Escherichia coli bacteriophage, bacteriophage compositions and uses thereof, bacteriophage preparations

Abstract

This invention proposes an *Escherichia coli* bacteriophage, a bacteriophage composition, its application, and a bacteriophage preparation, relating to the field of microbial technology. The *Escherichia coli* bacteriophage is named *Escherichia coli* bacteriophage GaoM7-14E, with accession number CGMCC NO.46188, deposited on September 20, 2024. Another *Escherichia coli* bacteriophage is named *Escherichia coli* bacteriophage JM3e, with accession number CGMCC NO.46186, deposited on September 18, 2024. The technical solution of this invention uses bacteriophage GaoM7-14E and bacteriophage JM3e as a bacteriophage composition, which can synergistically inhibit *Escherichia coli*, thereby inhibiting pathogenic *Escherichia coli*, and thus preventing and treating *Escherichia coli* diseases.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Escherichia coli bacteriophages, bacteriophage compositions and their applications, and bacteriophage preparations. Background Technology

[0002] Avian colibacillosis is an infectious disease in poultry caused by avian pathogenic Escherichia coli (APEC). Avian pathogenic Escherichia coli is a type of extraintestinal pathogenic Escherichia coli (ExPEC) that is primarily transmitted through the respiratory tract. It can cause disease in poultry such as chickens through virulence factors such as pili, invasive agents, iron acquisition systems, K1 capsules, and temperature-sensitive hemagglutinins. Symptoms include peripheral hepatitis, air sacculitis, pericarditis, peritonitis, and salpingitis; severe cases can lead to death in poultry.

[0003] Avian colibacillosis is one of the leading causes of morbidity and mortality in poultry, causing irreversible economic losses to the poultry industry, especially chicken farming, and posing an economic threat to the global poultry industry. Chicken farms have long used antibiotics to prevent and treat infections caused by pathogenic E. coli in poultry. However, the widespread use and even abuse of antibiotics has led to the continuous emergence and spread of multidrug-resistant strains (MDR), posing challenges not only to the treatment of avian colibacillosis but also adversely affecting human health. Summary of the Invention

[0004] The main objective of this invention is to provide an Escherichia coli bacteriophage, a bacteriophage composition and its application, and a bacteriophage preparation, with the aim of providing a bacteriophage capable of preventing and treating avian colibacillosis.

[0005] To achieve the above objectives, this invention proposes an Escherichia coli phage, which is taxonomically named Escherichia coli phage, specifically named Escherichia coli phage GaoM7-14E, with accession number CGMCC NO.46188 and accession date of September 20, 2024.

[0006] This invention proposes an Escherichia coli phage, which is taxonomically named Escherichia coli phage, specifically Escherichia coli phage JM3e, with accession number CGMCCNO.46186 and accession date of September 18, 2024.

[0007] This invention proposes a phage composition comprising Escherichia coli phage GaoM7-14E and Escherichia coli phage JM3e;

[0008] The *Escherichia coli* phage GaoM7-14E is the *Escherichia coli* phage GaoM7-14E as described above, and the *Escherichia coli* phage JM3e is the *Escherichia coli* phage JM3e as described above.

[0009] In one embodiment, the titers of both Escherichia coli phage GaoM7-14E and Escherichia coli phage JM3e are not less than 10. 7 PFU / mL.

[0010] In one embodiment, the infection multiplicity (MOI) is 0.001 to 10.

[0011] This invention proposes the application of bacteriophages in the preparation of antibacterial agents, wherein the bacteriophages include at least one of Escherichia coli bacteriophage GaoM7-14E and Escherichia coli bacteriophage JM3e;

[0012] The *Escherichia coli* phage GaoM7-14E is the *Escherichia coli* phage GaoM7-14E as described above, and the *Escherichia coli* phage JM3e is the *Escherichia coli* phage JM3e as described above.

[0013] In one embodiment, the antibacterial agent is used to inhibit Escherichia coli, including avian pathogenic Escherichia coli.

[0014] The present invention provides a phage preparation comprising the aforementioned phage composition.

[0015] In one embodiment, the phage preparation further includes poultry-acceptable excipients.

[0016] In one embodiment, the dosage form of the phage preparation includes at least one of the following: solution, powder, gel, granules, or lyophilized agent.

[0017] In the technical solution of this invention, by using Escherichia coli phage GaoM7-14E and Escherichia coli phage JM3e as a phage composition, the two phages can synergistically inhibit Escherichia coli, thereby inhibiting pathogenic Escherichia coli and preventing and treating Escherichia coli disease; at the same time, it avoids the phenomenon of Escherichia coli developing drug resistance due to the use of antibiotics. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a plaque result image of bacteriophage GaoM7-14E and bacteriophage JM3e in Example 1 of the present invention;

[0020] Figure 2 Electron micrographs of bacteriophage GaoM7-14E and bacteriophage JM3e in Example 2 of the present invention;

[0021] Figure 3 This is a schematic diagram of the whole genome of bacteriophage GaoM7-14E in Example 3 of the present invention;

[0022] Figure 4 This is a schematic diagram of the whole genome of bacteriophage JM3e in Example 3 of the present invention;

[0023] Figure 5 This is a graph showing the optimal MOI detection results for phage GaoM7-14E in Example 4 of the present invention;

[0024] Figure 6 This is a graph showing the optimal MOI detection results for bacteriophage JM3e in Example 4 of the present invention;

[0025] Figure 7 This is a one-step growth curve of bacteriophage GaoM7-14E in Example 5 of the present invention;

[0026] Figure 8 This is a one-step growth curve of bacteriophage JM3e in Example 5 of the present invention;

[0027] Figure 9 The figure shows the experimental results of the antibacterial effect of bacteriophage GaoM7-14E and bacteriophage JM3e in Example 7 of the present invention.

[0028] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them.

[0030] It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination should be considered non-existent and not within the scope of protection claimed by this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0031] Avian colibacillosis is an infectious disease in poultry caused by avian pathogenic Escherichia coli (APEC). Avian pathogenic Escherichia coli is a type of extraintestinal pathogenic Escherichia coli (ExPEC) that is primarily transmitted through the respiratory tract. It can cause disease in poultry such as chickens through virulence factors such as pili, invasive agents, iron acquisition systems, K1 capsules, and temperature-sensitive hemagglutinins. Symptoms include peripheral hepatitis, air sacculitis, pericarditis, peritonitis, and salpingitis; severe cases can lead to death in poultry.

[0032] Avian colibacillosis is one of the leading causes of morbidity and mortality in poultry, causing irreversible economic losses to the poultry industry, especially chicken farming, and posing a significant economic threat to the global poultry industry. Chicken farms have long used antibiotics to prevent and treat infections caused by pathogenic E. coli in poultry. However, the widespread use and even abuse of antibiotics has led to the continuous emergence and spread of multidrug-resistant strains (MDRs), posing challenges not only to the treatment of avian colibacillosis but also adversely affecting human health. With the increasing rate of antibiotic resistance, finding alternatives to antibiotics is urgently needed. To address the problem of antibiotic resistance in pathogenic E. coli in poultry farming and to find novel alternatives to antibiotics, bacteriophages have come into focus.

[0033] In view of this, the present invention utilizes sludge collected from chicken farms to screen and culture Escherichia coli bacteriophages. The Escherichia coli bacteriophages screened and cultured in this invention have been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The taxonomic name is *Escherichia coli* phage, specifically *Escherichia coli* phage GaoM7-14E, with accession number CGMCC NO.46188, and the deposit date is September 20, 2024.

[0034] This invention also proposes an Escherichia coli phage, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Its taxonomic name is Escherichia coli phage, specifically Escherichia coli phage JM3e, with accession number CGMCCNO.46186, and the deposit date is September 18, 2024.

[0035] The present invention also proposes a phage composition comprising Escherichia coli phage GaoM7-14E and Escherichia coli phage JM3e; wherein the Escherichia coli phage GaoM7-14E is the aforementioned Escherichia coli phage GaoM7-14E, and the Escherichia coli phage JM3e is the aforementioned Escherichia coli phage JM3e.

[0036] In the technical solution of this invention, by using Escherichia coli phage GaoM7-14E and Escherichia coli phage JM3e as a phage composition, the two phages can synergistically inhibit Escherichia coli, thereby inhibiting pathogenic Escherichia coli and preventing and treating Escherichia coli disease; at the same time, it avoids the phenomenon of Escherichia coli developing drug resistance due to the use of antibiotics.

[0037] In one embodiment, the titers of both Escherichia coli phage GaoM7-14E and Escherichia coli phage JM3e are not less than 10. 7 PFU / mL.

[0038] In one embodiment, the multiplicity of infection (MOI) is 0.001 to 10. Preferably, the MOI is 0.01 to 0.1.

[0039] This invention also proposes the application of bacteriophages in the preparation of antibacterial agents, wherein the bacteriophages include at least one of Escherichia coli bacteriophage GaoM7-14E and Escherichia coli bacteriophage JM3e; wherein the Escherichia coli bacteriophage GaoM7-14E is the aforementioned Escherichia coli bacteriophage GaoM7-14E, and the Escherichia coli bacteriophage JM3e is the aforementioned Escherichia coli bacteriophage JM3e.

[0040] In one embodiment, the antibacterial agent is used to inhibit Escherichia coli, including avian pathogenic Escherichia coli.

[0041] In the technical solution of the present invention, by using the antibacterial agent, pathogenic Escherichia coli in birds can be specifically inhibited, thereby preventing and treating avian colibacillosis.

[0042] The present invention also proposes a phage formulation comprising the aforementioned phage composition.

[0043] In the technical solution of the present invention, by preparing the phage composition into a phage preparation, it can be used to inhibit the growth and development of Escherichia coli, and at the same time expand the application range of the phage composition. In particular, Escherichia coli phage GaoM7-14E and Escherichia coli phage JM3e can specifically inhibit pathogenic Escherichia coli in birds, thereby preventing and treating avian colibacillosis.

[0044] In one embodiment, the phage preparation further includes poultry-acceptable excipients.

[0045] It should be noted that the excipients may be at least one of dispersants, stabilizers, fillers and solvents. The specific types are not limited here, as long as they are suitable for poultry farming.

[0046] In one embodiment, the dosage form of the phage preparation includes at least one of the following: solution, powder, gel, granules, or lyophilized agent.

[0047] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0048] The culture media, reagents, equipment, and materials used in the following examples are as follows:

[0049] LB (Luria-Bertani) liquid medium (g / L): YEAST EXTRACT 5g, TRYPTONE 10g, NaCl 10g, distilled water 1000mL. Weigh the above reagents, dissolve them in distilled water, and autoclave at 121℃ for 20min.

[0050] LB (Luria-Bertani) solid culture medium (g / L): YEASTE XTRACT 5g, TRYPTONE 10g, NaCl 10g, agar powder 20g, distilled water 1000mL. Weigh the above reagents, dissolve them in distilled water, autoclave at 121℃ for 20min, and pour into disposable sterile petri dishes under aseptic conditions for later use.

[0051] Phage buffer (200mL): NaCl 1.75g, Tris 1.21g, MgCl2·6H2O 0.41g, anhydrous CaCl2 0.04g; Weigh the above reagents, dissolve them in 200mL of distilled water, adjust the pH to 7.5 with dilute hydrochloric acid (6mol / L), and autoclave at 121℃ for 20min.

[0052] 0.5% water agar (g / L): 5g agar powder, 1000mL distilled water. Dissolve the agar powder in distilled water and autoclave at 121℃ for 20min.

[0053] Equipment and Instruments: Clean bench (Suzhou Purification Equipment Co., Ltd.), Full-temperature shaking incubator (Suzhou Peiying Experimental Equipment Co., Ltd.), DNP-9052 electric thermostatic incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.), Ultra-low temperature freezer (Changhong Meiling Co., Ltd.), Electric thermostatic drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.), METTLERAL204 electronic balance (Mettler-Toledo AG, Switzerland), MS100B magnetic stirrer (Shanghai Dam Industry Co., Ltd.), MINIB-100 mini metal bath (Hangzhou Mio Instrument Co., Ltd.), MTC-100 thermostatic mixer (Hangzhou Mio Instrument Co., Ltd.), DYY-6C double-stable electrophoresis apparatus (Beijing Liuyi Biotechnology Co., Ltd.), Life Express gradient PCR amplification instrument (Hangzhou Borui Technology Co., Ltd.), portable bacterial turbidity meter (Hangzhou Qiwei Instrument Co., Ltd.), vertical pressure steam sterilizer LDZX-50KBS (Shanghai Shenan Medical Machinery Factory), benchtop high-speed refrigerated centrifuge TGL-16M (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), transmission electron microscope JEM-1400 (Nippon Electron Co., Ltd.).

[0054] Example 1: Isolation and purification of Escherichia coli bacteriophage

[0055] Bacteriophages were isolated using *Escherichia coli* O157:H7 as the host bacterium. Chicken farm sludge was used to isolate the bacteriophages, which were stored at 4°C for later use.

[0056] Weigh 10g of sample into a sterilized Erlenmeyer flask containing 50mL of LB liquid medium. Add 10mL of phage buffer and 1mL of E. coli logarithmic phase suspension, mix well, and incubate overnight at 30°C. Centrifuge the liquid in the flask at 5000g at 4°C for 10min. Filter the supernatant through a 0.22μm filter to obtain the phage stock solution.

[0057] Take 500 μL of Escherichia coli logarithmic phase suspension, 500 μL of phage buffer, and 10 μL of phage stock solution and mix them into a test tube. Add 4 mL of water agar, mix well, spread evenly on a solid LB plate, and incubate overnight at 30°C.

[0058] Select single phage plaques with good growth and clear outlines and serially dilute them into 100 μL of phage buffer. Take 10 μL of each phage gradient solution and mix it thoroughly with 500 μL of *E. coli* suspension, 500 μL of phage buffer, and 4 mL of water agar. Spread the mixture evenly on LB agar plates and incubate overnight at 30°C. Repeat the above purification process 3-5 times until only a single phage plaque morphology appears on the same plate. The phage plaque results of the two phage strains on the bacterial growth are shown below. Figure 1 As shown. The bacteriophages were named GaoM7-14E and JM3e, respectively.

[0059] Example 2: Morphological observation of bacteriophages

[0060] 20 μL of the purified phage stock solution from Example 1 was dropped onto a copper grid and adsorbed for 10 min. Excess liquid was then blotted away with filter paper. Next, 20 μL of 2% phosphotungstic acid was added to the copper grid. After standing for 30 min, excess liquid was blotted away with filter paper, and the mixture was air-dried before observation and imaging under a transmission electron microscope. The electron microscopic morphology results of the two phage strains are as follows: Figure 2 As shown.

[0061] Example 3: Phage genome extraction, sequencing, and comparative genome analysis

[0062] After filtering the phage stock solution from Example 1 through a 0.22 μm filter, 500 μL of the solution was added to 10 μL of DNase I and incubated in a metal bath at 37 °C for 20 min to remove free DNA. After incubation, 500 μL of phage was added to 22 μL of EDTA to a final concentration of 20 mmol / L (0.5 M pH 8.0 EDTA) and incubated at 65 °C for 10 min. Then, 27.5 μL of 10% SDS was added to a final concentration of 0.5%, and 2.5 μL of 10 mg / L proteinase K was added to a final concentration of 50 mg / L. The solution was incubated at 55 °C for 1.5 h. Finally, 552 μL of DNA extraction phenol reagent was added, mixed, and centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was transferred to a new 1.5 mL centrifuge tube, an equal volume of DNA extraction phenol reagent was added, mixed, and centrifuged at 12000 rpm for 10 min at 4 °C. Transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal volume of chloroform-isoamyl alcohol (v / v 24:1), and centrifuge at 12000 rpm for 10 min at 4 °C. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 1 / 10 volume of 3M NaAc (pH 5.4) and 2 volumes of ice-cold anhydrous ethanol, mix well, and incubate at -20 °C for 2.5 h. Remove from -20 °C and centrifuge at 14000 rpm for 20 min at 4 °C, discarding the supernatant. Add 500 μL of ice-cold 70% ethanol, wash the precipitate, centrifuge at 12000 rpm for 3 min at 4 °C, discard the supernatant, and air-dry the precipitate in a clean bench for approximately 2 h. After air-drying, dissolve in 25 μL of ddH2O. Mix 5 μL of genomic sample with 2 μL of 6× Loading buffer and perform agarose gel electrophoresis for verification. Genomic samples showing bands are sent to Chengdu Life Baseline Technology Co., Ltd. for sequencing.

[0063] The whole genome pattern of bacteriophage GaoM7-14E is as follows Figure 3 As shown, the whole genome pattern of bacteriophage JM3e is as follows: Figure 4 As shown, the full-length genomes of bacteriophages GaoM7-14E and JM3e are 169,421 bp and 50,578 bp, respectively, and no known drug resistance genes or virulence genes were predicted in their genomes.

[0064] Comparative genomic analysis was performed on the two phages. The phage most closely related to GaoM7-14E was Enterobacteria phage QL01 (KT176190.1), with an ANI of 98.05% and an alignment coverage of 95%. The phage most closely related to JM3e was Escherichia phage Henu4_2 (PQ159479.1), with an ANI of 94.45% and an alignment coverage of 89%, indicating that JM3e is a novel Escherichia coli phage.

[0065] Example 4: Optimal MOI for bacteriophage

[0066] Dilute the bacteriophage and mix it with the overnight culture of host bacteria (10). 8 Mix 1 mL of phage and 1 mL of bacterial culture (CFU / mL) to achieve MOIs of 10, 1, 0.1, 0.01, and 0.001, respectively. Add 8 mL of LB liquid medium to each mixture, incubate at 37°C and 220 rpm for 12 h, centrifuge at 4°C and 8000 rpm for 10 min, filter the supernatant through a 0.22 μm filter, and measure the phage titer. The MOI corresponding to the highest phage titer is the optimal MOI for that phage. The optimal MOI results for the two phage strains are shown below. Figure 5 and Figure 6 As shown.

[0067] Example 5: Determination of one-step growth curve of bacteriophage

[0068] With the best MOI mixed bacterial solution (10 8 CFU / mL) and phage were incubated at 37°C for 10 min, then centrifuged at 8000 rpm for 5 min. The supernatant was discarded, and the phage was resuspended in 5 mL LB. The mixture was then incubated at 37°C for 200 rpm for 120 min. Sampling began 5 min later, with 90 μL samples taken each time. After all samples were collected, the phage titer was determined, and the phage latency and burst were calculated. Latency time is the shortest time required for infected cells to release their progeny viral particles. Burst yield is the phage titer at the end of the burst divided by the concentration of host bacteria at the start of infection. The one-step growth curves for the two phage strains are shown below. Figure 7 and Figure 8 As shown.

[0069] Example 6: Phage Host Profile Test

[0070] The host range of bacteriophages was determined using the dot-matrix assay. 500 μL of bacterial culture, 500 μL of phage buffer, and 5 mL of 0.5% agar were mixed and poured onto LB agar plates. After solidification, 10 μL of phage stock solution was added, and the plates were incubated at 37°C for 12 hours. The presence and transparency of phage plaques were observed. The strains required for the host range assay included 53 pathogenic Escherichia coli strains, 8 pathogenic Salmonella strains isolated from diseased chickens, and E. coli O157:H7 (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.) preserved in the laboratory.

[0071] The host spectrum test results of the two bacteriophages are shown in Table 1. Results with no plaques (-) and opaque plaques (+) were excluded; results with clear plaques (++) and strong lytic activity were selected for statistical analysis. It can be seen that the combination of bacteriophages GaoM7-14E and JM3e can lyse 85.48% (53 / 62) of the strains, indicating that the combination of these two bacteriophages can broadly and effectively lyse avian pathogenic Escherichia coli and pathogenic Salmonella.

[0072] Table 1. Results of lysis spectra determination of Escherichia coli bacteriophage assemblages GaoM7-14E and JM3e.

[0073]

[0074]

[0075]

[0076] Example 7: Evaluation of the inhibitory effect of bacteriophages on host bacteria

[0077] Mix 3 mL of 10 mL in the laboratory. 8 CFU / ml E. coli O157:H7 strain and 3mL 10 10 Phages at PFU / ml (MOI = 0.1) were incubated at 37°C for 12 h, with samples taken every 0.5 h. The absorbance at 600 nm was measured using a spectrophotometer. Bacterial suspension without phages was used as a control to evaluate the antibacterial effects of phages GaoM7-14E and JM3e on Escherichia coli O157:H7.

[0078] The results are as follows Figure 9 As shown, bacteriophages GaoM7-14E and JM3e can kill Escherichia coli O157:H7 in a short time, demonstrating excellent antibacterial properties.

[0079] Example 8: Evaluation of the effect of bacteriophage composition on purifying avian Escherichia coli in the environment

[0080] Nine randomly selected points (each 20cm × 20cm) were designated as experimental areas in the biological laboratory and marked. Three strains of avian pathogenic Escherichia coli were randomly selected from the laboratory's bacterial collection. The bacteria were cultured to a concentration of 1 × 10⁻⁶. 8 CFU / mL. Mix the bacterial suspensions of the two *E. coli* strains and spray them evenly over the test area in the biological laboratory. Culture bacteriophages GaoM7-14E and JM3e to obtain a concentration of 1×10⁻⁶ CFU / mL. 10Phage solution at PFU / mL was prepared. Two strains of E. coli phage were mixed and then sprayed on the test area for disinfection. One hour later, the test area was wiped with cotton balls, and bacterial residue was detected by culture method. The results showed that no E. coli strain residue was detected in any of the nine test areas.

[0081] Example 9: Clinical efficacy evaluation of phage composition in treating chicken Escherichia coli infection

[0082] One hundred and twenty one-day-old healthy, unvaccinated male broilers were randomly divided into four groups of 30 birds each. Each group was further divided into three parallel groups of 10 birds each. Water and antibiotic-free broiler feed were provided randomly throughout the experiment. One group served as the control group. All broilers in this group were not challenged with E. coli or treated with bacteriophages; they were only injected with 2 mL of phagebuffer. In the second group, all broilers were not challenged with E. coli; each broiler was only injected with 2 mL of 1×10⁻⁶ phagebuffer. 10 A combination of phages GaoM7-14E and JM3e at PFU / mL. In the third group, 0.5 mL of pathogenic Escherichia coli O157:H7 (10 9 All broilers were challenged with a combination of phages GaoM7-14E and JM3e (CFU / mL) for 2 hours after challenge. 10 The fourth group was treated with 0.5 mL of pathogenic Escherichia coli (PFU / mL). 9 All broiler chickens were challenged with E. coli (CFU / mL), and 2 mL of phage buffer was injected 2 hours after challenge. E. coli is mainly transmitted through the respiratory tract, and to prevent gastric acid from destroying the phages, bacterial culture, phage suspension, and phage buffer were all inoculated into the trachea of ​​1-day-old chicks using a feeding needle. The mortality rate and the protective rate of the phage combination on the chicks were observed and statistically analyzed after approximately 2 weeks of treatment.

[0083] The clinical efficacy evaluation results of this phage composition in treating chicken Escherichia coli infection are shown in Table 2. The results indicate that the combination of these two phage strains can effectively treat chicken infections caused by pathogenic Escherichia coli, achieving a protection rate of 86.67%.

[0084] Table 2. Clinical efficacy of Escherichia coli phage composition in treating chicken Escherichia coli infection.

[0085]

[0086] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A bacteriophage composition, characterized in that, The bacteriophage composition for inhibiting avian pathogenic E. coli and / or avian pathogenic Salmonella comprises E. coli bacteriophage GaoM7-14E and E. coli bacteriophage JM3e; wherein: The taxonomic name of the E. coli bacteriophage GaoM7-14E is Escherichia coli phage, the accession number is CGMCC NO. 46188, and the preservation time is September 20, 2024; The taxonomic name of the E. coli bacteriophage JM3e is Escherichia coli phage, the accession number is CGMCC NO. 46186, and the preservation time is September 18, 2024.

2. The phage composition of claim 1, wherein, The titer of the Escherichia coli phage GaoM7-14E and the titer of the Escherichia coli phage JM3e are both not less than 10 7 PFU / mL.

3. The phage composition of claim 1, wherein, The multiplicity of infection MOI is 0.001-10.

4. Use of a bacteriophage for the preparation of an antibacterial agent, characterized in that, The bacteriophage comprises at least one of the E. coli bacteriophage GaoM7-14E and the E. coli bacteriophage JM3e; The E. coli bacteriophage GaoM7-14E is the E. coli bacteriophage GaoM7-14E as claimed in claim 1, and the E. coli bacteriophage JM3e is the E. coli bacteriophage JM3e as claimed in claim 1.

5. Use of a bacteriophage according to claim 4 for the preparation of an antibacterial agent, characterized in that, The bacteriostatic agent is used for inhibiting E. coli, which comprises avian pathogenic E. coli.

6. A bacteriophage preparation, characterized in that, The bacteriophage preparation comprises the bacteriophage composition as claimed in any one of claims 1 to 3.

7. The bacteriophage preparation of claim 6, wherein the bacteriophage preparation is formulated as a liquid formulation. The bacteriophage preparation further comprises a poultry-acceptable adjuvant.

8. The phage preparation as described in claim 6, characterized in that, The dosage form of the bacteriophage preparation comprises at least one of a solution, a powder, a gel, a granule, or a lyophilized agent.

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