 8 Synergistic effect of bacteriophage PD2061 and antibiotics

[0106] Example 12 Treatment of Chicken Escherichia coli Disease with a Phage-Antibiotic Combination The challenge strain DC1, which is sensitive to bacteriophage PD2061, was selected. 160 one-day-old chicks were divided equally into the following eight groups, with 20 chicks in each group. The procedures were as follows: Group 1: First, the animals were challenged with toxicity, and then immediately treated with sulfadiazine suspension (0.2 ml per liter of water) for 5 consecutive days. Group 2: First, the virus was challenged, and then immediately treated with sodium sulfate gentamicin soluble powder (2g of sodium sulfate gentamicin soluble powder per liter of water) for 5 consecutive days. Experimental group 3: First, the virus was challenged, and then treated with spectinomycin hydrochloride and lincomycin hydrochloride soluble powder (the dosage is 0.5g spectinomycin hydrochloride and lincomycin hydrochloride soluble powder per 1L of water) for 5 consecutive days; Group 4: The patients were challenged with the virus first, and then immediately administered with compound sulfadiazine suspension (Guangdong Wenshi Dahuanong Biotechnology Co., Ltd., sulfadiazine 0.4g / ml, dosage: add 0.2ml compound sulfadiazine suspension per 1L of water), and simultaneously administered with bacteriophage PD2061 (dosage: add 10ml phage proliferation solution per 1L of water, phage titer: 1.0×10 7 PFU / ml) for 5 consecutive days; Group 5: The challenge treatment was performed first, and then sodium sulfate gentamicin soluble powder (Tai'an Haoxin Kangda Biological Co., Ltd., gentamicin 0.05g / g, dosage is 2g sodium sulfate gentamicin soluble powder in 1L water) was immediately used in combination with bacteriophage PD2061 (dosage is 10ml phage proliferation solution per 1L water, phage titer is 1.0×10 7 PFU / ml) for 5 consecutive days; In the sixth group, the challenge treatment was performed first, and then the patients were immediately treated with spectinomycin hydrochloride and lincomycin hydrochloride soluble powder (Guangdong Wenshi Dahuanong Biotechnology Co., Ltd., spectinomycin 0.4g / g, lincomycin 0.2g / g, dosage: 0.5g spectinomycin hydrochloride and lincomycin hydrochloride soluble powder per 1L of water) combined with bacteriophage PD2061 (dosage: 10ml phage proliferation solution per 1L of water, phage titer: 1.0×10 7 PFU / ml) for 5 consecutive days; Experimental group 7: After the challenge treatment, no treatment was given; Control group: No virus challenge was performed and the animals were raised normally as the control group.

[0107] The method of the challenge treatment is: using 0.2 ml of a concentration of 1×10 7 The chicks were challenged with Escherichia coli DC1 by intraperitoneal injection at 100 cfu / ml.

[0108] The total mortality of chicks in each group was counted every day for 5 consecutive days.

[0109] 2. Experimental Results As shown in Table 9, when treated with the same antibiotic, the number of chicken deaths in Experimental Groups 4-6, which included the addition of bacteriophage, was significantly lower than in Experimental Groups 1-3, which were treated with antibiotics alone. For example, the total mortality rate in Experimental Group 4 was lower than that in Experimental Group 1, the total mortality rate in Experimental Group 5 was lower than that in Experimental Group 2, and the total mortality rate in Experimental Group 6 was lower than that in Experimental Group 3. These results indicate that the combination of bacteriophage PD2061 and antibiotics is more effective in treating E. coli in chickens than the single antibiotic treatment.

[0110] Table 9 Deaths after combined use of phage and antibiotics

[0111] Example 13: Treatment of Mixed Infection of Chickens with Escherichia coli and Salmonella Using a Phage-Antibiotic Combination The challenge strains DC1 and SM1, which are sensitive to bacteriophage PD2061, were selected. 160 one-day-old chicks were divided equally into the following eight groups, with 20 chicks in each group. The procedures were as follows: Group 1: The animals were challenged first and then immediately treated with sulfadiazine (0.4 g / ml) in a 0.2 ml solution per 1 L of water. The solution was administered to the drinking water for 5 consecutive days. Group 2: The animals were challenged first and then immediately treated with sodium sulfate gentamicin soluble powder (Tai'an Haoxin Kangda Biological Co., Ltd., gentamicin 0.05 g / g) at a dosage of 2 g per liter of water for 5 consecutive days. Group 3: First, the animals were challenged with toxicity, and then treated with spectinomycin hydrochloride / lincomycin hydrochloride soluble powder (Guangdong Wenshi Dahuanong Biotechnology Co., Ltd., spectinomycin 0.4 g / g) at a dosage of 0.5 g spectinomycin hydrochloride / lincomycin hydrochloride soluble powder per 1 L of water for 5 consecutive days. Group 4: The patients were challenged with the virus first, and then immediately administered with compound sulfadiazine suspension (Guangdong Wenshi Dahuanong Biotechnology Co., Ltd., sulfadiazine 0.4g / ml, dosage: add 0.2ml compound sulfadiazine suspension per 1L of water), and simultaneously administered with bacteriophage PD2061 (dosage: add 10ml phage proliferation solution per 1L of water, phage titer 5.0×10 7 PFU / ml) for 5 consecutive days; Group 5: First, the virus was challenged, and then sodium sulfate gentamicin soluble powder (Tai'an Haoxin Kangda Biological Co., Ltd., gentamicin 0.05g / g, dosage: 2g per liter of water) was immediately used in combination with bacteriophage PD2061 (dosage: add 10ml of phage proliferation solution per 1L of water, phage titer: 5.0×10 7 PFU / ml) for 5 consecutive days; Group 6: First, the virus was challenged, and then immediately treated with spectinomycin hydrochloride and lincomycin hydrochloride soluble powder (Guangdong Wenshi Dahuanong Biotechnology Co., Ltd., spectinomycin 0.4g / g, lincomycin 0.2g / g, dosage: 0.5g per 1L of water) combined with bacteriophage PD2061 (dosage: 10ml phage proliferation solution per 1L of water, phage titer: 5.0×10 7 PFU / ml) for 5 consecutive days; Experimental group 7: After the challenge treatment, no treatment was given; Control group: No virus challenge was performed and the animals were raised normally as the control group.

[0112] The method of the challenge treatment is: using 0.2 ml of a concentration of 1×10 7 cfu / ml of E. coli DC1 and 0.2ml of the concentration of 1×10 7 The chicks were challenged by intraperitoneal injection of Salmonella SM1 at a concentration of cfu / ml.

[0113] The total mortality of chicks in each group was counted every day for 5 consecutive days.

[0114] 2. Experimental Results As shown in Table 10, when treated with the same antibiotics, the mortality rate in groups 4-6, which included phages, was significantly lower than in groups 1-3, which were treated with antibiotics alone. For example, the total mortality rate in group 4 was lower than that in group 1, with a 50% reduction in mortality. The total mortality rate in group 5 was lower than that in group 2, with a 55% reduction in mortality. The total mortality rate in group 6 was lower than that in group 3, with a 45% reduction in mortality. These results indicate that the combination of phage PD2061 and the three antibiotics mentioned above effectively controls mortality in the face of mixed E. coli and Salmonella infections, significantly reducing mortality rates and demonstrating a significantly better therapeutic effect than using antibiotics alone.

[0115] Table 10 Death rate after combined use of phage and antibiotics

[0116] Example 14 Safety Test of Bacteriophage 1. Experimental methods Sixty one-day-old chicks were selected and divided into a phage group of 30 and a control group of 30. The phage group was orally administered with 1×10 10 The animals were fed 0.2 mL of PFU of bacteriophage and observed for 7 days. The control group received an equal dose of sterile saline orally. Necropsy was performed to observe lesions in the heart, liver, spleen, lungs, kidneys, brain, and intestines. During the feeding period, the animals' mental state and feeding habits were observed. 2. Experimental Results and Analysis Throughout the dosing period, no symptoms of illness or toxicity were observed in the chickens in either the phage or control groups, and their mental state and feed intake remained normal. Detailed clinical autopsies revealed normal major organs and intestinal tracts in both the phage and control groups. This demonstrates the high safety of PD2061 phage, which has no adverse effects on the animal body.

[0117] Example 14 Whole genome analysis of Escherichia coli phage PD2061 The genome of phage PD2061 was extracted, and whole genome sequencing and sequence analysis were performed. The results are as follows: The genome is 166,913 bp long, with a G+C content of 35.49%. The C, G, A, and T base contents are 16.71%, 18.78%, 32.72%, and 31.77%, respectively. Whole-genome RAJD online annotation revealed 271 open reading frames (ORFs). Among these 271 ORFs, the highly conserved large subunit of the terminase (Phage terminase, large subunit) is found (see sequence 1 in the sequence listing). Analysis using the online CGE server tool revealed no resistance or virulence genes in the genome. PHAJDER analysis also revealed no lysogeny-related genes.

[0118] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solutions and concepts of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An Escherichia coli phage PD2061, characterized in that: Its deposit number is CGMCC NO.46163.

2. A phage composition, characterized in that: The invention comprises the Escherichia coli phage PD2061 according to claim 1.

3. Use of the Escherichia coli phage PD2061 according to claim 1 or the phage composition according to claim 2 in the preparation of a medicament for preventing and treating diseases caused by Escherichia coli and / or Salmonella infection.

4. A bacteriophage pharmaceutical preparation, characterized in that: The active ingredient comprises the Escherichia coli phage PD2061 according to claim 1 or the phage composition according to claim 2.

5. The bacteriophage pharmaceutical preparation according to claim 1, characterized in that The pharmaceutical preparation is in the form of an oral dosage form, an external dosage form or a parenteral dosage form.

6. The bacteriophage pharmaceutical preparation according to claim 1, characterized in that The bacteriophage drug preparation is a powder, and further comprises skim milk powder and lactose as protective agents.

7. A feed additive or poultry drinking water additive, characterized in that: The method comprises the Escherichia coli phage PD2061 according to claim 1 or the phage composition according to claim 2.

8. An environmental disinfectant, characterized in that The active ingredient comprises the Escherichia coli phage PD2061 according to claim 1 or the phage composition according to claim 2.

9. Use of the environmental disinfectant according to claim 7 in the disinfection of a chicken farm environment, characterized in that: The application method of the environmental disinfectant is: disinfecting the breeding environment and feeding equipment against Escherichia coli and / or Salmonella by spraying or soaking. The breeding environment includes chicken houses, chicken cages, drinking fountains, feeding troughs and other feeding tools, feces and bedding.

10. A chicken disinfectant, characterized in that: The method comprises the Escherichia coli phage PD2061 according to claim 1 or the phage composition according to claim 2.

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