Phage compositions for treating pathogenic e. coli infections and methods of making and use

Abstract

The application provides a bacteriophage composition for treating pathogenic E. coli infection, a preparation method and application, and belongs to the technical field of food safety. The application provides bacteriophages with lytic effects on pathogenic E. coli, and the bacteriophages include ECP2301, ECP2304 and ECP2307, and have important bacteriostatic effects on pathogenic E. coli host bacteria. The three bacteriophages in the bacteriophage composition have strong lytic effects on E. coli MQ220627, MQ1 and YS220605 respectively, and the three bacteriophages have a synergistic effect after being mixed, and have higher inhibitory effects than single bacteriophages, and can be used as pathogenic E. coli removal agents for milk and meat, and have good effects.

Description

Technical Field

[0001] This invention belongs to the field of food safety technology, specifically relating to a bacteriophage composition for treating pathogenic Escherichia coli infection, its preparation method, and its application. Background Technology

[0002] Escherichia coli is the most common commensal bacterium in the gastrointestinal tract of humans and animals, and also one of the most important pathogens. When the external environment affects the body, the phenomenon of livestock animals suffering from diarrhea, meningitis, and other diseases due to pathogenic E. coli is increasingly common in farms, causing huge losses to the livestock industry. Currently, the main control measure against enteropathogenic E. coli is the use of antibiotics, but due to the extensive use of antibiotics, drug resistance is a prominent problem. The demand for alternative antibiotics is constantly increasing; therefore, there is an urgent need to develop a new bactericidal method to replace antibiotics.

[0003] Phage therapy utilizes bacterial viruses or bacteriophages to target and destroy bacteria at different sites of infection. Recent advances in biotechnology have enabled the rapid expansion of existing phage libraries, resulting in potent and specific phages capable of targeting and destroying bacteria of interest. Pathogenic Escherichia coli can cause diarrhea in various animals. Furthermore, some pathogenic E. coli strains are antibiotic resistant, increasing the difficulty of treating E. coli-associated chronic infections. Phage therapy, which can bypass traditional antibiotic resistance mechanisms, avoid the toxic side effects of traditional small molecule therapies, and effectively combat biofilms, is particularly attractive.

[0004] Bacteriophages are a class of viruses that specifically infect target bacteria and replicate themselves. Highly virulent bacteriophages possess advantages such as host-specificity, self-replication, strong lytic ability, non-toxicity to plants and animals, environmental friendliness, and ease of production. Therefore, phage therapy holds great potential in the biocontrol of bacterial diseases. However, most bacteriophages have a relatively narrow host spectrum, infecting only one or a few strains. Therefore, the use of single-phage preparations can limit control efficacy and easily lead to the development of resistant bacteria. Summary of the Invention

[0005] The purpose of this invention is to provide a phage composition for treating pathogenic Escherichia coli infection, a method for its preparation, and its application. The phage composition prepared by mixing multiple different phages has more stable biological characteristics and lytic activity, and can also effectively solve the problems of bacterial resistance mutation and narrow phage host spectrum.

[0006] The present invention provides a bacteriophage that has a lytic effect on pathogenic Escherichia coli, the bacteriophage comprising at least one of the following: ECP2301, ECP2304 and ECP2307;

[0007] The accession number for ECP2301 is CCTCC NO:M 20241608, for ECP2304 it is CCTCC NO:M 20241609, and for ECP2307 it is CCTCC NO:M 20241610.

[0008] Preferably, the pathogenic Escherichia coli includes at least one of the following: MQ220627, MQ1 and YS220605;

[0009] The accession number for MQ220627 is CCTCC NO:M 20241664, the accession number for MQ1 is CCTCC NO:M20241665, and the accession number for YS220605 is CCTCC NO:M 20241666.

[0010] The present invention provides a group of phage compositions for treating pathogenic Escherichia coli infections, comprising at least two of the following phages: ECP2301, ECP2304 and ECP2307;

[0011] The accession number for ECP2301 is CCTCC NO:M 20241608, for ECP2304 it is CCTCC NO:M 20241609, and for ECP2307 it is CCTCC NO:M 20241610.

[0012] Preferably, the pathogenic Escherichia coli includes at least one of the following: MQ220627, XMCS2207-5, MQ1 and YS220605;

[0013] The accession number for MQ220627 is CCTCC NO:M 20241664, the accession number for MQ1 is CCTCC NO:M20241665, and the accession number for YS220605 is CCTCC NO:M 20241666.

[0014] The present invention provides the use of the above-mentioned bacteriophage or the above-mentioned bacteriophage composition in the preparation of a drug for inhibiting Escherichia coli infection in vitro.

[0015] The present invention also provides the use of the above-mentioned bacteriophage or the above-mentioned bacteriophage composition as a foodborne pathogenic Escherichia coli scavenger.

[0016] Preferably, the food includes dairy products or meat products.

[0017] The present invention provides a method for preparing the above-mentioned phage composition, comprising the following steps: (1) culturing phages ECP2301, ECP2304 and ECP2307 in Escherichia coli culture medium to obtain a culture medium;

[0018] (2) After centrifuging the culture medium described in step (1), take the supernatant containing phages and dilute it at different gradients. Mix each dilution with the Escherichia coli culture medium and incubate. Then, pour double-layer plates and culture them. Calculate the titers of the three phage strains by counting the phage plaques.

[0019] (3) The titers of the three bacteriophage strains were adjusted to (1.0±0.5)×10⁻⁶. 9 The phage composition was obtained by mixing the diluted phage solution with PFU / mL.

[0020] Preferably, the mixing ratio of the diluted phage fluid in step (3) is 1:1:1.

[0021] The present invention also provides a method for removing pathogenic Escherichia coli from food, comprising the following steps: mixing a food sample with the above-mentioned phage composition or a phage composition prepared by the above-mentioned preparation method, and reacting at 15-35°C for 2 hours to obtain food in which pathogenic Escherichia coli has been removed.

[0022] Beneficial Effects: This invention provides bacteriophages with lytic activity against pathogenic Escherichia coli. The bacteriophages include ECP2301, ECP2304, and ECP2307, and the host bacteria of the bacteriophage composition are pathogenic Escherichia coli MQ220627, MQ1, and YS220605, respectively, exhibiting significant antibacterial activity against these pathogenic Escherichia coli host bacteria. Verification through examples shows that the five bacteriophages in the bacteriophage composition of this invention all exhibit strong lytic activity against Escherichia coli MQ220627, MQ1, and YS220605. Furthermore, when three bacteriophages are mixed, a synergistic effect is observed, resulting in a more efficient inhibitory effect compared to individual bacteriophages. This mixture can be used as a pathogenic Escherichia coli scavenger in milk and meat, demonstrating good efficacy. Attached Figure Description

[0023] Figure 1 The complete genome loops of bacteriophages ECP2301, ECP2304, and ECP2307 are shown.

[0024] Figure 2 The graph shows the results of the temperature sensitivity test of bacteriophages.

[0025] Figure 3 This is a one-step growth curve of a bacteriophage;

[0026] Figure 4 The in vitro antibacterial curve of bacteriophage;

[0027] Figure 5 Figure showing the results of the pH tolerance experiment of bacteriophages;

[0028] Figure 6 The graph shows the MOI measurement results;

[0029] Figure 7 The results for the host bacterium Escherichia coli are shown in the figure. A: Colony morphology of Escherichia coli on eosin methylene blue agar plate; B: Gram staining microscopic morphology; C: Escherichia coli 16S rDNA sequence phylogenetic tree.

[0030] Figure 8 Plaques formed by bacteriophages;

[0031] Figure 9 The images show the morphology of bacteriophages under a transmission electron microscope; A: bacteriophage ECP2301, B: bacteriophage ECP2304, C: bacteriophage ECP2307.

[0032] Figure 10 The effect of bacteriophage preparations on the removal of Escherichia coli on the surface of meat samples;

[0033] Figure 11 The effect of phage preparations on the elimination of Escherichia coli in milk samples.

[0034] Biological Preservation Information

[0035] Escherichia coli YS220605 was deposited at the China Center for Type Culture Collection (CCTCC) on July 22, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20241666.

[0036] Escherichia coli MQ1 was deposited at the China Center for Type Culture Collection (CCTCC) on July 22, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241665.

[0037] Escherichia coli YS220627 was deposited at the China Center for Type Culture Collection (CCTCC) on July 22, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20241664.

[0038] Bacteriophage ECP2307 was deposited at the China Center for Type Culture Collection (CCTCC) on July 22, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241610.

[0039] Bacteriophage ECP2304 was deposited at the China Center for Type Culture Collection (CCTCC) on July 22, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241609.

[0040] Bacteriophage ECP2301 was deposited on July 22, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241608. Detailed Implementation

[0041] The present invention provides a bacteriophage that has a lytic effect on pathogenic Escherichia coli, the bacteriophage comprising at least one of the following: ECP2301, ECP2304 and ECP2307;

[0042] The accession number for ECP2301 is CCTCC NO:M 20241608, for ECP2304 it is CCTCC NO:M 20241609, and for ECP2307 it is CCTCC NO:M 20241610.

[0043] The bacteriophage ECP2307 described in this invention is preferably obtained from wastewater from cattle farms in Yushu Prefecture, Qinghai Province, while bacteriophages ECP2301 and ECP2304 are obtained from wastewater from cattle farms in Maqin County, Qinghai Province.

[0044] The pathogenic Escherichia coli of this invention preferably includes at least one of the following: MQ220627, MQ1, and YS220605; wherein the accession number of MQ220627 is CCTCC NO:M 20241664, the accession number of MQ1 is CCTCC NO:M20241665, and the accession number of YS220605 is CCTCC NO:M20241666. The pathogenic Escherichia coli of this invention is preferably isolated from diarrheal yaks in certain areas of Qinghai Province.

[0045] The host bacteria of the bacteriophages ECP2301, ECP2304 and ECP2307 described in this invention are pathogenic Escherichia coli MQ220627, MQ1 and YS220605, respectively, and they all have antibacterial effects against the host bacteria, exhibiting strong lytic activity. When the three bacteriophages are mixed, a synergistic effect is found among them, which has a more efficient inhibitory effect than a single bacteriophage.

[0046] The present invention provides a group of phage compositions for treating pathogenic Escherichia coli infections, comprising at least two of the following phages: ECP2301, ECP2304 and ECP2307;

[0047] The accession number for ECP2301 is CCTCC NO:M 20241608, for ECP2304 it is CCTCC NO:M 20241609, and for ECP2307 it is CCTCC NO:M 20241610.

[0048] The bacteriophage and pathogenic Escherichia coli described in this invention are preferably the same as those described above, and will not be repeated here.

[0049] The present invention provides the use of the above-mentioned bacteriophage or the above-mentioned bacteriophage composition in the preparation of a drug for inhibiting Escherichia coli infection in vitro.

[0050] This invention utilizes a method of co-culturing pathogenic Escherichia coli with the phage composition to identify the in vitro inhibitory effect of the phage composition on Escherichia coli. The results showed that the OD values ​​of the Escherichia coli cultures without the phage composition were significantly higher. 600 The average value increased over time, reaching 0.71 at 9 hours; while the OD of the E. coli culture containing the phage composition was... 600 The average value decreased over time, reaching 0.21 at 9 hours. Figure 4 ).

[0051] The present invention also provides the use of the above-mentioned bacteriophage or the above-mentioned bacteriophage composition as a foodborne pathogenic Escherichia coli scavenger.

[0052] The food products described in this invention preferably include dairy products or meat products.

[0053] The present invention provides a method for preparing the above-mentioned phage composition, comprising the following steps: (1) culturing phages ECP2301, ECP2304 and ECP2307 in Escherichia coli culture medium to obtain a culture medium;

[0054] (2) After centrifuging the culture medium described in step (1), take the supernatant containing phages and dilute it at different gradients. Mix each dilution with the Escherichia coli culture medium and incubate. Then, pour double-layer plates and culture them. Calculate the titers of the three phage strains by counting the phage plaques.

[0055] (3) The titers of the three bacteriophage strains were adjusted to (1.0±0.5)×10⁻⁶. 9 The phage composition was obtained by mixing the diluted phage solution with PFU / mL.

[0056] In this embodiment of the invention, preferably, 500 μL of purified bacteriophage is added to 50 mL of Escherichia coli culture medium (OD200). 600 =0.6) After incubation at 37℃ for 12h; (2) The next day, after centrifugation at 4℃ and 8000r / min for 15min, take 100μL of the supernatant containing the phage and dilute it to 10 -2 10 -4 10 -6 10 -8 10 -10 There are 5 gradients in total. Take 100 μL of phage diluted to each gradient and 100 μL of E. coli bacterial culture (OD200). 600 =1.2) Mix and incubate for 10 min, pour into double-layer plates and incubate overnight at 37℃. The next day, count the plaques and calculate the titers of the three phage strains (3 replicates per group); (3) Dilute the titers of the three phage strains to (1.0±0.5)×10 9 Take an appropriate amount of diluted phage solution at approximately PFU / mL and mix them in a 1:1:1 ratio to obtain an E. coli phage cocktail.

[0057] The present invention provides a method for removing pathogenic Escherichia coli from food, comprising the following steps: mixing a food sample with the above-mentioned phage composition or a phage composition prepared by the above-mentioned preparation method, and reacting at room temperature for 2 hours to obtain food in which pathogenic Escherichia coli has been removed.

[0058] In this invention, if the sample is liquid, 1 mL of the above-mentioned phage cocktail is added to every 100 mL of the sample to be tested; if the sample is solid, the above-mentioned phage cocktail is sprayed onto the surface of the sample to be tested and allowed to act at room temperature for 2 hours to eliminate pathogenic Escherichia coli in the sample. Room temperature as referred to in this invention means 15–35°C.

[0059] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the phage composition for treating pathogenic Escherichia coli infection, its preparation method, and its application, should not be construed as limiting the scope of protection of the present invention.

[0060] In this invention, the following abbreviations are used: MOI (multiplicity of infection): the ratio of virus to cells at infection. PFU (plaque forming unit): plaque forming unit. PFU / ml (Plaque Forming Unit per mL): infectious titer, expressed as the number of plaques formed per unit volume of virus. Burst rate (PFU / cell): the ratio of phage titer at the end of the burst (PFU / ml) to the host bacterial concentration at the beginning of lysis (cell / ml). ORF: open reading frame.

[0061] Example 1

[0062] 1. Isolation of bacteriophages

[0063] The collected wastewater was poured into an Erlenmeyer flask, and SM buffer (one-quarter volume of wastewater) was added. The mixture was shaken at 60 rpm at room temperature for 48 hours. The liquid was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. This process was repeated 3-4 times, and the supernatant was filtered through a 0.45 μm microfiltration filter to obtain the stock solution containing bacteriophages. 5 ml of 2×LB liquid medium was added, along with 5 ml of the stock solution and 200 μL of *E. coli* culture. The mixture was incubated at 180 rpm at 37°C for 18 hours. The co-culture solution was then filtered through a 0.22 μm microfiltration filter. 200 μL of the filtrate and 200 μL of *E. coli* culture were mixed 1:1 and incubated at 37°C for 10 minutes. The mixture was then added to 10 ml of 0.5% LB semi-solid medium and poured onto solidified LB medium to prepare a double-layer plate. The plate was incubated at 37°C for 18 hours, and clear, translucent phage plaques were observed on the double-layer plate.

[0064] 2. Purification of bacteriophages

[0065] Select a single, large-diameter, uniformly grown phage plaque and inoculate it into 1 ml of SM buffer. Incubate at 4°C for 18 h. Serially dilute the filtrate 10-fold and take 10 ml of each plaque. -2 10 -3 10 -4 10 -5 10 -6 Mix 100 μL of the filtrate at each concentration with 100 μL of E. coli culture, add 10 ml of LB semi-solid medium, pour onto solidified LB medium to prepare a double-layer plate, and incubate at 37°C for 18 h. Repeat the purification process 3–5 times until uniformly sized, clear, and transparent phage plaques appear on the plate, indicating that purification is complete.

[0066] Three bacteriophages, ECP2301, ECP2304, and ECP2307, were isolated and purified, with *Escherichia coli* strains MQ220627, MQ1, and YS220605 as hosts, respectively. After purification, all three strains exhibited uniformly sized, clear, and transparent phage plaques on agar plates. Figure 8 ); Genome sequencing results as follows Figure 1 As shown.

[0067] Table 1. Results of whole-genome analysis of bacteriophages

[0068]

[0069] 3. Phage titer determination

[0070] The phage stock solution was serially diluted 10-fold. LB semi-solid culture medium was added to a sterile test tube. 100 μL of *E. coli* bacterial culture and 100 μL of phage dilutions of different concentrations were mixed 1:1. 10 mL of 0.5% LB semi-solid medium was added, and the mixture was immediately poured into solidified agar medium to prepare a double-layer plate. The plates were incubated at 37°C for 12–16 h. Plates with clear and transparent phage plaques were selected for counting and titer calculation. Phage titer (PFU / mL) = average PFU / mL × dilution factor × 10.

[0071] The average titers of the three bacteriophages, determined by plate counting, were as follows: ECP2301 was 9.4 × 10⁻⁶. 11 PFU / mL, ECP2304 is 3.8 × 10 11 PFU / mL, ECP2307 is 9.8 × 10 11 PFU / mL.

[0072] 4. Determination of the host spectrum of bacteriophages

[0073] Select a single, large-diameter, uniformly grown phage plaque and inoculate it into 5 ml of LB liquid medium, adding 200 μL of *E. coli* bacterial suspension. Incubate at 37°C for 18 h at 180 rpm. Filter the co-culture solution through a 0.22 μm microfiltration filter for later use. Add 200 μL of *E. coli* bacterial suspension to 10 ml of LB semi-solid medium, pour the mixture onto solidified LB medium to prepare a double-layer plate. After drying, drop 10 μL of phage suspension onto the bacterial colony surface, adding an equal volume of physiological saline as a control. After the droplet dries, incubate at 37°C for 18 h and observe the results. The host spectrum determination of the phage composition was performed using the same method.

[0074] The plaque scoring criteria are based on "Phytophthology: From Theory to Practice", where "+4" represents complete lysis, "+3" represents complete lysis of the droplet area but with a faint background, "+2" represents a large amount of turbidity in the droplet area, and "+1" represents the presence of individual phages.

[0075] Host spectrum assays of the three bacteriophages and the composition showed that ECP2301, ECP2304 and ECP2307 had varying degrees of lytic activity against 34, 38 and 31 strains of Escherichia coli, respectively, while the bacteriophage composition had varying degrees of lytic activity against 58 strains of Escherichia coli.

[0076] Table 23 Host spectrum of bacteriophages and their combinations

[0077]

[0078]

[0079]

[0080] 5. Morphological observation of bacteriophages

[0081] Take 10 μL of phage suspension onto a copper grid, let it precipitate for 10 min, absorb the excess liquid with filter paper, then add 10 μL of phosphotungstic acid (PTA) for staining for 5–10 min, let it air dry, and observe the morphology of the phage under a transmission electron microscope (TEM).

[0082] Bacteriophage ECP2301 has a head length of approximately 55.0 nm and a tail length of approximately 20.0 nm; bacteriophage ECP2304 has a head length of approximately 80.0 nm and a tail length of approximately 87.0 nm; bacteriophage ECP2307 has a head length of approximately 76.0 nm and a tail length of approximately 116.0 nm. Figure 9 ).

[0083] 6. Determination of the optimal multiple of infection (MOI) for bacteriophages

[0084] A single colony of host *E. coli* was picked and inoculated into 5 mL of LB broth and incubated overnight. The number of *E. coli* was determined by plate count. The concentration of *E. coli* was adjusted and mixed with an equal volume of 100 μL of bacteriophage to achieve a multiplicity of infection (MOI) of 10⁻⁶. -5 10 -4 10 -3 10 -2 10 -1 1, 10, 10 2 10 3 10 4 and 10 5 Add to 10 mL LB liquid. Incubate at 37°C with gentle shaking for 5 hours, centrifuge at 8000 rpm for 10 minutes, filter, and serially dilute the filtrate to 10 mL. 12 The phage titer was determined, and the MOI with the highest titer was the optimal MOI.

[0085] The results are as follows Figure 6 As shown, the optimal MOI for phage ECP2301 is 0.0001, and the optimal MOI for phages ECP2304 and ECP2307 is 0.01.

[0086] 7. Isolation, screening, and purification of pathogenic Escherichia coli

[0087] A small amount of aseptically collected pathogen samples were inoculated into 10 mL of LB liquid medium and incubated overnight at 37°C and 180 rpm. The next day, the culture was streaked onto eosin methylene blue plates and incubated at 37°C for 18–24 h. The following day, single colonies with a purplish-black metallic sheen were selected and purified on plates 2–3 times. The purified single colonies were inoculated into LB liquid medium and incubated overnight at 37°C and 180 rpm. The following day, the bacterial culture was Gram-stained and examined under a microscope. The genome of the bacteria was then extracted, and PCR amplification and sequencing were performed using 16S rDNA universal primers. The sequencing results were compared with those from NCBI, and a phylogenetic tree was constructed.

[0088] The results showed that the three isolated strains formed purplish-black, metallic-lustered, smooth-edged colonies on eosin methylene blue plates. Microscopic examination revealed them to be red, non-spore-forming, blunt-ended, scattered or paired straight bacilli. 16S rDNA sequence alignment and phylogenetic analysis confirmed that the three strains were *Escherichia coli*. Figure 7 ).

[0089] 8. Determination of one-step growth curve

[0090] Using Escherichia coli MQ220627, XMCS2207-5, MQ1, YS220605, and MQ2206-3 as host bacteria, the host bacteria were cultured overnight and the bacterial concentration was adjusted to 1.00 × 10⁻⁶. 11 CFU / mL; Take 10 mL of LB liquid culture medium and add 1.00 × 10⁻⁶ CFU / mL. 8 PFU / mL phage solution and 1.00×10 8 100 μL of CFU / mL bacterial culture was incubated at 37℃ with gentle shaking. At 0 min, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, 3 h, 4 h, 5 h, and 6 h, 500 μL of culture was collected and filtered through a 0.22 μm microfiltration filter to determine the phage titer. A one-step growth curve was plotted to determine the phage latency, outbreak phase, and lysis rate. Lysis rate = phage titer at the end of the outbreak / host bacterial concentration at the initial stage of infection.

[0091] The results are as follows Figure 3 As shown, the incubation periods of the three bacteriophage strains were all 40 min; the outbreak periods were 60 min, 140 min, and 200 min, respectively; and the lysis rates were 680 PFU / cell, 680 PFU / cell, and 1450 PFU / cell, respectively.

[0092] 9. Temperature sensitivity of bacteriophages

[0093] Take 600 μL of phage solution and incubate it at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 30 min and 60 min respectively. Mix 100 μL of Escherichia coli bacterial solution with 100 μL of phage diluted at different temperatures at a ratio of 1:1, add 10 mL of 0.5% semi-solid LB, and immediately pour into solidified agar medium to make a double-layer plate. Incubate at 37℃ for 12-16 h, and then determine the phage titer.

[0094] The results are as follows Figure 2 As shown, the titer of bacteriophage ECP2301 did not change significantly under conditions of 20–70℃; the titers of ECP2304 and ECP2307 did not change significantly under conditions of 20–60℃; however, as the temperature gradually increased, the titers of the three bacteriophages dropped sharply and were completely inactivated at 80℃.

[0095] 10. pH stability of bacteriophages

[0096] Adjust the pH of sterile deionized water to 2.0–13.0 using 0.1 mol / L HCl and NaOH solutions, and obtain a titer of approximately 1.00 × 10⁻⁶. 8 Add 100 μL of PFU / mL phage solution to a sterile EP tube, then add 900 μL of deionized water at different pH values. After mixing, dilute to 10 for each pH value. 8 The phage titer was determined by taking the supernatant from the original host bacteria at a 1:1 ratio and using the double-layer agar plate method.

[0097] The results are as follows Figure 5 As shown, the titers of the three bacteriophages did not change significantly between pH 3.0 and 12.0. However, when pH > 12 or < 3, the bacteriophage activity decreased rapidly until it was completely inactivated. This indicates that SP3 is more tolerant to acidic or alkaline environments, but it will be rapidly inactivated under strong acid or strong alkaline conditions.

[0098] 11. Preparation of bacteriophage compositions

[0099] (1) Take 500 μL of purified bacteriophage and add it to 50 mL of Escherichia coli culture medium (OD200). 600 =0.6) After incubation at 37℃ for 12h; (2) The next day, after centrifugation at 4℃ and 8000r / min for 15min, take 100μL of the supernatant containing the phage and dilute it to 10 -2 10 -4 10 -6 10 -8 10 -10 There are 5 gradients in total. Take 100 μL of phage diluted to each gradient and 100 μL of E. coli bacterial culture (OD200). 600=1.2) Mix and incubate for 10 min, pour into double-layer plates and incubate overnight at 37℃. The next day, count the plaques and calculate the titers of the three phage strains (3 replicates per group); (3) Dilute the titers of the three phage strains to 1.0×10 9 Take an appropriate amount of diluted phage solution at approximately PFU / mL and mix them in a 1:1:1 ratio to obtain an E. coli phage cocktail.

[0100] 12. In vitro antibacterial test of bacteriophages

[0101] Fifty-eight pathogenic Escherichia coli strains were cultured overnight to achieve an initial OD value between 0.2 and 0.4. The control group was treated with 100 μL of E. coli and 100 μL of LLB; the experimental group was treated with 100 μL of E. coli and 100 μL of a bacteriophage combination. The OD value of the bacterial culture was measured every hour for 9 hours. The average value was calculated and plotted.

[0102] The results are as follows Figure 4 As shown, the OD values ​​of each group of E. coli bacterial suspensions without the addition of the phage composition are... 600 The average value increased over time, reaching 0.71 at 9 hours; while the OD of the E. coli culture containing the phage composition was... 600 The average value decreased over time, reaching 0.21 at 9 hours.

[0103] Table 358 Information on Escherichia coli strains

[0104]

[0105]

[0106] 13. Antibacterial test of bacteriophage preparations on meat products

[0107] Dilute the overnight culture of E. coli to 3.0 × 10⁻⁶. 7 Fresh beef samples were immersed in a diluent of E. coli for 1 min at CFU / mL. The samples were then divided into treated and untreated groups. The treated group was sprayed with a surface-sprayed solution containing 1.0 × 10⁻⁶ CFU / mL. 9 The phage preparation was administered at PFU / mL. Untreated groups were sprayed with an equal volume of SM buffer and allowed to stand at room temperature. At 0h, 1h, 2h, and 3h, both groups of meat samples were processed using a high-throughput tissue homogenizer. The E. coli load in the samples was then determined using the plate gradient dilution method, with three replicates per group. Results are as follows: Figure 10 As shown, the initial average bacterial load of Escherichia coli in the two groups of samples was 8.5 × 10⁻⁶. 7The CFU / mL E. coli load in meat samples treated with phage preparations decreased continuously over time. At 3 hours, the average bacterial load in the treated group was <100 CFU / mL, while the average bacterial load in the untreated group was >4.5 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0108] 14. Antibacterial test of bacteriophage preparations on dairy products

[0109] Dilute the overnight culture of E. coli to 3.0 × 10⁻⁶. 7 CFU / mL, take 1 mL of diluted E. coli solution and add it to 100 mL of fresh milk. Divide the milk sample into a treatment group and an untreated group. The treatment group is supplemented with a titer of 1.0 × 10⁻⁶ CFU / mL. 10 One mL of PFU / mL phage preparation was applied to the untreated group, and an equal volume of SM buffer was sprayed onto each group. The mixtures were incubated at room temperature, and milk samples were collected at 0 h, 1 h, 2 h, and 3 h. The E. coli load in the samples was determined using the plate serial dilution method, with three replicates per group. Results are as follows: Figure 11 As shown, the initial average bacterial load of Escherichia coli in the two groups of samples was 3.0 × 10⁻⁶. 5 The bacterial load of *E. coli* in milk samples treated with phage preparations decreased continuously over time. At 3 hours, the average bacterial load in the treated group was 240 CFU / mL, while the average bacterial load in the untreated group was 2.0 × 10⁻⁶ CFU / mL. 8 CFU / mL.

[0110] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A group of bacteriophage compositions for treating pathogenic Escherichia coli infections, characterized in that, Including the following bacteriophages: ECP2301, ECP2304, and ECP2307; The accession number of ECP2301 is CCTCC NO:M 20241608, the accession number of ECP2304 is CCTCC NO:M20241609, and the accession number of ECP2307 is CCTCC NO:M 20241610. The pathogenic Escherichia coli includes at least one of the following: MQ220627, MQ1 and YS220605; The accession number for MQ220627 is CCTCC NO:M 20241664, the accession number for MQ1 is CCTCC NO:M20241665, and the accession number for YS220605 is CCTCC NO:M 20241666.

2. The use of the phage composition of claim 1 in the preparation of a medicament for in vitro inhibition of Escherichia coli infection.

3. The use of the bacteriophage composition of claim 1 as a foodborne pathogenic Escherichia coli scavenger, characterized in that, The food products include dairy products or meat products.

4. The method for preparing the phage composition according to claim 1, characterized in that, The steps include: (1) culturing bacteriophages ECP2301, ECP2304 and ECP2307 in Escherichia coli culture medium to obtain culture medium; (2) After centrifuging the culture medium described in step (1), take the supernatant containing the phage and dilute it at different gradients. Mix each dilution with the Escherichia coli culture medium and incubate. Then, pour double-layer plates and culture them. Calculate the titer of the three phage strains by counting the phage plaques. (3) The titers of the three bacteriophage strains were adjusted to (1.0±0.5)×10. 9 The phage composition was obtained by mixing the diluted phage solution with PFU / mL at a ratio of 1:1:

1.

5. A method for removing pathogenic Escherichia coli from food, characterized in that, The process includes the following steps: mixing a food sample with the phage composition of claim 1 or the phage composition prepared by the preparation method of claim 4, and reacting it at 15-35°C for 2 hours to obtain food that has eliminated pathogenic Escherichia coli.

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