A broad lysis spectrum enterohemorrhagic Escherichia coli phage, bacteriostatic agent including the phage, and preparation method and application thereof

Abstract

The present invention discloses a broad lysis spectrum enterohemorrhagic Escherichia coli phage and its application. The phage strain has a deposition number of CCTCC NO: M 20211003 and was deposited in the China Center for Type Culture Collection of Wuhan University on August 10, 2021. It is classified and named as enterohemorrhagic Escherichia coli O157:H7 phage vB_EcoM_SQ17, Enterohemorrhagic Escherichia coli O157:H7 phage vB_EcoM_SQ17; it has a high bactericidal ability against enterohemorrhagic Escherichia coli, can lyse enterohemorrhagic Escherichia coli O157:H7, and can also lyse other pathogenic Escherichia coli. The present invention has a good antibacterial effect on enterohemorrhagic Escherichia coli in both whole milk and skim milk.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a broad-specification enterohemorrhagic Escherichia coli O157:H7 bacteriophage, including a bacteriophage antibacterial agent, a preparation method and an application thereof. Background Art

[0002] Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major foodborne zoonosis, primarily carried by poultry and livestock. It can colonize the intestinal tract and respiratory tract of both animals and humans, contaminating foods such as milk and causing food poisoning. This poses a potential threat of outbreaks and epidemics. The dangers of EHEC O157:H7 lie not only in contaminating foods like meat, eggs, and milk and causing diarrhea in animals, but also in causing diarrhea and hemorrhagic enteritis in humans, with subsequent symptoms such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). HUS and TTP are severe illnesses with high mortality rates. The harm caused by this pathogen has become a global problem.

[0003] Currently, studies have shown that antibiotics are ineffective and may even have side effects for infections caused by enterohemorrhagic Escherichia coli O157:H7, paving the way for their control and treatment. Furthermore, while antibiotics can inhibit bacterial infections to a certain extent, their long-term use can lead to the emergence of multidrug-resistant bacteria and superbugs, creating a vicious cycle. Lytic bacteriophages are viruses isolated from nature that specifically infect and lyse bacteria. Recent research has shown that bacteriophages are a new and safe antimicrobial agent, garnering increasing attention due to their high efficacy, rapid response, high specificity, high safety, resistance to developing bacteria, and ability to effectively lyse multidrug-resistant bacteria. A growing number of studies, both domestically and internationally, demonstrate that bacteriophages possess high bactericidal activity, a broad lytic spectrum, resistance to developing bacteria, high lytic activity against drug-resistant pathogens, and synergistic efficacy with other antimicrobial agents. Furthermore, antibodies produced in animals following in vivo administration of bacteriophages do not affect the efficacy of phages. Therefore, this research is expected to develop the EHEC O157:H7 phage into a new antibacterial agent for preventing and treating diseases caused by EHEC O157:H7 infection. Summary of the Invention

[0004] The purpose of the present invention is to provide a broad-spectrum enterohemorrhagic Escherichia coli O157:H7 phage, an antibacterial agent including the phage, and a preparation method and application. The phage of the present invention has a strong lytic effect on enterohemorrhagic Escherichia coli O157:H7 and can be used to inhibit enterohemorrhagic Escherichia coli O157:H7.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an enterohemorrhagic Escherichia coli O157:H7 phage with a broad lysis spectrum. The enterohemorrhagic Escherichia coli O157:H7 phage with a broad lysis spectrum is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20211003.

[0007] The present invention provides a method for culturing the enterohemorrhagic Escherichia coli O157:H7 phage described in the above scheme, comprising the following steps:

[0008] 1) Centrifuging the enterohemorrhagic Escherichia coli O157:H7 culture and collecting the precipitate;

[0009] 2) Resuspend the precipitate in step 1) using LB medium to obtain a bacterial suspension, and add the above-mentioned

[0010] The enterohemorrhagic Escherichia coli O157:H7 phage described in the protocol is cultured at 37° C. for 6 to 8 hours, centrifuged, and the supernatant is collected. The supernatant is filtered through a 0.22 μm filter membrane to obtain an enterohemorrhagic Escherichia coli O157:H7 phage lysate.

[0011] Preferably, the concentration of the enterohemorrhagic Escherichia coli O157:H7 culture in step 1) is 1×10 8 CFU / ml~1×10 10 CFU / ml.

[0012] The present invention provides an antibacterial agent, which comprises the enterohemorrhagic Escherichia coli O157:H7 phage described in the above scheme or a culture of the enterohemorrhagic Escherichia coli O157:H7 phage described in the above scheme.

[0013] Preferably, the antibacterial agent further comprises a pharmaceutically acceptable excipient.

[0014] Preferably, the pharmaceutically acceptable excipient comprises one of a buffer, a metal ion and a surfactant.

[0015] Kind or several.

[0016] The present invention provides the use of the enterohemorrhagic Escherichia coli O157:H7 phage or the antibacterial agent described in the above scheme in preparing a preparation for inhibiting and / or eliminating enterohemorrhagic Escherichia coli O157:H7.

[0017] Beneficial effects of the present invention: The present invention provides an enterohemorrhagic Escherichia coli O157:H7 phage (EHEC0157:H7 phage), which is deposited with the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20211003. The phage of the present invention has a strong lytic effect on enterohemorrhagic Escherichia coli O157:H7 (EHECO157:H7) and can be used to inhibit and / or eliminate enterohemorrhagic Escherichia coli O157:H7. The enterohemorrhagic Escherichia coli O157:H7 phage of the present invention can effectively prevent and control pathogenic bacteria contamination by inhibiting the excessive growth of enterohemorrhagic Escherichia coli O157:H7. The present invention provides a safe, non-toxic and side-effect-free source of bacteriophage products for preventing and controlling foodborne bacterial diseases caused by enterohemorrhagic Escherichia coli O157:H7.

[0018] Biological Deposit Description

[0019] Enterohemorrhagic Escherichia coli O157:H7 phage (Enterohemorrhagic Escherichia coli O157:H7 phage) vB_EcoM_SQ17 was deposited in the China Center for Type Culture Collection on August 10, 2021, located at Wuhan University, Wuchang District, Wuhan City, with the deposit number: CCTCC NO: M 20211003. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Results of purified phage detection on double-layer plates;

[0021] Figure 2 The results of electron microscopy observation of bacteriophages are shown in Figure 2.

[0022] Figure 3 The dynamic lysis effect of phage on EHEC O157:H7 within 6 hours at 37℃;

[0023] Figure 4 The lysis effects of phage on EHEC O157:H7 at 37℃ for 3h and 6h respectively;

[0024] Figure 5 The results are for the temperature tolerance of phage;

[0025] Figure 6 is the pH tolerance result of the phage;

[0026] Figure 7The results are for the antibacterial effect of bacteriophage in pasteurized whole milk;

[0027] Figure 8 The results show the antibacterial effect of bacteriophage in skimmed sterilized pure milk;

[0028] Figure 9 This is the result of the antibacterial effect of bacteriophage in whole-fat sterilized pure milk. DETAILED DESCRIPTION

[0029] The present invention provides a broad-host spectrum enterohemorrhagic Escherichia coli O157:H7 phage (vB_EcoM_SQ17), which is deposited with the China Center for Type Culture Collection under the deposit number CCTCC NO: M20211003. The enterohemorrhagic Escherichia coli O157:H7 phage of the present invention is derived from wastewater samples from pig farms.

[0030] The bacteriophage of the present invention has a strong lytic effect on EHEC O157:H7 and can be used to inhibit and / or eliminate EHEC O157:H7. The EHEC O157:H7 bacteriophage of the present invention can effectively prevent and control bacterial contamination by inhibiting the excessive growth of EHEC O157:H7.

[0031] The present invention provides a method for culturing the enterohemorrhagic Escherichia coli O157:H7 phage described in the above scheme, comprising the following steps:

[0032] 1) Centrifuging the enterohemorrhagic Escherichia coli O157:H7 culture and collecting the precipitate;

[0033] 2) Resuspending the precipitate described in step 1) in LB medium to obtain a bacterial suspension, adding the enterohemorrhagic Escherichia coli O157:H7 phage described in the above scheme to the bacterial suspension, culturing at 37°C for 6-8 hours, centrifuging, and taking the supernatant. Filtering the supernatant through a 0.22 μm filter membrane to obtain an enterohemorrhagic Escherichia coli O157:H7 phage suspension.

[0034] The present invention first centrifuges the enterohemorrhagic Escherichia coli O157:H7 culture and collects the precipitate; the concentration of the enterohemorrhagic Escherichia coli O157:H7 culture is preferably 1×10 8 CFU / ml~1×10 10 CFU / ml, more preferably 1×10 9 CFU / ml; the present invention has no special limitation on the centrifugation parameters, and conventional parameters in the art can be used.

[0035] After obtaining the precipitate, the present invention uses LB culture medium to resuspend the precipitate to obtain a bacterial suspension, and the enterohemorrhagic Escherichia coli O157:H7 phage described in the above scheme is added to the bacterial suspension. After culturing at 37°C for 6 to 8 hours, centrifugation is performed, and the supernatant is taken. The supernatant is filtered through a 0.22 μm filter membrane to obtain an enterohemorrhagic Escherichia coli O157:H7 phage lysate.

[0036] The present invention provides an antibacterial agent, which includes the enterohemorrhagic Escherichia coli O157:H7 phage described in the above scheme or the culture of the enterohemorrhagic Escherichia coli O157:H7 phage described in the above scheme; the antibacterial agent preferably also includes a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient preferably includes one or more of a buffer, metal ions and a surfactant.

[0037] The present invention provides the use of the enterohemorrhagic Escherichia coli O157:H7 phage or the antibacterial agent described in the above scheme in the preparation of a preparation for inhibiting and / or eliminating enterohemorrhagic Escherichia coli O157:H7; the enterohemorrhagic Escherichia coli O157:H7 preferably includes enterohemorrhagic Escherichia coli O157:H7 in livestock and poultry breeding or enterohemorrhagic Escherichia coli O157:H7 in the production environment.

[0038] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] The phage host bacteria Enterohemorrhagic Escherichia coli O157:H7 EO157-1 strain used in the embodiments of the present invention was isolated from wastewater samples from a pig farm and preserved in this laboratory.

[0040] Example 1: Isolation, Preparation and Purification of Enterohemorrhagic Escherichia coli O157:H7 Phage

[0041] 1. Phage Isolation

[0042] The sample of the present invention was collected from the wastewater sample of the pig farm. The sample was added to the peptone solution and shaken at room temperature for 2-4 hours. The supernatant was taken at 5000×g·min -1Centrifuge for 30 minutes and filter the supernatant with a 0.22 μm filter. Take 1 ml of the supernatant and add it to 50 ml of LB medium. Then add 1 ml of the overnight culture of enterohemorrhagic Escherichia coli O157:H7 EO157-1 strain (hereinafter referred to as EO157-1 strain) (2 times the LB overnight culture). Then place it at 37°C and shake at 150 rpm overnight. The next day, take the above culture and centrifuge it at 10,000 × g·min -1 , 4℃, centrifuge for 30min, take the supernatant; then filter the supernatant with a 0.22μm filter membrane to form a phage stock solution.

[0043] Take 2 ml of overnight culture solution and spread it on 20 ml LB agar plate. After absorption, take 0.01 ml of phage stock solution and drop it on the surface of the plate; after it is naturally dried, place it at 37℃ for anaerobically culture for 16-18 hours and observe the changes in the spotted area.

[0044] Take 0.1ml of phage stock solution, dilute it 10 times, and take 10 2 ~10 6 Mix 0.1 ml of each diluent with 0.1 ml of overnight cultured host bacterial solution, add about 5 ml of 0.6% LB agar medium, mix well and quickly pour into the upper layer of the LB plate, shake and let it stand for 5 minutes to solidify, place it at 37°C for 12 hours, and observe the plaque formation.

[0045] 2. Phage purification

[0046] Take 2 ml of freshly cultured overnight culture of EO157-1, centrifuge, resuspend in 1 ml of LB medium, add 0.1 ml of phage [at MOI (multiplicity of infection) ratios of 1:1, 1:10, and 1:100], add 100 ml of LB medium, and culture at 37°C with shaking for 6-8 h. Take the above culture and centrifuge at 13000 × g·min -1 , 4℃, centrifuge for 30 minutes, collect the supernatant; then filter the supernatant through a 0.22μm filter membrane to form a phage lysate. Add RNase A and DNase I to the phage suspension to a final concentration of 1μg / ml, incubate at 37℃ for 30 minutes; add 9.3g PEG 8000 and 5.8g NaCl, shake until dissolved, and place on ice for 1 hour; centrifuge at 10000×g·min at 4℃. -1 , 30min, remove the supernatant; add 5ml SM solution to thoroughly wash the tube wall and precipitate, and incubate at room temperature for 30min; add an equal volume of chloroform to extract PEG and cell debris in the phage suspension, and shake for 30s; 4℃, 3000×g·min -1 Centrifuge for 15 minutes, recover the hydrophilic phase containing phage particles, obtain purified phage, and detect the purified phage on a double-layer plate (e.g. Figure 1 shown).

[0047] 3. Phage electron microscopy detection

[0048] Take the purified phage suspension for electron microscopy observation, add 20 μl of sample and drop it on the copper grid, wait for it to precipitate for 15 minutes, absorb the excess liquid with filter paper, stain with 2% phosphotungstic acid for 30 minutes, and observe under electron microscopy after drying.

[0049] like Figure 2 As shown, the phage belongs to the family Myocaudaceae of the order Caudovirales. The head is symmetrical with a diameter of approximately 90.32±2 nm, the tail length is approximately 100±2 nm, and the tail diameter is 16.13±1 nm. The phage is named vB_EcoM_SQ17.

[0050] Example 2: Phage genome sequencing

[0051] High-throughput sequencing of phages was performed using the NovaSeq sequencer produced by Illumina.

[0052] Nucleotide sequence alignment of the vB_EcoM_SQ17 genome at NCBI revealed a full-length genome of 166,457 bp. Alignment with Escherichia phage vB_EcoM-ZQ3, Escherichia phageS143_2, and Shigella phage phi25-307 reached coverage of 91%, 92%, and 92%, respectively, with identities of 97.85%, 98.20%, and 97.90%, respectively. Analysis of the vB_EcoM_SQ17 genome for virulence and drug resistance genes revealed no genes encoding virulence or drug resistance. These results suggest that this phage poses no potential safety risks in the prevention and control of foodborne pathogens.

[0053] Literature indicates that phage short fiber protein determines host range and lysis spectrum. The amino acid sequence of the short fiber protein encoded by vB_EcoM_SQ17 differs significantly from the short fiber protein amino acid sequences of these highly similar phages, indicating that vB_EcoM_SQ17 is a new phage. The amino acid sequence of its short fiber protein ORF156 is:

[0054] MSNNTYQHVSNESVYVEFDPTGSNFDSSITNVQAALASISAYGVKGVPEASEAEKGVIQL

[0055] ASEQEVLDGFNSTKAVTPATLNARLQYPNASETQYGVTKYATQEEAIAGTLDTVSITPLKL

[0056] NQTIDNTFSTRYSTETTNGVIKIATQTAALAGSDDTTAMTPLKTQQLAIKLISQIAPNNDPAS

[0057] ESITGVVRLATVAQTRQGTLREGYAISPYTFMNSVATQEYKGVIRLGTQAEINSNLGDVAVT

[0058] GETLNGRGATGSMRGVVKLTTQAGIAPEGDSSGALAWNADVINTRGGQTINGSLNLDHLT

[0059] ANGIWSRGGMWKNGDQPVATERYASERVPVGTIQMFAGDSAPPGWVLCHGGTISGDQFP

[0060] DYRNVVGTRFGGDWNNPGIPDMRGLFVRGAGTGSHILNQRGQDGYGKDRLGVGCDGM

[0061] HVGGVQAQQMSYHKHAGAWGENGNSRGYAPFGASNGNGYLGNGKSADWDNHLFFTNDGFEMGGARDSFGTLNREGLIGYETRPWNISLNYIIKVHY.

[0062] Example 3: Phage vB_EcoM_SQ17 lysis spectrum detection

[0063] The experiment involved 15 E. coli strains and four Salmonella strains for lysis spectrum analysis. Overnight cultures of different strains were aspirated onto LB plates divided into several zones. The 15 E. coli strains included: eight EHEC O157:H7 strains (ATCC 43889, ATCC 35150, ATCC 43894, ATCC 700728, EO157-1, 86-24, 363, and 47); three enterotoxigenic E. coli (ETEC) strains (EK99-F41, C83698, and C83558); and four other E. coli strains (CVCC 249, ATCC 25922, ATCC 35218, and BL21). Take 0.1 ml of bacterial solution and drop it on the LB plate. Spread the bacterial solution evenly and wait for it to dry. Then take 0.01 ml of phage stock solution and drop it on the plates coated with different bacteria. After leaving it upright to dry naturally, place it upside down and culture it at 37℃ for 12 hours, and observe the phage lysis.

[0064] The results are shown in Table 1: phage vB_EcoM_SQ17 had transparent lysis zones for all 15 E. coli strains, but no lysis zones for 4 Salmonella strains, that is, the phage was positive for all 15 E. coli strains and negative for 4 Salmonella strains, indicating that the phage was specific for E. coli.

[0065] Table 1. Cleavage spectrum of enterohemorrhagic Escherichia coli O157:H7 phage vB_EcoM_SQ17

[0066]

[0067]

[0068] Note: In Table 1, “+” represents the lysis effect of phage on the strain. The more “+”, the higher the degree of phage lysis on the bacteria, and “–” represents no lysis.

[0069] Example 4: Lysis effect of bacteriophage vB_EcoM_SQ17 on EHEC O157:H7

[0070] The overnight culture of EO157-1 bacteria was adjusted to OD 600nm The value is about 0.23 (about 10 7 CFU / mL), the experimental groups were added with different dilutions of phage vB_EcoM_SQ17 (the stock solution was about 10 8 PFU / mL) was mixed with the bacterial solution and cultured in a 37°C incubator. A control group was set up at the same time, in which only bacterial solution without phage was added. The OD was measured every 30 minutes. 600nm The OD values ​​were measured for 6 hours and compared after adding phage. 600nm At the same time, bacterial liquid samples were taken at 3h and 6h, and the number of bacteria was measured by plate count method, and the bacterial concentration was calculated.

[0071] The results are as follows Figure 3 and Figure 4 As shown, in Figure 3 The figure shows that within 6 hours after the phage was added at 37℃, the OD values ​​of the phage groups with different concentrations were compared with those of the control group. 600nm The values ​​were all stable at a low level, which showed that the phage had a significant lysis effect on EO157-1, and different concentrations could effectively inhibit the growth of EO157-1.

[0072] exist Figure 4The results show that when the phage was exposed to the bacteria at 37℃ for 3 hours, compared with the initial bacterial concentration, the number of viable bacteria in the experimental group decreased by 4.16 Log CFU / mL, 4 Log CFU / mL, 3.30 Log CFU / mL and 3.88 Log CFU / mL at 3 hours; and decreased by 3.39 Log CFU / mL, 2.41 Log CFU / mL, 2.82 Log CFU / mL and 2.78 Log CFU / mL at 6 hours.

[0073] Example 5: Determination of temperature tolerance of bacteriophage vB_EcoM_SQ17

[0074] Dilute the phage stock solution to about 10 9 PFU / mL, and divided into 2 sterile centrifuge tubes, 1 mL in each tube, and placed the centrifuge tubes in a constant temperature water bath at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃, respectively, and measured the titer after 30min and 60min, respectively.

[0075] The results of the temperature tolerance test of bacteriophage vB_EcoM_SQ17 are as follows Figure 5 The results showed that phage vB_EcoM_SQ17 had good temperature tolerance and remained active at 30°C to 60°C.

[0076] Example 6: Determination of pH tolerance of bacteriophage vB_EcoM_SQ17

[0077] Use LB liquid medium as the medium and adjust the pH value (pH 2-13) with NaOH and HCl. Take 10 μL of phage stock solution with known titer (10 9 PFU / ml), added to 990 μL of peptone water with different pH values, and then bathed in 37°C water for 2 h to determine the titer of the phage in each centrifuge tube.

[0078] The pH tolerance test results of bacteriophage vB_EcoM_SQ17 are as follows Figure 6 As shown, phage vB_EcoM_SQ17 has good acid and alkali tolerance and remains active at pH 4 to 12.

[0079] Example 7: Antibacterial effect of bacteriophage vB_EcoM_SQ17 on EHEC O157:H7 in pasteurized whole milk

[0080] EHEC O157:H7 strain EO157-1 was cultured to the logarithmic growth phase, washed with PBS and centrifuged to remove the culture medium. The bacterial precipitate was retained and 5 ml of pasteurized whole milk was used to dilute the bacteria to a final concentration of 1.08×10 4CFU / mL, set up experimental group and control group. The experimental group added 1mL to a final concentration of about 10 8 PFU / mL of bacteriophage vB_EcoM_SQ17 was added to the control group without phage, and only an equal volume of SM buffer was added. The cells were cultured at 4°C, and samples were collected at 0, 4, 8, 12, and 24 h. The number of bacteria in the samples was measured by plate counting method, and the bacterial concentration in whole milk was calculated to draw the inhibition curve.

[0081] like Figure 7 As shown in the figure, the application test results of bacteriophage vB_EcoM_SQ17 in milk: at 4°C, compared with the control group, the number of viable bacteria decreased by 0.34 Log CFU / mL at 12 hours, and the number of viable bacteria decreased by 0.59 Log CFU / mL at 24 hours.

[0082] Example 8: Antibacterial effect of bacteriophage vB_EcoM_SQ17 on EHEC O157:H7 in skimmed sterilized milk

[0083] EHEC O157:H7 strain EO157-1 was cultured to the logarithmic growth phase, washed with PBS, centrifuged to remove the culture medium, and the bacterial pellet was retained. 5 ml of skim sterilized pure milk was used to dilute the bacteria to a final concentration of 1.5×10 4 CFU / mL, set up experimental group and control group. The experimental group added 1mL to a final concentration of about 10 8 PFU / mL of bacteriophage vB_EcoM_SQ17 was added to the control group without phage, and only an equal volume of SM buffer was added. The cells were cultured at 4°C, and samples were collected at 0, 4, 8, 12, 24, 48, and 72 h. The number of bacteria in the samples was measured by plate counting method, and the bacterial concentration in skim milk was calculated to draw the inhibition curve.

[0084] like Figure 8 As shown in the figure, the test results of the application of bacteriophage vB_EcoM_SQ17 in skimmed sterilized pure milk: at 4°C, compared with the initial bacterial concentration, the number of viable bacteria decreased by 3.51 Log CFU / mL at 12 and 24 hours, and no bacteria were detected at 48 and 72 hours, while the bacterial concentration in the control group was relatively stable and no significant changes were observed.

[0085] Example 9: Antibacterial effect of bacteriophage vB_EcoM_SQ17 on EHEC O157:H7 in whole-fat sterilized milk

[0086] EHEC O157:H7 strain EO157-1 was cultured to the logarithmic growth phase, washed with PBS, centrifuged to remove the bacterial culture medium, and the bacterial pellet was retained. 5 ml of full-fat sterilized milk was used to dilute the bacteria to a final concentration of 3.2×104 CFU / mL, set up experimental group and control group. The experimental group added 1mL to a final concentration of about 10 8 PFU / mL of bacteriophage vB_EcoM_SQ17 was added to the control group without phage, and only an equal volume of SM buffer was added. The cells were cultured at 4°C, and samples were collected at 0, 4, 8, 12, 24, 48, and 72 h. The number of bacteria in the samples was measured by plate counting method, and the bacterial concentration in whole milk was calculated to draw the inhibition curve.

[0087] like Figure 9 As shown in the figure, the application test results of bacteriophage vB_EcoM_SQ17 in whole-fat sterilized pure milk: at 4°C, compared with the initial bacterial concentration, the number of viable bacteria decreased by 2.4 and 2.89 Log CFU / mL at 12 and 24 hours, respectively, and the number of viable bacteria decreased by 3.41 Log CFU / mL at 48 and 72 hours, while the bacterial concentration in the control group was relatively stable and no significant changes were observed.

Claims

1. A broad-spectrum enterohemorrhagic Escherichia coli O157:H7 phage (Enterohemorrhagic Escherichiacoli O157:H7 phage), named vB_EcoM_SQ17, the phage preservation number is CTCC NO: M20211003, it was deposited in the China Center for Type Culture Collection on August 10, 2021, and was classified and named Enterohemorrhagic Escherichia coli O157:H7 phage vB_EcoM_SQ17.

2. A bacteriostatic agent, characterized in that The antibacterial agent comprises the enterohemorrhagic Escherichia coli O157:H7 phage according to claim 1 or a culture of the enterohemorrhagic Escherichia coli O157:H7 phage according to claim 1, and the culture method comprises the following steps: 1) Centrifuging the enterohemorrhagic Escherichia coli O157:H7 culture and collecting the precipitate; 2) resuspending the precipitate in step 1) using LB medium to obtain a bacterial suspension, adding the enterohemorrhagic Escherichia coli O157:H7 phage according to claim 1 to the bacterial suspension, culturing at 37° C. for 6 to 8 hours, centrifuging, taking the supernatant, and filtering the supernatant through a 0.22 μm filter membrane to obtain an enterohemorrhagic Escherichia coli O157:H7 phage lysate; wherein, The concentration of the enterohemorrhagic Escherichia coli O157:H7 culture in step 1) is 1×10 8 CFU / ml~1×10 10 CFU / ml.

3. Use of the enterohemorrhagic Escherichia coli O157:H7 phage according to claim 1 or the antibacterial agent according to claim 2 in the preparation of a preparation for inhibiting and / or eliminating enterohemorrhagic Escherichia coli O157:H7.

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