Escherichia coli bacteriophage and application thereof

Abstract

The invention relates to an Escherichia phage and application thereof, the Escherichia phage is named as PD348 and classified as Escherichia phage, the preservation number of the Escherichia phage is CGMCC NO.46171, the Escherichia phage is preserved in China General Microbiological Culture Collection Center on August 16, 2024, and the preservation number of the Escherichia phage is CGMCC NO.46171. The preservation number of the Escherichia phage is CGMCC NO.46171. The preservation number of the Escherichia phage is CGMCC NO.46171. The preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postcode is 100101. The bacteriophage composition has the advantages that the application range is wide, and tests prove that the bacteriophage provided by the invention is safe, green and free of toxic and side effects, so that the bacteriophage composition can be used for environmental modifiers, bacteriophage pharmaceutical preparations and the like, and can be used for preventing and treating diseases caused by escherichia coli.

Description

Technical Field

[0001] The invention relates to an Escherichia coli bacteriophage, belonging to the technical field of microorganisms. Background Art

[0002] Escherichia coli, also known as Escherichia coli. Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped, Escherichia coli bacterium with blunt ends, motile, and non-spore-forming. It is a warm-blooded organism commonly found in the lower part of the small intestine. Escherichia coli mainly parasitizes in the large intestine of humans and animals, accounting for about 1% of intestinal bacteria. According to different serotypes, the antigens of Escherichia coli are mainly divided into bacterial antigens (O antigens), flagellar antigens (H antigens) and capsule antigens (K antigens). Escherichia coli currently found in humans are divided into commensal type, pathogenic type (intestinal diarrhea type) and extraintestinal pathogenic type according to genetics and clinical studies. Escherichia coli can infect humans, poultry, pigs, sheep and many other animals, but its pathogens have strong specificity to the host. Human infection is mainly manifested in diarrhea, urinary tract infection, sepsis, pneumonia and meningitis, which pose a serious threat to human health.

[0003] Although most E. coli are considered harmless to humans, pathogenic E. coli causes a certain amount of mortality every year and is recognized as one of the main bacterial causes of infection and foodborne diarrhea. There are three main treatments for human diseases caused by E. coli infection, such as antibiotics, bacteriophages and traditional Chinese medicine. Since the discovery of antibiotics, they have been widely used and have occupied a clear dominant position, but over time, antibiotic resistance has gradually increased, and there are more and more reports of E. coli acquiring antimicrobial resistance. In addition, antibiotics abused in agriculture and aquaculture remain in our food, thus entering our bodies and not circulating in the food chain. The problem of antimicrobial resistance is now considered to be one of the important causes of human death by 2050.

[0004] The emergence of multi-drug resistant bacteria and superbugs that are not sensitive to any antibiotics has led people to re-examine the potential of bacteriophages as alternative agents to antibiotics. Bacteriophages also play a vital role in microbial communities, not only maintaining the ecological structure of the community but also promoting microbial evolution through horizontal gene transfer. Currently, bacteriophages have been proposed as an alternative therapy to antibiotics.

[0005] Phages are a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, or spirochetes. Phages grow and reproduce in bacterial host cells, can cause the lysis of pathogenic bacteria, reduce the density of pathogenic bacteria, thereby reducing or avoiding the chance of pathogenic bacteria infection or disease onset, and achieving the purpose of treating and preventing diseases. Phage therapy is a new approach to treating pathogenic bacterial infections by phage lysis of host bacteria. They are the most abundant and widespread organisms on Earth and play important roles in microbiology, physiology, evolution, and treatment. Compared with traditional antibiotics, phages have the advantages of wide distribution, easy screening, high host specificity, strong proliferation ability, high safety, and low R & D cost. Phage therapy has a very broad and attractive prospect as an alternative means to eliminate pathogenic bacteria and is worthy of in-depth exploration by researchers.

[0006] Therefore, there is an urgent need for further research in the existing technology. Summary of the Invention

[0007] To overcome the defects of the existing technology, the present invention provides an Escherichia coli phage. The technical solution of the present invention is:

[0008] An Escherichia coli phage, named PD348, classified as Escherichia coli bacteriophage, with a preservation number of CGMCC NO.46171, preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on August 16, 2024, at the preservation address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101.

[0009] The use of the Escherichia coli phage in lysing Escherichia coli or preparing a composition for lysing Escherichia coli.

[0010] The application of the Escherichia coli phage in preparing a drug for preventing and treating diseases caused by Escherichia coli infection.

[0011] The pH value during application does not exceed 10.

[0012] The temperature during application is not higher than 60 °C.

[0013] A phage pharmaceutical preparation, including the Escherichia coli phage; the content of phage in the environmental modifier is at least 107 PFU / mL.

[0014] An environmental modifier, including the Escherichia coli phage; the content of phage in the environmental modifier is at least 107 PFU / mL.

[0015] In the first aspect, the present invention provides an Escherichia coli phage, which is Escherichia coli phage PD348 with a preservation number of CGMCC NO. 46171. It was preserved on August 16, 2024 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. This phage PD348 has an elongated polyhedral head that encapsulates nucleic acid. The major axis of the head is about 140 nm and the minor axis is about 56 nm. It has a relatively short tail about 20 nm long, and the neck connects the head and the tail. The whole genome size of this phage PD348 is 77030 bp.

[0016] In the second aspect, an Escherichia coli phage provided by the present invention is Escherichia coli phage PD348. For 195 clinically isolated Escherichia coli strains with multidrug resistance, its lysis rate is 36%. The titer of the phage can maintain at a relatively high level of 10^8 - 10^9 PFU / mL after acting for 3 h under the condition of pH 4 - 10, showing good acid and alkali resistance. At the same time, the titer of phage PD348 maintains at a relatively high level of 10^8 - 10^9 PFU / mL after acting for 40 min at a temperature of 60 °C, showing good temperature resistance.

[0017] In the third aspect, based on the same inventive concept, an Escherichia coli phage PD348 provided by the present invention can be used to prevent and treat human diseases caused by Escherichia coli infection, such as pneumonia, cholecystitis, cystitis, peritonitis, liver abscess, lung abscess, kidney abscess, as well as arthritis, meningitis, etc.

[0018] In the fourth aspect, based on the same inventive concept, the present invention provides an environmental conditioner, and the active ingredient of the environmental conditioner includes the above-mentioned Escherichia coli phage; preferably, the content of the phage in the environmental conditioner is at least 10^7 PFU / mL. The objects for improvement include, but are not limited to, bathrooms, kitchens, and other possibly contaminated places, utensils, and items, to effectively control the growth and activity of Escherichia coli and prevent the contamination of Escherichia coli in the environment.

[0019] In the fifth aspect, based on the same inventive concept, the present invention provides a phage lysate, and the active ingredient of the phage lysate includes the above-mentioned Escherichia coli phage. It can be used for the treatment of patients infected with drug-resistant Escherichia coli.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The present invention screened an Escherichia coli phage PD348 with a novel structure. This phage PD348 has an elongated polyhedral head that encapsulates nucleic acid. The major axis of the head is about 140 nm and the minor axis is about 56 nm. It has a relatively short tail about 20 nm long.

[0022] 2. The Escherichia coli phage PD348 screened in the present invention has a titer of 109 PFU / mL; the one-step growth curve shows that its latent period is about 10 min and the burst size is about 20 PFU / cell; it has good thermal stability and still maintains high activity after being treated at 60 °C for 40 min; the titer remains basically stable after 3 h within the range of pH 4 - 10; through the accelerated storage test, it is predicted that it has good storage stability under the conditions of 4 °C and 37 °C.

[0023] 3. The phage involved in the present invention is obtained from nature, has a wide source and is easy to isolate, providing a source for drugs for preventing and treating diseases caused by Escherichia coli infection with Escherichia coli phage PD348 as the active ingredient.

[0024] 4. The phage composition involved in the present invention has a wide application. Tests have proved that the phage provided by the present invention is safe, green and has no toxic side effects. Therefore, it can be used as an environmental modifier, a phage pharmaceutical preparation, etc. for preventing and treating diseases caused by Escherichia coli. Description of the Drawings

[0025] Figure 1 It is a plaque map of Escherichia coli phage PD348;

[0026] Figure 2 It is an electron microscope photograph of Escherichia coli phage PD348;

[0027] Figure 3 It is a graph of the optimal multiplicity of infection of Escherichia coli phage;

[0028] Figure 4 It is a one-step growth curve graph of Escherichia coli phage;

[0029] Figure 5 It is a graph of the temperature stability of Escherichia coli phage;

[0030] Figure 6 It is a graph of the pH stability of Escherichia coli phage;

[0031] Figure 7 It is a graph of the ultraviolet irradiation stability of Escherichia coli phage;

[0032] Figure 8 It is a heat map of the lysis spectrum of Escherichia coli phage PD348. Detailed Embodiments

[0033] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not impose any limitation on the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0034] Example 1: Isolation and identification of Escherichia coli:

[0035] 1. Wipe the surface of the laminar flow hood with alcohol cotton and disinfect it with ultraviolet light for half an hour before starting the experiment. Prepare sampling cotton swabs in advance, dip them in patient samples (sputum, urine, feces) collected from the hospital, smear the first area on MacConkey medium, use an inoculation loop to streak the second and third areas, and observe the colony morphology and color on the plate after aerobic incubation at 37°C for 16 - 18 h. Pick colonies with a red, smooth, round, and neat edge morphology. As Figure 1 shown.

[0036] 2. Streak and inoculate on MacConkey medium, subculture and purify 3 - 5 times until colonies with uniform morphology are obtained. Pick single colonies and streak and inoculate them in NB agar medium. Pick colonies and identify them by PCR with Escherichia coli - specific primers. The identification result is Escherichia coli. After the identification is completed, the proliferated bacterial solution is placed in 60% glycerol broth and stored at -80°C.

[0037] Example 2: Isolation and purification of Escherichia coli phage:

[0038] 1. Sample soaking: Add appropriate samples such as fecal fluid, sewage, bedding, etc. into the sample soaking bottle, add 100 μL of bacterial solution per strain, then add appropriate amounts of NB liquid medium and nutrients, and incubate with shaking at 37°C and 170 rpm for 16 - 18 h.

[0039] 2. Verification of phage spot sensitivity: Equal amounts of the bubble-like samples were dispensed into 10 mL centrifuge tubes, 8 - 9 mL in each tube. Centrifuge at 11000 rpm for 10 min, pour the supernatant into a sterilized 10 mL centrifuge tube, and centrifuge again at 11000 rpm for 5 min for standby. Take the above centrifuged liquid, dilute it to 0 and -3 gradients, draw grids on the plate and make marks (write the strain name, dilution gradient, and experiment time respectively). Take 100 μL of the selected strain and add it to the upper layer of NB medium (5 mL, agar concentration 0.7%), quickly shake the test tube to mix evenly, pour it into a petri dish containing the lower layer of NB medium (agar concentration 1.5%), and wait for it to solidify; Dilute the bubble-like filtrate to an appropriate gradient at a 10-fold ratio, pipette 1.5 - 2 μL of each sample dilution and drop it onto the corresponding grids on the surface of the medium according to the pre-marked positions; After the liquid on the spotted plate has dried, incubate it upright in an incubator at 37°C under specific conditions for an appropriate time to cultivate clear phage plaques, and observe and record the results.

[0040] 3. Leaching phages: Use sterilized forceps to push out a single phage plaque together with the attached upper layer of medium, put it into a 1.5 mL centrifuge tube containing 1 mL of specific medium, and use forceps to crush the attached medium. Place the centrifuge tube on a shaker at 37°C and 170 rpm for at least 30 min for standby.

[0041] 4. Phage purification: Use sterilized forceps to push out a single phage plaque together with the attached upper layer of medium, put it into a 1.5 mL centrifuge tube containing 1 mL of specific medium, and use forceps to crush the attached medium. Place the centrifuge tube on a shaker at 37°C and 170 rpm for at least 30 min for standby.

[0042] 5. Phage proliferation: Take 100 μL of the leached supernatant and put it into a 5 mL NB liquid medium containing 100 μL of host bacteria in advance (add the bacterial liquid first and then the phage leaching solution, 100 μL of phage leaching solution + 100 μL of bacterial liquid + 5 mL of specific medium), and at the same time set up a bacterial liquid control group (100 μL of bacterial liquid + 5 mL of specific medium). Incubate on a shaker at 37°C and 170 rmp for 2.5 - 4 h. Pay attention to controlling the time well. Observe once every 1.5 h first, and then observe once every 0.5 h until the proliferation liquid becomes clear - turbid - clear again, and the phage proliferation liquid is clearer than the control group. Observe and record the time when it becomes clear. Centrifuge the phage proliferation liquid at 11000 rpm for 10 min, take the supernatant, and filter it using a 0.22 μm sterile microporous filter membrane to obtain the final phage proliferation liquid and place it in a refrigerator at 4°C for standby.

[0043] 6. Phage preservation: The filtrate of Escherichia coli phage was mixed with glycerol at a concentration of 60% in a volume ratio of 1:1 and stored at -80°C.

[0044] Example 3 Electron Microscopic Observation of Phage:

[0045] Take 20 μL of the liquid containing crude phage particles and drop it on a copper grid. Let it precipitate naturally for 15 min, and then suck off the excess liquid from the side with filter paper. Add a drop of 2% (W / V) phosphotungstic acid (PTA) to the copper grid to stain the phage for 10 min, and then suck off the staining solution from the side with filter paper. After the sample is dried, observe the phage morphology with an electron microscope.

[0046] The morphology of the phage was observed by transmission electron microscopy as Figure 2 shown. The head of this phage has a polyhedral structure and a short tail. According to the morphological classification of phages, the phage morphology conforms to the characteristics of the family Podoviridae and belongs to the Podovirus. The major axis of the phage head is about 140 nm, the transverse diameter is about 56 nm, and the tail length is about 20 nm.

[0047] Example 4 Phage Genome Sequencing and Analysis:

[0048] Send 10 mL of the phage liquid to Beijing University of Chemical Technology for sequencing. Use the Megahit software for sequence splicing to obtain the complete phage genome sequence; use the CARD and VFDB databases to predict online whether the phage PD348 contains antibiotic resistance genes and virulence factors.

[0049] Example 4 Analysis of Phage Biological Characteristics:

[0050] 1. Determination of Phage Titer: Take 100 μL of the phage proliferation liquid or concentrated liquid (prepared in Example 2) and mix it evenly with 900 μL of NB liquid medium for 10-fold serial dilution. Here, the dilution factor is 10-1, and dilute it successively to an appropriate multiple. Take 100 μL of the phage dilution in a 1.5 mL centrifuge tube, then add 100 μL of the host bacteria proliferation liquid (the Escherichia coli liquid prepared in Example 2), mix well, incubate at 37 °C for 5 min, mix again, add 200 mL of the mixed liquid to the 50 °C liquid upper layer medium, mix well, pour it into the lower layer medium culture dish, shake the culture dish to mix well, and after solidification, place it at 37 °C for 16 - 18 h until the plaques are transparent. Take 2 parallels for each dilution, and calculate the phage titer.

[0051] Phage titer = average number of plaques × dilution factor × 10 (PFU / mL)

[0052] As detected by the double-layer plate method as Figure 1 shown, the titer of the Escherichia coli phage PD348 proliferated in liquid is 2.3×10 9 -7.85×10 9 PFU / mL.

[0053] 2. Determination of the Optimal Multiplicity of Infection of Phage

[0054] MOI refers to the ratio of the number of phages to the number of host bacteria at the initial infection. Adjust the concentration of the host bacteria culture in the exponential phase to 10 8 CFU / mL. Set MOIs to 0.0001, 0.001, 0.01, and 0.1 respectively. Add the phage liquid with the determined titer to the bacterial liquid according to the proportion. Each of the bacterial liquid and the phage liquid is 500 μL. Mix well and incubate at 37°C with shaking at 200 r / min for 5 h. Centrifuge the mixed culture at 10000 r / min for 10 min and measure the phage titer. The infection multiplicity with the highest titer is the optimal infection multiplicity.

[0055] The results are as Figure 3 shown: The optimal MOI of phage PD348 is 0.001 PFU / mL.

[0056] 3. Determination of the one-step growth curve of phages

[0057] Mix 1 mL of the phage proliferation liquid and 1 mL of the fresh proliferation liquid of the host bacteria thoroughly in the ratio required for the optimal infection multiplicity. Incubate at 37°C for 5 min, centrifuge at 12000 rpm for 30 s, aspirate the supernatant as much as possible with a micropipette, then wash once with 5 mL of NB broth (centrifuge at 12000 rpm for 30 s), and discard the supernatant. Suspend the precipitate with pre-warmed NB broth (total volume is 5 mL) and mix well. Quickly place it in a shaking incubator at 37°C with shaking at 170 rpm. Take out 150 μL at the 0 moment and every 10 min, centrifuge at 10000 rpm for 1 min. According to the phage titer determination, make 2 parallels and take the average value of the results. Use the infection time as the abscissa and the phage titer in the infection system as the ordinate to plot the one-step growth curve, obtain the latent period and the burst period of the phages, and calculate the burst size.

[0058] The results are as Figure 4 shown: The latent period of phage PD348 is 10 min, the burst period is from 10 to 80 min, and its burst size is 20.4.

[0059] 4. Determination of the temperature stability of phages

[0060] Take 200 μL of the phage proliferation liquid and aliquot it into sterile EP tubes. Incubate at 60°C and 70°C in a water bath for 20 min and 40 min respectively. Measure 2 parallels at each temperature. After the water bath treatment, take samples and immediately place them in an ice bath for cooling. Measure the phage titer after treatment at different temperatures according to the phage titer determination operation. Calculate the phage titers at different temperatures and different treatment times. Use the water bath time as the abscissa and the logarithm of the phage titer as the ordinate to plot the phage thermal stability curve.

[0061] The results are as Figure 5As shown, phage PD348 can maintain a high infectivity at 60°C. The phage titer gradually decreases as the temperature increases. After acting for 40 min at 60°C, its titer still remains above 10 9 PFU / mL.

[0062] 5. Determination of the acid-base stability of phages

[0063] Add 4.5 mL of NB broth with different pH values (4 and 10) to sterile test tubes, 6 tubes for each pH value. Then place the test tubes in a 37°C incubator. After the temperature stabilizes, add 500 μL of phage proliferation solution to each tube, mix well, and incubate in the 37°C incubator for 1 h, 2 h, and 3 h. After the reaction, add an appropriate amount of 1 mol / L HCl or NaOH to the mixture to adjust the pH value of the mixture to about 7, then perform 10-fold serial dilutions, and determine the phage titer under different pH values according to the phage titer determination method. Set 2 replicates for each pH value. Calculate the phage titer at each time period, and draw a phage pH stability curve with the reaction time as the abscissa and the logarithm of the phage titer as the ordinate.

[0064] The results are as Figure 6 shown. The pH tolerance range of phage PD348 is 4 - 10, and it maintains a high titer within this range. At pH 4 and pH 10, after acting for 1 - 3 h, the phage titer remains above 10 9 PFU / mL.

[0065] 6. Influence of ultraviolet light on phages:

[0066] Take 4 mL of phage concentrate (prepared in Example 2) in a 90 mm sterile petri dish, place the petri dish 40 cm away from the ultraviolet lamp in the laminar flow hood, and turn on the ultraviolet lamp for continuous irradiation. Take 100 μL of the irradiated phage concentrate every 30 min, perform 10-fold serial dilutions, and then determine the phage titer. Continuously measure for 120 min. Draw a curve of the change in phage titer with ultraviolet irradiation time with time as the abscissa and the logarithm of the phage titer as the ordinate, and analyze the influence of ultraviolet light on phage activity.

[0067] The results are as Figure 7 shown. Phage PD348 has a weak tolerance to ultraviolet light and loses its activity after 80 min of ultraviolet irradiation.

[0068] 7. Accelerated storage test of phages

[0069] According to the stability of phages to temperature, group the detection temperatures and reaction times; select at least 3 effective temperatures, and detect the phage titer at 4 time points for each temperature;

[0070] 40℃ <![CDATA[2h1.08×10 8 > <![CDATA[4h9.5×10 8 > <![CDATA[6h9.0×10 8 > <![CDATA[8h7.55×10 8 > 50℃ <![CDATA[1h6.0×10 8 > <![CDATA[2h3.85×10 8 > <![CDATA[3h1.70×10 8 > <![CDATA[4h8.2×10 7 > 60℃ <![CDATA[0.5h1.03×10 7 > <![CDATA[1h7.35×10 6 > <![CDATA[1.5h7.35×10 5 > <![CDATA[2h4.7×10 5 >

[0071] Plot the number of surviving phages N (logarithmic value) against time, and obtain the slope of the linear equation from the trend line of the data points in the figure, LogN = Kt + b;

[0072] 40℃ y = -0.0245x + 9.0833 50℃ y = -0.2948x + 9.114 60℃ y = -1.0044x + 7.6099

[0073] Plot the slope at the above temperature with log(k) against the reciprocal of the temperature (absolute temperature). According to the linear equation of the trend line of the data points in the figure, deduce the degradation rate constants at other temperatures, and obtain a quadratic regression:

[0074] Under the same storage temperature condition, the number of survivors in the sample conforms to the first-order reaction kinetic equation: LnN 0 -LnN = Kt, where N0 is the initial number of live phages in the test (mL -1 ), N is the number of live phages at time t (mL -1 ), k is the rate constant (1 / h), and t is the sampling time (h).

[0075] Calculate through the above equation that the effective times under any conditions of 37°C and 4°C are 2 months and 28 years respectively.

[0076] 8. Determination of phage lysis rate

[0077] The spot assay method was used to determine the lysis rate of phages: Take 1 mL of fresh phage proliferation solution and centrifuge at 10,000 rpm for 10 min to sediment bacterial debris. Separate single colonies of 195 Escherichia coli strains clinically isolated in the laboratory were inoculated into 5 mL of NB liquid medium respectively and cultured at 37°C for 16 - 18 h to obtain the bacterial suspensions of each strain. Take 100 μL of the bacterial suspension and add it to the upper layer of NB medium. After thorough mixing, spread it evenly on the lower layer of agar plates. After solidification, take 1.5 μL of phage proliferation solution and drop it on the plate respectively. When adding the samples, there should be no contact between various phage proliferation solutions to avoid affecting the test results. After natural drying, culture at 37°C for 16 - 18 h and observe the results.

[0078] Through the lysis spectrum determination experiment, it was found that the phages in the present invention had a lysis rate of 36% against 195 Escherichia coli strains clinically isolated with multi-drug resistance (carbapenems, cephalosporins, fluoroquinolones, penicillins, aminoglycosides).

[0079] As described above, it is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. An Escherichia coli phage, characterized in that: The Escherichia coli phage is named PD348, classified as Escherichia coli phage, and its preservation number is CGMCC NO.46171. It was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 16, 2024, and the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

2. Use of the Escherichia coli phage according to claim 1 in lysing Escherichia coli or preparing a composition for lysing Escherichia coli.

3. Use of the Escherichia coli phage according to claim 1 in the preparation of medicines for preventing and treating diseases caused by Escherichia coli infection.

4. The use according to claims 1-3, characterized in that: The pH value should not exceed 10 during application.

5. The use according to claims 1-3, characterized in that: The temperature during application should not exceed 60℃.

6. A bacteriophage pharmaceutical preparation, characterized in that: The environmental improver comprises the Escherichia coli phage as claimed in claim 1; the content of the phage in the environmental improver is at least 107 PFU / mL.

7. An environmental improver, comprising the Escherichia coli bacteriophage according to claim 1; the content of bacteriophage in the environmental improver is at least 107 PFU / mL.

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