Wide-spectrum bacteriophage EFH2 capable of eliminating enterococcus faecalis suffering from humans and animals

Abstract

The invention discloses a wide-spectrum bacteriophage EFH2 capable of eliminating enterococcus faecalis suffering from humans and animals, and relates to the technical field of biology. The wide-spectrum bacteriophage is preserved in the China Center for Type Culture Collection on September 26, 2024, and the preservation number of the wide-spectrum bacteriophage is CCTCC NO: M 20242087. The wide-spectrum bacteriophage EFH2 is a newly found bacteriophage, has good tolerance under the conditions that the pH is 3-9 and the temperature is 4-60 DEG C, and has the titer as high as 2.31 * 10 < 8 > PFU / mL after being cultured for 8 hours under the condition that the MOI is 0.01. The wide-spectrum bacteriophage EFH2 is wide in antibacterial spectrum, short in incubation period, high in outbreak amount, capable of rapidly killing host bacteria, small in toxic and side effect, high in safety, wide in temperature and acid-base tolerance range, good in hydrophilicity and capable of well killing and disinfecting animals infected with multi-drug-resistant enterococcus and environments and food polluted by enterococcus.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a broad-spectrum bacteriophage EFH2 capable of eliminating zoonotic Enterococcus faecalis. Background Art

[0002] Enterococcus is a Gram-positive bacterium that is commonly found in the gastrointestinal tract of humans and various animals (mammals, reptiles, birds and some invertebrates). In addition, it is also widely found in the living environment, such as water, soil, vegetables and fruits, and food of animal origin (including meat, eggs and dairy products). At present, this genus includes about 50 species, mainly Enterococcus faecalis and Enterococcus faecium, which account for more than 80% of the isolates and often cause animal endocarditis, sepsis and amyloid encephalopathy. Enterococci are the second most common pathogen associated with acquired infections, and in catheter-related urinary tract infections, Enterococcus faecalis ranks fifth; among aerobic Gram-positive cocci, Enterococci have become an important pathogen of nosocomial infections after Staphylococci; it is also the third most common microorganism causing bloodstream infections. This species is often associated with the etiology of various human diseases, such as bacteremia, endocarditis and urinary tract infections (UTIs), and Enterococci are the second most common cause of Gram-positive related bacteremia.

[0003] Bacteriophage (phage) is a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes or spirochetes. The abundance of phages in the environment is extremely high. The total number of phages in the biological world is about 1×10 31 , and can specifically lyse bacteria. As early as 1919, scientists used bacteriophages for the prevention and control of avian typhoid, and achieved good results. At present, there are reports of successful applications of phage therapy in livestock and poultry farming, aquaculture, human diseases, and food. Most of the isolated natural phage strains are highly specific and only target some strains of a certain type of bacteria, which is not suitable for clinical application. For this reason, broadening the lysis spectrum of phages can ensure the effectiveness of treatment.

[0004] Chinese patent CN116574691A discloses a strain of Enterococcus faecalis phage vB_EfS_L1 and its application. The deposit number of the Enterococcus faecalis phage vB_EfS_L1 is CCTCC NO: M20221675, which was deposited in the China Center for Type Culture Collection on October 27, 2022. The phage is a typical tailed phage and a long-tailed phage from the electron microscopic morphology, and is named Enterococcus faecalis phage vB_EfS_L1; through the isolation and identification of phage vB_EfS_L1 and whole genome sequencing, its functional genes and proteins are further explored to expand the host spectrum. The phage can specifically lyse Enterococcus faecalis.

[0005] Chinese patent CN117264910A discloses a high-temperature resistant broad-spectrum Salmonella phage and its clinical application, which relates to the biological field. The phage was deposited in the China Center for Type Culture Collection on August 14, 2023, with a deposit number of CCTCC NO: M20231469. The phage has strong lysis ability, no incubation period, fast lysis speed, short lysis cycle, and can lyse 6 different serotypes of Salmonella, including Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Derby, Salmonella Panama, Salmonella Agona, and Salmonella Indiana. It is a broad-spectrum phage; in addition, the phage has high heat resistance and acid and alkali resistance, strong bactericidal ability, and can significantly inhibit the growth of Salmonella.

[0006] Research on enterococcal phages mainly focuses on the study of enterococcal lysis, but there are not many reports on the ability of enterococcal phages to lyse enterococci in a broad spectrum. Therefore, it is very necessary to find a new enterococcal phage that can lyse enterococci in a broad spectrum, with high stability, good safety and high titer. Summary of the invention

[0007] The purpose of the present invention is to provide a broad-spectrum bacteriophage EFH2 that can eliminate zoonotic Enterococcus faecalis to solve the problems of the above-mentioned prior art. The broad-spectrum bacteriophage EFH2 has a wide antibacterial spectrum, a short incubation period, a high burst volume, can quickly kill host bacteria, has low toxic and side effects, is highly safe, has a wide temperature and acid-base tolerance range, and has good hydrophilicity, and has a good killing and disinfecting effect on animals infected with multidrug-resistant Enterococci and environments and foods contaminated by Enterococci.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a broad-spectrum phage that can eliminate zoonotic Enterococcus faecalis. The broad-spectrum phage is Enterococcus faecalis phage (Enterococcus facalis phage) EFH2, which was deposited in the China Center for Type Culture Collection on September 26, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20242087.

[0010] The present invention also provides a use of the above-mentioned broad-spectrum bacteriophage in the preparation of a drug for preventing and / or treating Enterococcus faecalis infection.

[0011] The present invention also provides an application of the above-mentioned broad-spectrum bacteriophage in the preparation of an Enterococcus faecalis bactericide.

[0012] Furthermore, the Enterococcus faecalis is a multidrug-resistant Enterococcus faecalis.

[0013] The present invention also provides a medicine for preventing and / or treating Enterococcus faecalis infection, wherein the active ingredient comprises the above-mentioned broad-spectrum bacteriophage.

[0014] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the dosage form of the drug is injection, powder, gel, granule or lyophilized agent.

[0016] Furthermore, the drug also includes other active ingredients that have an antibacterial effect on Enterococcus faecalis.

[0017] The present invention also provides an enterococcus faecalis bactericide, wherein the active ingredient comprises the above-mentioned broad-spectrum bacteriophage.

[0018] Furthermore, the Enterococcus faecalis fungicide is in the form of a spray, powder, gel, granule or lyophilized agent.

[0019] The present invention discloses the following technical effects:

[0020] (1) The Enterococcus faecalis phage EFH2 is a newly discovered phage that has good tolerance at pH 3-9 and temperature 4-60°C. When cultured for 8 h under MOI = 0.01, its titer was as high as 2.31×10 8 The phage not only has a good disinfecting effect on the host enterococcus HX12-4 isolated from chickens, but also has a strong killing effect on zoonotic enterococci isolated from poultry (12 strains), pigs (3 strains) and humans (2 strains). It has a broad antibacterial spectrum and can be used to prevent and control the harm caused by the spread of enterococci between humans and animals.

[0021] (2) The faecal enterococcal phage EFH2 has a short incubation period and a high burst volume, can quickly kill the host bacteria, has low toxic side effects, is highly safe, has a relatively wide temperature and acid-base tolerance range, and has good hydrophilicity. It is easy to make into an injection and has a good killing and disinfecting effect on animals infected with multidrug-resistant enterococci and environments and foods contaminated by enterococci. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 is the plaque image of phage EFH2;

[0024] Figure 2 Transmission electron microscopy image of bacteriophage EFH2; scale bar is 100 nm;

[0025] Figure 3 is the optimal infection multiplicity graph of bacteriophage EFH2;

[0026] Figure 4 This is the host spectrum heat map of bacteriophage EFH2;

[0027] Figure 5 is the one-step growth curve of bacteriophage EFH2;

[0028] Figure 6 Schematic diagram of the effect of temperature on the activity of bacteriophage EFH2;

[0029] Figure 7 Schematic diagram of the effect of pH on the activity of bacteriophage EFH2;

[0030] Figure 8 Schematic diagram of the UV tolerance of bacteriophage EFH2;

[0031] Fig. 9 Schematic diagram of the genome of bacteriophage EFH2. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The reagents and culture medium involved in the following examples are as follows:

[0038] SM buffer (1 L): Take 50 mL of Tris-HCl (pH 7.4) buffer, weigh 5.8 g of sodium chloride and 2 g of magnesium chloride, dissolve them completely in 950 mL of distilled water, and sterilize them at 121°C for 20 min before use.

[0039] BHI agar medium (400 mL): Weigh 27.6 g of BHI agar medium powder, dissolve it in 400 mL of distilled water, sterilize it by high pressure at 121°C for 20 min, cool it to about 60°C, pour it into a plate, dry it, and keep it for use at room temperature.

[0040] 0.6% BHI semi-solid medium (200 mL): Weigh BHI broth powder and dissolve it in 200 mL of distilled water. Autoclave at 121° C. for 20 min and set aside.

[0041] 2×BHI medium (400 mL): Weigh 12 g of BHI powder, dissolve it in 400 mL of distilled water, sterilize it by high pressure at 121°C for 20 min, and set aside.

[0042] Example 1

[0043] 1. Isolation of Enterococcus facalis phage EFH2

[0044] The host bacteria Enterococcus faecalis HX12-4 (deposited in Wuhan Institute of Virology, Wuhan Branch of Chinese Academy of Sciences) was streaked on an agar medium. After culturing overnight, a single clone was picked and inoculated into 5 mL of liquid culture medium. After shaking culture at 37°C for 12 h, it was used as a fresh bacterial liquid for isolating phages.

[0045] Method for isolating Enterococcus faecalis phage from sewage samples: First, sewage was collected from the chicken house of Huazhong Agricultural University, a livestock and poultry farm in Hubei Province and Jiangxi Province. The samples were collected in March and August 2022, respectively, and stored in sterile containers at 4°C and transported to the laboratory. After vortex mixing, place it at 4°C overnight, and add an appropriate amount of SM buffer to release the phage in the sample. Centrifuge the water sample overnight at 10000r / min for 10min, discard the precipitate, and filter the supernatant with a 0.22μm filter membrane. Take 10mL of 2×BHI culture medium and mix it with the filtered filtrate sample, add 100μL of the revived host bacteria, and place it in a 37°C constant temperature shaking incubator at 100r / min for 24h. Take out the enriched filtrate sample and centrifuge it at 10000r / min for 10min, then take the supernatant and filter it with a 0.22μm filter membrane.

[0046] The double agar overlay method was used to detect bacteriophages: 200 μL of fresh bacterial solution was mixed with 4 mL of 0.6% BHI semi-solid culture medium at about 50°C, and poured evenly on the BHI agar plate. After cooling and solidification, 10 μL of enriched water sample was added to the culture medium containing different bacterial solutions. After drying, it was inverted in a 37°C ordinary incubator overnight, and the phage plaques were observed the next day.

[0047] The above-mentioned spare phages were tested by double-layer plate method, and the results were as follows Figure 1 As shown, the phage can form round plaques in agar culture medium with no halo around and clear and regular edges, which is a typical lytic phage.

[0048] 2. Amplification and Purification of Enterococcus faecalis Phage EFH2

[0049] After the BHI plate containing double-layer agar was cultured overnight, the plaques on the plate were removed with a sterile pipette tip, suspended in 2.5mL SM buffer, and incubated at 4℃ for 24h. After incubation, centrifuged at 10000r / min for 5min, and the supernatant was filtered through a 0.22μm filter membrane, and the phage was purified by the double-layer plate method. Take 100μL of the diluted filtrate and mix it with 100μL of fresh Enterococcus faecalis host bacterial solution, add 4mL 0.6% BHI semi-solid medium and pour it on the BHI solid plate. After cooling and solidification, culture at 37℃ for 24h. The next day, a single plaque was picked up with a sterile pipette tip and placed in 2.5mL SM buffer, vortexed and mixed, and purified by the double-layer plate method. Repeated 5 times until the morphology and size of the plaques were consistent, and the purification was completed. Pick a single plaque and suspend it in SMbuffer for the preparation of high-concentration phage.

[0050] Pick a single colony of the host bacteria, inoculate it into BHI broth liquid culture medium, and place it in constant temperature shaking culture at 37℃ for 6-8h. Use McFarland turbidimeter to adjust the bacterial solution concentration to about 10 8 CFU / mL. Take 2mL of bacterial solution and 2mL of phage solution, mix and incubate for 20min, then add to a 200mL conical flask and incubate at 37℃ for 24h. The next day, centrifuge the culture at 4℃ and 10000r / min for 10min, collect the supernatant and filter it with a 0.22μm filter membrane to obtain the phage stock solution, which is stored at 4℃ for later use. Dilute the phage stock solution 10 times with SM buffer for 6 gradients, take 100μL of phage dilution and mix it with 100μL of host bacterial solution, add 4mL of 0.6% BHI at about 50℃ and pour it on the BHI solid plate. After cooling and solidification, incubate at 37℃ for 24h, count the formed plaques and calculate the initial phage concentration to get the phage titer. The specific calculation formula is phage titer (PFU / mL) = number of plaques × dilution factor × 10. When MOI = 0.01, the titer is the highest, which can reach 2.31×10 8 PFU / mL.

[0051] 3. Transmission electron microscopy observation of Enterococcus faecalis phage EFH2

[0052] The purified phage suspension was observed under an electron microscope. The purified phage suspension was dropped on a copper grid and allowed to settle for 15 minutes. The excess liquid was removed with filter paper and stained with a drop of 2% (pH 7.0) phosphotungstic acid (PTA, 2% w / v). After drying at room temperature, the suspension was observed under a transmission electron microscope. The results were as follows: Figure 2(100kV) shows that the head diameter of EFH2 is about 45.38±1.08nm, and the tail length is about 158.95±1.07nm. According to the latest classification criteria of bacteriophages by the International Committee on Taxonomy of Viruses (ICTV), EFH2 belongs to the family Siphoviridae.

[0053] 4. Biological Deposit

[0054] Enterococcus facalis phage EFH2 was deposited in the China Center for Type Culture Collection on September 26, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242087.

[0055] Example 2

[0056] Determination of the optimal infection multiplicity of Enterococcus faecalis phage EFH2 (the infection multiplicity is the ratio of the number of phages to the number of host bacteria at the initial stage of infection)

[0057] The host bacteria (same as in Example 1, "1. Isolation of Enterococcus faecalis phage EFH2") were cultured to the logarithmic growth phase (OD 600 =0.6) was mixed with phage and cultured in 5 mL BHI broth liquid culture medium according to the phage concentration / host bacteria concentration of 100, 10, 1, 0.1, and 0.01, and cultured at 37°C for 8 h. The supernatant was centrifuged at 10000 r / min for 10 min, filtered through a 0.22 μm filter membrane for sterilization, and then diluted to a suitable gradient. The titer of phage in different supernatants was determined by the double-layer plate method, and compared with the double-layer agar plate without phage. The infection multiplicity with the highest titer was the optimal infection multiplicity. The results are shown in FIG. Figure 3 The results showed that the optimal infection multiplicity of Enterococcus faecalis phage EFH2 was 0.01.

[0058] Example 3

[0059] Analysis of host spectrum of Enterococcus faecalis phage EFH2

[0060] The titer of the phage purified in Example 1 was adjusted to 10 9 PFU / mL was used for standby use. The host spectrum of phages was analyzed using 36 bacterial strains isolated from different animals. The specific operation was as follows:

[0061] First, the above 36 bacterial strains were revived. Staphylococcus aureus, Salmonella and Enterococcus faecalis were revived using BHI plates, and Escherichia coli was revived using LB plates. After resuscitation, single colonies were picked and inoculated into BHI or LB liquid culture medium, and cultured at 37°C with constant temperature shaking for 8 hours until the logarithmic growth phase. The double-layer plate method was used to determine whether the phage had a lysis effect on the strain. 100 μL of freshly cultured enterococcal culture liquid or 200 μL of culture liquid of other species was mixed with 4 mL of 0.6% BHI or LB semi-solid culture medium at about 50°C, and poured evenly on BHI or LB agar plates. After cooling and solidification, 10 μL of phage stock solution was added to the culture medium. After drying, it was inverted and cultured at 37°C overnight. The plaques were observed on the second day, and the double-layer agar plate without phage filtrate was used as a blank control. The experiment was repeated 3 times, and the experimental results were observed. If plaques were produced, it was recorded as "+", otherwise it was recorded as "-". The results are as follows Figure 4 As shown, the results showed that the Enterococcus faecalis phage EFH2 was able to completely lyse 17 strains of multidrug-resistant Enterococci collected and isolated clinically.

[0062] Table 1. Phage EFH2 lysis spectrum

[0063]

[0064]

[0065]

[0066]

[0067] Note: ERY: erythromycin; CLI: clindamycin; ENR: enrofloxacin; OFL: ofloxacin; CEF: ceftiofur; CFX: cefoxitin; SF: sulfamethoxazole; OXA: oxacillin; DOX: doxycycline; SXT: trimethoprim-sulfamethoxazole; FFC: florfenicol; TIA: tiamulin; TIL: tilmicosin; GEN: gentamicin; BAC: bacitracin; TET: tetracycline; KIT: kitasamycin; BAM: flavomycin; AVI: enramycin.

[0068] Example 4

[0069] Determination of one-step growth curve of Enterococcus faecalis phage EFH2:

[0070] 100 μL of phage EFH2 (10 8PFU / mL) was mixed with 900 μL of fresh host bacteria cultured to the logarithmic growth phase (same as in Example 1 "1. Isolation of Enterococcus faecalis phage EFH2" section), incubated at 37°C for 15 min, centrifuged at 10,000 r / min for 1 min, the supernatant was discarded and the precipitate was washed twice with BHI. Subsequently, 10 mL of BHI liquid culture medium preheated at 37°C was added to resuspend the precipitate, and cultured at 37°C with constant temperature shaking at 160 r / min. Samples were taken every 10 min for 120 min. After centrifugation and dilution, the phage titer was immediately determined by the double-layer plate method. The experiment was repeated 3 times. A one-step growth curve was drawn with time as the horizontal axis and the phage titer as the vertical axis, and the burst volume was calculated. The calculation formula is burst volume = number of phages at the end of the burst / number of host bacteria at the beginning of infection. The results of the one-step growth curve are shown in the figure. Figure 5 As shown, the phage EFH2 has a latent period of 0-10 minutes, a rapid increase in titer from 10 to 80 minutes, an outbreak period of about 70 minutes, and the phage titer tends to stabilize from 90 minutes to 120 minutes, with an average outbreak amount of about 29.19 PFU / cell.

[0071] Example 5

[0072] Temperature, pH and UV tolerance test of Enterococcus faecalis phage EFH2:

[0073] 1. Phage temperature tolerance

[0074] Take 7 1 mL portions of phage solution (10 8 PFU / mL) were placed in centrifuge tubes, and the centrifuge tubes were incubated at 4, 25, 40, 50, 60, 70, and 80°C. The phage titer was measured at 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min. The experiment was repeated 3 times. Figure 6 As shown: the phage EFH2 was incubated at 4℃ and 25℃ for 60min, and the phage titer did not decrease. After incubation at 50℃ for 30min, the titer began to decrease, and stabilized after 30min, with a decrease of about 2 titers. After incubation at 60℃ for 10min, the titer began to decrease, and stabilized after 20min, with a decrease of about 3 titers. After incubation at 70℃ for 10min, the titer began to decrease, and the phage was completely inactivated at 50min. After incubation at 80℃ for 10min, EFH2 was completely inactivated.

[0075] 2. Phage pH tolerance

[0076] The pH values ​​of the sterile buffer were adjusted to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, respectively. The sterile buffers of different pH values ​​were sterilized by filtration with a 0.22 μm filter. 900 μL of the sterile buffer and 100 μL of the phage solution (10 8 PFU / mL) and placed in a 37°C water bath for 1 hour. Take 100 μL to measure the titer and observe the tolerance of phages under different pH conditions. Figure 7 As shown: EFH2 was stable after 1 hour of incubation at pH 3-9. At pH 10, 11 and 12, the titer dropped by 2 titers after 1 hour. At pH 1, 2, 12 and 13, all of them were inactivated after 1 hour.

[0077] 3. Ultraviolet tolerance test of Enterococcus faecalis phage EFH2

[0078] Take 6 1 mL portions of phage solution (10 8 PFU / mL) was placed in a centrifuge tube, which was placed about 40 cm below the UV lamp in the biosafety cabinet. It was left to stand for 1 hour under UV lamp irradiation. Samples were taken at 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min to measure the change in phage titer. The experiment was repeated 3 times. Figure 8 As shown: EFH2 has good resistance to ultraviolet rays.

[0079] Example 6

[0080] Genome of Enterococcus faecalis phage EFH2:

[0081] The phage stock solution was concentrated by the PEG / NaCl method. 40 mL of the phage stock solution was added with 4 g of PEG 8000. After dissolution, it was placed at 4°C overnight. Centrifuged at 10,000 r / min for 10 min, the supernatant was discarded, and the precipitate was dissolved with 1 mL of SM buffer and added to a 2 mL centrifuge tube. 1 mL of chloroform was added at a ratio of 1:1, and the mixture was centrifuged at 5,000 r / min for 15 min. The supernatant was taken and the phage DNA was extracted.

[0082] The phenol-chloroform-isoamyl alcohol method was used to extract the complete genome of the phage from the purified and concentrated lysate. The specific steps are as follows:

[0083] (1) Take 600 μL of phage concentrate and place it in a 1.5 mL centrifuge tube. Add 3 μL of DNase I and 3 μL of RNase A to a final concentration of 1 μg / mL and incubate at 37°C overnight. The next day, inactivate the enzyme in an 80°C water bath for 15 min to fully inactivate the enzyme.

[0084] (2) Add reagents to the centrifuge tube in sequence: 24 μL of 0.5% EDTA solution, 1.5 μL of 20 μg / mL proteinase K solution, and 30 μL of 10% SDS solution. The total volume is 660 μL. Incubate in a 56°C water bath for 1 h.

[0085] (3) Add an equal volume (660 μL) of balanced phenol solution, gently invert and mix for 1 min, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0086] (4) Add an equal volume of phenol-chloroform-isoamyl alcohol solution (25:24:1), mix by inversion for 1 min, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0087] (5) Add an equal volume of chloroform, mix thoroughly by inversion, and centrifuge at 10,000 rpm for 5 min. Transfer the supernatant to a new 1.5 mL centrifuge tube. Repeat this step twice.

[0088] (6) Add 400 μL of isopropanol and let stand at -20°C for more than 1 hour. Centrifuge at 4°C, 12,000 rpm for 20 minutes and slowly discard the supernatant.

[0089] (7) Add 1 mL of 75% ethanol (precooled at -20°C) to wash the DNA precipitate. Slowly pipette to resuspend and let stand for 1 min. Centrifuge at 12,000 rpm for 10 min. Slowly pour off the ethanol and let stand at room temperature for 1 to 3 min to allow the ethanol to evaporate completely.

[0090] (8) Add 50 μL of ultrapure water (preheated at 56°C) to dissolve the DNA precipitate, seal the tube, and store at -20°C.

[0091] (9) The extracted DNA was verified by 0.6% agarose gel electrophoresis and then sent to BGI Biotechnology Co., Ltd. for phage genome sequencing. The test results were as follows Fig. 9 shown.

[0092] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A broad-spectrum bacteriophage capable of eliminating zoonotic Enterococcus faecalis, characterized in that: The broad-spectrum phage is Enterococcus facalis phage EFH2, which was deposited in the China Center for Type Culture Collection on September 26, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20242087.

2. Use of the broad-spectrum bacteriophage according to claim 1 in the preparation of a drug for preventing and / or treating Enterococcus faecalis infection.

3. Use of the broad-spectrum bacteriophage according to claim 1 in the preparation of an Enterococcus faecalis bactericide.

4. The use according to claim 2 or 3, characterized in that: The Enterococcus faecalis is a multi-drug resistant Enterococcus faecalis.

5. A drug for preventing and / or treating Enterococcus faecalis infection, characterized in that: The active ingredient comprises the broad-spectrum bacteriophage according to claim 1.

6. The drug according to claim 5, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that The dosage form of the medicine is injection, powder, gel, granule or lyophilized agent.

8. The drug according to claim 5, characterized in that The drug also includes other active ingredients having an antibacterial effect on Enterococcus faecalis.

9. A faecal enterococcus bactericide, characterized in that: The active ingredient comprises the broad-spectrum bacteriophage according to claim 1.

10. The Enterococcus faecalis bactericide according to claim 9, characterized in that The formulation of the Enterococcus faecalis bactericide is a spray, a powder, a gel, a granule or a lyophilized agent.

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