WO2022048061A1 - Phage de salmonella présentant une tolérance élevée à la température et un large spectre de lyse et composition associée - Google Patents

Phage de salmonella présentant une tolérance élevée à la température et un large spectre de lyse et composition associée Download PDF

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WO2022048061A1
WO2022048061A1 PCT/CN2020/134390 CN2020134390W WO2022048061A1 WO 2022048061 A1 WO2022048061 A1 WO 2022048061A1 CN 2020134390 W CN2020134390 W CN 2020134390W WO 2022048061 A1 WO2022048061 A1 WO 2022048061A1
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phage
salmonella
sg8p3
composition
pullorum
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PCT/CN2020/134390
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Chinese (zh)
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黄杰
费文斌
胡怿林
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菲吉乐科(南京)生物科技有限公司
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Priority to KR1020217033423A priority Critical patent/KR20210142679A/ko
Publication of WO2022048061A1 publication Critical patent/WO2022048061A1/fr

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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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • 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
    • 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
    • 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
    • C12N2795/10131—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
    • 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
    • C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to the technical field of bacteriophage, in particular to a high-temperature-resistant and wide-splitting-spectrum Salmonella bacteriophage and its composition, kit and application.
  • Salmonella is generally colonized in the intestinal tract of animals. If it is not handled properly in food processing, it can easily contaminate food, cause food safety hazards and endanger human health. In recent years, the abuse of antibiotics by farmers and feed manufacturers has led to the emergence of more and more drug-resistant strains of the bacteria, rendering many antibiotics used to treat Salmonella infections ineffective. According to reports, the resistance of Salmonella in China to sulfonamide antibiotics is close to 100%, and people have to seek other effective prevention and control methods against the infection of the bacteria
  • Bacteriophages are a class of viruses that obligately parasitize or infect bacteria and are widely found in nature. The virulent phage can proliferate in the sensitive host bacteria, and finally lyse the host bacteria to release progeny phages, thereby achieving the effect of destroying the host bacteria. In production, virulent phages are widely used to kill various pathogenic bacteria, especially the use of them to treat infections caused by pan-drug-resistant "super bacteria", and have achieved good results.
  • the existing domestic invention patent with the publication number of CN104830806B discloses a Salmonella phage and its bacteriostatic application. Its preservation number is: CCTCC NO: M2014145, and the preservation date is April 24, 2014.
  • the bacteriophage has a strong lytic effect on Salmonella.
  • the phage of this strain belongs to the family Musculophage according to the morphological analysis of electron microscope, and is named as Salmonella phage STP4-a; it can survive under the conditions of 40 ⁇ 60°C and pH 4 ⁇ 12; -20 The activity is stable after being stored at °C for 1 year; the protective agent for phage storage is a culture solution containing 20% glycerol.
  • Using the phage of the invention to specifically lyse Salmonella can kill drug-resistant Salmonella.
  • the domestic invention patent with the announcement number of CN106497888B discloses Salmonella phage, phage antibacterial composition and its application.
  • the bacteriophages provided by the invention are Salmonella enteritidis bacteriophage LPSE1 and Salmonella typhimurium bacteriophage LPST10. Whether it is bacteriophage LPSE1 or LPST10, both can have a good inhibitory effect on Salmonella, and the application examples in food systems also confirm that when they are used for inhibiting It has a good effect when used as a bacterial agent.
  • the above-mentioned prior art solutions have the following defects: the lysis range of the above-mentioned Salmonella phage and the tolerance to high temperature are both limited, and there is an urgent need to provide a high temperature-resistant Salmonella phage with a wide lysis spectrum.
  • one of the objects of the present invention is to provide a Salmonella bacteriophage with high temperature resistance and wide lysis spectrum.
  • a high-temperature-resistant and wide-splitting-spectrum Salmonella phage the Salmonella phage is Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), and the deposit number is CCTCC NO:M 2020205 .
  • a novel Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a wide lysis spectrum is provided, and the phage has good tolerance to high temperature and can efficiently kill Salmonella.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has an icosahedral head, a length of 80-100 nm, a transverse diameter of 50-70 nm, a contractile muscle sheath, and a tail. Length 90 ⁇ 110nm, diameter 15 ⁇ 25nm.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has good high temperature resistance characteristics at 75°C.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a broad-spectrum bactericidal ability against Salmonella.
  • the second object of the present invention is to provide a composition containing the above-mentioned high-temperature-resistant and wide-splitting-spectrum Salmonella phage. Phages can be used in combination or mixed with other substances to meet specific needs.
  • the second purpose of the present invention is achieved through the following technical solutions: a composition containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage, the composition containing the Salmonella pullorum phage (Salmonella pullorum phage SG8P3) and other Salmonella phages at least one of them.
  • the other Salmonella phage is Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and the deposit number is: CCTCC M2018765; Myotail phage BP-66 (Myoviridae sp.BP- 66), the deposit number is CCTCC NO: M 2015146; the muscle tail bacteriophage BP-63 (Myoviridae sp.BP-63) deposit number is CCTCC NO: M 2015145; or the long tail bacteriophage BP-12 (Chilikevirus sp.BP-12) The deposit number is one of CCTCC NO: M 2015141.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is used in combination with other phages to obtain a better killing effect on the target bacteria.
  • the proportional relationship between the Salmonella phage SG8P3 and other phages can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
  • the composition further includes one of bisdecyl dimethyl ammonium chloride with a concentration of 0.05% chemical bactericide or a final concentration of 0.01% of sanjierzil.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is mixed with other antibacterial agents to obtain broad-spectrum antibacterial and specific killing of Salmonella.
  • Antibacterial agents that can be used herein in conjunction with the phage in this protocol include, but are not limited to, antibiotics and chemical antibacterial agents.
  • the proportional relationship between Salmonella phage SG8P3 and other antibacterial agents can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
  • the third object of the present invention is to provide the application of a composition containing the above-mentioned high-temperature-resistant and wide-splitting-spectrum Salmonella phage.
  • the Salmonella pullorum phage (SG8P3) composition can be used as an active ingredient of a biological disinfectant or biological medicament to prevent but not limited to bacterial diseases caused by Salmonella.
  • the third object of the present invention is achieved through the following technical solutions: a composition containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage, the composition containing the Salmonella pullorum phage (Salmonella pullorum phage SG8P3) and other bacteriophages at least one of them.
  • the other bacteriophages are Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), the deposit number is CCTCC NO: M2018765; Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1), The deposit number is CCTCC NO: M 2020438; the Staphylococcus aureus phage J1P2, the deposit number is one of CCTCC NO: M2016285.
  • Salmonella pullorum phage SG4P1 Salmonella pullorum phage SG4P1
  • the deposit number is CCTCC NO: M2018765
  • Escherichia coli phage EC35P1 Esscherichia coli phage EC35P1
  • the deposit number is CCTCC NO: M 2020438
  • the Staphylococcus aureus phage J1P2 the deposit number is one of CCTCC NO: M201628
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used for the treatment and prevention of bacterial infections caused by Salmonella but not limited to Salmonella; Biopharmaceuticals for bacterial diseases caused by Salmonella.
  • the proportional relationship between the Salmonella phage SG8P3 and other phages can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
  • the fourth object of the present invention is to provide a reagent or a kit containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage or the composition of Salmonella phage.
  • the fourth above-mentioned purpose of the present invention is achieved through the following technical solutions: a reagent or kit containing the composition of Salmonella bacteriophage with high temperature resistance and wide cleavage spectrum, in the reagent or the kit, the Salmonella bacteriophage SG8P3 ( A composition of Salmonella pullorum phage SG8P3) or Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3).
  • the fifth object of the present invention is to provide an application of the above-mentioned Salmonella phage and its composition.
  • the fifth object of the present invention is achieved through the following technical solutions: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used as biological fungicides.
  • it can be used as a daily fungicide, can specifically kill Salmonella in the environment, and is not limited to Salmonella, and improve the distribution of microorganisms in the environment; it can also be used in livestock and poultry product breeding, transportation and preservation It can also be mixed with other fungicides and sprayed in food production workshops to prevent and control Salmonella and other bacterial contamination in the process of livestock and poultry breeding, transportation and storage. contamination by other bacteria.
  • the sixth object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used to provide potential therapeutic drugs for bacterial infections caused by Salmonella but not limited to Salmonella or health products.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used for the treatment and prevention of bacterial infection caused by Salmonella, but not limited to Salmonella.
  • the seventh object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
  • the seventh purpose of the present invention is achieved through the following technical solutions: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used for the treatment and prevention of bacterial infections caused by Salmonella, but not limited to Salmonella. instrument.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be approved and registered as medical devices for the treatment and prevention of bacterial infections caused by Salmonella, but not limited to Salmonella.
  • the eighth object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition are used as feed additives.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used to add to feed, and can specifically and continuously prevent and control the survival and reproduction of Salmonella in feed, and not limited to Salmonella, prevent and control feed storage and Contamination caused by, but not limited to, Salmonella in animal breeding.
  • the present invention includes at least one of the following beneficial technical effects:
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has extremely strong acid resistance, can specifically partially or completely inactivate Salmonella, and provides phage strain source for industrialized production of phage bactericide;
  • Salmonella pullorum phage SG8P3 of the present invention (Salmonella pullorum phage SG8P3) is a potent phage isolated from nature, and has been proved safe and without any side effects through toxicological experiments; the test phage does not contain virulence genes or bad genes; Any genetic modification of the test phage;
  • the Salmonella phage SG8P3 of the present invention has good high temperature resistance, and still has a survival rate of 80% in a water bath at 65°C for 2 hours, and has a titer of 10 6 PFU/mL in a water bath at 75°C for 2 hours;
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wider host range when used alone; when used in combination with other phages, its composition can crack more Salmonella, with a cracking rate of more than 98%, and has stronger cracking sex;
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and non-host pathogenic bacteria can not identify any one of the 28 non-host pathogenic bacteria for test, and the specificity is good;
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has a sterilization rate of 10 4 to 10 6 PFU/mL of phage in a Salmonella culture medium with a concentration of 10 2 to 10 3 cfu/mL.
  • Salmonella phage SG8P3 can be used as an active ingredient in various products for environmental disinfection , for example, including but not limited to disinfection and decontamination of water distribution systems, medical facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with aqueous carriers, which can effectively control target bacteria. growth and activity.
  • the liquid soaking and spraying forms include but are not limited to detergents, disinfectants, detergents, etc.; the aqueous carriers include but are not limited to phosphate buffer, LB medium, chlorine free water, etc.;
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can effectively kill the Salmonella on the chicken surface and prevent and control the contamination of the chicken during the preservation process of Salmonella; the composition of Salmonella phage SG8P3 can effectively kill the Salmonella on the chicken surface, the large intestine Bacillus, Staphylococcus aureus, and prevent the contamination of Salmonella, Escherichia coli, and Staphylococcus aureus in meat such as chicken during storage. It can be used as an active ingredient in various products for food defense.
  • the present invention includes, but is not limited to, the prevention of food spoilage caused by the infection of Salmonella, Escherichia coli, and Staphylococcus aureus in the form of liquid immersion, spraying, combined use with synthetic components, etc., and is especially suitable for cooked food or unsuitable sterilization.
  • liquid soaking, spraying forms include but are not limited to food sterilizers, food disinfectants, food preservatives, etc.;
  • synthetic components in the present invention include but are not limited to benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, calcium acid, etc.;
  • Salmonella pullorumphage SG8P3 (Salmonella pullorumphage SG8P3) of the present invention can be used to prepare compositions, reagents, or test kits; it can be applied to the rapid detection of Salmonella, including but not limited to testing in target samples in the form of test paper, test kit, etc.
  • the detection of Salmonella, or the screening of target pathogenic bacteria in clinical samples, can effectively ensure the sensitivity of the detection;
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can be used alone or in combination, as a biological bactericide, feed additive, or because of Salmonella, Escherichia coli, staphylococcus aureus for the treatment of bacterial infections, health care products and medical devices; Salmonella phage SG8P3 and its compositions can be used to treat or prevent infectious diseases caused by Salmonella, Escherichia coli, and Staphylococcus aureus; Livestock (pig, cattle, sheep, etc.), poultry (chicken, duck, geese, etc.), and various beasts, fish, rodents, etc.;
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can be applied to industrial production, can be specifically amplified by host bacteria, can be highly purified by applying standard virus purification methods, and has excellent promotion prospects.
  • Those skilled in the art can prepare the Salmonella phage SG8P3 of the present invention or its composition according to the description of the present invention and common knowledge in the art to prepare various products for medical treatment, detection, disinfection and food protection, etc. for industrial application.
  • the product form may include, but is not limited to, application to the body surface, mouth, rectum, interior of the pleura, etc.
  • the carrier-carrying form includes but Not limited to oral aqueous carriers, oral anhydrous carriers, cream preparations, etc.
  • concentrated injection forms include but are not limited to vaccine injection, pleural injection, meridian injection, etc.
  • medicament soaking forms include but are not limited to aerosols, rinses, etc.;
  • Salmonella phage SG8P3 of the present invention (Salmonella pullorum phage SG8P3) and compositions thereof can be prepared by those skilled in the art according to the records of the application and common sense in the art, and can be applied to prevent and treat diseases caused by Salmonella and are not limited to the diseases caused by Salmonella. Biopharmaceuticals.
  • Figure 1 is a photograph of the plaque morphology of Salmonella phage SG8P3.
  • Figure 2 is a schematic diagram of the morphological structure of Salmonella phage SG8P3 under a transmission electron microscope.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), the deposit number is CCTCC NO: M2020205, the deposit unit is the China Center for Type Culture Collection, and the deposit time is June 12, 2020.
  • Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1)
  • the preservation number is CCTCCNO: M2018765
  • the preservation unit is the China Center for Type Culture Collection
  • the preservation time is November 09, 2018.
  • Myoviridae BP-66 (Myoviridae sp.BP-66), the deposit number is CCTCC NO: M 2015146, the deposit unit is the China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei province, and the deposit date is March 23, 2015.
  • Myoviridae BP-63 (Myoviridae sp.BP-63), the deposit number is CCTCC NO: M 2015145, the deposit unit is China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei province, and the deposit date is 2015 March 23, 2008.
  • the long-tailed bacteriophage BP-12 (Chilikevirus sp.BP-12) has the deposit number CCTCC NO:M 2015141, the deposit unit is the China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei province, and the deposit date is 2015 March 23, 2008.
  • Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) has the deposit number CCTCC NO:M 2020438, the deposit unit is China Type Culture Collection Center, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei province, and the deposit time is August 20, 2020 day.
  • Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) has the deposit number CCTCC NO: M2016285, the deposit unit is China Type Culture Collection Center, the address is Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei province, and the deposit date is May 2016 26th.
  • LB liquid medium The formula of LB liquid medium is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, distilled water 1000 mL, pH 7.0.
  • the formula of LB solid medium is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar 15 g, distilled water 1000 mL, pH 7.0.
  • the semi-solid agar medium formula is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar 7 g, distilled water 1000 mL, pH 7.0.
  • SM buffer solution sodium chloride 5.8g, magnesium sulfate 2g, 1mol/L Tris-HCl 50mL, gelatin 0.25g, distilled water 1000mL.
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Myotail phage BP-63 (Myoviridae sp.BP-63), Myoviridae phage BP-66 (Myoviridae sp.BP-636 ), long-tailed bacteriophage BP-12 (Chilikevirus sp.BP-12), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) compositions 1 to 9 are all in accordance with Prepared by the method described in Example 6.
  • the samples in the present invention are collected from farm sewage and nearby farmland soils in Shandong, Henan and Jiangsu.
  • the collected samples were centrifuged at 5000 r/min for 10 min and passed through a 0.22 ⁇ m filter.
  • the above filtrate was mixed with 2 times LB liquid medium at a ratio of 1:1, inoculated with 100 ⁇ L of the target Salmonella strain, and enriched overnight.
  • the plaque morphology of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is shown in Figure 1; the morphology of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) under transmission electron microscope is shown in Figure 2, and the shape is an icosahedron head with a length of 80 ⁇ 100nm, transverse diameter 50-70nm, with contractile muscle sheath, tail length 90-110nm, diameter 15-25nm.
  • the head length of SG8P3 is 87 nm
  • the transverse diameter is 66 nm
  • the tail length is 95 nm
  • the diameter is 18 nm.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) was amplified by the host bacteria, after the medium was clarified, centrifuged at 8000g for 10min to remove impurities, and solid polyethylene glycol (PEG8000) was added to a final concentration of 10% (w/v) , stirred to dissolve, overnight at 4 °C, centrifuged at 8000g for 20 min at 4 °C, and the pellet was resuspended in SM buffer. The obtained phage suspension was stored at 4°C until use.
  • PEG8000 solid polyethylene glycol
  • Example 2 Determination of the optimal multiplicity of infection (MOI) of Salmonella by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3)
  • Phage counting method Dilute the obtained Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) sample by a 10-fold gradient, take 100 ⁇ L of the sample with a certain dilution ratio, and spread it according to the method in step (4) described in Example 1. Layer plates, take the appropriate proportion of plates to count the number of plaques, one plaque represents one phage monomer.
  • Table 1 The titers of Salmonella phage SG8P3 under different multiplicities of infection
  • Example 3 Detection test for deletion of virulence genes or undesirable genes of Salmonella phage SG8P3
  • the Salmonella phage SG8P3 of the present invention does not contain virulence genes or defective genes, wherein the absence of virulence genes or defective genes refers to the virulence genes or defective genes described in Table 2.
  • mice half male and half male, were randomly divided into two groups (phage group and control group) after three days of adaptive feeding, 10 mice in each group (5 males and 5 females), and the dose to the phage group was 10 10 pfu/kg
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3)
  • the control group was given the same amount of normal saline for 15 days, and the experimental mice were sacrificed by severed neck, and the internal organs were examined.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) had no effect on the daily behavior of mice. Anatomical examination of internal organs showed no abnormality.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has biological safety and can be applied to feed additives.
  • Salmonella bacteriophage SG8P3 can also be used as a health care product or a drug, and it has also been proved to be safe in this toxicological test.
  • Example 5 Thermostability test of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3)
  • Phage counting method the same as that described in Example 2.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) was more likely to survive at 55°C.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has good high temperature resistance.
  • the 65°C water bath for 2h still has 80% survival rate
  • the 75°C water bath for 2h still has 10 6 PFU/mL titer.
  • Example 6 Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) to Salmonella lysis range test and preparation and lysis range test of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) compositions
  • Phage lysis profiles were determined using the spot method. Select a large number of Salmonella strains, mix 5 mL of the LB semi-solid medium containing Salmonella, and pour it on a petri dish filled with LB solid medium, and use the Salmonella phage SG8P3 isolated in Example 1 after the semi-solid medium is solidified. (Salmonella pullorum phage SG8P3) for instillation.
  • Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) stock solution: inoculate the Salmonella pullorum phage SG4P1 host bacteria into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37 ° C to cultivate to an OD value of 0.2, and then add to the 1000 PFU/mL of Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) was added to it, and it was shaken and cultured at 150 rpm at 37° C. for 12 h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 ⁇ m filter to obtain the Salmonella pullorum phage SG4P1 stock solution.
  • Escherichia coli phage EC35P1 stock solution inoculate the Escherichia coli phage EC35P1 host bacteria into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37°C to culture to an OD value of 0.2, add 1000PFU/mL Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1), shake and culture at 150rpm at 37°C for 12h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 ⁇ m filter to obtain a stock solution of Escherichia coli phage EC35P1.
  • Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) stock solution: Inoculate the Staphylococcus aureus phage J1P2 host into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37 °C to cultivate to an OD value of 0.2 , 1000 PFU/mL of Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) was added to it, and it was shaken and cultured at 150 rpm at 37° C. for 12 h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 ⁇ m filter to obtain a stock solution of Staphylococcus aureus phage J1P2.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) with a titer of 1 ⁇ 10 8 PFU/mL, respectively, and mix the two strains of phages in the SM solution in equal volumes to prepare Salmonella A 1:1 composition of phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) (composition 1).
  • composition 2 The stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and myotail phage BP-63 (Myoviridae sp.
  • the 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and Myoviridae phage BP-63 (Myoviridae sp.BP-63). : 1 composition (composition 2).
  • the stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and muscle tail phage BP-66 (Myoviridae sp.
  • the 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and muscle tail phage BP-66 (Myoviridae sp.BP-66).
  • Composition of 1 Composition 3).
  • the stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and long-tailed phage BP-12 (Chilikevirus sp.
  • the 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and long-tailed phage BP-12 (Chilikevirus sp.BP-12).
  • the composition of 1 composition 4).
  • the titers were taken as 1 ⁇ 10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63) and muscle tail phage
  • BP-66 Myoviridae sp.BP-666
  • the 4 strains of phages were uniformly mixed in equal volumes to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and muscle tail phage BP A 1:1:1:1 composition of -63 (Myoviridae sp. BP-63) and Myoviridae phage BP-66 (Myoviridae sp
  • the titers were taken as 1 ⁇ 10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63) and long-tailed phage
  • BP-12 Chovirus sp.BP-12
  • the 4 strains of phages were uniformly mixed in equal volumes to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and muscle tail phage BP A 1:1:1:1 composition of -63 (Myoviridae sp. BP-63) and long-tailed bacteriophage BP-12 (Chilikevirus sp.
  • the titers of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Myoviridae BP-66 (Myoviridae sp.
  • the stock solution of phage BP-12 (Chilikevirus sp. BP-12), the 4 strains of phage were uniformly mixed in equal volumes to prepare Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-66 (Myoviridae sp. BP-66 ) and a 1:1:1:1 composition of long-tailed bacteriophage BP-12 (Chilikevirus sp. BP-12) (composition 7).
  • the titers were taken as 1 ⁇ 10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63), muscle tail phage
  • BP-66 Myoviridae sp.BP-66
  • long-tailed bacteriophage BP-12 Chilikevirus sp.BP-12
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3)
  • chicken Salmonella pullorum phage SG4P1 Salmonella pullorum phage SG4P1
  • muscle tail bacteriophage BP-63 Myoviridae sp.
  • composition 8 composition 8
  • Myoviridae sp. BP-66 Myoviridae sp. BP-66
  • long tail bacteriophage BP-12 Cholikevirus sp. BP-12 1:1:1:1:1 composition (composition 8).
  • the titers were taken as 1 ⁇ 10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 ( Staphylococcus aureus phage J1P2), 4 strains of phages were uniformly mixed in equal volume to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and a 1:1:1:1 composition
  • composition 11 Take Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1 ⁇ 10 8 PFU/mL and a final concentration of 0.01% sanjierzil respectively, and mix them in equal volumes to prepare a 1:1 composition (composition 11).
  • composition 12 Take Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1 ⁇ 10 8 PFU/mL, bisdecyl dimethyl ammonium chloride with a final concentration of 0.05%, and a final concentration of 0.01% Syringe, and uniformly mix the three in equal volumes. A 1:1:1 composition was made (Composition 12).
  • Single colonies of 160 Salmonella strains belonging to 34 serotypes were picked and inoculated into test tubes containing 3 mL of LB, respectively, and cultured at 160 rpm for 8 h to obtain bacterial liquid of each strain.
  • After natural air-drying incubate at 37°C for 6-8h, and observe the results. The test was repeated three times.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wide host range.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) can lyse 153 strains of Salmonella, with a lysis rate of 95.6%.
  • the composition culture solution of SG8P3 can generally identify at least 157 strains of Salmonella, and the lysis rate is at least 98%. It shows that the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wide host range and can identify Salmonella from different sources and different serotypes.
  • the composition of SG8P3 can make up for the limitation of the host spectrum in the single application of phage, and has great potential in phage therapy. great application potential.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition showed excellent bactericidal ability against a large number of Salmonella strains of different serotypes in different regions, which proved that it has a wide lysis spectrum and can be used as a substitute for breeding. choose.
  • Example 7 Lysis test of Salmonella phage SG8P3 against non-host pathogenic bacteria
  • Example 8 Bactericidal effect of Salmonella phage SG8P3 and its composition in liquid
  • BS Salmonella selective medium was used, counted by the method of dilution coating, cultured at 37°C for 24h, and the colonies with black metallic luster were positive colonies.
  • a control group and a blank group (CK) were set at the same time.
  • the control group was given Salmonella with a final concentration of 1 ⁇ 10 3 cfu/mL; the blank group was given the same amount of normal saline.
  • the residual amount of Salmonella was detected after 4 hours. See Table 6 for the results.
  • the test results in Table 6 show that when the concentration of Salmonella phage SG8P3 is 10 4 to 10 6 PFU/mL, the bactericidal effect of phage SG8P3 on Salmonella in liquid LB medium is the best, and the killing rate is above 99%. It indicated that Salmonella phage SG8P3 has the potential to be used as a biocide.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) composition 1 to composition 8 and composition 10 to composition 12, the preparation method thereof is shown in Example 6.
  • the test results in Table 7 show that when the Salmonella phage SG8P3 concentration is a high concentration of 1 ⁇ 10 6 PFU/mL, the composition with it not only has a good bactericidal effect, but also has no antagonistic effect on other composition components, and can be used in combination with chemical bactericidal substances.
  • the composition of Salmonella phage SG8P3 has the potential for application as a biocide.
  • Example 9 Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) for the prevention and control of Salmonella infection during chick breeding by Salmonella phage SG8P3 and its composition
  • the preparation methods of compositions 1 to 8 refer to Example 6.
  • a total of 550 experimental chicks were randomly divided into 11 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1 ⁇ 10 8 PFU/kg of test phage and 1 ⁇ 10 8 cfu/kg of Salmonella; the control group was given 1 ⁇ 10 8 cfu/kg of Salmonella; the blank group was given the same amount of normal saline for 15 days. The daily growth and survival rate of chicks were counted, and the results are shown in Table 8.
  • the results in Table 8 show that the chickens in the control group had a lot of diarrhea and white diarrhea, and all the chicks died on the 7th day; the survival rate of the blank group was 95%; the survival rate of the chickens in the single Salmonella phage SG8P3 group was 79%; Salmonella phage SG8P3 composition 8 The chick survival rate was 95%, and the chick survival rate of other compositions was also over 83%, indicating that Salmonella phage SG8P3 could be used synergistically with other components without affecting its performance.
  • This test also shows that Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) and its composition can be used as biological fungicides or feed additives, and can effectively prevent and control Salmonella infection during chicken breeding.
  • Example 10 Prevention and control of Salmonella contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
  • Phage counting method the same as that described in Example 2.
  • Salmonella enumeration method the same as the method described in Example 8.
  • composition 1 The preparation method of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) composition 1 to composition 8: the same as the method described in Example 6.
  • Table 9 show that a large amount of Salmonella grows on the chicken surface of the control group 8 hours after inoculation; while in each experimental group, due to the addition of the Salmonella phage SG8P3 and its composition, the Salmonella on the chicken surface is always controlled at a very low level. Salmonella bacteriophage SG8P3 and its composition can be used as biocides to effectively kill Salmonella on chicken surfaces and prevent Salmonella contamination during chicken storage.
  • Example 11 Prevention and treatment of Escherichia coli infection during chick breeding by Salmonella phage SG8P3 and its composition
  • a total of 350 experimental chicks were randomly divided into 7 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1 ⁇ 10 8 PFU/kg of test phage and 1 ⁇ 10 8 cfu/kg of Escherichia coli; the control group was given 1 ⁇ 10 8 cfu/kg of Escherichia coli; the blank group was given the same amount of normal saline for 15 days. . The daily growth and survival rate of chicks were counted, and the results are shown in Table 10.
  • the results in Table 10 show that all the chicks of the control group died on the 8th day; the survival rate of the blank group was 97%; the chicks of the single Staphylococcus aureus phage J1P2 group, the single Salmonella phage SG8P3 group and the composition 8 treatment group were respectively on the challenge day. All died on days 11 and 12; the survival rate of chicks in the single-coliphage EC35P1 group was 89%; and the survival rate of chicks in the composition 9 treatment group was 95%.
  • Salmonella phage SG8P3 can be used synergistically with other components without affecting its performance; meanwhile, Salmonella phage SG8P3 and its composition can be used as biological fungicides or feed additives to control bacterial diseases caused by Escherichia coli.
  • Example 12 Prevention and treatment of Staphylococcus aureus infection by Salmonella phage SG8P3 and its composition during chick rearing
  • a total of 350 experimental chicks were randomly divided into 7 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1 ⁇ 10 8 PFU/kg of test phage and 1 ⁇ 10 8 cfu/kg of Staphylococcus aureus; the control group was given 1 ⁇ 10 8 cfu/kg of Staphylococcus aureus; the blank group was given the same amount of normal saline, Continuous administration for 15d. The daily growth and survival rate of chicks were counted, and the results are shown in Table 11.
  • the results in Table 11 show that all the chicks of the control group died on the 9th day; the survival rate of the blank group was 97%; the chicks of the single Salmonella phage SG8P3 group, the single Escherichia coli phage EC35P1 group and the composition 8 treatment group were on the 10th day of challenge respectively. All died on the 12th day; the survival rate of chicks in the single Staphylococcus aureus phage J1P2 group was 86%; and the survival rate of chicks in the composition 9 treatment group was 94%.
  • Salmonella phage SG8P3 can be used synergistically with other components without affecting its performance; meanwhile, Salmonella phage SG8P3 and its composition can be used as biological fungicides or feed additives to control bacterial diseases caused by Staphylococcus aureus.
  • Example 13 Prevention and control of Escherichia coli contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
  • Phage counting method the same as that described in Example 2.
  • Counting method of Escherichia coli using eosin methylene blue selective medium, counting by the method of dilution coating, culturing at 37°C for 24h, the colonies with dark purple metallic luster on the plate are Escherichia coli colonies.
  • Table 12 show that a large amount of Escherichia coli grew on the chicken surface of the control group after 8h; the phage J1P2 group, the phage SG8P3 group and the composition 8 had weak inhibitory effects on Escherichia coli, and there were still 10 3 cfu/ mL of Escherichia coli; the phage EC35P1 group had better inhibitory effect on Escherichia coli; and the addition of composition 9 kept the Escherichia coli on the chicken surface at a very low level all the time. Salmonella bacteriophage SG8P3 and its composition can be used as biological fungicides to effectively kill Escherichia coli on the surface of chicken and prevent the contamination of Escherichia coli during the preservation of chicken.
  • Example 14 Prevention and control of Staphylococcus aureus contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
  • Phage counting method the same as that described in Example 2.
  • Staphylococcus aureus enumeration method use high-salt mannitol agar selective medium, apply dilution coating method to count, cultivate at 37°C for 24h, and yellow colonies on the plate are Staphylococcus aureus colonies.
  • Table 13 show that: after 8h, a large amount of Staphylococcus aureus grew on the chicken surface of the control group; the phage EC35P1 group, the phage SG8P3 group and the composition 8 had relatively weak inhibitory effects on Staphylococcus aureus, and 8 hours after inoculation, the chicken surface still remained 10 3 cfu/mL Staphylococcus aureus; Phage J1P2 group had better inhibitory effect on Staphylococcus aureus; and the addition of composition 9 kept Staphylococcus aureus on the chicken surface at a very low level. Salmonella bacteriophage SG8P3 and its composition can be used as biological fungicides to effectively kill Staphylococcus aureus on the chicken surface and prevent Staphylococcus aureus contamination during the preservation of chicken meat.
  • Example 15 Preparation and use of reagents or kits of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof
  • the reagent or kit contains 5-10 mL of a composition of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) or Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1 ⁇ 10 7 PFU/mL, 1 L LB semi-solid medium , 1L LB solid medium.
  • the method for using the reagent or kit is as follows: taking a liquid with a titer of 1 ⁇ 10 7 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) phage or a liquid of the composition of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), using a double layer
  • the plate drop method was used to determine the lysis profile of the test phage. Picking a single colony to be detected, inoculating it into a target liquid medium, and performing shaking culture at a target temperature in combination with the growth characteristics of the strain to be detected, to obtain a bacterial liquid of the strain to be detected.

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Abstract

L'invention concerne un phage de Salmonella ayant une tolérance élevée à la température et un large spectre de lyse, une composition de celui-ci et son utilisation. Le phage de Salmonella est un phage de Salmonella Pullorum SG8P3, et le numéro de dépôt est CCTCCNO : M2020205.
PCT/CN2020/134390 2020-09-05 2020-12-08 Phage de salmonella présentant une tolérance élevée à la température et un large spectre de lyse et composition associée WO2022048061A1 (fr)

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