Escherichia coli bacteriophage, bacteriophage composition and application thereof

Abstract

The invention discloses an Escherichia coli bacteriophage, a bacteriophage composition and application thereof, the Escherichia coli bacteriophage is an Escherichia coli bacteriophage D4 or an Escherichia coli bacteriophage D10, the preservation number of the Escherichia coli bacteriophage D4 is CGMCC NO.46363, the preservation number of the Escherichia coli bacteriophage D10 is CGMCC NO.46366, and the preservation number of the The strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 2, 2025, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bacteriophage composition comprises the escherichia coli bacteriophage and other bacteriophages. The Escherichia coli phage D4 or D10 shows excellent splitting performance on clinically separated multi-drug-resistance Escherichia coli, can be used as an active component independently or in combination with other phages, and is used for preparing environment-friendly biological bactericides, pharmaceutical preparations or disinfectants. The compound is applied to the aspects of sterilization, bacteriostasis and disinfection of multi-drug-resistant escherichia coli, treatment or prevention of infectious diseases caused by escherichia coli and the like.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to an Escherichia coli bacteriophage, its bacteriophage composition, and its applications. Background Technology

[0002] There are six main types of pathogenic Escherichia coli: enteropathogenic Escherichia coli (EPEC) that can cause gastrointestinal infections, enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli (EHEC), enteroaggregative Escherichia coli (EAEC), and Escherichia coli (ESIES) that produces Shiga-like toxins and has a certain degree of invasiveness, which has been discovered in recent years. In addition, there is urethral pathogenic Escherichia coli (UPEC) that can cause urinary tract infections, and the newly named enteroaggregative adherent Escherichia coli (EAggEC).

[0003] With the widespread use of antibiotics in clinical bacterial infection control, resistance to many commonly used antimicrobial agents among Enterobacteriaceae has gradually increased, and carbapenem-resistant Enterobacteriaceae (CRE) have emerged, clinically characterized by multidrug resistance or pan-drug resistance, and are rapidly spreading globally. CRE typically exhibits resistance to cephalosporins, β-lactam+enzyme inhibitors, monocyclic β-lactams, fluoroquinolones, and aminoglycosides. The resistance mechanisms of CRE mainly include alterations to bacterial cell wall penicillin-binding proteins, increased efflux pump expression, decreased membrane permeability, and carbapenemase production. Compared to carbapenemase-sensitive strains, CRE often carries multiple resistance genes, limiting treatment options, requiring longer treatment durations, increasing treatment costs, and resulting in more severe symptoms and higher mortality rates, posing a significant challenge to clinical practice.

[0004] Currently, phage therapy is a promising alternative treatment for preventing and treating CRE infection. Phages ( bacteriophage Bacteriophages are a class of viruses that use bacteria, fungi, and other microorganisms as hosts, and are widely distributed in the natural environment and in the human body. The genome of a bacteriophage encodes and synthesizes various enzymes, such as endolysins, depolymerases, and peptidoglycan hydrolases associated with viral particles. These enzymes degrade bacterial peptidoglycans, capsular polysaccharides, and other substances, assisting bacteriophages in disrupting the integrity of bacterial biofilms and thus promoting efficient biofilm penetration. Numerous high-profile and well-described clinical case reports, coupled with more widely available bacteriophage identification and production technologies, have led to the increased use of bacteriophages in clinical medicine in recent years.

[0005] However, the currently developed bacteriophage resources for the effective prevention and control of CRE infection are still relatively limited, and their lysis spectra are narrow and vary. Therefore, the prevention and control of the latest CRE Escherichia coli requires the continuous development of new bacteriophages and their preparations.

[0006] Therefore, existing technologies need further improvement. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides an Escherichia coli bacteriophage, a bacteriophage composition thereof, and its applications. This Escherichia coli bacteriophage exhibits excellent lytic performance and a broad lytic spectrum against multidrug-resistant pathogens (CRE). This bacteriophage and its composition with other bacteriophages can be used as active ingredients for the inhibition of CRE and the prevention and treatment of diseases caused by CRE.

[0008] To address the above problems, this application provides the following technical solution: Firstly, this application provides a strain of Escherichia coli bacteriophage, which is an Escherichia coli bacteriophage ( Escherichia virus D4 or E. coli phage ( Escherichia virus The preservation number of Escherichia coli phage D4 is CGMCC NO. 46363, and the preservation number of Escherichia coli phage D10 is CGMCC NO. 46366.

[0009] Phage D4 was collected from sediments at the bottom of the Yaojiang River in Ningbo City, Zhejiang Province, and deposited on January 2, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Under a microscope, bacteriophage D4 is tadpole-shaped, consisting of a head and a tail. The head has a polyhedral structure, and the tail sheath is retractable. Its total length is 175–185 nm, and its transverse diameter (i.e., head width) is 75–85 nm. The tail length is 85–95 nm. It belongs to the Myotail Phage family.

[0011] In this application, phage D4 or D10 includes mutant strains with point mutations, deletion mutations, or addition mutations exhibiting homology higher than 98% or 99% and maintaining substantially the same bactericidal activity. Since phages are highly susceptible to mutation during replication, mutants of the aforementioned phages are also within the scope of protection claimed in this application. The sequences of phage D4 or D10 can be obtained by sequencing the biological material preserved according to this invention using known methods. For those skilled in the art, screening for mutants with extremely similar characteristics to the phages provided by this invention requires no inventive effort.

[0012] This bacteriophage D4 strain exhibits a certain degree of acid-base stability. Experiments showed that its titer remained largely unchanged from its initial titer within a pH range of 3 to 9, maintaining relatively stable activity. This has significant application value in the treatment of diseases using bacteriophages. The bacteriophage's good tolerance to acid and high titer ensure that a sufficient number of bacteriophages can pass through the digestive tract.

[0013] In addition, Escherichia coli phage D4 maintained high activity after being bathed in water at 50℃ and 60℃ for 2 hours, with little change from the initial titer; after being bathed in water at 70℃ for 2 hours, the activity decreased by only one order of magnitude, indicating good heat resistance.

[0014] Furthermore, the currently prevalent drug-resistant Escherichia coli exhibits good lysis performance. A lysis experiment using E. coli phage D4 against 16 strains of E. coli with different drug resistance at the Second Hospital of Ningbo City demonstrated that this phage can specifically lyse four of the multidrug-resistant E. coli pathogens (16-B037, 16-B043, 19-F-131, and 19-WJ-533). Clinically, this phage shows promise in inhibiting the growth of these four multidrug-resistant pathogens and in preventing and treating diseases caused by them.

[0015] This application also provides a strain of Escherichia coli bacteriophage ( Escherichia virus D10, with accession number CGMCC NO.46366, was also collected from sediments at the bottom of the Yaojiang River in Ningbo City, Zhejiang Province. It was deposited on January 2, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] Under a microscope, E. coli bacteriophage D10 is tadpole-shaped, consisting of a head and a tail. Its total length is 215–225 nm, and its transverse diameter (head width) is 75–85 nm; the tail length is 115–125 nm. The head has a polyhedral structure, and the tail sheath is retractable. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), this bacteriophage belongs to the Myocaudidae family. This bacteriophage also exhibits good temperature and acid-base stability.

[0017] Secondly, this application provides a phage composition comprising the aforementioned Escherichia coli phage D4 / D10 and other phages.

[0018] In practical applications, in order to further broaden the lysis spectrum of phage preparations, give full play to the differences in the lysis spectra of different phages, and achieve complementary advantages, the above-mentioned Escherichia coli phage D4 / D10 can be used in combination with other phages.

[0019] Optionally, the phage composition includes: the aforementioned Escherichia coli phage D4 and Escherichia coli phage D9, wherein the preservation number of Escherichia coli phage D9 is CGMCC NO. 46365.

[0020] Escherichia coli phage D9 was also collected from sediments at the bottom of the Yaojiang River in Ningbo City, Zhejiang Province, and deposited on January 2, 2025, at the China General Microbiological Culture Collection Center, at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

[0021] In addition to lysing the aforementioned seven multidrug-resistant Escherichia coli pathogens (16-B037, 16-B043, 19-F-131, 18-F-25, 19-WJ-533, 18-WJ-340, and 18-F-18), this phage composition also lyses four other multidrug-resistant Escherichia coli pathogens: 18-WJ-84, 18-F-67, 19-WJ-739, and 19-WJ-755, significantly expanding the lysis range. In particular, this composition exhibits specific lysis performance against Escherichia coli 18-WJ-84, which is difficult to lyse with other phage compositions.

[0022] Optionally, the phage composition comprises: the aforementioned Escherichia coli phage D4 and Escherichia coli phage 10.

[0023] This phage composition exhibits specific and excellent lysis performance against pathogenic Escherichia coli 18-F-67, 19-WJ-739 and 19-WJ-755, significantly expanding the lysis range.

[0024] Optionally, the phage composition includes: the aforementioned *E. coli* phage D4 and *E. coli* phage 5, wherein the accession number of *E. coli* phage D5 is CGMCC NO.46364. *E. coli* phage D5 was also collected from sediments at the bottom of the Yaojiang River in Ningbo City, Zhejiang Province, and deposited on January 2, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] Optionally, the phage composition includes: any three of the aforementioned Escherichia coli phage D4, Escherichia coli phage D5, Escherichia coli phage D9, and Escherichia coli phage D10.

[0026] For example, the phage composition may be: a composition of Escherichia coli phages D4, D5 and D9, a composition of Escherichia coli phages D4, D9 and D10, a composition of Escherichia coli phages D4, D5 and D10, or a composition of these four phages: Escherichia coli phages D4, D5, D9 and D10.

[0027] Experiments have shown that the different phage compositions exhibit different lysis profiles based on their different synergistic effects. Therefore, the different phage compositions can be rationally selected for application according to the actual situation of the pathogen.

[0028] Thirdly, this application also provides the use of the aforementioned Escherichia coli phage D4 or Escherichia coli phage D10 or the aforementioned phage composition in the antibacterial activity of Escherichia coli.

[0029] Fourthly, this application also provides a biological bactericide, the active ingredient of which includes the aforementioned Escherichia coli bacteriophage or the aforementioned bacteriophage composition.

[0030] Fifthly, this application also provides the use of the above-mentioned Escherichia coli phage D4 or Escherichia coli phage D10 or the aforementioned phage composition in the preparation of a medicament for the prevention and treatment of diseases caused by Escherichia coli infection.

[0031] The prevention and treatment include both prevention and treatment. The term "prevention" as used herein refers to all behaviors that suppress or delay the disease by administering the composition. The term "treatment" as used herein refers to all behaviors that improve or alleviate the disease by administering the composition.

[0032] Based on the aforementioned lytic properties of the phage, the aforementioned Escherichia coli phage or the aforementioned phage composition can be used in the preparation of drugs for the prevention and treatment of diseases caused by Escherichia coli infection. These diseases include various illnesses caused by Escherichia coli infection, such as urinary tract infections, wound infections, bacteremia, or endocarditis caused by Escherichia coli infection.

[0033] Sixthly, this application also provides a phage drug formulation, the active ingredient of which includes the aforementioned Escherichia coli phage D4 / D10 or the aforementioned phage composition.

[0034] Optionally, the phage drug formulation further comprises a pharmaceutically acceptable carrier in the form of a solution, powder, gel, granule, or lyophilized product. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered active ingredient. For the pharmaceutical composition to be formulated into a liquid formulation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, and ethanol. They can be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers, and antibacterial agents, may be added if desired. The compositions of the present invention can also be formulated into injections (e.g., aqueous solutions, suspensions, and emulsions), or pills, capsules, granules, or tablets, when also combined with diluents, dispersants, surfactants, binders, and / or lubricants.

[0035] Seventhly, this application also provides an environmental disinfectant, the active ingredient of which includes the aforementioned Escherichia coli bacteriophage or the aforementioned bacteriophage composition.

[0036] The environmental disinfectant can be used in hospitals, clinics, and health centers to prevent hospital-acquired infections. It can also be used for disinfection of public places and patient living environments to prevent the growth of pathogens. The environmental disinfectant can be applied to the object to be disinfected by spraying or soaking.

[0037] The present invention has the following beneficial effects: 1. The *E. coli* bacteriophages D4 and D10 provided by this invention, after whole-genome sequencing, do not contain virulence genes or drug resistance genes. This invention does not involve any genetic modification of the tested bacteriophages, indicating their safety and lack of toxic side effects. *E. coli* bacteriophage D4 is a bacteriophage isolated from nature and can be produced using the non-pathogenic *E. coli* standard strain ATCC11775. It can undergo large-scale fermentation culture, and its culture medium can survive stably at room temperature, exhibiting high biocompatibility.

[0038] 2. Escherichia coli phage D4 has good resistance to high temperature and acid, and the phage titer is high, which can ensure that a sufficient number of phages pass through the digestive tract.

[0039] 3. The Escherichia coli phage D4 can infect multiple strains of multidrug-resistant prevalent pathogenic Escherichia coli isolated clinically. It can be used alone or mixed with other phages as a phage composition. As an active ingredient, it can be used to prepare biological bactericides, pharmaceutical preparations or disinfectants, and applied to the sterilization and bacteriostasis of MDR Escherichia coli, disinfection, and treatment or prevention of infectious diseases caused by Escherichia coli.

[0040] 4. Escherichia coli phage D4 can be combined with phages D5, D9, and D10 to form phage compositions. These different compositions exhibit different lytic spectrum characteristics based on their varying synergistic effects. Therefore, the appropriate phage composition can be selected for application based on the specific pathogen. Attached Figure Description Figure 1 The images shown are electron micrographs of the Escherichia coli phages of the present invention; A is Escherichia coli phage D4, B is Escherichia coli phage D5, C is Escherichia coli phage D9, and D is Escherichia coli phage D10. Figure 2 This is a schematic diagram illustrating the thermal stability of Escherichia coli phages according to the present invention; A represents Escherichia coli phage D4, B represents Escherichia coli phage D5, C represents Escherichia coli phage D9, and D represents Escherichia coli phage D10. Figure 3 This is a schematic diagram illustrating the acid-base stability of the Escherichia coli phages of the present invention; A represents Escherichia coli phage D4, B represents Escherichia coli phage D5, C represents Escherichia coli phage D9, and D represents Escherichia coli phage D10. Figure 4 This is a schematic diagram illustrating the optimal infection multiple of the Escherichia coli phage of the present invention; A represents Escherichia coli phage D4, B represents Escherichia coli phage D5, C represents Escherichia coli phage D9, and D represents Escherichia coli phage D10. Figure 5 This is a schematic diagram of the one-step growth curve of the Escherichia coli phage of the present invention; A is Escherichia coli phage D4, B is Escherichia coli phage D5, C is Escherichia coli phage D9, and D is Escherichia coli phage D10. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0042] In the following examples, the strain codes used are all based on our company's naming convention.

[0043] coli phage ( Escherichia virusD4, with accession number CGMCC46363, was deposited on January 2, 2025, at the China General Microbiological Culture Collection Center, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0044] coli phage ( Escherichia virus D5, with accession number CGMCC46364, was deposited on January 2, 2025, at the China General Microbiological Culture Collection Center, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0045] coli phage ( Escherichia virus D9, with accession number CGMCC46365, was deposited on January 2, 2025, at the China General Microbiological Culture Collection Center, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0046] coli phage ( Escherichia virus D10, with accession number CGMCC46366, was deposited on January 2, 2025, at the China General Microbiological Culture Collection Center, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0047] Example 1: Isolation and purification of Escherichia coli bacteriophage 1. Experimental Methods In this invention, the source sample for isolating Escherichia coli bacteriophage was collected from sediments at the bottom of the Yaojiang River in Ningbo City, Zhejiang Province. After being filtered through double-layer filter paper, the sample was centrifuged at low speed at room temperature, and then the supernatant was filtered through a 0.22 μm filter membrane.

[0048] (1) Isolation of bacteriophages: Take 10 mL of the filtered supernatant and add it to 10 mL of BHI medium. At the same time, add 1 mL of the host bacteriophage bacterial culture in the logarithmic phase. Incubate at 37℃ for 24 h. Take the culture and centrifuge at 8000 rpm for 10 min. Filter the supernatant through a 0.22 μm filter membrane and set aside. Take 100 μL of the filtrate and mix it evenly with 300 μL of the host Escherichia coli bacterial culture. Let it stand for 15 min to allow it to fully bind to the receptors on the bacterial surface. Add the above mixture to 4 mL of MH semi-solid agar medium cooled to 50℃. Mix well and immediately spread it on the solidified MH plate. After the agar solidifies, incubate upside down at 37℃ for 8-12 h and observe the growth of phage plaques.

[0049] (2) Phage purification: On a double-layer plate where phage plaques have formed, large and clear phage plaques are picked up with a sterile pipette tip, shaken in 1 mL of SM solution to desorb, and then filtered through a 0.22 μm microporous membrane to obtain phage filtrate. This filtrate is inoculated into 5 mL of MH liquid medium, 100 μL of the corresponding host *E. coli* culture is added and mixed well, and the mixture is incubated overnight at 37°C and 180 rpm. After centrifugation at 5000 rpm for 10 min, the supernatant is collected and filtered through a bacterial filter membrane. The morphology of the phage plaques is observed using the double-layer plate method. After repeating the operation 3-5 times, phage plaques with consistent shape and size can be obtained.

[0050] 2. Experimental Results Finally, a strain of Escherichia coli bacteriophage was isolated and purified, and named Escherichia coli bacteriophage D4. Its plaque formation on a double-layer agar plate is shown in the image below. Figure 1 As shown.

[0051] Escherichia coli phage D5, Escherichia coli phage D9 and Escherichia coli phage D10 were isolated using the same method described above.

[0052] Example 2: Electron microscopic observation of Escherichia coli bacteriophages 1. Experimental Methods The supernatant of the cultures of phages D4, D5, D9 and D10 prepared in Example 1 was used for electron microscopy observation: 20 μL of sample was dropped onto a copper grid and allowed to settle naturally for 15 min. Excess liquid was absorbed from the side with filter paper. One drop of 2% phosphotungstic acid (PTA) was added to the copper grid and stained for 10 min. The staining solution was then absorbed from the side with filter paper and dried for electron microscopy observation.

[0053] 2. Experimental Results and Analysis like Figure 1 As shown in A, Enterobacterial phage D4 is tadpole-shaped, consisting of a head and a tail. Its total length is 175–185 nm, and its transverse diameter (i.e., head width) is 75–85 nm. The tail length is 85–95 nm. The tail sheath is retractable. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), this phage belongs to the Myocaudidae family.

[0054] like Figure 1 As shown in B, Escherichia coli phage D5 is tadpole-shaped, consisting of a head and a tail. Its total length is 165–175 nm, and its transverse diameter (i.e., head width) is 60–70 nm. The tail length is 85–95 nm. The head has a polyhedral structure, and the tail sheath can retract. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), this phage belongs to the Myocaudae family.

[0055] like Figure 1As shown in C, Escherichia coli phage D9 is tadpole-shaped, consisting of a head and a tail. Its total length is 180–190 nm, and its transverse diameter (i.e., head width) is 70–80 nm. The tail length is 90–100 nm. The head has a polyhedral structure, and the tail sheath can retract. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), this phage belongs to the Myocaudae family.

[0056] like Figure 1 As shown in Figure D, Escherichia coli phage D10 is tadpole-shaped, consisting of a head and a tail. Its total length is 215–225 nm, and its transverse diameter (i.e., head width) is 75–85 nm. The tail length is 115–125 nm. The head has a polyhedral structure, and the tail sheath is retractable. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), this phage belongs to the Myocaudae family.

[0057] Example 3 Determination of phage titer 1. Experimental Method: The cultured phage stock solution was serially diluted 10-fold. 10 μL of the appropriately diluted phage and 1 mL of the host bacterial suspension were mixed and added to sterile EP tubes, then thoroughly mixed. After incubation at 37°C for 5 min, the tubes were plated using the double-layer agar method. After solidification with agar, the tubes were inverted and incubated at 37°C for 24 h. The results were then observed. Each dilution gradient was performed in triplicate. For plaque counting, dilutions with plaque counts between 30 and 300 were used as the baseline. The average of the three replicates for each dilution was used to calculate the phage titer.

[0058] Phage titer (pfu / mL) = average number of phage plaques × dilution factor × 100.

[0059] 2. Experimental Results and Analysis The titer of phage D4 was 2.45 × 10⁻⁶. 7 PFU / mL, phage D5 titer is 2.95 × 10⁻⁶. 7 PFU / mL, phage D9 titer is 1.2 × 10⁻⁶. 7 PFU / mL, phage D10 titer is 4.5 × 10⁻⁶. 6 PFU / mL.

[0060] Example 4: Determination of the optimal multiple of infection (MOI) of Escherichia coli phage against Escherichia coli 1. Experimental Methods The host bacterium *Escherichia coli* standard strain ATCC11775 was mixed with bacteriophages at MOI ratios of 10, 1, 0.1, 0.01, and 0.001, respectively. The bacterial concentration was determined by OD600, and the MOI corresponding to the lowest final bacterial concentration was the optimal MOI. The experiment was repeated three times.

[0061] The optimal multiple of infection (MOI) of Escherichia coli phages D9 and D10 was determined according to the above method.

[0062] 2. Experimental Results and Analysis The results are as follows Figure 4 As shown in A, the MOI of Escherichia coli phage D4 is 1; Figure 4 The results of B indicate that the MOI of E. coli phage D5 is 1. Figure 4 As shown in C, the MOI of E. coli phage D9 is 1. Figure 4 As shown in D, the MOI of Escherichia coli phage D10 is 0.1.

[0063] Example 5: Determination of the one-step growth curve of bacteriophage 1. Experimental Methods A suspension of *E. coli* in the logarithmic growth phase was inoculated with *E. coli* ATCC11775 and *Escherichia virus* D4 / D5 / D9 / D10 bacteriophages at the optimal multiplicity of infection ratio. The mixture was incubated in a water bath at 37°C for 5 min, followed by centrifugation at 12000 rpm for 5 min at room temperature. The supernatant was discarded to remove unadsorbed free bacteriophages. This process was repeated three times with BHI broth. The precipitate was then resuspended in BHI broth at 37°C and immediately placed in a shaker at 200 rpm at 37°C for incubation, with timing maintained. Samples were taken at different time points, filtered, and the bacteriophage titer was determined using the double-layer plate method. A one-step growth curve of the bacteriophage was plotted with time on the x-axis and the logarithmic value of the bacteriophage titer on the y-axis. The latency and lysis phases of the bacteriophage were determined, and the outbreak size was calculated.

[0064] Outbreak volume = Phage titer at the end of the outbreak / Host bacterial concentration at the beginning of infection.

[0065] 2. Experimental Results and Analysis The results of the one-step growth curve are as follows Figure 5 As shown in Figure A, the latency period of D4 was 15 min, the burst period was 105 min, the burst dose was 43 PFU / cell, and the curve tended to stabilize after 120 min, entering the plateau phase.

[0066] like Figure 5 As shown in Figure B, the incubation period of phage D5 was 10 min, the outbreak period was 80 min, the outbreak amount was 217 PFU / cell, and the curve tended to stabilize after 90 min, entering the plateau phase.

[0067] like Figure 5As shown in C, the incubation period of phage D9 was 15 min, the outbreak period was 75 min, the outbreak amount was 126 PFU / cell, and the curve tended to stabilize after 90 min, entering the plateau phase.

[0068] like Figure 5 As shown in Figure D, the incubation period of phage D10 was 5 min, the outbreak period was 105 min, the outbreak amount was 622 PFU / cell, and the curve tended to stabilize after 120 min, entering the plateau phase.

[0069] Example 6: Determination of the thermal stability of bacteriophages 1. Experimental Methods The valence is 2.45 × 10 7 PFU / mL, 2.95×10 7 PFU / mL, 1.2×10 7 PFU / mL, 4.5×10 6 PFU / mL phage D4 / D5 / D9 / D10 stock solution was aliquoted into 50 mL sterile centrifuge tubes and incubated at 37℃, 50℃, 60℃, 70℃, 80℃ and 90℃ for 2 h, respectively. Then, it was serially diluted 10-fold with physiological saline and plated on double plates to determine its potency.

[0070] 2. Experimental Results and Analysis like Figure 2 As shown in Figure A, Escherichia coli phage D4 maintained high activity after being incubated in water at 50°C and 60°C for 2 hours, with essentially no change from the initial titer; after being incubated in water at 70°C for 2 hours, the activity decreased by only one order of magnitude.

[0071] like Figure 2 As shown in B, Escherichia coli phage D5 maintained high activity after being incubated in water at 50℃ and 60℃ for 2 hours, with essentially no change from the initial titer; after being incubated in water at 70℃ for 2 hours, the activity decreased by only one order of magnitude; and it remained active after being incubated in water at 80℃ and 90℃ for 2 hours.

[0072] like Figure 2 As shown in Figure C, Escherichia virus D9, a bacteriophage of Escherichia coli, maintained high activity after being bathed in water at 50°C to 70°C for 2 hours, with essentially no change from its initial titer.

[0073] like Figure 2 As shown in D, Escherichia virus D10, a bacteriophage of Escherichia coli, maintained high activity after being in a water bath at 50°C for 2 hours, with essentially no change from the initial titer; after being in a water bath at 60°C for 2 hours, it decreased by only one order of magnitude, and after being in a water bath at 70°C for 2 hours, it decreased by one order of magnitude.

[0074] The above results demonstrate that Escherichia coli bacteriophages D4, D5, D9, and D10 possess excellent heat resistance properties, which have significant application value in the production, transportation, and storage of bacteriophages.

[0075] Example 7: Determination of the acid-base stability of bacteriophages 1. Experimental Methods The pH of physiological saline was adjusted using dilute hydrochloric acid and dilute NaOH solution to prepare buffer solutions with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. 100 μL of a buffer solution with a titer of 1.2 × 10⁻⁶ was added to 900 μL of the prepared buffer solution. 7 Phage proliferation broth at PFU / mL was incubated in a water bath at 37°C for 2 hours, then diluted 10-fold with physiological saline and plated on double plates for titer determination. Results were statistically analyzed after incubation at 37°C upside down for 4-6 hours.

[0076] 2. Experimental Results and Analysis The results are as follows Figure 3 As shown, under pH 3-9 conditions, the titers of phages D4, D5, D9, and D10 were similar to their initial titers, indicating that these four phage strains have good acid resistance. This has significant application value in phage therapy, as the phages' good acid tolerance and high titer ensure a sufficient number of phages can pass through the digestive tract.

[0077] Example 8: Determination of the lytic performance of Escherichia coli bacteriophage against clinically pathogenic Escherichia coli 1. Experimental Methods Host bacteria: Sixteen monoclonal strains of pathogenic Escherichia coli with different drug resistance were selected from the Second Hospital of Ningbo (see Tables 1 and 2 for details). These strains are non-patented and can be obtained by the public from the CCTCC Collection Center.

[0078] (1) Each host bacterium was inoculated into a centrifuge tube containing 3 mL of BHI (brain and heart infusion medium) and cultured at 37°C and 180 rpm for 8 h to obtain bacterial suspensions. 300 μL of bacterial suspension and 100 μL of Escherichia coli phage proliferation solution were mixed with MH semi-solid medium and spread onto the prepared MH plates. After air drying, the plates were cultured at 37°C for 8-12 h, and the results were observed. The standard strain of Escherichia coli ATCC11775 was used as a control.

[0079] (2) Follow the method in (1) except that the filtrate of Escherichia coli phage D4, D5, D9 and D10 phages is a mixture of two, three or four phages (1:1 / 1:1:1 / 1:1:1:1), see Table 3 for details.

[0080] Table 1 Information on clinically pathogenic Escherichia coli

[0081] Table 2 Drug resistance information of clinically pathogenic Escherichia coli

[0082] 2. Experimental Results and Analysis The experimental results are shown in Table 3. Escherichia coli phage D4 can lyse four multidrug-resistant bacteria, namely 16-B037, 16-B043, 19-F-131 and 19-WJ-533 (drug resistance information is shown in Table 2). Clinically, these four specific multidrug-resistant pathogens have promising applications in inhibiting their growth and preventing and treating diseases caused by them.

[0083] On the other hand, the combination of Escherichia coli phage D4 and Escherichia coli phage D9 lysed 10 pathogenic Escherichia coli strains: 16-B037, 16-B043, 19-F-131, 18-F-25, 19-WJ-533, 18-WJ-340, 19-F-160, 18-F-67, 19-WJ-739, and 18-F-50; while the combination of Escherichia coli phage D4 and Escherichia coli phage D10 lysed 16-B037, 16-B043, 19-F-131, 19-WJ-533, and 18-WJ-340. Nine pathogenic bacteria, namely 40, 18-F-67, 19-WJ-739, 19-WJ-755, and 18-F-50, were lysed by a combination of Escherichia coli phages D4, D5, and D9; eleven pathogenic bacteria, namely 16-B037, 16-B043, 19-F-131, 18-F-25, 19-WJ-533, 18-WJ-340, 18-F-18, 18-F-67, 19-WJ-739, 19-WJ-755, and 18-F-50, were lysed by a combination of the above four phages; and ten pathogenic bacteria were lysed by a combination of the above four phages.

[0084] The results indicate that different synergistic effects occur among the phages in the above-mentioned phage composition, resulting in different phage lysis specificities and lysis spectra of the composition. This significantly expands the lysis spectrum of Escherichia coli phage D4 on the original basis, broadens its application range, and makes it more promising for application.

[0085] Table 3. Lysis results of Escherichia coli bacteriophages on clinically pathogenic Escherichia coli.

[0086] Example 9: Detection of Escherichia coli bacteriophage virulence genes and drug resistance genes 1. Experimental Methods Based on the CARD resistance gene database and the VFDB virulence factor database, bioinformatics analysis was performed on the whole genome of Escherichia coli phage D4 to determine whether it contains the aforementioned virulence genes and drug resistance genes.

[0087] 2. Experimental Results and Analysis The results showed that E. coli phage D4 does not contain any of the 660 virulence genes and 640 drug resistance genes in the aforementioned database. Therefore, this phage poses no risk of virulence or drug resistance gene contamination in clinical use. This phage is safe to use.

[0088] Example 10 Phage Genome Analysis 1. Experimental Methods After enriching and culturing a single strain of bacteriophage D4, Guangzhou Guangdong Meggene Technology Co., Ltd. was commissioned to perform whole-genome sequencing on the phage. Sequence analysis was then performed on the sequencing results of this bacteriophage.

[0089] 2. Analysis Results (1) The genome is 68691 bp in length, with a G+C content of 46.17%, and the base contents of C, G, A, and T are 22.75%, 23.42%, 27.38%, and 26.45%, respectively. Online RAST annotation of the whole genome showed that it contains 103 open reading frames (ORFs). Among these 103 ORFs, 16 structural proteins were found, mainly including phage structural and packaging proteins (head proteins, tail proteins, tail fibrin, substrate proteins, and terminal enzyme large subunits, etc.), phage lysis-related proteins (lysins), DNA replication and modification-related proteins (DNA polymerase, DNA ligase, DNA exonuclease, etc.), and other functional proteins (oxidoreductases). Furthermore, among the 103 ORFs, 98 start codons are ATG, 5 are GTG, and 0 are TTG. Analysis using tRNAscan-SE software showed that the genome does not contain tRNA genes. Analysis of the CARD and VFDB databases showed that the genome did not contain drug resistance genes or virulence genes.

[0090] (2) In the genome of bacteriophage: the gene sequence of the tail fiber protein related to bacteriophage host recognition is shown in sequence 1 of the sequence listing; the sequence of the highly conserved terminal enzyme large subunit protein gene is shown in sequence 2 of the sequence listing; the sequence of the DNA polymerase gene is shown in sequence 3 of the sequence listing; the sequence of the lysozyme gene related to lysis ability is shown in sequence 4 and sequence 5 of the sequence listing. The relevant information of these sequences is shown in Table 4 below.

[0091] Table 4 Gene Sequence Information

[0092] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A strain of Escherichia coli bacteriophage, characterized in that, Escherichia coli bacteriophage ( Escherichia virus D4 or E. coli phage ( Escherichia virus The preservation number of the Escherichia coli phage D4 is CGMCC NO.46363, and the preservation number of the Escherichia coli phage D10 is CGMCC NO. 46366.

2. A bacteriophage composition, characterized in that, Includes Escherichia coli phage D4 or Escherichia coli phage D10 as described in claim 1.

3. A bacteriophage composition, characterized in that, include: The Escherichia coli bacteriophage as described in claim 1 ( Escherichia virus D4 and Escherichia coli phage ( Escherichia virus The contents include D10 and any two or three of Escherichia coli phage D5 and Escherichia coli phage D9; the preservation number of Escherichia coli phage D5 is CGMCC NO. 46364, and the preservation number of Escherichia coli phage D9 is CGMCC NO. 46365.

4. The use of the Escherichia coli phage according to claim 1 or the phage composition according to any one of claims 2 to 3 in the inhibition of Escherichia coli or in the preparation of medicaments for the prevention and treatment of diseases caused by Escherichia coli infection.

5. A biological antibacterial agent, characterized in that, Its active ingredients include the Escherichia coli phage as described in claim 1 or the phage composition as described in any one of claims 2 to 3.

6. A bacteriophage drug formulation, characterized in that, Its active ingredients include the Escherichia coli phage as described in claim 1 or the phage composition as described in any one of claims 2 to 3.

7. An environmental disinfectant, characterized in that, The active ingredient includes Escherichia coli phage D4 as described in claim 1 or the phage composition as described in any one of claims 2 to 3.

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