Broad-spectrum bacteriophage composition against escherichia coli and use thereof

Abstract

This invention discloses a broad-spectrum bactericidal Escherichia coli phage ensemble and its application, belonging to the field of microbial technology. The broad-spectrum bactericidal Escherichia coli phage ensemble consists of Escherichia coli phages (… Escherichia coliphage The bacteriophage combination consists of WYS69, Escherichia coli phage WYS60-2, and Escherichia coli phage A70. The preservation number of Escherichia coli phage WYS69 is CCTCC NO: M 2026876; the preservation number of Escherichia coli phage WYS60-2 is CCTCC NO: M 2026877; and the preservation number of Escherichia coli phage A70 is CCTCC NO: M 2026878. Verification showed that this broad-spectrum bacteriophage combination can effectively lyse 95 different serotypes of pathogenic Escherichia coli, including the main pathogenic serotypes prevalent in animal clinical settings, food environments, and human clinical settings.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a broad-spectrum bactericidal Escherichia coli phage combination and its application. Background Technology

[0002] Pathogenic Escherichia coli (E. coli) is an important zoonotic pathogen with a broad host spectrum, capable of infecting humans and various animal hosts, and is one of the most threatening foodborne pathogens. Epidemiological data shows that pathogenic E. coli infection is the third leading cause of diarrhea-related mortality. Common zoonotic pathogenic E. coli serotypes include enterohemorrhagic E. coli (EHEC) serotypes such as O157:H7, O26, O111, O103, and O145, as well as avian pathogenic E. coli (APEC)-related serotypes such as O78, O2, and O1. Poultry are important hosts for pathogenic E. coli infection. Poultry infected with most pathogenic E. coli serotypes often exhibit asymptomatic carrier states, and this latent infection characteristic often leads to its being overlooked during prevention and control. However, these pathogenic Escherichia coli strains carried by poultry can enter the food chain through contamination of poultry meat and egg products, posing a potential threat to food safety and public health. Therefore, purifying the intestinal tracts of poultry infected or colonized with pathogenic Escherichia coli is of great significance. The overuse of antibiotics has led to an increasingly serious problem of multidrug resistance in avian pathogenic Escherichia coli, challenging traditional control methods.

[0003] Bacteriophages offer unique advantages as novel antibacterial agents; however, the serotypes of pathogenic Escherichia coli causing clinical infections are diverse, and the bactericidal spectrum of a single phage cannot effectively cover multiple serotypes. Furthermore, the action of a single phage on bacteria easily leads to phage resistance, resulting in decreased or even ineffective efficacy. Therefore, there is an urgent need to develop phage combinations with broad-spectrum antibacterial capabilities that are less prone to inducing phage resistance. Summary of the Invention

[0004] The purpose of this invention is to provide a pathogenic Escherichia coli phage combination and its application to solve the problems existing in the prior art. This phage combination can effectively lyse a variety of pathogenic Escherichia coli with different serotypes (including O157:H7, O78, O2, O1, O26, O111, etc.), providing a green, safe, and efficient biological control method for the prevention and control of pathogenic Escherichia coli infection in poultry farming. At the same time, it also shows good application prospects for the control of pathogenic Escherichia coli contamination in poultry production environments.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a broad-spectrum bactericidal Escherichia coli phage ensemble, wherein the broad-spectrum bactericidal Escherichia coli phage ensemble comprises Escherichia coli phages (… Escherichia coliphageIt is composed of WYS69, Escherichia coli phage WYS60-2 and Escherichia coli phage A70; The preservation number of the Escherichia coli bacteriophage WYS69 is CCTCC NO: M 2026876; The preservation number of the Escherichia coli bacteriophage WYS60-2 is CCTCC NO: M 2026877; The preservation number of the Escherichia coli bacteriophage A70 is CCTCC NO: M 2026878; The deposit dates of the Escherichia coli phage WYS69, the Escherichia coli phage WYS60-2, and the Escherichia coli phage A70 were all April 30, 2026. The depository institution for all of them was the China Center for Type Culture Collection (CCTCC), and the depository address was Wuhan University, Wuhan, China.

[0006] Optionally, the ratio of the number of bacteriophage WYS69, the number of bacteriophage WYS60-2 and the number of bacteriophage A70 is 1:1:1.

[0007] The present invention also provides the use of the aforementioned broad-spectrum bactericidal Escherichia coli phage combination in the preparation of medicaments for the prevention and / or treatment of pathogenic Escherichia coli infections.

[0008] This invention also provides the application of the aforementioned broad-spectrum bactericidal Escherichia coli phage combination in the preparation of pathogenic Escherichia coli bactericides.

[0009] The present invention also provides a drug for preventing and / or treating pathogenic Escherichia coli infection, wherein the active ingredient of the drug is the aforementioned broad-spectrum bactericidal Escherichia coli phage combination.

[0010] Optionally, the drug may also include pharmaceutically acceptable excipients.

[0011] Optionally, the dosage form of the drug is an injection, an aqueous solution, a powder, a gel, granules, or a lyophilized preparation.

[0012] The present invention also provides a pathogenic Escherichia coli bactericide, wherein the active ingredient of the pathogenic Escherichia coli bactericide is the aforementioned broad-spectrum bactericidal Escherichia coli phage combination.

[0013] Optionally, the formulation of the Escherichia coli bactericide is a spray, powder, gel, granule, or lyophilized agent.

[0014] The present invention discloses the following technical effects: This invention provides a broad-spectrum bacteriophage combination for killing Escherichia coli and its application. The combination consists of three bacteriophages with highly efficient lytic activity: WYS69, WYS60-2, and A70. WYS69 belongs to... Tequatrovirusgenus, WYS60-2 belongs to Mosigvirus A70 belongs to the genus. Phapecoctavirus The three genera exhibit good complementarity in their lytic properties. This phage ensemble can maintain stable activity under a wide range of environmental conditions, tolerate temperatures below 70°C, and has a pH tolerance range of 4–10, demonstrating strong adaptability and ease of operation.

[0015] Verification has shown that the phage ensemble provided by this invention can effectively lyse 95 different serotypes of pathogenic Escherichia coli, including serotypes prevalent in animal clinical settings, food environments, and human medical settings. Therefore, the phage ensemble provided by this invention offers a green, safe, and efficient biological control method for preventing E. coli infection in animal husbandry, disinfecting food contaminated with E. coli, and treating clinically pathogenic E. coli. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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 drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Transmission electron microscope images of bacteriophages WYS69, WYS60-2, and A70; Figure 2 This refers to the temperature tolerance of bacteriophages; Figure 3 This refers to the pH tolerance of bacteriophages. Figure 4 The results of disinfection for food contamination with E. coli; Figure 5 The image shows the results of an experiment on Escherichia coli infection and colonization in the chicken intestine and bacteriophage treatment. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1: Identification and preservation of Escherichia coli bacteriophages 1. Phage identification 1.1 Phage morphological characteristics In the early stages of this invention, three bacteriophage strains, WYS69, WYS60-2, and A70, were isolated. The particles of the three bacteriophage strains were observed under a transmission electron microscope, as shown... Figure 1 As shown, WYS69 is Tequatrovirus It belongs to the bacteriophage family, with a polyhedral head and four tail filaments; WYS60-2 is... Mosigvirus It belongs to the bacteriophage family and is similar in shape and size to WYS69; A70 is... Phapecoctavirus It belongs to the bacteriophage family, with a head that is icosahedral in shape and approximately 90 nm in diameter.

[0024] 1.2 Molecular biological identification Using SEQ ID NO.1 and SEQ ID NO.2 as a primer pair, bacteriophage WYS69 was amplified by PCR. The resulting PCR product was 523 bp, and the nucleotide sequence is shown in SEQ ID NO.7. Using SEQ ID NO.3 and SEQ ID NO.4 as a primer pair, bacteriophage WYS60-2 was amplified by PCR. The resulting PCR product was 495 bp, and the nucleotide sequence is shown in SEQ ID NO.8. A pair of primers composed of SEQ ID NO. 5 and SEQ ID NO. 6 is used to perform PCR amplification on bacteriophage A70. The obtained PCR product is 764 bp, and the nucleotide sequence is shown in SEQ ID NO. 9.

[0025] SEQ ID NO. 1: 5'-AATGCCGATGGCACTGAAC-3'; SEQ ID NO. 2: 5'-CCATTGCTCCAGTGGAACCG-3'; SEQ ID NO. 3: 5'-GTGTATCAACTGCCGCCG-3'; SEQ ID NO. 4: 5'-ACGTGGTGTAATCTGTCTGG-3'; SEQ ID NO. 5: 5'-AAGGTGTGCACACGAAGATG-3'; SEQ ID NO. 6: 5'-CGGACAACCAGTGCCATG-3'.

[0026] SEQ ID NO. 7: 5'-AATGCCGATGGCACTGAACGTGGCGTTATATATGCTCGTCCTCAAACTACAACTGCCGGTGAAATACGCCTTAGGGTTAGACAAGGAACAGGAAGCACTACCAATAGTGAATTCTATTTCCGCTCTATAAATGGAGGCGAATTTCAGGCTAACCGTATTTTAGCTTCGGATGCATTAGTAACAAAACGCATTGCGGTTGATACTGTTATTCACGATGCCAAAACGTTTGGACAATATGATTCTCACTCTTTGGTCAACTATGTTTATCCTGGAACAGGTGAAACAAACGGAATCAATTACCTTCGCAAATTCCGTGCTAAATCTGGTGGTACAATTTATCATGAATTAGCTTCTGCGCAAACCGGAAAAAGTGATGAACTTTCTTGGTGGACTGGTAATACAGCGGTTAATAAACAAATGGGCCTTCGCAATGATGGATCTTTAGTATTACGACGTTCACTTGCAATTGGTACAATTACAACAGATGAAAACATCAATAACTACGGTTCCACTGGAGCAATGG-3'.

[0027] SEQ ID NO.8: 5'-GTGTATCAACTGCCGCCGAAGGCTTCGAGAGATCCGATTGGGTATATACCGAAATATTTGAATCATTTCAGACCAACGCTTATACATTTCAACATATGATTGAAATGCAATTGGCTAATACTTTTATTAGAGAAAAATTTACAAATAACAACATGGCTTATGTCGATTTTAATAACGACACTATAATGGCAGCTCTAATGAATGAATCGTTCCAATTTAGCCCTGCTTATGCGGATATTTCATCTATTAAAAATTTCATTATTGGCGAAAATGAGTATCATGAAATTCAAGGTTCGATTCAACAGGTGTGTCATGATATAAACCGTGTTTATTTGATGGAATCCGAAGGAATTCTTTATTTGATTGATGAGCGTATCAGCCTGTGGTTAAAGTCTCTAATGATAAGGGACAAACCTGGAAATCGGTTAAATTGTTTAATGACCGTGTAGGGTACCCTGTGTATCAGCCTGTGTATTACCAGACACACACCACGT-3'。

[0028] SEQ ID NO.9: 5'--3'.

[0029] 2. Phage preservation coli phage ( Escherichia coliphage WYS69 was deposited at the China Center for Type Culture Collection (CCTCC) on April 30, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026876. coli phage ( Escherichia coliphageWYS60-2 was deposited at the China Center for Type Culture Collection on April 30, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026877. coli phage ( Escherichia coliphage A70 was deposited at the China Center for Type Culture Collection (CCTCC) on April 30, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026878.

[0030] Example 2: In vitro bactericidal effect of bacteriophage combination In this embodiment, the lysis of phage combinations on 95 different serotypes of Escherichia coli strains was detected. The 95 Escherichia coli strains were provided by the Jiangsu Provincial Research Center for Antimicrobial Resistance and Phage Engineering and have been published in the literature: Epidemiological Trends and Antimicrobial Resistance Analysis of Animal-Derived Pathogenic Escherichia coli in China from 2005 to 2024, Chinese Journal of Veterinary Medicine, 2025, 61(12): 29-40.

[0031] The lysis of 95 different serotypes of *Escherichia coli* strains by single bacteriophages and bacteriophage combinations (WYS69, WYS60-2, and A70 in a 1:1:1 ratio) was determined, and the results are shown in Table 1. The bacteriophage combinations lysed all 95 typical strains of different serotypes, with a lysis rate of 100%. However, a single bacteriophage could not completely lyse all 95 different serotypes.

[0032] Table 1. Lysis results of phage combinations on 95 different serotypes of pathogenic Escherichia coli. Example 3: Temperature tolerance experiment of bacteriophages This embodiment tests the temperature tolerance of three bacteriophage strains.

[0033] Take 100 μL of phage solution into each of six 1.5 mL sterile EP tubes (initial titer 10). 9 ~10 10 PFU / mL). Centrifuge tubes were incubated at 30℃~80℃ (10℃ intervals) for 1 h, and 900 μL of SM buffer was added to each tube. The titer was determined by the bilayer plate method, repeated 3 times, and plotted using GraphPad Prism 9.5.0.

[0034] The results are as follows Figure 2As shown, all three phage strains maintained stable activity at temperatures ranging from 30℃ to 60℃, began to lose activity at 60℃ to 70℃, and were completely inactivated after treatment at 70℃ for 1 h.

[0035] Example 4: Phage pH Tolerance Experiment This embodiment tests the pH tolerance of three bacteriophage strains.

[0036] Take 100 μL of phage fluid from each of the 11 sterile 1.5 mL EP tubes (initial titer 10). 8 ~10 9 (PFU / mL), add 900 μL of SM buffer (pH 2-12) sequentially, and incubate at room temperature for 1 h. Then, serially dilute 10-fold with SM buffer of the same pH, determine the titer using the bilayer plate method, repeat 3 times, and plot using GraphPad Prism 9.5.0.

[0037] The results are as follows Figure 3 As shown, the three bacteriophages can tolerate a pH range of 4–10; when pH < 4 or > 10, the bacteriophage activity begins to be lost; when pH > 12, the bacteriophage activity is completely lost; when pH < 2, bacteriophage WYS69 still retains some activity.

[0038] Example 5: Disinfection Experiment of Food Contamination by Escherichia coli Using *Escherichia coli* O157 as the host bacterium, after culturing to the logarithmic growth phase, it was uniformly coated onto the surface of sterile beef chunks to construct a meat contamination model. Individual spraying of bacteriophages WYS69, WYS60-2, and A70, as well as a mixture of the three (ratio 1:1:1, titer 10) were also performed. 5 Four treatment groups were established (PFU / mL). 1 mL of suspension from each group was sprayed onto the meat surface and incubated at 37°C. Meat samples were collected every 1 hour starting from 0 h, washed and diluted with 1 mL of PBS, spread onto LB agar plates, and incubated at 37°C for 8 h for colony counting to assess the amount of viable bacteria remaining on the meat surface.

[0039] The results are as follows Figure 4 As shown, the initial bacterial count of the beef surface contamination was approximately 10. 7 CFU / mL, A70 alone showed significant bactericidal effect within 0-6 hours (reducing to approximately 10 CFU / mL). 3 (CFU / mL), but rebounded from the 7th hour, rising back to about 10 by the 8th hour. 4 CFU / mL; WYS69 and WYS60-2 alone showed poor efficacy, with bacterial counts exceeding the initial value at 4 h. In contrast, the mixed phage group maintained continuous antibacterial activity throughout 8 h, with the final bacterial count reduced by approximately 5 log₂ / mL from the initial value. 10The magnitude of the effect, and the absence of a significant rebound, indicate that the combination of the three bacteriophages has a synergistic and efficient advantage in continuous disinfection.

[0040] Example 6: Colonization of Chicken Enteric Pathogenic Escherichia coli and Phage Therapy Experiment The experimental chickens were divided into a healthy group, an E. coli infection group, and four phage treatment groups (WYS69 treatment group, WYS60-2 treatment group, A70 treatment group, and phage combination treatment group, in which the ratio of WYS69, WYS60-2, and A70 in the phage combination treatment group was 1:1:1). On the second day, all chickens except the healthy group were given E. coli O78 by gavage, with each chicken receiving 500 μL of 1 ... 9 CFU / mL, once daily, 500 μL each time, for 7 consecutive days. On day 11, start feeding phage, 500 μL per animal. 10 PFU / mL, once a day, for 3 consecutive days. Weigh the chickens every 7 days to calculate their weight gain and count E. coli.

[0041] The results of the E. coli count are as follows: Figure 5 As shown, the results indicated that at the end of the eighth day of force-feeding, the level of E. coli O78 in the chick feces was detected at 10. 8 CFU / g. After phage administration on day 11, the content of E. coli O78 began to decrease, and by day 15, the single-strain phage treatment group had decreased by approximately 1-2 log. 10 The order of magnitude decrease was approximately 3 log in the phage ensemble treatment group. 10 Order of magnitude. As time progressed, the content of *E. coli* O78 in feces began to decrease slowly. By day 30, the content of *E. coli* O78 in the phage combination treatment group was undetectable. On day 35, the content of *E. coli* O78 in the phage A70 treatment group and the phage WYS69 treatment group was also 0, while the phage WYS60-2 treatment group had 5 CFU / g. The *E. coli* infection group still contained 10... 3 Escherichia coli O78 with a concentration of approximately CFU / g.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A broad-spectrum bactericidal Escherichia coli phage assemblage, characterized in that, The broad-spectrum bactericidal E. coli phage assemblage consists of E. coli phages ( Escherichia coliphage It is composed of WYS69, Escherichia coli phage WYS60-2 and Escherichia coli phage A70; The preservation number of the Escherichia coli bacteriophage WYS69 is CCTCC NO: M 2026876; The preservation number of the Escherichia coli bacteriophage WYS60-2 is CCTCC NO: M 2026877; The preservation number of the Escherichia coli bacteriophage A70 is CCTCC NO: M 2026878.

2. The broad-spectrum bactericidal Escherichia coli phage combination as described in claim 1, characterized in that, The ratio of the number of bacteriophages WYS69, WYS60-2, and A70 is 1:1:

1.

3. The use of a broad-spectrum bactericidal Escherichia coli phage combination as described in claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of pathogenic Escherichia coli infections.

4. The application of a broad-spectrum bactericidal Escherichia coli phage combination as described in claim 1 or 2 in the preparation of a bactericide for pathogenic Escherichia coli.

5. A drug for the prevention and / or treatment of pathogenic Escherichia coli infection, characterized in that, The active ingredient of the drug is the broad-spectrum bactericidal Escherichia coli phage combination as described in claim 1 or 2.

6. The drug as described in claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The drug as described in claim 6, characterized in that, The dosage form of the drug is injection, aqueous solution, powder, gel, granule or lyophilized form.

8. A bactericide for pathogenic Escherichia coli, characterized in that, The active ingredient of the pathogenic Escherichia coli bactericide is the broad-spectrum bactericidal Escherichia coli phage combination described in claim 1 or 2.

9. The pathogenic Escherichia coli bactericide according to claim 8, characterized in that, The formulation of the Escherichia coli bactericide is a spray, powder, gel, granule, or lyophilized agent.

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