Phage capable of splitting pathogenic escherichia coli and application thereof

Abstract

The invention discloses a bacteriophage capable of splitting pathogenic escherichia coli and application of the bacteriophage. The phage is named as Escherichia coli phage vBEcoMYS1, and is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the phage is GDMCC No: 67465-B1vBEcoMYS1. Experiments prove that the bacteriophage has relatively strong splitting capacity, relatively strong thermal stability and relatively strong pH stability, and can keep high titer under the conditions that the temperature is 4-40 DEG C and the pH is 5-9. The bacteriophage can be used for splitting multiple escherichia coli, including enterotoxigenic escherichia coli (ETEC) and shiga toxin-producing escherichia coli (STEC), has a relatively wide splitting spectrum, and can be used as an antibiotic substitute. In addition, the genome of the bacteriophage does not contain virulence genes and antibiotic resistance genes, so that the bacteriophage has good biological safety.

Description

Technical Field

[0001] This invention relates to a bacteriophage capable of lysing *Escherichia coli* and its applications, particularly to a bacteriophage capable of lysing pathogenic *Escherichia coli* and its applications. This invention belongs to the field of biotechnology. Background Technology

[0002] Escherichia coli (E. coli) is a common Gram-negative bacillus found in the environment and in organisms. Pathogenic Escherichia coli is a common zoonotic pathogen that seriously endangers public health. Enterotoxigenic Escherichia coli (ETEC) is one of the main pathogens causing diarrhea in infants and young animals. Enterotoxigenic ETEC produces enterotoxins by adhering to small intestinal epithelial cells, causing watery diarrhea in the infected host, and in severe cases, endangering the host's life. Shiga toxin-producing Escherichia coli (STEC) can infect people of all ages and livestock and poultry. It is a foodborne pathogen with high pathogenicity and lethality. Shiga toxin-producing E. coli damages intestinal epithelial cells through shiga toxin, causing damage to the host's intestinal mucosa. This can lead to mild intestinal diseases such as watery diarrhea and hemorrhagic enteritis in humans. In severe cases, it can lead to hemolytic uremic syndrome. Shiga toxin-producing E. coli can exist in various farm environments, contaminating the farming environment and spreading to multiple hosts through food, water and other routes.

[0003] As companion animals, dogs have long-term close contact and communication with their owners. Due to the lifestyle characteristics of dogs, they can easily transmit drug-resistant pathogenic E. coli from the environment and the dogs themselves to their owners. When the dogs and their owners have weakened immune systems, these drug-resistant pathogenic E. coli from dogs begin to multiply in large numbers in the host's body and release bacterial toxins.

[0004] With the widespread use of antibiotics, the increasing resistance of E. coli and antibiotic residues in animals have led to a growing trend in antibiotic reduction and replacement in livestock farming for the treatment of pathogenic E. coli infections. Bacteriophages, viruses that use bacteria as hosts, are characterized by high specificity and safety. Phage therapy can effectively target specific bacterial strains without affecting the host's microbiome and can be applied to the treatment of antibiotic-resistant bacteria. It does not promote the development of drug-resistant bacteria or antibiotic residues, and can effectively prevent the spread of veterinary drug-resistant bacteria during livestock treatment.

[0005] Currently, phage therapy is used to treat infections caused by enterotoxigenic Escherichia coli and Shiga toxin-producing Escherichia coli, but there is limited research on phage therapy for treating canine enterotoxigenic Escherichia coli and Shiga toxin-producing Escherichia coli. Summary of the Invention

[0006] The purpose of this invention is to provide a bacteriophage capable of lysing pathogenic Escherichia coli and its applications.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] This invention isolates a canine Escherichia coli bacteriophage from wastewater in a dog breeding farm in Changchun City, Jilin Province. The bacteriophage is named Escherichia coli phage vB_EcoM_YS1 and classified as Escherichia coliphage. It is deposited at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, with accession number GDMCC No: 67465-B1, and the deposit date is December 11, 2025.

[0009] Furthermore, the present invention also proposes the use of the canine Escherichia coli phage in any of the following aspects:

[0010] (1) Preparation of microbial agents for lysing Escherichia coli bacteriophages;

[0011] (2) To prepare pharmaceutical preparations for the prevention or treatment of Escherichia coli infection;

[0012] (3) Prepare feed or feed additives for the prevention or treatment of Escherichia coli infection;

[0013] (4) Prepare health products for the prevention or treatment of Escherichia coli infection;

[0014] (5) Prepare cleaning agents or disinfectants for livestock and poultry breeding environments to prevent Escherichia coli infection.

[0015] Preferably, the Escherichia coli includes enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing Escherichia coli (STEC).

[0016] Furthermore, the present invention also proposes a microbial preparation for lysing Escherichia coli, wherein the microbial preparation contains the canine Escherichia coli phage.

[0017] A pharmaceutical preparation for the prevention or treatment of Escherichia coli infection, wherein the active ingredient of the pharmaceutical preparation includes the canine Escherichia coli bacteriophage.

[0018] A feed additive containing the canine Escherichia coli bacteriophage.

[0019] A cleaning agent or disinfectant containing the canine Escherichia coli bacteriophage.

[0020] Preferably, the dosage forms of the microbial preparation, the pharmaceutical preparation, and the feed additive include one or more of the following: solution, powder, gel, granule, emulsion, suspension, and lyophilized agent.

[0021] In this document, the term "prevention" refers to all actions that suppress or delay the disease by administering the phage; the term "treatment" refers to all actions that improve or alleviate the disease by administering the phage.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention provides an Escherichia coli bacteriophage vB_EcoM_YS1, whose optimal multiplicity of infection is 0.01. Under these conditions, bacteriophage vB_EcoM_YS1 can replicate up to 3 × 10⁻⁶. 9 The above-mentioned phage exhibits strong lytic ability, as well as excellent thermal and pH stability, maintaining high titer under conditions of 4-40℃ and pH 5-9. One-step growth curve experiments show that the phage's incubation period for infecting *E. coli* is 0-20 min, the outbreak period is 20-50 min, and it enters a stationary phase after 50 min. This phage can lyse various *E. coli* species, including enterotoxigenic *E. coli* (ETEC) and Shiga toxin-producing *E. coli* (STEC), exhibiting a broad lytic spectrum. It can serve as a safe antibiotic alternative, leaving no residue, and does not induce drug resistance, making it environmentally friendly. It shows promising potential in clinical applications for the prevention and treatment of *E. coli* infections, effectively addressing the problem of drug resistance in some *E. coli* species. Furthermore, the *vB_EcoM_YS1* phage genome does not contain virulence genes or antibiotic resistance genes, and it has been safely used in mouse experiments, demonstrating good biocompatibility. Attached Figure Description

[0024] Figure 1 Morphological characteristics of Escherichia coli bacteriophage vB_EcoM_YS1 purified in this invention;

[0025] In the image, A shows a plaque image of bacteriophage vB_EcoM_YS1 obtained using the double-layer plate method; B shows a transmission electron microscope image of bacteriophage vB_EcoM_YS1.

[0026] Figure 2 This is a one-step growth curve of the bacteriophage vB_EcoM_YS1 of this invention;

[0027] Figure 3This is a temperature tolerance diagram of the bacteriophage vB_EcoM_YS1 of the present invention;

[0028] Figure 4 This is a pH tolerance diagram of the bacteriophage vB_EcoM_YS1 of this invention;

[0029] Figure 5 This is the genome map of bacteriophage vB_EcoM_YS1. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] Unless otherwise specified, all reagents and equipment used in the following examples were purchased from commercial sources.

[0032] The strains, reagents, and culture media involved in the examples: The host bacteria involved in the examples are canine Escherichia coli, which are preserved by the Institute of Special Products of the Chinese Academy of Agricultural Sciences.

[0033] Example 1: Isolation and purification of canine Escherichia coli phage vB_EcoM_YS1

[0034] 1. Isolation and purification of canine Escherichia coli bacteriophage

[0035] The collected wastewater samples were centrifuged at 5000×g for 30 min at 4℃. The supernatant was passed through a 0.45 μm filter membrane and then filtered through a 0.22 μm filter membrane to remove all bacteria. 1 mL of the filtered wastewater and 100 μL of pathogenic *Escherichia coli* EC-YS1 culture in the logarithmic growth phase (OD600 = 0.3–0.6) were mixed with 5 mL of Brain Heart Infusion Broth (BHI) liquid medium. The mixture was incubated overnight at 37℃ with shaking at 180 r / min. The culture was then filtered through a 0.45 μm filter membrane and then through a 0.22 μm filter membrane to remove bacteria, resulting in an enriched bacteriophage filtrate.

[0036] First, the droplet method was used for verification, followed by isolation and purification using the double-layer plate method. 100 μL of logarithmic-phase *E. coli* culture was evenly spread onto BHI solid medium. Then, 10 μL of enriched phage filtrate was dropped into the center of the plate, and the plate was incubated at 37°C for 4 h to observe the presence of plaques. For the droplet method verification, the phage stock solution with plaques was diluted 10-fold. 100 μL of each gradient was mixed 1:1 with the logarithmic-phase bacterial solution, incubated at 37°C for 20 min, then 5 mL of 1% BHI semi-solid medium was added and mixed thoroughly at 50°C. This mixture was then poured into 2% BHI solid medium to solidify, and incubated upside down at 37°C for 8–12 h to observe plaque formation. Clear plaques were picked and added to a mixture of 50 μL of logarithmic-phase bacterial culture and 5 mL of BHI liquid medium. The double-layer plate experiment was repeated 5 times to purify the plaques into uniformly sized plaques.

[0037] 2. Morphological observation of bacteriophages

[0038] The size and brightness of phage plaques on double-layer plates were observed. High-titer purified phages were precipitated onto a copper grid, stained with phosphotungstic acid (PTA, 2% w / v), and dried. The morphology of the phages was observed using a transmission electron microscope at an accelerating voltage of 80 kV and a magnification of 70 k. Figure 1 ).

[0039] Based on the classification of the International Committee on Taxonomy of Viruses (ICTV) and the morphology of the bacteriophage, the isolated bacteriophage was formally classified as a member of the Straboviridae family. The isolated canine Escherichia coli bacteriophage was named *Escherichia coli phage* vB_EcoM_YS1 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No: 67465-B1.

[0040] Example 2: Host range identification of bacteriophage vB_EcoM_YS1

[0041] 1. Typing of canine Escherichia coli

[0042] After thawing the frozen canine Escherichia coli strains (Table 1) preserved in the laboratory, they were inoculated onto BHI agar plates. Single strains were picked and inoculated onto BHI liquid medium and cultured overnight at 37°C and 180 r / min. Molecular identification as Escherichia coli was confirmed by 16S rRNA sequencing. Subsequently, PCR amplification of the canine Escherichia coli genes uidA, escV, eae, bfpB, stx1, stx2, lt, stp, sth, invE, ipaH, aggR, pic, and astA confirmed the presence of ETEC and STEC strains (Table 1).

[0043] 2. Identification of the host range of bacteriophage vB_EcoM_YS1

[0044] Twenty potential host bacteria were selected for phage host spectrum determination, including 17 Escherichia coli, 2 Enterococcus faecalis, and 1 Staphylococcus saprophyticus. The lysis effect was verified by the spot method, and the results are shown in Table 1.

[0045] Table 1. Host profile determination of Escherichia coli bacteriophage vB_EcoM_YS1

[0046]

[0047] Note: "-" indicates no cleavage; "+" indicates cleavage.

[0048] As can be seen from the results in Table 1, the Escherichia coli phage vB_EcoM_YS1 isolated by the present invention has a lytic effect on enterotoxigenic Escherichia coli (ETEC), Shiga toxin-producing Escherichia coli (STEC), and some non-enteric pathogens.

[0049] Example 3: Determination of the optimal multiple of infection (MOI) of bacteriophage vB_EcoM_YS1

[0050] Different titers (10000, 1000, 100, 10, 1, 0.1, 0.01) of bacteriophage were mixed with pathogenic Escherichia coli EC-YS1 bacterial culture to determine the optimal MOI, with bacterial cultures without bacteriophage and bacteriophage stock solution serving as controls. Each ratio was repeated three times.

[0051] Results: The optimal multiplicity of infection (MWI) of phage vB_EcoM_YS1 against Escherichia coli was 0.01. Under these conditions, phage vB_EcoM_YS1 could replicate up to 3 × 10⁻⁶. 9 The above demonstrates strong pyrolysis capabilities.

[0052] Example 4: One-step growth curve of bacteriophage vB_EcoM_YS1

[0053] To determine the one-step growth curve, 1 mL of phage vB_EcoM_YS1 lysis buffer was mixed with 1 mL of logarithmically growing pathogenic Escherichia coli EC-YS1 bacterial culture at the optimal MOI of 0.01. The mixture was shaken at 37°C and 180 rpm for 10 min. The precipitate was washed three times with PBS and resuspended in BHI liquid medium, then incubated on a shaker at 37°C and 180 rpm. 100 μL samples were taken every 10 min during the first hour, and then every 30 min thereafter. Results were measured using the bilayer plate method, and the experiment was repeated three times. A one-step growth curve of the phage was plotted with the logarithm of the phage titer on the ordinate and time on the abscissa.

[0054] Results: The one-step growth curve of bacteriophage vB_EcoM_YS1 is as follows: Figure 2 As shown, the incubation period for this bacteriophage to infect Escherichia coli is 0-20 min, the outbreak period is 20-50 min, and it enters the stationary phase after 50 min.

[0055] Example 5: Determination of the temperature and pH tolerance of bacteriophage vB_EcoM_YS1

[0056] 100 μL of phage vB_EcoM_YS1 lysis buffer was mixed with 100 μL of logarithmically growing pathogenic Escherichia coli EC-YS1 bacterial culture at the optimal MOI of 0.01, and incubated for 1 h at 4℃, 15℃, 25℃, 40℃, 50℃, 60℃, 70℃, and 80℃, respectively. Phage titer was determined using the double-layer plate method, with each experiment repeated three times.

[0057] SM buffer solutions with pH values ​​of 1.0, 3.0, 5.0, 7.0, 9.0, 11.0, and 13.0 were prepared using 1 M HCl and 1 M NaOH. The phage stock solution was then diluted with SM buffer solutions of different pH values ​​and incubated in 37 °C water for 1 h. The phage titer was determined using the bilayer plate method, with each experiment repeated three times.

[0058] Result: As Figure 3 , 4 As shown, this result indicates that bacteriophage vB_EcoM_YS1 has strong thermal and pH stability, and can still maintain high titer under conditions of 4-40℃ and pH 5-9.

[0059] Example 6: Genome-wide analysis and safety analysis of bacteriophage vB_EcoM_YS1

[0060] Phage DNA was extracted using the TaKaRa Viral DNA Extraction Kit according to the instructions and sequenced by shotgun sequencing (WGS) on the Illumina NovaSeq platform. The data were assembled using A5-MiSeq v20160825 and SPAdesv 3.12.0.

[0061] Sequences were compared for homology, and phage sequences with high homology were identified using Blasten (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Virulence genes and antibiotic resistance genes in the phages were retrieved by comparing and querying the Virulence Factor Database (VFDB) (http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi?func=VFanalyzer) and the Comprehensive Antibiotic Resistance Database (CARD) (https: / / card.mcmaster.ca / ).

[0062] Twenty BALB / c mice weighing approximately 20g were randomly divided into an experimental group and a control group. The experimental group was injected with 200μL of purified phage vB_EcoM_YS1 solution, while the control group was injected with 200μL of sterile PBS. The mortality of the mice was observed.

[0063] Whole-genome sequencing results showed that the genome length of bacteriophage vB_EcoM_YS1 was 169,025 bp. The A, C, G, and T base contents were 28.80%, 19.18%, 21.45%, and 30.57%, respectively, and the overall GC content was 40.62%. Figure 5 ).

[0064] Based on the phage genome map and annotation, vB_EcoM_YS1 has 272 coding sequences (CDS), which are mainly involved in phage structure, host cell lysis, replication, and metabolism. Furthermore, the vB_EcoM_YS1 genome does not contain virulence genes or antibiotic resistance genes, and no mice in either the experimental or control groups died.

Claims

1. A canine-derived Escherichia coli (E. coli) bacteriophage, characterized in that, The bacteriophage was named Escherichia coli phage vB_EcoM_YS1 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67465-B1.

2. Use of the canine Escherichia coli (E. coli) bacteriophage according to claim 1 in any of the following aspects: (1) Preparation of microbial agents for lysing Escherichia coli bacteriophages; (2) To prepare pharmaceutical preparations for the prevention or treatment of Escherichia coli infection; (3) Prepare feed or feed additives for the prevention or treatment of Escherichia coli infection; (4) Prepare health products for the prevention or treatment of Escherichia coli infection; (5) Prepare cleaning agents or disinfectants for livestock and poultry breeding environments to prevent Escherichia coli infection.

3. The use as described in claim 2, characterized in that, The Escherichia coli mentioned include enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing Escherichia coli (STEC).

4. A microbial preparation for lysing Escherichia coli, characterized in that, The microbial preparation contains the canine Escherichia coli phage as described in claim 1.

5. A pharmaceutical preparation for the prevention or treatment of Escherichia coli infection, characterized in that, The active ingredient of the pharmaceutical preparation includes the canine Escherichia coli phage as described in claim 1.

6. A feed additive, characterized in that, The feed additive contains the canine Escherichia coli phage as described in claim 1.

7. A cleaning agent or disinfectant, characterized in that, The cleaning agent or disinfectant contains the canine Escherichia coli phage as described in claim 1.

8. The microbial preparation according to claim 4, the pharmaceutical preparation according to claim 5, and the feed additive according to claim 6, characterized in that, The dosage forms of the microbial preparations, pharmaceutical preparations, or feed additives include one or more of the following: solutions, powders, gels, granules, emulsions, suspensions, and lyophilized agents.

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