CN111057681B - Bacteriophage and application thereof - Google Patents

Bacteriophage and application thereof Download PDF

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CN111057681B
CN111057681B CN201911213163.7A CN201911213163A CN111057681B CN 111057681 B CN111057681 B CN 111057681B CN 201911213163 A CN201911213163 A CN 201911213163A CN 111057681 B CN111057681 B CN 111057681B
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phage
k88ac
bacteriophage
etec
escherichia coli
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CN111057681A (en
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唐青海
阳坤
杨海
滕威
杨灿
王芳宇
易诚
何丽芳
刘会敬
唐姣玉
曹丽敏
唐斯萍
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Hengyang Normal University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A23B4/22—Microorganisms; Enzymes; Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A23B7/155—Microorganisms; Enzymes; Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The Phage Phage-HY19 is separated from the environment of a farm, has the characteristic of wide host range on main pathogen ETEC-K88ac causing animal diarrhea, is nontoxic, can be safely used for preventing and treating animal diarrhea, adopts Phage specificity to crack ETEC-K88ac, can avoid ETEC-K88ac evolution and drug-resistant strain generation caused by antibiotic use, and can also be used for sterilizing the breeding environment and keeping food fresh.

Description

Bacteriophage and application thereof
The technical field is as follows:
the invention relates to a bacteriophage and application thereof, and belongs to the technical field of biology.
Background art:
colibacillosis is an acute intestinal infectious disease caused by pathogenic escherichia coli, which is a common clinical pathogen causing animal diarrhea and seriously harms animal health. In recent years, antibiotics are used in clinical treatment of colibacillosis without caution and unreasonable, so that colibacillosis has drug resistance and is more and more serious in drug resistance, and the colibacillosis is more and more difficult to prevent and treat. With the emergence of various multi-drug-resistant strains and the occurrence of the defects of drug resistance transfer and the like, the search for an effective, green, safe and pollution-free antibacterial or bacteriostatic preparation to replace antibiotics to control pathogenic escherichia coli is imperative. Bacteriophage is a type of bacteria-dependent virus, and after a lytic bacteriophage infects a bacterium, the bacteriophage can be rapidly proliferated in a host bacterium and is lysed. The bacteriophage is very effective in dissolving specific host bacteria, has a mechanism for killing bacteria different from that of antibiotics, is not easy to generate drug resistance and residue, and is very suitable to become an ideal novel antibacterial agent. Thus, the use of bacteriophages for the treatment of infections is a promising new approach to anti-infective treatment.
The research of phage preparation is generally concerned in recent years, for example, the invention patent ZL200910029306.9 relates to a Shigella flexneri phage strain developed by farm of Jiangsu province, and the strain has strong cracking effect on Shigella flexneri; the invention patent ZL2009102632950 is also separated from agricultural hospital of Jiangsu province to obtain a bacteriophage EK99-C of enterotoxigenic escherichia coli, and is applied to treatment of diarrhea of young stock, bacterial infection and environmental purification and disinfection; the invention patent ZL201510226544.4 relates to a composition containing a bacteriophage EK88P-1, which is a bacteriophage having a wide antibacterial activity on Escherichia coli and is separated from Yintirong Biotech, korea, as an active ingredient, and a method for preventing and treating Escherichia coli infection by using the composition. However, the existing phage with lysis effect on pathogenic Escherichia coli and related phage preparation products are not abundant and are difficult to meet the market demand.
The invention content is as follows:
in view of the above problems, the present invention aims to provide a pathogenic escherichia coli bacteriophage, which can effectively lyse enterotoxigenic escherichia coli K88ac, and further process the enterotoxigenic escherichia coli K88ac into a bacteriophage preparation for preparing a medicament for preventing and treating diarrhea caused by enterotoxigenic escherichia coli, in particular, piglet diarrhea caused by enterotoxigenic escherichia coli ETEC-K88ac, and purify a breeding environment as a bactericide, or a fresh meat preservative for killing enterotoxigenic escherichia coli ETEC-K88ac.
In order to solve the technical problems and enrich the types of phage preparations, the invention adopts the following technical scheme.
One Phage, named Phage-HY19, classified and named: t4-like bacteriophage T-evens subgroup, wherein the bacteriophage is preserved in China general microbiological culture Collection center (address: no. 3 Xilu No.1 Hospital, beijing, chaoyang, north Chen) 24.04.2019, and the preservation number is CGMCC No.17584.
The phage has strong lytic effect on enterotoxigenic escherichia coli ETEC-K88ac.
The invention also claims the application of the bacteriophage in preparing a medicament for preventing and treating diarrhea caused by enterotoxigenic escherichia coli.
Preferably, the diarrhea is piglet diarrhea caused by enterotoxigenic escherichia coli ETEC-K88ac infection.
The invention also claims the application of the bacteriophage in purifying a culture environment.
Preferably, the application is spraying the culture solution or preparation containing the bacteriophage Phage-HY19 on the surface of the culture environment to be decontaminated.
The invention also requests to protect the application of the Phage Phage-HY19 in preparing food preservatives, and the Phage Phage-HY19 is used for killing enterotoxigenic escherichia coli on the surface of food.
Preferably, the food product is fresh meat or fruit, preferably fresh pork.
Preferably, the enterotoxigenic escherichia coli is enterotoxigenic escherichia coli ETEC-K88ac.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
the Phage Phage-HY19 with strong cracking effect on ETEC-K88ac is separated from a farm environment, has the characteristic of wide host range on main pathogen ETEC-K88ac causing animal diarrhea, is nontoxic, can be safely used for preventing and treating animal diarrhea, can avoid ETEC-K88ac evolution and drug-resistant strain generation caused by antibiotic use due to the adoption of Phage specific cracking ETEC-K88ac, and can be used for killing a breeding environment and keeping food fresh.
Drawings
FIG. 1: phage-HY19 plaque map, A, 12h after inoculation; b, 24h after inoculation; c, 48 hours after inoculation.
FIG. 2: graph of the determination of the optimal multiplicity of infection (MOI) of phage.
FIG. 3: phage-HY19 Phage temperature sensitivity determination results.
FIG. 4: phage-HY19 Phage in vitro bactericidal activity, wherein: group 1, E.coli-HuNHY19+ Phage-HY19; group 2, E.coli-HuNHY19+ PBS; group 3: CVCC1524+ Phage-HY19; group 4: CVCC1524+ PBS.
Detailed Description
Example 1: separation and purification of enterotoxigenic escherichia coli ETEC-K88ac phage
1. Separation of phage:
in a medium-sized farm in suburbs in Heyang city in 2016, a plurality of piglets continuously suffer severe diarrhea within 2 months, the disease condition cannot be controlled by antibiotic treatment, the mortality rate reaches 20 percent, a highly toxic enterotoxigenic escherichia coli E.coli-HuNHY19 is obtained by separation, and the highly toxic enterotoxigenic escherichia coli E.coli-HuNHY19 is preserved in the China general microbiological culture Collection center (address: no.1 Hospital No. 3, west Siro-Kyoho in the Inward region in Beijing city) 24 days in 2019, the preservation number is CGMCC No.17622, and the classification and the naming are as follows: escherichia coli (Escherichia coli). And E.coli-HuNHY19 is O8: k91 K88ac, indicating that the predominant pathogenic e.coli prevalent is enterotoxigenic e.coli K88ac.
To this end, a sewage sample was further collected at the discharge port of the sewage treatment system of the pig farm, filtered with gauze to remove large-particle impurities, centrifuged at 12000 Xg for 10 minutes, and the supernatant was filtered with a sterile filter having a pore size of 0.22 μm, and the filtrate was left at 4 ℃ for further use.
Inoculating a strain of enterotoxigenic escherichia coli E.coli-HuNHY19 (with the preservation number of CGMCC No. 17622) which is separated and identified into an LB culture medium, carrying out shake culture at 37 ℃ to logarithmic phase, inoculating the strain into a treated sewage sample according to the proportion of 1%, carrying out shake culture at 37 ℃ for 24 hours, amplifying specific phages in the sewage sample, then centrifuging the cultured sample at 12000 Xg at 4 ℃ for 10 minutes, and passing the supernatant through a sterile filter with the aperture of 0.22 mu m, wherein the obtained filtrate can be used for separating the specific phages.
And detecting whether the specific phage exists in the filtrate by adopting a spotting experiment and a double-layer plate experiment. And (3) a sample application method: and uniformly coating the freshly cultured escherichia coli on an LB (lysogeny broth) plate, naturally drying in the air, dripping 10 mu L of filtrate, standing until the filtrate is completely absorbed by a culture medium, inverting the plate at 37 ℃, standing overnight for culture, and observing whether transparent spots appear at the dripping filtrate on the next day. Double-layer plate method: heating a sterile LB semisolid culture medium to be completely melted, then preserving heat at 45 ℃, adding 100 muL of phage filtrate and 200 muL of freshly cultured Escherichia coli, fully and uniformly mixing, pouring the mixture into the upper layer of a solid LB flat plate, naturally solidifying, standing at 37 ℃ for overnight culture, and observing whether plaques appear the next day. If the results of both methods are positive, the presence of phage in the filtrate is confirmed.
Purification of bacteriophages
Purifying the Phage by using a double-layer plate method, picking a single clear plaque with large diameter on a double-layer plate by using a sterilized 10 mu L gun head, soaking the single clear plaque in a liquid LB culture medium, standing for 6h at 4 ℃, then continuously diluting the single plaque by using a sterile liquid LB culture medium by multiple times, repeating a double-layer plate experiment, repeating the continuous passage for more than 10 generations until the plaque with uniform shape and size is formed, and finally, mixing the Phage with SM Buffer (30%) uniformly and storing the mixture at-80 ℃ for preservation, namely Phage-HY19. Specific plaques are shown in FIG. 1.
Determination of phage optimal multiplicity of infection (MOI)
Culturing a host bacterium E.coli-HuNHY19 (with the preservation number of CGMCC No. 17622) to a logarithmic growth phase, and then culturing the host bacterium E.coli-HuNHY19 to a logarithmic growth phase according to the phage: host bacterium =10, 1, 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 Transferring into liquid LB culture medium, shaking culturing at 37 deg.C for 10 h, centrifuging at 4 deg.C for 15 min at 10,000 Xg, filtering the supernatant with 0.22 μm sterile filter, and determining titer by double-layer plate method to obtain the best MOI.
As shown in FIG. 2, the most preferred MOI was determined to be 10 for Phage-HY19 -6 When the phage gains the most effective proliferation and produces the maximum number of progeny phage, the titer can reach 5.5 × 10 9 pfu/mL。
Amplification, concentration and purification of phage particles
2mL of overnight-cultured host bacterium E.coli-HuNHY19 (preservation number CGMCC No. 17622) was added to 200mL of sterilized liquid LB medium, and the mixture was shake-cultured at 37 ℃ and 150rpm until the initial logarithmic growth stage. Inoculating Phage Phage-HY19 into bacterial liquid according to optimal MOI, adding CaCl 2 Culturing at 37 deg.C and 150rpm under shaking for 12-169h, 12000 Xg, and 4 deg.C to a final concentration of 1mM, and separating at 4 deg.CTaking the supernatant after 10min, and amplifying to obtain phage lysate.
And pouring the supernatant into a clean conical flask, adding DNaseI and RNaseA until the final concentration is 1 microgram/mL, incubating for 30 min at 37 ℃, adding NaCl with the final concentration of 1 mol/L to promote the separation of the phage particles from the bacterial fragments, and uniformly stirring and then carrying out ice bath for 1 hour. Centrifuging at 4 deg.C and 10000 rpm for 10min, removing cell debris, collecting supernatant, adding PEG8000 with plastid ratio of 10%, stirring gently or inverting to dissolve, avoiding vigorous stirring, and ice-cooling for 2 hr to make bacteriophage particles and PEG8000 fully combine to form precipitate. Centrifuging at 4 deg.C and 10,000rpm for 10min, discarding supernatant, recovering precipitated phage particles, which are attached to the inner wall of the centrifugal barrel, inverting the barrel for 5 min, draining off liquid, sucking off residual liquid with filter paper, and resuspending the precipitate with 2mL of SM Buffer. Adding equal volume of chloroform for extraction, recovering water phase containing phage, and repeatedly extracting
The concentrated phage particles were stored at 4 ℃ until free of impurities.
Observation with a transmission electron microscope
Centrifuging with CsCl density gradient, dropping concentrated phage particles on copper sheet, precipitating for 3min, sucking off excessive liquid with filter paper, dropping 2% phosphotungstic acid, staining for 10min, drying at room temperature, and observing with transmission electron microscope.
Phage-HY19 has a typical icosahedral head and a flexible tail observed by a transmission electron microscope, the head size is 98X 82nm, the tail size is about 103X 24nm, and the Phage-HY19 belongs to the Myoviridae in the order of tailed Phage and belongs to T4-like Phage according to Phage taxonomy. It was further typed to determine it as a T4-like phage T-evens subpopulation.
Phage preservation
The purified Phage Phage-HY19 is deposited in China general microbiological culture Collection center (address: no. 3 of Xilu 1 on North Chen of the Korean district, beijing) at 24.4.2019 with the preservation number of CGMCC No.17584.
Example 2:
1. temperature sensitivity of Phage Phage-HY19
The titer of 600 mu L is 10 8 pfu/mL phage solution is put into 5 sterile EP tubes, and respectively acted for 20min, 40min and 60 min under the conditions of 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃, each temperature is set to be 3 times, sampling is carried out after the action time is over, the samples are immediately put into a water bath for cooling, then the titer of the phage is measured, and the experiment is repeated for 3 times. The temperature sensitivity of Phage-HY19 is shown in figure 3, and the activity of the Phage Phage-HY19 is not obviously changed after the Phage Phage-HY19 is acted for 1 hour at 50-70 ℃; after 40min of action at 80 ℃, the phage is completely inactivated; after 20min at 90 ℃, the phage were completely inactivated.
Determination of host spectra of Phage Phage-HY19
2.1 Test materials:
coli-HuNHY19 (preservation number CGMCC No.17622, O8: K91, K88ac, self-separation identification), CVCC1524 (O8: K91, K88ac, obtained from China veterinary microorganism strain preservation management center CVCC), CVCC1510 (O9: K88ac, obtained from China veterinary microorganism strain preservation management center CVCC), CVCC200 (O149: K91, K88ac, obtained from China veterinary microorganism strain preservation management center CVCC), ATCC25922 (O1 serotype, obtained from ATCC), CVCC222 (O141: K99, obtained from China veterinary microorganism strain preservation management center CVCC), salmonella enteritidis (laboratory separation identification) and Shigella dysenteriae (laboratory separation identification).
The test method comprises the following steps:
the method is completed by adopting a double-layer flat plate method. The strains listed in the test material 2.1 were selected and the host range of the isolated phage was determined by double-plate experiments (as described above), which showed that the phage could lyse the strain if any plaques appeared. See table 1 below for specific results.
TABLE 1 determination of the Phage Phage-HY19 host Spectrum
Figure RE-DEST_PATH_IMAGE001
The above results show that: the obvious plaque formation is observed on the plates of Escherichia coli E.coli-HuNHY19, CVCC1524, CVCC1510 and CVCC200, and Salmonella enteritidis, shigella dysenteriae, CVCC222 and ATCC25922 grow well on the upper medium of the double-layer plate without the plaque formation, which indicates that the Phage Phage-HY19 has the lysis effect on ETEC-K88ac host bacteria and has no cross lysis effect.
Example 3: activity test of Phage Phage-HY19
3.1 Toxicity test
Collecting total 20 Kunming mice for 5-week-old female experiment, randomly dividing into two groups of 10 mice, wherein group 1 is experimental group, and oral concentration is 10 9 2mL of pfu/mL phage culture solution, group 2 as a control group, orally taking PBS with the same amount, continuously orally taking 14d, removing neck to kill mice, and observing the change conditions of viscera, digestive tract and mucous membrane. The result shows that the Phage Phage-HY19 has no influence on the daily behavior of the mice, and the anatomical examination is abnormal.
In vitro bactericidal activity
1.0mL (the bacterial content is 1 multiplied by 10) of each of the Escherichia coli E.coli-HuNHY19 (the preservation number is CGMCC No. 17622) and CVCC1524 (O8: K91, K88 ac) bacterial liquid which are cultured overnight 9 cfu/mL) and OD was added with LB 600nm The value was adjusted to 1.0 (the bacteria content was about 1X 10) 9 cfu/mL), phage-HY19 culture medium (Phage titer 1X 10) 8 pfu/mL), and finally adjusting the OD with LB 600nm About 0.5, and the corresponding strain was incubated at 37 ℃ while keeping the control group as it was without adding phage. OD detection at 0, 1, 2, 3, 4, 5h of culture 600nm A change in value. See figure 4 for specific results.
As is clear from the results shown in FIG. 4, OD of the bacterial suspension after inoculation with Phage Phage-HY19 600nm The value decreased from 1 hour later, and after 3 hours, the value was significantly decreased, while the OD of the bacterial solution to which Phage Phage-HY19 was not added 600nm Values show a continuously rising trend. Test results show that the Phage Phage-HY19 can effectively inhibit the growth of ETEC-K88ac bacteria.
3.3 In vivo Sterilization test
Collecting 20 Kunming mice (tested negative for toxigenic Escherichia coli in vivo before experiment) for 5-week female experiment, randomly dividing into two groups, respectively test group and control groupColi E.coli-HuNHY19 was inoculated into LB liquid medium and activated, and its concentration was adjusted to 10 8 cfu/ml; feeding Kunming mice for experiment at a dose of 1mL each, and taking Phage Phage-HY19 culture solution orally 1h after challenge (Phage titer is 1 × 10) 8 pfu/mL) 1mL; in contrast, after 1h of detoxification in the control group, 1mL of PBS was orally administered, at 12h, 24h and 36h, two mice were randomly selected from each group for cervical dislocation and death, 1g of each intestinal tract was taken, 2mL of PBS homogenate was added, centrifugation was performed at 6000rpm at 4 ℃ for 15 minutes, 0.1mL of supernatant was diluted and applied to LB plates at different dilutions to calculate the concentration of escherichia coli in the homogenate, and the specific results are as follows in table 2:
TABLE 2 in vivo phage Sterilization test results in mice
Figure RE-DEST_PATH_IMAGE002
Based on the results of Table 2 above, it can be seen that the concentration of Escherichia coli in intestinal homogenate was 9.2X 10 after the Phage Phage-HY19 culture solution was orally administered for 12 hours under the same challenge conditions 3 cfu/mL, while the concentration of the control group was 8.5X 10 5 cfu/mL, no toxigenic E.coli could be detected after 24 and 36h of oral administration, while E.coli concentration in the control group showed a sustained increase.
Environmental Disinfection test
The bacterial liquid (the bacterial content is 1 multiplied by 10) of Escherichia coli CVCC1524 (O8: K91, K88 ac) 8 cfu/mL) 50ml/m 2 Uniformly mixing, spraying onto the surface of a trough of a farm, and adding Phage Phage-HY19 culture solution (Phage titer is 1 × 10) 8 pfu/mL) at 50mL/m 2 The spraying amount of (2) is the amount of spraying and killing the surface of the trough, and after 2 hours, detecting the residue of escherichia coli.
The detection result shows that the concentration of the escherichia coli CVCC1524 on the surface of the silo after 2h is 3 multiplied by 10 3 CFU/ m 2 And after 4h, no escherichia coli CVCC1524 is detected on the surface of the trough, which shows that the bacteriophage Phage-HY19 culture solution can effectively kill escherichia coli ETEC-K88ac in the culture environment.
Food sterilization test
Cutting fresh pork into 5cm square (5 cm × 5cm × 5 cm) lean meat blocks, and mixing Escherichia coli CVCC1524 (O8: K91, K88 ac) bacterial solution according to 10% 8 The cfu/kg is uniformly sprayed on the surface of each fresh pig lean meat, and then the treated group is treated with Phage Phage-HY19 culture solution (Phage titer is 1 × 10) 8 pfu/mL) was sprayed on the surface of the pork with a spray amount of 5mL/kg, the control group was sprayed with PBS with the same spray amount, and the E.coli content was measured after 2 h.
The detection result shows that the content of Escherichia coli CVCC1524 on the surface of the lean meat of the post-treatment group of 2h is 65CFU/g, and the content of Escherichia coli CVCC1524 on the surface of the lean meat of the control group of pigs is 3 multiplied by 10 5 CFU/g; after 4h, the content of Escherichia coli CVCC1524 on the surface of the lean meat of the treated group is 0 CFU/g, and the content of Escherichia coli CVCC1524 on the surface of the lean meat of the control group is 8 multiplied by 10 5 CFU/g. The test surface shows that the Phage Phage-HY19 culture solution can effectively kill Escherichia coli ETEC-K88ac on the surface of food.

Claims (6)

1. The Phage is named as Phage-HY19, is preserved in China general microbiological culture Collection center at 24.04.2019 with the preservation number of CGMCC No.17584, and has a cracking effect on ETEC-K88ac host bacteria without cross-cracking effect.
2. Use of the bacteriophage of claim 1 for the preparation of a medicament for the prevention of diarrhea caused by enterotoxigenic e.coli, said diarrhea being piglet diarrhea caused by enterotoxigenic e.coli ETEC-K88ac infection.
3. Use of a bacteriophage according to claim 1 for decontaminating a culture environment by spraying a culture solution or formulation comprising the bacteriophage Phage-HY19 onto the surface of the culture environment to be decontaminated.
4. Use of the bacteriophage of claim 1 in the preparation of a food preservative for killing enterotoxigenic escherichia coli ETEC-K88ac on the surface of food.
5. Use according to claim 4, wherein the food product is fresh meat or fruit.
6. Use according to claim 5, wherein the fresh meat is fresh pork.
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