CN120683060A

CN120683060A - Escherichia coli phage PD2061, phage composition and application thereof

Info

Publication number: CN120683060A
Application number: CN202510978763.1A
Authority: CN
Inventors: 任慧英; 刘广芹; 魏凯; 王翠; 王思寒; 李宁; 孙虎芝; 潘姝雅; 陈婷婷
Original assignee: Qingdao Phagepharm Bio Tech Co ltd
Current assignee: Qingdao Phagepharm Bio Tech Co ltd
Priority date: 2025-07-16
Filing date: 2025-07-16
Publication date: 2025-09-23

Abstract

The invention belongs to the technical field of phages, and discloses an escherichia coli phage PD2061, a phage composition and application thereof, wherein the escherichia coli phage PD2061 has a preservation number of CGMCC NO.46163, is preserved in the China general microbiological culture Collection center of the China general microbiological culture Collection system for 8 months and 16 days in 2024, and has a preservation address of North Star Xishu No. 1 and No.3 in the Korean region of Beijing city. The escherichia coli phage PD2061 not only has high cracking rate on escherichia coli, but also has certain cracking performance on salmonella, and has greater advantages in preventing and treating diseases of escherichia coli and salmonella mixed infection. The phage can be used for preparing a pharmaceutical preparation for treating or preventing animal-derived colibacillosis and/or salmonellosis, so that the survival rate of birds suffering from colibacillosis/salmonellosis or mixed infection is improved, the utilization rate of feed is improved, and the death and panning rate of birds in cultivation is reduced.

Description

Escherichia coli phage PD2061, phage composition and application thereof

Technical Field

The invention relates to the technical field of phages, in particular to an escherichia coli phage PD2061, a phage composition and application thereof.

Background

The escherichia coli belongs to enterobacteriaceae, is widely distributed on animal intestinal tracts, air suspension particles, surfaces of clothes, farm environments, rivers, lakes and the like, and has the characteristics of wide distribution and the like. In recent years, with the gradual expansion of chicken raising scale and the increase of raising density, and the problems of low management level, poor environmental sanitation level, more stress-inducing factors and the like of part of chicken farms, the occurrence probability of colibacillosis of chickens is obviously improved.

Coli can be spread vertically or horizontally, and can mainly cause bird navel inflammation, egg yolk malabsorption, pericarditis, air sac inflammation, perihepatic inflammation, diarrhea, septicemia, etc., and part of the colibacillus can cause arthritis, wet eye circles, subcutaneous abscess, etc. Meanwhile, the feed additive can reduce food consumption of animals, prevent feed conversion, reduce feed rewards and reduce production performance.

Antibiotics are generally used for treating colibacillosis in livestock and poultry production, but unscientific use of antibiotics brings a series of problems including generation of multi-drug resistant strains, damage of intestinal flora balance, reduction of animal immunity, animal poisoning caused by excessive dosage of antibiotics, environmental pollution, residual antibiotics in animal products and the like, so that a need exists to find efficient, safe and residue-free bactericidal preparations to replace antibiotics.

The phage is a virus with high specificity for infecting microorganisms such as bacteria and fungi, and is used as a natural antibacterial agent, the phage has the advantage incomparable with antibiotics, and the phage has a long history of clinical treatment, has better effect and is an antibiotic substitute with wide prospect.

However, the existing coliphage has a certain limitation on its cleavage spectrum based on the cleavage specificity of its host, and it is generally required to use a plurality of phage in combination to expand the cleavage spectrum, so that the existing coliphage resources are still to be further developed.

Disclosure of Invention

Aiming at the problems, the invention provides an escherichia coli phage PD2061, a phage composition and application thereof, wherein the escherichia coli phage PD2061 has a wide cracking spectrum and a certain cracking performance on escherichia coli and salmonella, can be used for preparing a pharmaceutical preparation for treating or preventing chicken colibacillosis and/or salmonella, improves the survival rate of chicken suffering from colibacillosis/salmonella or mixed infection, and can be combined with antibiotics to be used as a combined preparation for preventing and treating the diseases.

In order to solve the problems, the application provides the following technical scheme:

In a first aspect, the present application provides an escherichia coli (ESCHERICHIA COLI BACTERIOPHAGE) phage PD2061, which has a preservation number of CGMCC No.46163, and is preserved in the China general microbiological culture Collection center (mcm) for 8 months and 16 days of 2024, with a preservation address of North Star, no. 1, no. 3, and a preservation number of CGMCC No.46163.

As observed by an electron microscope, the phage PD2061 has a polyhedral head structure and a contractile tail, the head is 60-65 nm wide and 78-83 nm long, the tail is about 90-93 nm long, and the shape of the phage PD2061 accords with the characteristics of the myophagidae and belongs to the myophagidae according to the classification method of the International Commission on virus classification (ICTV).

The titer of the coliphage PD2061 is high and can reach 3X 10 ¹¹ pfu/ml.

The phage has good high temperature resistance, and can be applied in high temperature and high humidity environments such as sheds.

The escherichia coli phage PD2061 has good acid resistance, has low potency loss when passing through gastric acid, and can enter the intestinal tract to fully exert the effect. Experiments prove that the phage is stable at pH 4.0-10.0, and the potency can still be detected under the conditions of pH 1.0 and pH 13.0 for 3 hours.

The coliphage PD2061 has broad-spectrum cracking performance on colibacillus, and has a cracking rate of 90.00% on colibacillus, and in addition, the phage has a certain cracking performance on salmonella, and the cracking rate of the phage reaches 20%. Thus, the coliphage PD2061 is of greater advantage in the control of diseases in which E.coli and Salmonella are co-infected.

In the present application, phage PD2061 includes a mutant strain having a homology of more than 98% or 99% by performing a point mutation or a deletion mutation or an addition mutation and maintaining substantially the same bactericidal activity. Since phages are very prone to mutation during replication, the above mutants of phages are also within the scope of the claimed application. The genomic sequence of phage PD2061 can be sequenced by known methods from the biological material deposited according to the application. Screening of phage for mutants that are extremely similar to their trait according to the present application does not require inventive effort for those skilled in the art.

In a second aspect, the present application also provides a phage composition comprising the aforementioned E.coli phage PD2061.

In practical application, in order to further widen the lysis spectrum of the phage, fully exert the difference of the lysis spectrums of different phages and perform advantage complementation, the escherichia coli phage PD2061 and other phages can be used in combination, such as one or more of PD328 (disclosed in patent publication No. CN115717126 a), PD06 and PD114 (both of which are disclosed in patent publication No. CN 111349618B) which are identical to the escherichia coli phage, so as to expand the sterilization spectrum of the escherichia coli, kill all escherichia coli pathogens in the environment as much as possible, and be used for preventing and treating escherichia coli diseases. In addition, the coliphage PD2061 can be matched with other phages of different types (for inhibiting different pathogenic bacteria causing similar diseases, such as the existing salmonella phage) for preventing and treating salmonella, and can also be used for preventing and treating escherichia coli and salmonella combined infection.

In a third aspect, the application also provides the use of the coliphage PD2061 or the phage composition described above in the preparation of a medicament for preventing and treating diseases caused by infection with Escherichia coli and/or Salmonella. The control includes prevention and treatment. The term "preventing" is meant herein to include all actions that inhibit or delay the disease by administering the composition. The term "treatment" is meant herein to include all actions that result in improvement or improvement of the disease by administration of the composition.

Alternatively, the diseases caused by the E.coli infection include acute sepsis caused by chicken-origin E.coli, navel inflammation, air sac inflammation, egg yolk peritonitis, salpingitis, etc.

Alternatively, the Salmonella infection-induced diseases include pullorum disease, fowl typhoid disease, fowl paratyphoid disease, etc. caused by chicken-origin E.coli.

The coliphage PD2061 or the phage composition has obvious effect of preventing and treating diseases caused by chicken-origin escherichia coli and/or salmonella infection.

In a fourth aspect, the present application also provides a phage pharmaceutical preparation, the active ingredient of which comprises the aforementioned coliphage PD2061 or the aforementioned phage composition.

Preferably, the phage pharmaceutical preparation also comprises other antibacterial or bactericidal active ingredients, and the pharmaceutical preparation is in the form of oral administration dosage form, external administration dosage form or parenteral administration dosage form. The phage preparation is applied by adding phage or its composition as therapeutic drug into drinking water or feed, or by drenching chicken, subcutaneously injecting, intramuscular injecting, and can prevent and treat colibacillosis, improve survival rate, etc.

Optionally, the phage pharmaceutical formulation 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 abrogate the biological activity and properties of the administered active component. In order to formulate the pharmaceutical composition into a liquid formulation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, ringer's solution, buffered saline, albumin infusion, dextrose solution, maltodextrin solution, glycerol, ethanol, various types of media, and the like. They may be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers, bacteriostats, and the like, may be added as desired. When combined with diluents, dispersants, surfactants, binders and/or lubricants, the compositions of the present invention can also be prepared in injection and oral dosage forms (e.g., aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilisates.

Optionally, the phage pharmaceutical formulation is a powder, which further comprises skimmed milk powder and lactose as protective agents.

Optionally, the phage powder comprises the following components in parts by weight, namely skimmed milk powder, lactose and phage suspension.

The phage powder is prepared by mixing skimmed milk powder, lactose and phage suspension in the same amount, and spray drying.

Preferably, the spray drying condition is that the inlet temperature is 120 ℃, the air flow speed is 120L/min, the spray rate is 100%, the size of a spray head cover is 4.0 mu m, and the spray drying is about 1 h.

In a fifth aspect, the application also provides a feed additive comprising the aforementioned coliphage PD2061 or the aforementioned phage composition. Preferably, the concentration of phage is above 10 ¹⁰ PFU/mL.

The drinking water additive or the feed additive is added into water or mixed with feed to feed chickens, so that drinking water and feed of chickens are disinfected and sterilized, and the acid resistance of the phage can survive in gastric acid environment, and the colibacillosis is effectively prevented and treated.

The application also provides an additive for poultry drinking water, which comprises the escherichia coli phage PD2061 or the phage composition. Preferably, the concentration of phage is above 10 ¹⁰ PFU/mL.

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

In a sixth aspect, the present application also provides an environmental disinfectant, the active ingredient of which comprises the aforementioned coliphage PD2061 or the aforementioned phage composition.

Preferably, the concentration of phage is above 10 ⁸ PFU/mL. Preferably, the environmental disinfectant also contains other active ingredients for the inhibition or elimination of bacteria in the environment.

The application also provides an application of the environment disinfectant in the environment disinfection of chicken farms, wherein the application method of the environment disinfectant comprises the steps of disinfecting escherichia coli and/or salmonella of a breeding environment and a breeding device through spraying and soaking, and the breeding environment comprises feeding tools such as chicken houses, chicken cages, drinking vessels, food tanks and the like, excrement and padding.

The application method comprises, but is not limited to, sterilizing and decontaminating water distribution systems of chicken farms, breeding facilities, breeding appliances or other environmental surfaces in the forms of liquid soaking, spraying, combined use with aqueous carriers and the like, and sterilizing and preserving feed, wherein the environmental disinfectant can be used for replacing antibiotics or traditional sterilizing products to exert a sterilizing effect, is safe to use and does not damage human bodies and poultry.

In a seventh aspect, the application also provides a chicken disinfectant comprising the aforementioned coliphage PD2061 or the aforementioned phage composition.

The chicken disinfectant can be used for achieving the sterilization of escherichia coli and/or salmonella by spraying or soaking the surface of the raw chicken after the treatment in a slaughterhouse.

In addition, studies have shown that when antibiotics are combined with Phage, they have a synergistic effect (Phage-Antibiotic Synergy, PAS), and when Phage is combined with antibiotics, phage can cause bacterial cell elongation or filiform action through certain mechanisms, such as certain antibiotics, so as to enhance sensitivity of the bacteria to Phage lysis, increase plaque size, accelerate Phage amplification so as to enhance Phage lysis capability, and meanwhile Phage can increase sensitivity of the bacteria to certain antibiotics, thereby reducing Minimum Inhibitory Concentration (MIC) of the antibiotics and reducing antibiotic usage. Therefore, the development of antibiotic and phage combination formulations is of market application value.

Therefore, in the eighth aspect, the application also provides a phage antibiotic combined preparation, the active ingredient of which is the coliphage PD2061 or the mixture of the phage composition and antibiotics, wherein the antibiotics are any one or more than two of compound sulfadiazine, gentamycin sulfate, spectinomycin hydrochloride and lincomycin hydrochloride.

Experiments prove that after phage PD2061 is added, the MIC value of the compound sulfadiazine suspension is reduced by 64 times, the MIC value of gentamycin sulfate powder is reduced by 32 times, and the MIC value of spectinomycin hydrochloride lincomycin hydrochloride mixed powder is reduced by 8 times. Furthermore, in the case of treatment with the same antibiotic, there was a significant reduction in the number of dead panning in the group of chickens suffering from colibacillosis compared to the group without phage addition, demonstrating that phage PD2061 works better in combination with the antibiotic than in the case of the antibiotic alone.

The results show that the use of the phage antibiotic combined preparation can greatly reduce the dosage of antibiotics and has better control effect on the colibacillosis of poultry. The antibiotics with different formulations can be selected to be directly mixed with phage or diluted and then mixed with phage.

Alternatively, the active ingredient of the phage antibiotic combination preparation is the aforementioned coliphage PD2061 or a mixture of the aforementioned phage composition and antibiotic. When diluted, the content of phage PD2061 in 1L of water is 1.0X10 ^7~10⁸ PFU, and the antibiotic is compound sulfadiazine or gentamycin sulfate or mixture of spectinomycin hydrochloride and lincomycin hydrochloride.

Wherein, in each 1L of water, the content of the compound sulfadiazine is 0.08-0.18 g (or 0.2-0.45 ml of compound sulfadiazine suspension (the concentration is 0.4 g/ml)), the content of the sodium gentamicin sulfate is 0.1-0.15 g (or 2-3 g of sodium gentamicin sulfate soluble powder (0.05 g/g of spectinomycin hydrochloride lincomycin soluble powder)), the content of the spectinomycin hydrochloride and the lincomycin hydrochloride is 0.2-0.4 g of the spectinomycin, and 0.1-0.2 g of the lincomycin hydrochloride (or 0.5-1 g of the spectinomycin hydrochloride lincomycin soluble powder (0.4 g/g, 0.2g/g of the lincomycin hydrochloride)).

Preferably, when the phage PD2061 is used, the dosage of the phage PD2061 is 1.0X10 ⁸ PFU/L, the content of the compound sulfadiazine is 0.08g/L (or 0.2ml/L of compound sulfadiazine suspension (0.4 g/ml), the content of the sodium gentamicin sulfate is 0.1g/L (or 2g/L of sodium gentamicin sulfate soluble powder (0.05 g/g of spectinomycin hydrochloride lincomycin soluble powder)), the content of the spectinomycin hydrochloride and the lincomycin hydrochloride is 0.2g/L of the spectinomycin, and the content of the compound sulfadiazine is 0.1g/L of the lincomycin hydrochloride soluble powder (0.4 g/g of the spectinomycin) or 0.5g of the spectinomycin hydrochloride is 0.2g/g of the lincomycin).

The invention has the following beneficial effects:

1. The invention provides an escherichia coli phage PD2061 with a wide lysis spectrum, which not only has high lysis rate on escherichia coli, but also can lyse salmonella, and has greater advantages in preventing and treating diseases of escherichia coli and salmonella mixed infection. The phage can be used for preparing a pharmaceutical preparation for treating or preventing colibacillosis and/or salmonellosis of chickens, so that the survival rate of colibacillosis/salmonellosis or mixed infection of chickens is improved, the utilization rate of feed is improved, and the death and panning rate of chickens in cultivation is reduced.

The phage can be used as an active ingredient of an environment disinfectant, a feed additive and a chicken raw meat disinfectant, and solves the problems of antibiotic residue and pathogen resistance caused by using antibiotics while solving the problem of pathogen infection.

2. The phage is obtained from nature, is easy to carry out industrial production, and the medicine or disinfectant prepared from the phage not only can reduce cost, but also has the advantage of green and environment-friendly, and cannot cause harm to beneficial bacteria in the environment.

3. The invention also provides a phage antibiotic combined preparation, which can effectively reduce the dosage of antibiotics in practical application, reduce the production cost of yield and improve the control effect on corresponding diseases.

Drawings

FIG. 1 is a plaque photograph of E.coli phage;

FIG. 2 is an electron microscope image of E.coli phage;

FIG. 3 is a graph showing the results of a test for pH stability of E.coli phage;

FIG. 4 is a one-step growth curve of E.coli phage;

FIG. 5 shows the results of a test for the temperature stability of E.coli phage.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

EXAMPLE 1 isolation and purification of E.coli phage PD2061

1. Experimental method

(1) Resuscitating and culturing host bacteria

Selecting 20 strains of escherichia coli (JD 1-JD 20) stored in a laboratory, carrying out streak resuscitating on the sterilized inoculated bad dip frozen stock solution on a MAC (media access control) culture medium, culturing for 18-24 hours at a constant temperature of 37 ℃ to obtain single bacterial colonies, selecting the single bacterial colonies, inoculating the single bacterial colonies into 5mL of NB broth, and carrying out shake culture for 16 hours at the speed of 37 ℃ and 170rpm to obtain fresh escherichia coli bacterial solution.

(2) Isolation of phages

Taking a proper amount of chicken feather, fecal water and other samples from Shandong region, adding a proper amount of broth culture medium into a sample soaking bottle, adding 20 strains of escherichia coli bacterial liquid obtained by multiplication, placing the mixed liquid into a dish with the concentration of 37 ℃ and 170 rpm/min for shake culture for 12h, centrifuging for 5min at 11000 rpm, filtering with a sterile microporous filter membrane with the concentration of 0.22 mu m to obtain phage multiplication liquid, diluting phage stock solution by 10 times, taking proper gradient phage dilution liquid, respectively mixing the phage stock solution with 20 strains of escherichia coli (JD 1-JD 20) one by one in 1, incubating for 5min at the temperature of 37 ℃, absorbing 200 mu L of mixed liquid, placing the mixture into an upper agar (with the concentration of agar being 0.7%), pouring the mixture into a dish with the concentration of agar being 1.5% after the mixture is mixed uniformly, shaking and evenly placing the agar onto a culture medium for solidification, and placing the dish into a 37 ℃ incubator for inversion culture for 4-6 h to obtain a double-layer plate for forming bacterial plaques.

(3) Purification of phages

Individual plaques were picked up on double agar medium to form plaques and incubated in 1mL of NB broth on a thermostated shaker at 37 ℃,170rpm for about 30min to give phage extract. Uniformly mixing phage leaching liquid and corresponding spotted escherichia coli (hereinafter referred to as host bacteria) proliferation liquid in a ratio of 1:1 (incubating for 5min at 37 ℃), sucking 200 mu L of the mixture, uniformly mixing the mixture with upper agar, rapidly pouring the mixture onto a lower agar plate, shaking the mixture, uniformly flattening the mixture until a culture medium is solidified, and reversely culturing the mixture in a 37 ℃ incubator for 4-6 h to obtain a double-layer plate for forming the plaques again. Single plaques were picked with sterile forceps on double-layered medium to form plaques and placed in 1mL NB broth and incubated on a thermostatic shaker at 37℃and 170rpm for about 30min to give phage extract. Repeating the steps for 3 times to obtain the purified phage leaching solution.

(4) Phage proliferation and potency assay

And (3) taking an equal amount of purified phage leaching solution and host bacteria proliferation solution, culturing in 5mL of liquid NB medium at 37 ℃ with shaking at 170rpm until the liquid becomes clear, centrifuging the clear liquid at 11000rpm for 10min, taking the supernatant, filtering the supernatant by using a sterile microporous filter membrane with the size of 0.22 mu m to obtain phage proliferation solution, and measuring the titer of the newly separated phage by adopting a double-layer flat plate method. As shown in FIG. 1, E.coli phage formed clear plaques on double-layered agar medium plates, no halos around, and clear edges.

2. Experimental results and analysis

12 E.coli phages were selected using 20 E.coli isolates according to the above experimental procedure, numbered PD2061-PD2070, respectively. The 12 coliphage forms transparent plaques on a double-layer agar culture medium plate, no halo is arranged around the coliphage, the edge is clearly visible, the diameter is about 0.2 mm-1.0 mm, the titer is 2.8X10 ⁹PFU/mL-3.0×10¹¹ PFU/mL, and the results are shown in the following table 1.

TABLE 1 titers of 12 E.coli phages newly isolated (pfu/mL)

Example 2 morphological observations and identification of E.coli phage PD2061

1. The experimental method comprises the steps of copper mesh manufacturing and electron microscope observation, namely, taking 20 mu L of phage sample, dripping the phage sample on the copper mesh with the carbon coating film, after natural precipitation of the phage sample is 15 min, slightly drying by using filter paper, then dyeing the phage sample for 1-2 min by using 2% (W/V) phosphotungstic acid (PTA), slightly drying by using the filter paper, and after the phage sample is dried, observing and photographing by using a transmission electron microscope.

2. Identification result

As shown in FIG. 2, it is observed by electron microscopy that the phage PD2061 has a polyhedral head structure and a contractile tail, the head is 60-65 nm wide, the head is 78-83 nm long, and the tail is about 90-93 nm long, and the shape of the phage PD2061 of the present application accords with the characteristics of the myophagidae and belongs to myophagides according to the classification method of the International Commission for virus classification (ICTV).

Example 3 determination of the cleavage spectrum of E.coli phage PD2061

1. Experimental materials

The host bacteria include 20 clinical isolates (JD 1-JD 20) of E.coli in example 1 and 60 E.coli from different animal sources stored in laboratory, including chicken-origin E.coli, duck-origin E.coli, pigeon-origin E.coli, goose-origin E.coli, pig-origin E.coli, donkey-origin E.coli, etc. (see Table 2).

2. The experimental method comprises the following steps:

the 80 pathogenic strains of Escherichia coli from different sources isolated from different regions stored in a laboratory are selected, and the lysis rate of the Escherichia coli phage PD2061 on the 80 pathogenic strains is measured by a double-layer plate method.

3. Experimental results and analysis

From the results of the cleavage spectrum experiments in Table 2, it can be seen that:

(1) The coliphage PD2061 can lyse 72 strains in 80 strains of colibacillus of different animal species sources, the lysis rate is up to 90.00%, and the coliphage belongs to a broad-lysis-spectrum phage.

(2) The coliphage PD2061 has higher lysis rate on colibacillus of different animal sources. Wherein, the cracking rate of the chicken-derived escherichia coli is 90 percent (27/30), and the cracking rate of other zoo escherichia coli is basically 90 percent.

TABLE 2 results of cleavage spectrum of E.coli phage PD2061 against 80 E.coli

EXAMPLE 4 cleavage assay of phage PD2061 against Salmonella

1. Experimental method

In this example, 10 Salmonella gallinarum strains were selected and the cleavage spectrum of the E.coli phage PD2061 was measured in accordance with the cleavage spectrum measurement method in example 3.

2. Experimental results and analysis

As shown in the results of Table 3, the cleavage rate of the Escherichia coli phage PD2061 on the 10 strains of salmonella is 20%, and the results show that the phage PD2061 can cleave part of salmonella, can play a role in salmonella control, and has application advantages in control of mixed infection of Escherichia coli and salmonella.

TABLE 3 results of cleavage spectra of E.coli phage PD2061 against 10 Salmonella strains

Example 5 tolerance of E.coli phage PD2061 to pH

1. Experimental method

4.5 ML of NB broth with different pH values (1, 2,3,4, 5, 6, 7, 8, 9, 10, 11, 12 and 13) is added into a sterile test tube, three parts are taken as one part, the total of two parts are taken, then the test tube is placed into a water bath kettle with the temperature of 37 ℃, after the temperature is stable, 500 mu L of 3X 10 ¹¹ pfu/mL of phage PD2061 proliferation liquid is added, and the mixture is uniformly mixed for the water bath with the temperature of 37 ℃ for 1h, 2h and 3h. Immediately after the completion of the action, a proper amount of 1mol/L HCl or NaOH was added to the mixture to give a pH of the mixture of about 7, and 10-fold dilution was performed, and the titer was measured by taking a proper dilution gradient, and 3 replicates were set for each pH test tube. And drawing a phage pH stability curve by taking the pH value as an abscissa and the logarithmic value of phage titer as an ordinate.

2. Experimental results and analysis

As can be seen from FIG. 3, the titer of the phage PD2061 does not change significantly within the pH range of 4.0-10.0, which indicates that the phage can withstand a certain range of acid-base environments, and the titer of the phage can still be detected after 3 hours of action at the pH of 1.0 and the pH of 13.0, which proves that the phage has strong acid-base resistance and can adapt to a certain range of acid-base environments within a certain period of time. In addition, the phage has good acid resistance, lower potency loss when passing through gastric acid, and can fully play a role when entering into intestinal tracts.

Example 6 temperature stability of phages

1. The experimental method comprises the following steps:

the phage multiplication liquid with the same volume titer of 3X 10 ¹¹ pfu/mL is placed in each container at 60 ℃, 70 ℃, 80 ℃ and 90 ℃ respectively, two parallel samples are kept at each temperature, 10 min, 20 min and 60 min are respectively kept, sampling is carried out after the action is finished, each sample is immediately placed in an ice bath for cooling, 10-fold ratio dilution is carried out, and the titer is measured by taking a proper dilution gradient. And drawing a thermal stability curve of the phage by taking the temperature as an abscissa and taking the logarithmic value of phage titer as an ordinate.

2. Experimental results and analysis:

As shown in FIG. 5, the phage remained substantially active after 60℃at 60 min, remained at a titer above 10 ¹⁰ pfu/mL after 70℃at 60 min, remained at a titer above 10 ⁵ pfu/mL after 80℃at 60 min, and remained active after 90℃at 60 min. Thus, the phage was found to have higher thermostability.

Example 6 one-step growth curve of E.coli phage PD2061

1. Experimental method

Phage multiplication solution with a multiplicity of infection of 0.001 and fresh host bacteria multiplication solution were each 1mL, mixed well (timing was started at this time), incubated at 37℃for 5min, centrifuged at 13000rpm for 30s, the supernatant was aspirated as much as possible with a micropipette, washed 1 time with 5mL NB broth (centrifuged at 13000rpm for 30 s), and the supernatant was discarded. Suspending the precipitate (total volume of 5 mL) with preheated NB broth, mixing thoroughly, rapidly shaking and culturing in shaking table at 37deg.C and 170rpm, taking 150 μl out at 0 time and every 10min, centrifuging 10000 rpm for 1min,

The titer of phage is measured by a double-layer plate method after 10-fold dilution of NB broth, 3 parallels are made, the result is averaged, the infection time is taken as an abscissa, the titer of phage in an infection system is taken as an ordinate, a one-step growth curve is drawn, the incubation period and the outbreak period of phage are obtained, and the outbreak amount is calculated.

Burst amount = total number of phage at end of burst/total number of bacteria at beginning of burst

2. Experimental results and analysis

As can be seen from the results of FIG. 4, after the infection of the host bacteria with phage PD2061, the phage lysis period was about 80min, the incubation period was about 10min, the phage burst period was about 70min, and after 80min, the number of phages was substantially unchanged, and the phage had entered the stationary phase, at which time the titer could reach 3X 10 ¹¹ pfu/mL, and the burst amount of phage PD2061 was 150.

Example 7 determination of optimal multiplicity of infection (MOI) of Escherichia coli by Escherichia coli phage PD2061

1. Experimental method

And (3) selecting a single escherichia coli colony, inoculating the single escherichia coli colony into 5ml of NB broth culture medium, and culturing at 37 ℃ and 170rpm for 12-16 hours to obtain bacterial liquid. The bacterial concentration was measured by the pouring method, and the bacterial concentration was adjusted to 1X 10 ⁹cfu/mL、1×10⁸cfu/mL...1×10⁵ cfu/mL based on the measurement result. The phage concentration obtained by isolation in example 1 was adjusted to 1X 10 ⁵～1×10⁷ PFU/mL. Phage and bacterial fluids were added to NB medium according to the phage number to bacterial number ratios of Table 4 and propagated in a shaker at 37℃at 170rpm until the fluid became clear and the time to proliferation was recorded. Taking a proper amount of clear liquid, centrifuging at 11000rpm for 10min, taking supernatant, filtering with a sterile microporous filter membrane of 0.22 mu m, and determining the phage titer in the filtrate by a double-layer flat-plate method, wherein the MOI (phage number/bacterial number) with the highest phage titer is the optimal infection complex of the phage.

2. Experimental results

As shown in Table 4, the result shows that the PD2061 titer was the highest at a MOI of 0.001:1 and was 3.0X10 ¹¹ PFU/mL, and this result revealed that the highest propagation yield was achieved with a small initial input of phage PD2061 at the time of production, which was advantageous for mass industrial production.

TABLE 4 titers of E.coli phage PC817 at different infection complex numbers

Example 8 use of E.coli phages for environmental decontamination

1. Experimental method

A large amount of escherichia coli is detected in a certain broiler hatchery in the coastal city of Shandong province, and the sterilization effect is not obvious by using chemical disinfectants and the like. The experimental design uses coliphage PD2061 to prepare disinfectant to spray disinfect the hatching workshop, so as to determine the disinfection effect.

The preparation method of the phage environment disinfectant comprises the steps of taking phage PD2061 proliferation liquid and water to dilute the phage PD2061 proliferation liquid and water by 1:1000 times, and using the phage PD2061 proliferation liquid and water with the titer of 1.0X10 ⁷ PFU/mL. Meanwhile, the benzalkonium bromide disinfectant was diluted 1:25 with water as a control.

(1) The air disinfection method comprises the following steps:

Two hatching workshops with the same size are respectively found, the natural sedimentation method is adopted to sample the air before and after the hatching workshops, 5 points are taken as test points at the center point and the 4 points of the corner, the sampling points are 0.3 m away from the ground, the four points of the corner are 1m away from the wall, and XLD agar plates with the diameter of 9cm are placed at each point. Before disinfection, each sampling point uses 2 XLD agar plates, a petri dish cover is opened, sampling is carried out for 10min, a foggy line carried by a chicken farm is used for disinfection, an experimental group and a control group are arranged, the experimental group is treated by using phage environment disinfectants (10 mL/m ³), and the control group is disinfected by using benzalkonium bromide (1:25 dilution).

After 30min of sterilization, 2 XLD agar plates were placed at each of the five sampling points, the petri dish lid was opened, and sampling was performed for 10min. And placing the sampling culture dishes before and after disinfection in a 37 ℃ constant temperature incubator, culturing for 12-24 hours, and counting the cultured bacteria.

Counting the total number of colonies according to the Orthomson formula, wherein C is the total number of colonies per cubic meter (CFU/m 3), N is the number of colonies per dish, A is the area of the culture dish (cm 2), and T is the sampling time (min).

2. Experimental results and analysis

The results are shown in Table 5, in the control group, the extinction rate of E.coli in the hatching workshop after being sterilized by the benzalkonium bromide disinfectant is 47.47%, and in the experimental group, the extinction rate of E.coli in the hatching workshop after being sterilized by the phage mixture preparation is 89.52%. Therefore, the environmental disinfection effect of the phage mixed preparation is obviously better than that of the prior benzalkonium bromide chemical disinfectant, and the use is safer.

TABLE 5 bacterial colony counts of E.coli before and after the disinfection of phage disinfectants and benzalkonium bromide disinfectants

Therefore, the result shows that the phage mixed preparation has excellent sterilizing effect on escherichia coli in a hatching workshop, and can be popularized and applied as a novel biological environment disinfectant.

EXAMPLE 9 therapeutic Effect of E.coli phages on colibacillosis in chicken farms

1. Experimental method

Selecting a certain laying hen field in north, wherein the death rate in the field is up to 2.91%, abnormal egg soft shell eggs appear, the laying rate is reduced by about 15%, dissecting the dead and panning chicken on site, finding that the surfaces of organs such as livers are covered with a layer of yellow cheese-like substances, aseptically collecting liver samples, carrying the liver samples back to a laboratory for detection, finding that the chicken field is mainly the rising of the death rate and the reduction of the egg quality caused by escherichia coli infection, and immediately selecting two 1200 feather chicken houses as a test group and a control group respectively.

(1) Treatment of test groups the phage preparation prepared was added to the waterline to give a total phage concentration of 1X 10 ⁸ pfu/mL in the waterline for five consecutive days. No phage was added to the control treated waterline. Other immunization and administration procedures were performed following the farm procedure.

(2) And simultaneously counting the daily dead-panning number, laying rate and the like of the chicken houses of the test group and the control group, performing split inspection on the dead-panning chickens of the test group and the control group, and separating and identifying the bacterial strains in the livers of the dead-panning chickens.

2. Experimental results and analysis

(1) As shown in Table 6, the test group using the coliphage PD2061 can effectively reduce the death and panning number of chickens, reduce the mortality of colibacillus, gradually increase the laying rate, and has good treatment effect on colibacillus infection.

TABLE 6 statistics of therapeutic effect of phage preparations

EXAMPLE 10 prevention effect of phage preparation on colibacillosis in chickens

1. Experimental method

Selecting a certain broiler farm in the south and 1 day old chicks, wherein 100 chicks are adopted in a test group and 100 chicks are adopted in a control group, and performing experiments according to the following method:

(1) The phage preparation adopted in the embodiment adopts a freeze-dried powder formulation, and the preparation method comprises the steps of selecting skimmed milk powder and lactose as protective agents, adding the phage preparation, wherein the specific ratio of the skimmed milk powder to the lactose to the phage is 1:1:8, drying by using a Swiss BUCHIB-90 nanometer spray dryer, collecting spray-dried particles after spray drying for about 1 hour, and finally obtaining phage powder, and the total concentration of phage is 1X 10 ¹⁰ PFU/g.

Wherein, the technological parameters of spray drying are that the inlet temperature is 120 ℃, the air flow speed is 120L/min, the spray rate is 100 percent, and the size of a nozzle cover is 4.0 mu m.

(2) Experimental group treatment the phage powder prepared was used by adding 500 g/ton to feed. Control group treatment, wherein phage powder is not added into the feed. Other immunization and administration procedures were performed following the farm procedure.

(3) The total death rate after week 1 and week 2 was measured, the strain in the liver of the dead-spotted chickens was measured, and the total weight gain rate was counted.

2. Experimental results and analysis

As shown in Table 7, the overall mortality rate of the phage group was lower than that of the control group to which no phage was added, the detection rate of E.coli in the phage group-killed-at-the-time chicken was far lower than that of the control group, and the weight gain rate (1.25%) of the phage group was higher than that of the control group (100%), and the isolation rate (12.5%) of E.coli was far lower than that of the control group (69.23%), which revealed that the phage preparation of the present invention had excellent effect in E.coli prevention, and was able to improve the feed utilization rate and the weight gain of chicks.

TABLE 7 statistics of the results of the prophylactic effect of phage cocktail preparations

EXAMPLE 11 in vitro synergy of phage preparations with antibiotics

1. Experimental method

(1) A strain of Escherichia coli DC1 isolated from chicken farm sensitive to phage PD2061 was selected, and after proliferation, the bacterial concentration was adjusted to 1X 10 ⁷ cfu/ml.

(2) Dissolving the antibiotic compound sulfadiazine suspension, gentamicin sulfate powder and spectinomycin hydrochloride lincomycin hydrochloride soluble powder by using sterile water in a ratio of 1:9, and then putting the mixture into a shaking table at 170rpm and 37 ℃ to fully and uniformly mix for 30min to obtain various antibiotic solutions.

(3) The minimum inhibitory concentration MIC value (MIC value is expressed as a dissolution multiple of powder/suspension) of the antibiotics is measured by a 96-well plate method, the dilution multiple of the MIC of the antibiotics is diluted by a 2-fold ratio for 10 times, 50ul of each of all dilutions is added into the 96-well plate, 50ul of phage PD2061 is added into each well (the titer of added phage is 1.0X10 ⁷ PFU/ml), 100ulNB broth is taken as a culture solution adding hole, the culture broth is placed into a microbial growth curve analyzer, the parameters are set to 37 ℃ and 800rpm are set for 12 hours, and the observation and statistics result on the next day are compared.

2. Experimental results

(1) The results are shown in Table 8, and it is clear from the results that the MIC value of the antibiotic compound sulfadiazine suspension to the Escherichia coli DC1 was 0.025mg/ml when no phage was added, and the MIC value was reduced by 64-fold when the compound sulfadiazine suspension was diluted 0.00039mg/ml after phage PD2061 was added.

The MIC value of the antibiotic gentamycin sulfate powder to the escherichia coli DC1 is 0.0015mg/ml, and after phage PD2061 is added, the gentamycin sulfate powder still has an antibacterial effect when diluted to 0.000048mg/ml, and the MIC value is reduced by 32 times.

The antibiotic spectinomycin hydrochloride has a MIC value of 0.2mg/ml for the escherichia coli DC1, and after phage PD2061 is added, the spectinomycin hydrochloride lincomycin hydrochloride (which is a finished product mixed antibiotic) still has an antibacterial effect when diluted to 0.025, and the MIC value is reduced by 8 times.

(2) After phage PD2061 is added into various antibiotics, the minimum antibacterial concentration of the antibiotics is greatly reduced, and the combined use of phage and antibiotics can effectively reduce the use amount of the antibiotics, reduce the use cost of the antibiotics and reduce the antibiotic residues in the environment.

TABLE 8 synergistic effects of phage PD2061 with antibiotics

EXAMPLE 12 treatment of colibacillosis in chickens with phage antibiotic combination preparations

The virulent strain DC1 sensitive to phage PD2061 was selected, 160 chickens of 1 day old were selected, and the 160 chickens were equally divided into the following eight groups of 20 chickens each, each operating as follows:

In the test 1 group, firstly, the toxicity counteracting treatment is carried out, then, the compound sulfadiazine suspension is immediately used for treatment, the dosage is that 0.2ml of the compound sulfadiazine suspension is added into 1L of water, and drinking water is used for 5 days;

The test 2 groups are treated by first carrying out toxicity eliminating treatment, then immediately treating by using the soluble powder of the gentamicin sodium sulfate (the dosage of the use is that 2g of the soluble powder of the gentamicin sodium sulfate is added into each L of water) and continuously using for 5 days;

test 3 groups, namely, carrying out toxicity attack treatment firstly, and then using the spectinomycin hydrochloride lincomycin hydrochloride soluble powder for treatment (the dosage of the treatment is that 0.5g of the spectinomycin hydrochloride lincomycin hydrochloride soluble powder is added into 1L of water) for continuous use for 5 days;

Test 4 groups, namely, firstly carrying out toxicity counteracting treatment, then immediately using compound sulfadiazine suspension (sulfadiazine 0.4g/ml of water of Wen Shida Hua of the biological technology limited company of Guangdong, the usage amount is that 0.2ml of compound sulfadiazine suspension is added into 1L of water), and simultaneously using phage PD2061 (the usage amount is that 10ml of phage proliferation liquid is added into 1L of water, and the phage titer is 1.0X10 ⁷ PFU/ml) for treatment, and continuously using for 5 days;

Test 5 groups, namely, firstly carrying out toxicity counteracting treatment, then immediately treating by using soluble powder of gentamicin sulfate (gentamicin 0.05g/g of Teanhao Xinkangda biological Co., ltd., the dosage of which is 1L of water, adding 2g of soluble powder of gentamicin sodium sulfate) and using phage PD2061 (the dosage of which is 10ml of phage proliferation liquid is added to every 1L of water, and the phage titer is 1.0X10 ⁷ PFU/ml) for 5 days;

The test 6 groups are treated by first carrying out toxicity attack, then immediately using the spectinomycin hydrochloride lincomycin soluble powder (0.4 g/g of spectinomycin and 0.2g/g of lincomycin by the Guangdong Wen Shida Huanong biotechnology Co., ltd.) and using the spectinomycin hydrochloride lincomycin soluble powder of which the dosage is 0.5g per 1L of water together with the phage PD2061 (the dosage is 10ml of phage proliferation liquid per 1L of water and the phage titer is 1.0X10 ⁷ PFU/ml) for treatment, and then using for 5 days;

Test 7 groups, after the treatment of counteracting toxic substances, do no treatment;

and in the control group, the virus elimination is not carried out, and normal breeding is used as the control group.

The method for the detoxification treatment comprises the step of performing intraperitoneal injection detoxification on the chicks by using 0.2ml of escherichia coli DC1 with the concentration of 1 multiplied by 10 ⁷ cfu/ml.

Total mortality was counted for each group of chicks daily for 5 days.

2. Experimental results

As shown in table 9, in the case of treatment with the same antibiotic, the number of dead chicks in the test groups 4 to 6 to which phage was added was significantly reduced compared to the test groups 1 to 3 to which no phage was added and only the antibiotic was used, for example, the total number of dead chicks in the test group 4 was lower than the test group 1, the total number of dead chicks in the test group 5 was lower than the test group 2, and the total number of dead chicks in the test group 6 was lower than the test group 3. The results demonstrate that the phage PD2061 in combination with the antibiotic was better for the treatment of chicken colibacillosis than the single antibiotic group.

Table 9 phage antibiotic combination treatment of death and panning

Implementation example 13 treatment of Mixed infection with phage antibiotics in combination preparations for E.coli and Salmonella in chickens

The virulent strains DC1 and SM1 sensitive to the phage PD2061 are selected, 160 chickens with the age of 1 day are selected, the 160 chickens are equally divided into the following eight groups, 20 chickens in each group, and the operations are respectively as follows:

The test 1 group comprises the steps of firstly carrying out toxicity counteracting treatment, then immediately using 0.4g/ml of sulfadiazine of compound sulfadiazine suspension of Wen Shida Hua biological technology Co., guangdong, adding 0.2ml of compound sulfadiazine suspension into 1L of water, and drinking water for 5 days;

The test 2 groups, namely, firstly carrying out toxicity counteracting treatment, then immediately using soluble powder of gentamicin sodium sulfate for treatment (0.05 g/g of gentamicin of Thailand Kangda biological Co., ltd.) and adding 2g of soluble powder of gentamicin sodium sulfate into each L of water for continuous use for 5 days;

Test 3 groups, namely, firstly carrying out toxicity counteracting treatment, then using spectinomycin hydrochloride lincomycin soluble powder (0.4 g/g of spectinomycin) of Wen Shida Hua of agricultural biotechnology limited company in Guangdong for treatment, wherein 0.5g of the spectinomycin hydrochloride lincomycin soluble powder is added into 1L of water for continuous use for 5 days;

Test 4 groups, namely, firstly carrying out toxicity counteracting treatment, then immediately using compound sulfadiazine suspension (sulfadiazine 0.4g/ml of water of Wen Shida Hua of biological technology Co., guangdong, the dosage of which is 0.2ml of compound sulfadiazine suspension is added into 1L of water), and simultaneously using phage PD2061 (the dosage of which is 10ml of phage proliferation liquid is added into 1L of water, and the phage titer is 5.0X10 ⁷ PFU/ml) for treatment, and continuously using for 5 days;

test 5 groups, namely, firstly carrying out toxicity attack treatment, then immediately treating by using soluble powder of gentamicin sulfate (gentamicin 0.05g/g, the dosage of which is 1L of water 2 g) and using phage PD2061 (10 ml of phage proliferation liquid is added into 1L of water, and the phage titer is 5.0X10 ⁷ PFU/ml) for 5 days;

Test 6 groups, namely, firstly carrying out toxicity counteracting treatment, then immediately using spectinomycin hydrochloride lincomycin soluble powder (Guangdong Wen Shida Huanong biotechnology Co., ltd., spectinomycin 0.4g/g, lincomycin 0.2 g/g) and using phage PD2061 (using amount is 10ml phage proliferation liquid per 1L water, phage titer is 5.0X10 ⁷ PFU/ml) for treatment, and using for 5 days;

The method for the virus-killing treatment comprises the step of carrying out intraperitoneal injection virus-killing on chickens by using 0.2ml of escherichia coli DC1 with the concentration of 1X 10 ⁷ cfu/ml and 0.2ml of salmonella SM1 with the concentration of 1X 10 ⁷ cfu/ml.

Total mortality was counted for each group of chicks daily for 5 days.

2. Experimental results

As shown in Table 10, in the case of using the same antibiotic, the number of the dead pigs in the test group 4 to 6 added with phage was significantly reduced compared with the test group 1 to 3 not added with phage but with antibiotic, for example, the total number of the dead pigs in the test group 4 was lower than that in the test group 1, the rate of the dead pigs was reduced by 50%, the total number of the dead pigs in the test group 5 was lower than that in the test group 2, the rate of the dead pigs was reduced by 55%, the total number of the dead pigs in the test group 6 was lower than that in the test group 3, and the rate of the dead pigs was reduced by 45%. The results show that the phage PD2061 can effectively control the death and the panning rate and obviously reduce the death and panning rate when being combined with the three antibiotics in the face of mixed infection of escherichia coli and salmonella, and the treatment effect is obviously better than that of single use of the antibiotics.

Table 10 phage antibiotic combination treatment of death and panning

Example 14 safety test of phage

1. Experimental method

60 Chicks of 1 day old are selected and divided into 30 phage groups and 30 control groups, wherein 0.2 mL of 1X 10 ¹⁰ PFU phage are orally taken by the phage groups, and the chicks are fed and observed for 7 days. The control group was orally administered with the same dose of sterile physiological saline. And (3) performing a section examination to observe pathological changes of heart, liver, spleen, lung, kidney, brain, intestinal tract, and observing states such as spirit, ingestion and the like in the feeding process. 2. Experimental results and analysis

No disease or toxic symptoms, mental states and normal feeding were observed in chickens from phage and control groups throughout the dosing period. Through detailed clinical section observation, the main organs and intestinal tracts of the chicks are normal no matter in a phage group or a control group. This indicates that phage PD2061 is highly safe and has no adverse effect on the animal organism.

EXAMPLE 14 Whole genome analysis of E.coli phage PD2061

The genome of phage PD2061 was extracted, subjected to whole genome sequencing and sequence analysis, and the results were as follows:

The genome has the total length of 166913 bp, the G+C content of 35.49%, and the base C, G, A, T content of 16.71%, 18.78%, 32.72% and 31.77% in sequence. The results of online annotation of the whole genome RAJD showed that the genome contained 271 Open Reading Frames (ORFs). Meanwhile, 271 ORFs contain a highly conserved terminal enzyme large subunit (PHAGE TERMINASE, large subunit) which is shown as a sequence 1 in a sequence table, and the genome is analyzed by an online tool CGE SERVER to be free of drug resistance genes and virulence genes. The genome was analyzed PHAJDER to be free of lysogenic related genes.

It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.

Claims

1. An Escherichia coli phage PD2061, characterized in that its deposit number is CGMCC No. 46163.

2. A phage composition, characterized in that it 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 phage pharmaceutical preparation, characterized in that its active ingredient comprises the Escherichia coli phage PD2061 according to claim 1 or the phage composition according to claim 2.

5 . The phage pharmaceutical preparation according to claim 1 , wherein 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 pharmaceutical 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 it 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. The use of the environmental disinfectant as claimed in claim 7 in the disinfection of a chicken farm environment is 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, wherein 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 it comprises the Escherichia coli phage PD2061 according to claim 1 or the phage composition according to claim 2.