Escherichia coli bacteriophage D9, and bacteriophage composition and application thereof

Abstract

The invention discloses an Escherichia coli bacteriophage D9, a bacteriophage composition and application thereof, the preservation number of the Escherichia coli bacteriophage D9 is CGMCC (China General Microbiological Culture Collection Center) NO. 46365, and the Escherichia coli bacteriophage D9 is preserved in China General Microbiological Culture Collection Center on January 2, 2025, and the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The bacteriophage composition comprises an escherichia coli bacteriophage D9 and other bacteriophages. The coliphage D9 has better high-temperature resistance and acid resistance, is high in breeding speed, is suitable for industrial production, has excellent splitting performance on multiple strains of multi-drug-resistant clinical escherichia coli, can be used as an active ingredient of a biological bactericide, a pharmaceutical preparation or a disinfectant, and has a wide application prospect. 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 a strain of Escherichia coli bacteriophage D9, its bacteriophage composition, and its applications. Background Technology

[0002] 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 patient symptoms and higher mortality rates, posing a significant challenge to clinical practice.

[0003] bacteriophage ( bacteriophage Phages 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. In 1919, De Hele successfully treated four patients with bacterial dysentery using bacteriophages at a children's hospital in Paris, pioneering bacteriophage therapy. Subsequently, people began to expand bacteriophage therapy trials, including the treatment of cholera, dysentery, bubonic plague, conjunctivitis, and skin infections. In 1921, the application of bacteriophages in the treatment of human infections was first reported.

[0004] 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 and capsular polysaccharides, assisting bacteriophages in disrupting the integrity of bacterial biofilms and thus promoting efficient biofilm penetration. Phage therapy is a promising alternative treatment for the prevention and treatment of CRE infection. Numerous high-profile and well-described clinical case reports, coupled with more widely available phage identification and production technologies, have led to the wider clinical use of bacteriophages 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, the existing technology needs further improvement. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a strain of Escherichia coli phage D9, its phage composition, and its applications. This Escherichia coli phage D9 exhibits excellent lytic performance and a broad lytic spectrum against multidrug-resistant pathogens (CRE), and can be used as an active ingredient 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 ( Escherichia virus D9, with accession number CGMCC NO. 46365.

[0009] Phage D9 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 D9 is tadpole-shaped, consisting of a head and a tail. The head has a polyhedral structure, and the tail sheath can retract. The total length of the bacteriophage is 180–190 nm, the transverse diameter (i.e., head width) is 70–80 nm, and the tail length is 90–100 nm. It belongs to the Myotail Phagesaceae family.

[0011] In this application, bacteriophage D9 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 bacteriophages are highly susceptible to mutation during replication, mutants of the aforementioned bacteriophages are also within the scope of protection claimed in this application. The sequence of bacteriophage D9 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 bacteriophages provided by this invention requires no inventive effort.

[0012] 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.

[0013] This demonstrates that bacteriophage D9 has a high burst rate and excellent reproductive capacity, making it suitable for industrial production and capable of increasing yield.

[0014] This bacteriophage exhibits a certain degree of acid-base stability. Experiments have shown that it maintains relatively stable activity within a pH range of 3-9. This has significant application value in the treatment of diseases using bacteriophages. The bacteriophage demonstrates good tolerance to acid and possesses high potency, ensuring a sufficient quantity of bacteriophages can pass through the digestive tract.

[0015] In addition, Escherichia coli phage D9 also has a certain degree of temperature stability. It maintains high activity after being bathed in water at 50℃ and 70℃ for 2 hours, with little change from the initial titer; and it only decreases by one order of magnitude after being bathed in water at 60℃ for 2 hours.

[0016] Furthermore, the currently prevalent drug-resistant Escherichia coli exhibits good lysis performance. A lysis experiment using E. coli phage D9 against 16 strains of E. coli with different drug resistance at the Second Hospital of Ningbo City demonstrated that this phage can specifically lyse 6 of the multidrug-resistant E. coli pathogens (16-B037, 16-B043, 18-WJ-340, 19-F-131, 19-WJ-533, and 18-F-25), showing a broad lysis spectrum. Clinically, this phage shows promise in inhibiting the growth of these 6 groups of multidrug-resistant pathogens and in preventing and treating diseases caused by these pathogens.

[0017] Secondly, this application provides a phage composition comprising the aforementioned Escherichia coli phage D9 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 D9 can be used in combination with other phages.

[0019] Optionally, the phage composition includes: the aforementioned Escherichia coli phage D9 and Escherichia coli phage D10, wherein the preservation number of Escherichia coli phage D10 is CGMCC NO.46366.

[0020] This phage composition, in addition to lysing Escherichia coli ( Escherichia coli In addition to 16-B037 and 16-B043, it also lyses E. coli ( Escherichia coli 18-F-50, Escherichia coli ( Escherichia fergusonii 18-F-67, Escherichia coli ( Escherichia coli )19-F-160, Escherichia coli ( Escherichia fergusonii19-WJ-739 ) and Escherichia coli ( Escherichia coli 19-WJ-755 significantly expanded the pyrolysis range.

[0021] Optionally, the phage composition further includes: Escherichia coli phage D5, the preservation number of which is CGMCC NO.46364.

[0022] The lysis spectrum of the phage composition consisting of Escherichia coli phages D5, D9 and D10 has been further broadened, and based on the synergistic effect of these three phages, their lysis specificity is different from that of any one of the phages, which further improves the flexibility of the application of these three phages.

[0023] Escherichia coli bacteriophages D10 and D5 were collected from sediments at the bottom of the Yaojiang River in Ningbo City, Zhejiang Province, and were deposited on February 26, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0024] Thirdly, this application also provides the use of the aforementioned Escherichia coli phage D9 or the aforementioned phage composition in the inhibition of Escherichia coli.

[0025] Fourthly, this application also provides a biological bactericide, the active ingredient of which includes the aforementioned Escherichia coli bacteriophage or the aforementioned bacteriophage composition.

[0026] Fifthly, this application also provides the use of the above-mentioned Escherichia coli phage D9 or the aforementioned phage composition in the preparation of medicaments for the prevention and treatment of diseases caused by Escherichia coli infection.

[0027] 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.

[0028] Based on the aforementioned lytic properties of phage D9, 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.

[0029] Sixthly, this application also provides a phage drug formulation, the active ingredient of which includes the aforementioned Escherichia coli phage D9 or the aforementioned phage composition.

[0030] 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.

[0031] Seventhly, this application also provides an environmental disinfectant, the active ingredient of which includes the aforementioned Escherichia coli phage D9 or the aforementioned phage composition.

[0032] 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 environments to prevent the growth of pathogens. The environmental disinfectant can be applied to the object to be disinfected by spraying or soaking.

[0033] The present invention has the following beneficial effects: 1. The Escherichia coli phage D9 provided by this invention does not contain virulence genes or drug resistance genes after whole-genome sequencing. This invention does not perform any genetic modification on the tested phage, indicating that it is safe and has no toxic side effects.

[0034] 2. Escherichia coli phage D9 is a phage isolated from nature. It can be produced using the non-pathogenic Escherichia coli standard strain ATCC11775 and can be cultured on a large scale. Its culture medium can survive stably at room temperature and has high biosafety.

[0035] 3. Escherichia coli phage D9 has a rapid reproduction rate, making it suitable for industrial production and increasing yield. Furthermore, it still exhibits strong lytic activity after being bathed in a 70℃ water bath for 3 hours. Under pH 3 conditions, the phage titer is not significantly different from the initial titer, indicating that the phage has good heat and acid resistance and a high titer, which ensures that a sufficient number of phages can pass through the digestive tract.

[0036] 4. The Escherichia coli phage D9 can infect multiple strains of MDR Escherichia coli isolated clinically. It can be used alone or in combination with other phages as an active ingredient to prepare biological bactericides, pharmaceutical preparations or disinfectants, and can be applied to the sterilization and bacteriostasis of MDR Escherichia coli, disinfection, and treatment or prevention of infectious diseases caused by Escherichia coli. Attached Figure Description

[0037] Figure 1 This is a photograph of the morphology of the D9 phage plaque of Escherichia coli in this invention; Figure 2 This is an electron microscope image of Escherichia coli bacteriophage D9 of the present invention; Figure 3 This is a schematic diagram illustrating the thermal stability of Escherichia coli phage D9 according to the present invention. Figure 4 This is a schematic diagram illustrating the acid-base stability of Escherichia coli bacteriophage D9 of the present invention; Figure 5 This is a schematic diagram illustrating the optimal multiplicity of infection for Escherichia coli phage D9 of the present invention; Figure 6 This is a schematic diagram of the one-step growth curve of Escherichia coli phage D9 in this invention; Figure 7 This is a photograph of the morphology of the D5 plaque of Escherichia coli bacteriophage in this invention; Figure 8 This is an electron microscope image of Escherichia coli bacteriophage D5 of the present invention; Figure 9 This is a schematic diagram illustrating the thermal stability of Escherichia coli phage D5 according to the present invention. Figure 10 This is a schematic diagram illustrating the acid-base stability of Escherichia coli bacteriophage D5 of the present invention; Figure 11 This is a schematic diagram illustrating the optimal multiplicity of infection for Escherichia coli phage D5 of the present invention; Figure 12 This is a schematic diagram of the one-step growth curve of Escherichia coli phage D5 in this invention; Figure 13 This is a photograph of the morphology of the D10 plaque of Escherichia coli bacteriophage in this invention; Figure 14 This is an electron micrograph of Escherichia coli bacteriophage D10 of the present invention; Figure 15 This is a schematic diagram illustrating the thermal stability of Escherichia coli phage D10 of the present invention. Figure 16 This is a schematic diagram illustrating the acid-base stability of Escherichia coli phage D10 of the present invention; Figure 17 This is a schematic diagram illustrating the optimal multiplicity of infection for Escherichia coli phage D10 of the present invention; Figure 18This is a schematic diagram of the one-step growth curve of Escherichia coli phage D10 of the present invention. Detailed Implementation

[0038] 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.

[0039] In the following examples, the strain codes used are all based on our company's naming convention.

[0040] 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.

[0041] coli phage ( Escherichia virus The Escherichia coli phage D5 has the accession number CGMCC NO.46364. This phage 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.

[0042] coli phage ( Escherichia virus The accession number of D10 is CGMCC NO.46366. This bacteriophage 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.

[0043] Example 1: Isolation and purification of Escherichia virus D9 from Escherichia coli bacteriophage 1. Experimental Methods The source sample for isolating Escherichia virus D9 bacteriophage in this invention 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 0.22μm filter membrane.

[0044] (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℃. After mixing, immediately spread it on the solidified MH plate. After the agar solidifies, incubate it upside down at 37℃ for 8-12 h and observe the growth of the phage plaques.

[0045] (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.

[0046] 2. Experimental Results Finally, a strain of Escherichia coli bacteriophage was isolated and purified, and named Escherichia coli bacteriophage D9. Its plaque formation on a double-layer agar plate is shown in the image below. Figure 1 As shown.

[0047] Escherichia coli phage D5 and Escherichia coli phage D10 were isolated using the same method described above.

[0048] Example 2: Electron microscopic observation of Escherichia coli bacteriophages 1. Experimental Methods The supernatants of phage cultures D9, D5 and D10 prepared in Example 1 were 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 before electron microscopy observation.

[0049] 2. Experimental Results and Analysis like Figure 2As shown, 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.

[0050] like Figure 8 As shown, 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 Myocaudidae family.

[0051] like Figure 14 As shown, 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 Myocaudidae family.

[0052] Example 3: Determination of phage titer 1. Experimental Method: The cultured phage D9, D5, and D10 stock solutions were serially diluted 10-fold. 10 μL of each appropriately diluted phage and 1 mL of 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 plate 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.

[0053] Phage titer (pfu / mL) = average number of phage plaques × dilution factor × 100.

[0054] 2. Experimental Results and Analysis The titer of phage D9 is 1.2 × 10⁻⁶. 7 PFU / mL. The titer of phage D5 was 2.95 × 10⁻⁶. 7 PFU / mL, phage D10 titer is 4.5 × 10⁻⁶. 6 PFU / mL.

[0055] Example 4: Determination of the optimal multiple of infection (MOI) of Escherichia coli by E. coli phage D9 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.

[0056] 2. Experimental Results and Analysis The results are as follows Figure 5 As shown, this result indicates that the MOI of E. coli phage D9 is 1. The results are as follows... Figure 11 As shown, this result indicates that the MOI of E. coli phage D5 is 1; Figure 17 As shown, this result indicates that the MOI of Escherichia coli phage D10 is 0.1.

[0057] Example 5: Determination of the one-step growth curve of bacteriophage D9 1. Experimental Methods A suspension of *E. coli* in the logarithmic growth phase was inoculated with *E. coli* ATCC11775 and *Escherichia virus D9* bacteriophage 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 phage. This process was repeated twice 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 phage titer was determined using the double-layer plate method. A one-step growth curve of the phage was plotted with time on the x-axis and the logarithm of the phage titer on the y-axis. The phage latency and lysis period were determined, and the outbreak size was calculated.

[0058] Outbreak volume = Phage titer at the end of the outbreak / Host bacterial concentration at the beginning of infection.

[0059] 2. Experimental Results and Analysis The results of the one-step growth curve are as follows Figure 6 As shown, 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.

[0060] like Figure 12 As shown, 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.

[0061] like Figure 18As shown, 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.

[0062] Example 6: Determination of the thermal stability of bacteriophages 1. Experimental Methods With a valence of 1.2 × 10 7 PFU / mL phage D9 stock solution was aliquoted into 50 mL sterile centrifuge tubes and incubated at 37℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 2 h each. The solutions were then serially diluted 10-fold with physiological saline and plated on double plates to determine their titer. The same method was used for phage D5 (stock solution titer 2.95 × 10⁻⁶). 7 The experiment was conducted using PFU / mL and D10 (4.5 × 10⁻⁶). 6 PFU / mL).

[0063] 2. Experimental Results and Analysis The results are as follows Figure 3 As shown, Escherichia coli phage D9 maintained high activity after being incubated in a water bath at 50℃ to 70℃ for 2 hours, with virtually no change in its initial titer. These results demonstrate that Escherichia coli phage D9 possesses excellent heat resistance, a characteristic with significant application value in the production, transportation, and storage of phages.

[0064] like Figure 9 As shown, E. 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. Figure 15 As shown, Escherichia virus D10, a bacteriophage of Escherichia coli, maintained high activity after being in a water bath at 50°C for 2 hours, with virtually 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.

[0065] 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.

[0066] 2. Experimental Results and Analysis The results are as follows Figure 4 As shown, under pH 3-9 conditions, the titer of Escherichia virus D9 is similar to its initial titer, indicating that Escherichia virus D9 has good acid resistance. This has significant application value in phage therapy, as the phage's good acid tolerance and high titer ensure a sufficient number of phages can pass through the digestive tract. Under pH 3-9 conditions, the titers of Escherichia virus D5 and Escherichia virus D10 are also similar to their initial titers.

[0067] Example 8: Determination of the lytic performance of Escherichia coli D9 against clinically pathogenic Escherichia coli 1. Experimental Methods Host bacteria: 15 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.

[0068] (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 D9 phage proliferation solution were mixed with MH semi-solid medium and spread on 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.

[0069] (2) Follow the method in (1) except that the filtrate of E. coli D9, D5 and D10 phages is a mixture of two or three phages (1:1 / 1:1:1), see Table 3 for details.

[0070] 2. Experimental Results and Analysis The experimental results are shown in Table 3. Escherichia coli D9 can not only lyse carbapenem-resistant Escherichia coli 16-B037 and 16-B043, but also lyse ESBL-resistant Escherichia coli 18-F-25, 19-WJ-533, 18-WJ-340 and 19-F-131 and other multidrug-resistant bacteria (resistance information is shown in Table 2). It has a broad spectrum of lysis and shows promise for clinical application in inhibiting the growth of these six multidrug-resistant pathogens and preventing and treating diseases caused by these pathogens.

[0071] Compared to D9, the combination of E. coli phage D9 and E. coli phage D10 exhibits additional lysis of 19-F-160, 18-F-67, 19-WJ-739, 19-WJ-755, and 18-F-50. This result indicates that the phages in the two compositions fully utilize synergistic effects, thereby significantly expanding the original phage lysis spectrum and demonstrating greater application potential.

[0072] Furthermore, the lysis spectrum of the combination of E. coli phages D9, D5, and D10 differs from that of the mixtures of the aforementioned single and double phages. This reflects that the synergistic cooperation between different multi-strain phages is relatively effective, and different drug-resistant pathogenic E. coli can be controlled through the cooperation of different phages.

[0073] Table 1 Information on clinically pathogenic Escherichia coli

[0074] Table 2 Drug resistance information of clinically pathogenic Escherichia coli

[0075] Table 3. Lysis results of Escherichia coli D9 on clinically pathogenic Escherichia coli

[0076] 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 D9 to determine whether it contains virulence genes and drug resistance genes.

[0077] 2. Experimental Results and Analysis The results showed that E. coli phage D9 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.

[0078] Example 10 Phage Genome Analysis 1. Experimental Methods After enriching and culturing a single strain of bacteriophage D9, 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.

[0079] 2. Analysis Results (1) The genome is 68415 bp in length, with a G+C content of 46.23%, and the base contents of C, G, A, and T are 27.26%, 26.50%, 23.43%, and 22.80%, respectively. Online RAST annotation of the whole genome showed that it contains 103 open reading frames (ORFs). Among these 103 ORFs, 26 structural proteins were found, mainly including phage structural and packaging proteins (head proteins, tail proteins, tail fibrils, substrate proteins, capsid proteins, and terminal enzyme large subunits, etc.), phage lysis-related proteins (lysins), DNA replication and modification-related proteins (DNA polymerases, DNA ligases, etc.), and other functional proteins (oxidoreductases). Furthermore, among the 103 ORFs, 97 start codons are ATG, 5 are GTG, and 1 is TTG. Analysis using tRNAscan-SE software showed that the genome does not contain tRNA genes. Analysis using the online tool CGE server showed that the genome did not contain drug resistance genes or virulence genes. PHASTER analysis showed that the genome did not contain lysogenic genes.

[0080] (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.

[0081] Table 4 Gene Sequence Information Table

[0082] 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. An Escherichia coli bacteriophage (PhiD9) characterized by, Escherichia virus ) a genome comprising a nucleic acid sequence of SEQ ID NO: 1, The preservation number of which is CGMCC NO. 46365.

2. A bacteriophage composition, characterized in that, The bacteriophage D9 of Escherichia coli as claimed in claim 1.

3. The phage composition of claim 2, wherein, The bacteriophage D9 of Escherichia coli as claimed in claim 1. The bacteriophage D9 of Escherichia coli as claimed in claim 1 and the bacteriophage D10 of Escherichia coli, the preservation number of which is CGMCC NO. 46366.

4. The phage composition of claim 3, wherein, The bacteriophage D9 of Escherichia coli as claimed in claim 1 and the bacteriophage D10 of Escherichia coli, the preservation number of which is CGMCC NO. 46366. The bacteriophage D5 of Escherichia coli, the preservation number of which is CGMCC NO. 46364.

5. The bacteriophage D9 of Escherichia coli as claimed in claim 1 or the bacteriophage composition as claimed in any one of claims 2-5 for use in bacteriostasis of Escherichia coli.

6. The bacteriophage of Escherichia coli as claimed in claim 1 or the bacteriophage composition as claimed in any one of claims 2-5 for use in the preparation of a medicament for preventing and treating diseases caused by Escherichia coli infection.

7. A biocide, characterized in that, The effective component of which comprises the bacteriophage of Escherichia coli as claimed in claim 1 or the bacteriophage composition as claimed in any one of claims 2-5.

8. A bacteriophage pharmaceutical formulation, characterized in that, The effective component of which comprises the bacteriophage of Escherichia coli as claimed in claim 1 or the bacteriophage composition as claimed in any one of claims 2-5.

9. The bacteriophage pharmaceutical preparation of claim 8, wherein, The bacteriophage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier, and the dosage form is a solution, a powder, a gel, a granule or a lyophilized agent.

10. An environmental disinfectant, characterized in that, The active ingredient comprises the bacteriophage D9 of Escherichia coli as claimed in claim 1 or the bacteriophage composition as claimed in any one of claims 2-5.

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