WO2013191363A1 - Nouveau bactériophage et son utilisation pour l'inhibition de la prolifération de bactéries pathogènes - Google Patents

Nouveau bactériophage et son utilisation pour l'inhibition de la prolifération de bactéries pathogènes Download PDF

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WO2013191363A1
WO2013191363A1 PCT/KR2013/003334 KR2013003334W WO2013191363A1 WO 2013191363 A1 WO2013191363 A1 WO 2013191363A1 KR 2013003334 W KR2013003334 W KR 2013003334W WO 2013191363 A1 WO2013191363 A1 WO 2013191363A1
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culture
kctc
caudoviral
phage
myoviride
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PCT/KR2013/003334
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English (en)
Korean (ko)
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성기홍
최대건
김재훈
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주식회사 씨티씨바이오
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Priority claimed from KR1020120067606A external-priority patent/KR101466620B1/ko
Priority claimed from KR1020120067811A external-priority patent/KR101381336B1/ko
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    • 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
    • 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
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • C12N2795/10031—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
    • C12N2795/10032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to novel phages newly isolated and identified, compositions for inhibiting or killing bacterial growth, compositions for preventing or treating bacterial infectious diseases, compositions for treating ballast water, antibiotics, disinfectants, and feed additives. will be.
  • Vibrio parahaemolyticus is a Gram-negative bacillus belonging to the family Vibrionaceae , and is mainly a fish and shellfish contaminated by the bacteria of a person with an underlying disease with reduced immune conditions such as liver disease, alcoholism and diabetes. It is known to cause infections when ingested after reproduction or incomplete cooking or when the wound is exposed to contaminated seawater. In many countries in Asia, including Korea and Japan, the eating habits of fish and shellfish do not cease every year due to their eating habits. Recently, as more and more fish and shellfish are reproduced in western countries, the occurrence of diseases caused by bacterial infections in the United States and Europe is spreading around the world. This infectious disease is known to be closely related to oysters, especially among seafood. As the bacteria grow actively when the water temperature rises, domestically prohibits the sale and distribution of oysters especially during the summer epidemic season (June-September). In particular, due to the recent increase in group food, the pandemic of the disease is predicted, and attention is required.
  • ah Monad motile aeromonas
  • Monastir Heathrow Pilar (Aeromonas hydrophila) is susceptible to a wide variety of fish, causing sepsis, bleeding, ulcers, etc., and this Collective mortality caused serious economic damage.
  • the degree of densities of breeding is severe, so infectivity is strong during the development of aeromonas hydrophila.
  • koi and goldfish in the fish farming market of Jincheon, Chungbuk are the most common ornamental fish that are seriously damaged by aeromonas hydrophila.
  • aeromonas hydrophila is an underwater flora, antibiotics are often treated for the purpose of controlling them. As a result, aeromonas hydrophila is resistant to antibiotics. In addition to threatening health, the damage caused by the emergence of resistant bacteria is increasing.
  • Bacteriophage is a bacterium that is specifically infected with bacteria and was discovered in 1915 by a British bacteriologist Twort in a study of the microscopic melting of Staphylococcus aureus colonies. Also, in 1917, French bacteriologist d'Herelle discovered that the filtrate of stool from foreign patients had a function of dissolving Shigella disentriae. In the sense, they named it bacteriophage. Phage treatment has been continuously studied only in Eastern Europe and the former Soviet Union since the development of antimicrobial agents. However, due to the recent misuse of antibiotics, the incidence of multidrug-resistant pathogenic bacteria and superbacteria has increased, and many problems of conventional antibiotics have emerged. As an alternative to antibacterial agents, it is in the spotlight. Moreover, bacteriophages take shorter development time and lower development costs than antibacterial agents, and are recognized for their superiority in terms of bacterial selectivity, persistence and side effects.
  • the problem to be solved by the present invention is to isolate a new bacteriophage having lytic activity on Vibrio parahemoliticus, Vibrio parahemoliticus growth inhibition or killing composition or Vibrio parahemoliticus growth comprising the same Methods for inhibiting or killing, compositions for treating ballast water or methods for treating vessel equilibrium, pharmaceutical compositions for preventing or treating Vibrio parahemolyticus infectious diseases or methods for preventing or treating Vibrio parahemolyticus infectious diseases, antibiotics, disinfectants, and It is to provide a feed additive for fish and shellfish.
  • the problem to be solved by the present invention is to isolate a new bacteriophage having lytic activity on aeromonas hydrophila, aeromonas hydrophila growth inhibition or killing composition or aeromonas hydrophila growth comprising the same A method of inhibiting or killing, providing a pharmaceutical composition for preventing or treating aeromonas hydrophilic infectious disease, or a method for preventing or treating aeromonas hydrophilic infectious disease, an antibiotic, a disinfectant, and a feed additive for fish.
  • the invention relates to phages having specific killing capacity for Vibrio parahaemolyticus .
  • the phage is characterized in that the phage of the accession number KCTC 12130BP.
  • the present inventors collected sediment samples collected from seawater of Namhae oyster farm in Korea, and isolated and identified phages having specific killing ability against Vibrio parahaemolyticus , which was identified as PVP-1. It was named and deposited on February 3, 2012 with accession number KCTC 12130BP to BRC.
  • the present inventors isolated Vibrio parahaemolyticus ATCC 33844 as a host to isolate the phage (hereinafter referred to as PVP-1), PVP-1 to Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) Plaques were formed and confirmed to have bacteriolytic activity.
  • PVP-1 is a vibrio. It was proved to be the first bacteriophage belonging to the saporitrophic family that has the ability to infect parahemolyticus.
  • Phage PVP-1 according to the present invention can be cultured in large quantities by conventional phage culture methods.
  • the culture medium may be a medium consisting of a carbon source, nitrogen source, vitamins and minerals.
  • NB nutrient broth
  • TLB tryptic soy broth
  • TSA tryptic soy agar
  • the culture can be carried out under conventional culture conditions. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the concentrated cells, and this step may be performed according to the needs of those skilled in the art.
  • the concentrated cells can be preserved so as not to lose their activity by freezing or freeze drying according to conventional methods.
  • PVP-1 is susceptible to Vibrio parahaemolyticus .
  • the Vibrio parahemoliticus Vibrio parahemoliticus ATCC27969, ATCC33844, ATCC17802, VP01, VP02, VP03, VP04, VP05, VP06, VP07, VP08, VP09, VP10, VP11, VP13, VP14, VP17 , VP18, VP20, VP21, VP22, VP23, VP24, VP25, VP26, or VP27.
  • the Vibrio parahemoliticus is a multidrug resistant strain.
  • the present invention is Vibrio parahaemolyticus growth inhibition or killing composition
  • a phage PVP-1 or a culture thereof as an active ingredient or by treating it Vibrio parahemority A method of inhibiting or killing the proliferation of couss.
  • the present invention relates to an antibiotic, a disinfectant, and a shellfish feed additive comprising phage PVP-1 or a culture thereof, or an active ingredient thereof.
  • the present invention relates to antibiotics, disinfectants, and shellfish feed additives for inhibiting or killing phage PVP-1 or its culture, or Vibrio parahaemolyticus comprising the same as an active ingredient.
  • Vibrio parahemoliticus growth inhibition or killing composition, antibiotics, disinfectants, and feed additives according to the present invention has a very high specificity for Vibrio parahemoliticus compared to the existing ones, so that the fungus does not kill vibrio parahemoli It can kill only ticus and has no side effects, and can kill antibiotic-resistant and multi-drug-resistant pathogens, and the effect is excellent, and it does not induce resistance or resistance of pathogens, and thus has a long product life.
  • antibiotic is meant to include preservatives, fungicides, and antibacterial agents.
  • the feed additive may be added to the shellfish feed.
  • PVP-1 can be directly prepared during the manufacture of the feed without being separately prepared as a feed additive and mixed in the feed. Alternatively, it may be prepared by adding a culture thereof.
  • the fish and shellfish may include, but are not limited to, oysters, shellfish, crabs, shrimp, or fish.
  • the present invention relates to a composition for treating ballast water (ballast water) comprising phage PVP-1 or a culture thereof, or an active ingredient thereof, and a biological treatment method of ballast water using the same.
  • ballast water comprising phage PVP-1 or a culture thereof, or an active ingredient thereof, and a biological treatment method of ballast water using the same.
  • phage PVP-1 or its culture, or Vibrio parahaemolyticus containing the same as an active ingredient composition for treating ballast water for inhibiting or killing proliferation and biological treatment of ballast water using the same It is about a method.
  • the composition for treating ballast water may kill Vibrio parahaemolyticus or may inhibit the growth thereof that may exist in the ballast water.
  • Ship ballasts are loaded or spilled in the tanks below the ship to balance the load from loading and unloading the ship, or to prevent the screw propeller from floating above sea level and making it impossible to navigate. I say sea water.
  • the 'ship ballast water' is drawn up through a pump before being put into a tank storing water and ballast water in a ballast water tank. Refers to both water.
  • the term 'ship' means all types of ships that are operated in a marine environment, and are submersible vessels, floating vessels, floating platform, floating storage facility, and supporting and storing and unloading products. Includes facilities for
  • the Vibrio parahemoliticus growth inhibiting or killing composition may be prepared in a liquid state, such as solutions, suspensions, emulsions, powder based on the dry phage It may also be prepared in solid form such as granules, tablets, or capsules.
  • the composition for inhibiting or killing Vibrio parahemolyticus growth, a composition for treating ballast water, an antibiotic, a disinfectant, and a fish feed additive may further include an acceptable carrier, and may be formulated with the carrier.
  • the acceptable carrier refers to a carrier that does not stimulate an organism and does not inhibit biological activity and properties. It may be prepared in the form of a hydrating agent, a granulating agent, a liquid hydrating agent, a liquid, a water soluble granule, or an encapsulant using a surfactant or extender for the purpose of stable formulation suitable for actual packaging.
  • the surfactant examples include alkyl (C 8 -C 12 ) arylsulfonate, dialkyl (C 3 -C 6 ) arylsulfonate, dialkyl (C 8 -C 12 ) sulfosuccinate, ligninsulfonate, naphthalene Sodium salts of sulfonate compounds such as sulfonate condensates, naphthalenesulfonate formalin condensates, alkyl (C 8 -C 12 ) naphthalenesulfonate formalin condensates, polyoxyethylene alkyl (C 8 -C 12 ) phenylsulfonates, or Calcium salts, alkyl (C 8 -C 12 ) sulfates, alkyl (C 8 -C 12 ) arylsulfates, polyoxyethylene alkyl (C 8 -C 12 ) sulfates, polyoxyethylene alkyl (C 8
  • Extenders include bentonite, talc, clay, kaolin, calcium carbonate, silica sand, pumice, diatomaceous earth, acidic clay, zeolite, pearlite, white carbon, ammonium sulfate, urea, glucose, dextrin, soy flour, rice, wheat It can be easily understood by those skilled in the art that ocher, glucose and starch, water, etc. may be used alone or in combination of two or more thereof, and other extenders may be used. will be.
  • Phage concentration from the composition for inhibiting or killing Vibrio parahemoliticus growth, composition for treating ballast water, antibiotics, disinfectants and fish feed additives may be arbitrarily adjusted to suit the purpose of use. It will be readily understood by those of ordinary skill in the art. For example, in the case of a composition for treating ballast water, the amount of ballast water, the number of Vibrio parahemoliticus populations per unit volume, and the content of PVP-1 in the composition (or antibiotics, disinfectants, or shellfish feed additives), Various adjustments can be made by such factors as the formulation method or administration method of the composition.
  • composition for inhibiting or killing Vibrio parahemoliticus growth may further include an effective bacteriophage in addition to PVP-1, and the effective bacteriophage is preferably Vibrio Phages having lytic activity against parahemolyticus, but not limited thereto, may be included.
  • composition for inhibiting or killing the Vibrio parahemoliticus growth when the composition for inhibiting or killing the Vibrio parahemoliticus growth, the composition for treating ballast water, antibiotics, disinfectants and fish feed additives, it was conventionally used to suppress or kill the growth of Vibrio parahemoliticus. It can be used in combination with a method.
  • the present invention is Vibrio parahaemolyticus comprising a phage or culture thereof as an active ingredient ( Vibrio parahaemolyticus ) infectious disease preventing or treating pharmaceutical composition using the Vibrio parahaemolyticus infectious disease It relates to a method of preventing or treating.
  • the phage according to the present invention has a specific killing ability against Vibrio parahemolyticus, and thus is effective in preventing or treating various diseases caused by Vibrio parahemolyticus infection.
  • the Vibrio parahemolyticus infectious disease is, but is not limited to, gastroenteritis, food poisoning, or wound infection.
  • the Vibrio parahemolyticus infectious disease also includes symptoms expressed from such a disease, for example, vomiting, abdominal pain, fever, chills, or watery diarrhea.
  • Such 'prophylaxis or treatment' includes the prevention, inhibition, and alleviation of Vibrio parahemolyticus infectious diseases.
  • composition of the present invention may be administered to an animal in the form of a pharmaceutical preparation, or may be administered by a method of feeding it by mixing with feed or drinking water.
  • the route of administration of the composition of the present invention may be administered via various routes orally or parenterally as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, It may be administered in a conventional manner via transdermal, nasal, inhalation, and the like.
  • the prophylactic or therapeutic method of the present invention includes administering a composition of the present invention in a pharmaceutically effective amount. It will be apparent to one of ordinary skill in the art that a suitable total daily usage may be determined by the physician within the scope of good medical judgment.
  • the specific therapeutically effective amount for a particular subject, together with the type and severity of the response to be achieved, the age, body weight, general health, sex and diet, time of administration, route of administration and rate of composition, duration of treatment, and the specific composition of the subject It is desirable to apply differently depending on various factors including drugs used or co-used and similar factors well known in the medical field.
  • the present invention relates to bacteriophages having specific killing ability against Aeromonas hydrophila.
  • the present inventors collected a sample from the Seoseong Seoam stream, and separated the phage (hereinafter referred to as PAH1-C) from which the Aeromonas hydrophila JUNAH was dissolved as a host and collected at the Nanji Water Reclamation Center.
  • PAH1-C phages lysing Aeromonas hydrophila JUNAH
  • PAH6-C phages lysing Aeromonas hydrophila JUNAH
  • the 'PAH1-C or PAH6-C' includes not only phage but also phage culture.
  • Phage PAH1-C or PAH6-C phage according to the present invention can be cultured in large quantities by conventional phage culture methods.
  • the culture medium may be a medium consisting of a carbon source, nitrogen source, vitamins and minerals.
  • NB nutrient broth
  • TLB tryptic soy broth
  • TSA tryptic soy agar
  • the culture can be carried out under conventional culture conditions. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the concentrated cells, and this step may be performed according to the needs of those skilled in the art.
  • the concentrated cells can be preserved so as not to lose their activity by freezing or freeze drying according to conventional methods.
  • PAH1-C and PAH6-C are susceptible to Aeromonas hydrophila.
  • the PAH1-C is susceptible to aeromonas hydrophila AH1, AH6, AH9, AH16, AK1 and AK9
  • the PAH6-C is aeromonas hydrophila AH7, AH16, AK1 and AK9 Show sensitivity
  • Aeromonas hydrophila is an antibiotic resistant strain or a multidrug resistant strain.
  • the present invention is a composition for inhibiting or killing Aeromonas hydrophila proliferation comprising PAH1-C or PAH6-C as an active ingredient, or by treating the growth of Aeromonas hydrophila It relates to a method for suppressing or killing the bacteria.
  • the present invention relates to an antibiotic, a disinfectant and a feed additive for fish comprising PAH1-C or PAH6-C, or an active ingredient thereof.
  • the present invention relates to antibiotics, disinfectants and fish feed additives for phage PAH1-C or PAH6-C, or aeromonas hydrophila growth inhibition or killing comprising the same as an active ingredient.
  • antibiotic is meant to include preservatives, fungicides, and antibacterial agents.
  • the feed additive may be added to the feed for fish.
  • the feed additives may be prepared by adding PAH1-C or PAH6-C directly in preparation of the feed without mixing them into the feed.
  • the fish includes carp, cichlids, sea bass, cod, crucian carp, salmon, rockfish, loach. Catfish or nettlefish may be included, but are not limited thereto.
  • compositions for inhibiting or killing Aeromonas hydrophila growth, antibiotics, disinfectants, and feed additives according to the present invention have a very high specificity for Aeromonas hydrophila compared to the conventional ones.
  • the fungi can kill only certain pathogens without killing them, and there are no side effects, and they can kill multidrug-resistant pathogens, and the effect is not only excellent, but also does not induce resistance or resistance of pathogens and thus has a long product life.
  • the aeromonas hydrophila growth inhibiting or killing composition, antibiotics, disinfectants and feed additives may be prepared in the form of a liquid, such as solutions, suspensions, emulsions, powders, granules, tablets, or capsules based on the dry phage It can also be produced in the same solid phase.
  • the composition for inhibiting or killing aeromonas hydrophila growth, an antibiotic, a disinfectant, and a feed additive may further include an acceptable carrier, and may be formulated together with the carrier.
  • the acceptable carrier refers to a carrier that does not stimulate an organism and does not inhibit biological activity and properties. It may be prepared in the form of a hydrating agent, a granulating agent, a liquid hydrating agent, a liquid, a water soluble granule, or an encapsulant using a surfactant or extender for the purpose of stable formulation suitable for actual packaging.
  • the surfactant examples include alkyl (C8 ⁇ C12) arylsulfonate, dialkyl (C3 ⁇ C6) arylsulfonate, dialkyl (C8 ⁇ C12) sulfosuccinate, ligninsulfonate, naphthalenesulfonate condensate, naphthalene Sodium or calcium salts of alkyl sulfonate compounds such as sulfonate formalin condensates, alkyl (C8 to C12) naphthalene sulfonate formalin condensates, polyoxyethylene alkyl (C8 to C12) phenyl sulfonates, and alkyl (C8 to C12) sulfates Sodium salts or calcium salts, polyoxyalkylene succinates of sulfate compounds such as alkyl (C8 to C12) aryl sulfates, polyoxyethylene alkyl (C8 to C12)
  • Extenders include bentonite, talc, clay, kaolin, calcium carbonate, silica sand, pumice, diatomaceous earth, acidic clay, zeolite, pearlite, white carbon, ammonium sulfate, urea, glucose, dextrin, soy flour, rice, wheat It can be easily understood by those skilled in the art that ocher, glucose and starch, water, etc. may be used alone or in combination of two or more thereof, and other extenders may be used. will be.
  • Phage concentration to dry matter content in the composition for inhibiting or killing aeromonas hydrophila growth, antibiotics, disinfectants and feed additives can be arbitrarily adjusted according to the purpose of use to those skilled in the art. It will be easily understood.
  • composition for inhibiting or killing aeromonas hydrophila growth, antibiotics, disinfectants, and feed additives may include PAH1-C or PAH6-C, that is, only one phage, or both phages.
  • an effective bacteriophage may be additionally included in addition to the phage, and the effective bacteriophage preferably includes phage having lytic activity against aeromonas hydrophila, but not limited thereto, and phage having lytic activity against other bacteria. Can be.
  • composition for inhibiting or killing aeromonas hydrophila growth when treating the composition for inhibiting or killing aeromonas hydrophila growth, antibiotics, disinfectants and feed additives, it can be used in combination with conventional methods for inhibiting or killing bacteria growth including aeromonas hydrophila. Preferably, it can be used together with the method conventionally used for suppressing or killing aeromonas hydrophila proliferation.
  • the present invention is a pharmaceutical composition for preventing or treating aeromonas hydrophila infectious disease comprising a phage or a culture thereof as an active ingredient or by using the aeromonas hydrophila (Aeromonas hydrophila) ) A method for preventing or treating an infectious disease.
  • the phage according to the present invention has a specific killing ability against Aeromonas hydrophila, and therefore, various phages caused by Aeromonas hydrophila infection It is effective for prevention or treatment.
  • the Aeromonas hydrophila infectious disease is sepsis, Areromonas disease or ulcer, but is not limited thereto.
  • the Vibrio parahemolyticus infectious disease also includes symptoms expressed from such a disease, for example, diarrhea or bleeding, but is not limited thereto.
  • the 'prophylaxis or treatment' includes the prevention, inhibition, and alleviation of Aeromonas hydrophila infectious diseases.
  • composition of the present invention may be administered to an animal in the form of a pharmaceutical preparation, or may be administered by a method of feeding it by mixing with feed or drinking water.
  • the route of administration of the composition of the present invention may be administered via various routes orally or parenterally as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, It may be administered in a conventional manner via transdermal, nasal, inhalation, and the like.
  • the prophylactic or therapeutic method of the present invention includes administering a composition of the present invention in a pharmaceutically effective amount. It will be apparent to one of ordinary skill in the art that a suitable total daily usage may be determined by the physician within the scope of good medical judgment.
  • the specific therapeutically effective amount for a particular subject, together with the type and severity of the response to be achieved, the age, body weight, general health, sex and diet, time of administration, route of administration and rate of composition, duration of treatment, and the specific composition of the subject It is desirable to apply differently depending on various factors including drugs used or co-used and similar factors well known in the medical field.
  • the newly isolated phage PVP-1 according to the present invention has the effect of inhibiting or killing Vibrio parahaemolyticus , and especially inhibiting or killing the growth of multidrug-resistant (to antibiotic resistance) Vibrio parahaemolyticus. It is effective in preventing or treating Vibrio parahemoliticus infectious diseases such as gastroenteritis, food poisoning, or wound infection, and can be used as a composition for treating ballast water, as well as feed, antibiotics or disinfectants. .
  • the newly isolated phage PAH1-C or PAH6-C according to the present invention is aeromonas hydrophila ( Aeromonas hydrophila ) Proliferation inhibitory or killing effect, especially against antibiotic-resistant aeromonas hydrophila, and therefore has antiproliferative or killing ability, such as aeromonas, such as sepsis, aeromonas disease or ulcer It is effective in preventing or treating hydrophilic infectious diseases, and can be used not only as a feed but also as an antibiotic or disinfectant.
  • FIG. 1 shows the pulse-field gel electrophoretogram of the degradation products by PVP-1 gDNA and twelve restriction endonucleases.
  • M line shows low range PFG marker (new England Biolab).
  • Line 1 shows undigested phage gDNA.
  • Lines 2-13 show phage gDNA degradation patterns generated by SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI, respectively (New England Biolab).
  • the genome size of PVP-1 was estimated to be about 100 kb.
  • Figure 2 is observed by electron microscopy of negatively stained PVP-1. Each shows A magnification of PVP-1 alone, B shows PVP-1 as a group, and C shows PVP-1 inhibiting the growth of Vibrio parahemoliticus. It is shown.
  • Figure 4 shows the pulse-field gel electrophoretograms of the degradation products by PAH1-C gDNA (left) and PAH6-C gDNA (right) and 12 restriction endonucleases. It is shown.
  • M line shows low range PFG marker (new England Biolab).
  • Line 1 shows undigested phage gDNA.
  • Lines 2-13 show phage PAH1-C and PAH6-C degradation patterns generated by SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI, respectively (New England Biolab).
  • the genome size of both phage gDNAs was estimated to be about 50 kb.
  • PAH1-C left
  • PAH6-C right
  • Figure 6 Aeromonas hydrophila It shows the lytic activity of PAH1-C and PAH6-C against AH16.
  • Muliplicity of infection (MOI) was co-cultured with PAH1-C or PAH6-C at 0.01, 1, and 100. The results are described as mean ⁇ standard deviation value through three tests.
  • Figure 7 shows the therapeutic effect of PAH1-C and PAH6-C phage on A. hydrophila infection of loach.
  • the double-layered method commonly used for phage separation and the number of plaque-forming units (PFUs) thereof (AdamsM (1959) Bacteriophages. Interscience Publishers, New York) was used.
  • Vibrio parahaemolyticus ATCC 33844 was used as a host bacterium for phage isolation. Plaque morphology was observed after 18-24 hours of incubation. The separated material was incubated at 20 ° C. for 36 hours, centrifuged at 10,000 ⁇ g for 20 minutes, filtered through a 0.45 ⁇ m filter to add 10 ml of 10 ⁇ TSB to 90 ml, and host bacteria 10 6-10 7 CFU. Was incubated for 12 hours or more.
  • the supernatant was collected by centrifugation at 5,000 rpm for 20 minutes, the host bacteria 10 8 were applied to the surface of TSA agar medium, and 5-10 ul of the supernatant was dropped and incubated at 25 ° C. for 18 hours.
  • the titration was diluted to determine the titer and plaque appearance.
  • Pure phage strains were obtained through three series of single-plaque separations and named PVP-1.
  • the filtered phage lysate was precipitated with 10% (wt / vol) polyethylene glycol 8000 in 1M NaCl at 4 ° C. for 12 hours and recovered by centrifugation at 10,000 ⁇ g for 10 minutes at 4 ° C.
  • Purified phage was subjected to CsCl step gradient ultracentrifugation (density gradient: 1.15, 1.45, 1.50 and 1.70 g / ml; 250,000 ⁇ g; 22 h; 4 ° C. and SM buffer (10-mM NaCl, 50-mMTris [pH 7.5]). And 10-mM MgSO 4 ) and stored in the dark at 4 ° C. until use.
  • the host region of PVP-1 was 10 ⁇ l diluted phage suspension (3.3 ⁇ 0 7 PFU / ml) in a bilayered agar plate inoculated with Vibrio parahaemolyticus and Vibrio vulnificus. ) was dropped. The bacteria were then incubated for 20 hours at 37 ° C. and checked for plaque formation.
  • gDNA Purified phage genomic DNA
  • DNase I (20U / ⁇ l) (Takara)
  • RNase A (5U / ⁇ l) (intron)
  • capsids of 10 9 PFU / ml or more of phage stock were removed with Proteinase K, then the supernatant treated with PCI (phenol-chloform-isoamyl alcohol) was treated with isopropanol and then with 70% ethanol. After washing, the genome was finally concentrated in a TE buffer. The genome of the concentrated phage was electrophoresed in 0.7% agarose gel (1X TAE buffer) and used as a control. Each phage group was treated with DNase I, RNase A, and Mung bean nuclease, and then positive band. The result was judged by the presence or absence of.
  • PCI phenol-chloform-isoamyl alcohol
  • the flocs were punched through the mold into a small amount of digestion buffer (500 mM EDTA, 10 mM Tris [pH 8.0], 1% SDS [wt / vol] and 1 mg / ml proteinase K) and incubated overnight at 50 ° C. Drain the digestion buffer, wash the sample three times with TE buffer, and then 10 U of SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI (New England Biolabs) ) At 37 ° C. for 1 h each.
  • digestion buffer 500 mM EDTA, 10 mM Tris [pH 8.0], 1% SDS [wt / vol] and 1 mg / ml proteinase K
  • the flocs were washed three times with TE buffer, placed in wells of 1.2% Pulsed Field Certified agarose (Bio-Rad) in 0.5X TBE and covered with molten 0.5% NuSieve GTG agarose.
  • the samples were electrophoresed using the CHEF-DR III System (Bio-Rad) at 6 V / cm with pulse ramps for 5-15 seconds at 14 ° C. for 16 hours in 0.5 ⁇ TBE buffer.
  • TEM Transmission electron microscopy
  • Genomic DNA is extracted according to a method known in "Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.” Sequencing Regent and 454 GSFLX Titanium Sequencing System (Roche) (about 50 ⁇ coverage).
  • Whole genome sequences were obtained by sequence assembly using SeqMan II sequence analysis software (DNAStar).
  • Estimated ORFs open reading frames
  • Glimmer 3.02 and GeneMark.hmm were analyzed using BLASTP and InterProScan. Putative tRNA genes were found using tRNAscan-SE (v. 1.21) software.
  • Phage was isolated in a total of 11 separation attempts. Phage were isolated purely through three cloning procedures. Phage, named PVP-1, was isolated from the seawater of the Southern Sea Oyster Farm and formed plaque in Vibrio parahaemolyticus ATCC 33844.
  • Vibrio vulnificus as well as 30 isolates of Vibrio parahaemolyticus , were examined and finally determined by examining their susceptibility and host area. All 30 isolates of Vibrio parahemoliticus are multidrug resistant strains. Of the 30 weeks of Vibrio parahemoliticus, 27 weeks of Vibrio parahemoliticus formed plaques. Therefore, it is judged that PVP-1 exhibits susceptibility to Vibrio parahemolyticus. However, PVP-1 did not show susceptibility to the one type of Vibrio bulnipicus used in this study (Table 1).
  • the separated phages were stored at 4 ° C. in the dark.
  • sipori hypertrophy (Siphoviridae) phage are known as having a double helix (ds) DNA genome.
  • ds double helix DNA genome.
  • gDNA of PVP-1 was completely degraded by DNase I but not completely by RNase A or Mung bean nuclease. Therefore, it was assumed to be ds (double strand) DNA.
  • gDNA was degraded by SphI, NcoI, EcoRI, and its size was estimated to be approximately 100 kb (FIG. 1).
  • Phage were classified according to the classification Ackermann, in the form of a surface enlargement sipori (Siphoviridae) and (family) kawoodo Bilal less (Caudovirales) neck (order) (Fig. 2).
  • the double-redundant nonredundant DNA genome of PVP-1 was 111,506 bp in length and had a G + C content of 39.71%.
  • a total of 157 ORFs and 19 tRNAs (including 1 pseudogene) were identified, including 40 or more amino acid residues, indicating that PVP-1 is the first member of the family of saporitrophids that has the ability to infect Vibrio parahemolyticus. Show bacteriophage. Proteins homologous to 45 ORFs were not found in the GenBank database, and 69 and 40 of the remaining ORFs encode proteins that are homologous to known phage- and bacterium-related proteins, respectively. It turned out.
  • Bioinformatic analyses were performed to estimate the function of phage-related ORFs, and those ORFs were grouped into at least three functional roles, namely DNA metabolism ( orf2 , orf3 , orf4 , orf6 , orf7 , orf12 , orf14 , orf15 , orf16 , orf17 , orf18 , orf21 , orf28 , orf32 , orf42 , and orf52 ), viral morphogenesis ( orf139 , orf141 , orf143 , orf144 , orf148 , orf149 , orf153 , orf155 , orf156 , and orf157 ), and lytic properties ( orf73 , orf82 , and orf83 ).
  • ORFs including DNA metabolism and viral morphogenetic genes, were clustered together at each end of the genome sequenced by functional role and were similar to T5 or T5-like phage ( ⁇ 9%). This shows a close genetic association between PVP-1 and the phages. However, it showed no sequence similarity with marine vibrio phages (phiHSIC and SIO-2) belonging to the siporitrophic family, and most of the sequences of genes in PVP-1 are novel and therefore similar to other sequenced phages or bacteria. could know.
  • Vibrio phage PVP-1 The genome sequence of Vibrio phage PVP-1 was deposited in the Genbank database as accession number JQ340389.
  • Vibrio parahemolyticus ATCC 33844 cultures Bacterial lysis activity of PVP-1 was tested in Vibrio parahemolyticus ATCC 33844 cultures (FIG. 3). When the Vibrio parahemolyticus culture was not infected with PVP-1 (control), the OD 600 value continued to increase during incubation. However, until 24 hours after PVP-1 infection, survival of Vibrio parahemolyticus ATCC 33844 was significantly inhibited at MOI 0.01, 1 and 100.
  • Samples were collected from sewage treatment plants, rivers, and rivers around fish farms. After sampling the dates and samples, more than 500ml were collected and 100ml were used for bacteriophage separation. Specifically, bacteriophages were isolated from the Seoseong Seoamcheon and Nanji Water Reclamation Center. At the same time, bacteria were isolated from the farm or river. Isolated bacteria were identified using PCR and Vitek II.
  • the supernatant was collected by centrifugation at 5,000 rpm for 20 minutes, and the host bacteria 108 CFU were applied to the surface of TSA agar medium, and then 5-10 ul of the supernatant was dropped and incubated at 25 ° C. for 18 hours.
  • the titration was diluted to determine the titer and plaque appearance.
  • Pure phage strains were obtained through three series of single-plaque separations and named PAH1-C and PAH6-C.
  • the filtered phage lysate was precipitated with 10% (wt / vol) polyethylene glycol 8000 in 1M NaCl at 4 ° C. for 12 hours and recovered by centrifugation at 10,000 ⁇ g for 10 minutes at 4 ° C.
  • Purified phage was subjected to CsCl step gradient ultracentrifugation (density gradient: 1.15, 1.45, 1.50 and 1.70 g / ml; 250,000 ⁇ g; 22 h; 4 ° C. and SM buffer (10-mM NaCl, 50-mMTris [pH 7.5]). And 10-mM MgSO4) and stored at 4 ° C. in the dark until use.
  • the host regions of PAH1-C and PAH6-C were 10 ⁇ l of diluted phage suspension (3.3) in double-layered agar plates inoculated with Aeromonas hydrophila and Aeromonas sorbia. 10 ⁇ 10 7 PFU / ml) was dropped. The bacteria were then incubated for 20 hours at 37 ° C. and checked for plaque formation.
  • gDNA Purified phage genomic DNA
  • DNase I (20U / ⁇ l) (Takara)
  • RNase A (5U / ⁇ l) (intron)
  • capsids of 10 9 PFU / ml or more of phage stock are removed with Proteinase K.
  • PCI phenol-chloform-isoamyl alcohol
  • isopropanol washed with 70% ethanol.
  • the genome was finally concentrated in a TE buffer.
  • the genome of the concentrated phage was electrophoresed in 0.7% agarose gel (1X TAE buffer) and used as a control.
  • Each phage group was treated with DNase I, RNase A, and Mung bean nuclease, and then positive band. The result was judged by the presence or absence of.
  • the flocs were punched through the mold into a small amount of digestion buffer (500 mM EDTA, 10 mM Tris [pH 8.0], 1% SDS [wt / vol] and 1 mg / ml proteinase K) and incubated overnight at 50 ° C. Decompose the digestion buffer, wash the sample three times with TE buffer, then 10 U of SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI (New England Biolabs) ) At 37 ° C. for 1 h each.
  • digestion buffer 500 mM EDTA, 10 mM Tris [pH 8.0], 1% SDS [wt / vol] and 1 mg / ml proteinase K
  • the flocs were washed three times with TE buffer, placed in wells of 1.2% Pulsed Field Certified agarose (Bio-Rad) in 0.5X TBE and covered with molten 0.5% NuSieve GTG agarose.
  • the samples were electrophoresed using the CHEF-DR III System (Bio-Rad) at 6 V / cm with pulse ramps for 5-15 seconds at 14 ° C. for 16 hours in 0.5 ⁇ TBE buffer.
  • TEM Transmission electron microscopy
  • PAH1-C and PAH6-C against Aeromonas hydrophila were evaluated.
  • PAH1-C (1.1x10 10 PFU / ml) and PAH6-C (1.1x10 10 PFU / ml) and Aeromonas hydrophila AH16 (8.8x10 6 CFU / ml) strains were performed, and purified phage and Aeromonas hydrophila AH16 was divided into three groups of 0.01, 1, and 100 (multiplicity of infection, quantitative ratio of bacteria and bacteriophages) in 10 ml of clean TSB.
  • the preparation was cocultured at 20 ° C. with 250 rpm shaking. Bacteria inoculated in phage-free TSB were used as controls. Absorbance (OD 600 ) was measured until 24 h after inoculation.
  • Aeromonas hydrophila used for artificial infection was aeromonas hydrophila AH16 strain that caused mass mortality in loach, phage used PAH1-C and PAH6-C. Infections were intramuscular injection (IM) and therapeutic phages were administered in the same way.
  • IM intramuscular injection
  • the experimental group consisted of 4 groups that did not hold phage after bacterial infection (20 rice), PAH1-C phage treatment after bacterial infection (20 rice), PAH6-C phage treatment after bacterial infection (20 rice), bacteria
  • Post-infection group consisted of PAH1-C and PAH6-C phages (20 US).
  • the AH16 strain used for bacterial infection was diluted artificially in PBS at a concentration of 3.3x10 7 CFU / fish, and then artificially infected.
  • PAH1-C used for phage treatment was 1.1x10 9 PFU / fish and PAH6-C 1.0x10 9 Inoculated at a concentration of PFU / fish.
  • the prognosis was observed for 7 days in a 25 ° C water bath.
  • Phage Two phages were isolated in a total of 11 separation attempts. Phage were isolated purely through three cloning procedures. Phage, named PAH1-C, was isolated from the sedimentary Seoamcheon and formed plaques in Aeromonas hydrophila JUNAH. Phage, named PAH6-C, was also isolated at the Nanji Water Reclamation Center and formed plaques in Aeromonas hydrophila JUNAH.
  • PAH1-C formed plaques in 6 weeks of Aeromonas hydrophila of 7 weeks of Aeromonas hydrophila and 1 of Aeromonas sorbia of 10 weeks of Aeromonas sorbia.
  • PAH6-C formed plaque in 4 weeks of Aeromonas hydrophila of 7 weeks of Aeromonas hydrophila and 1 of Aeromonas sorbia of 10 weeks of Aeromonas sorbia.
  • both PAH1-C and PAH6-C are judged to be susceptible to aeromonas hydrophila (Table 2).
  • All Aeromonas hydrophila of Table 4 below are antibiotic resistant strains, among which Aeromonas hydrophila Aeromonas hydrophila JUNAH used as host bacteria is a multidrug resistant strain.
  • the separated phages were stored in 4 ° C dark.
  • myobiride phages are known to have a double helix (ds) DNA genome.
  • ds double helix
  • the gDNAs of PAH1-C and PAH6-C were completely degraded by DNase I but not completely by RNase A or Mung bean nuclease. Therefore, it was assumed to be ds (double strand) DNA.
  • gDNA was cleaved by SacII, Sau3AI, MspI, SmaI, NcoI, HpaII and EcoRI, and its size was estimated to be approximately 50 kb (FIG. 4).
  • PAH1-C and PAH6-C were classified according to the Ackermann classification into myoviridae and Caudovirales orders in morphology (FIG. 5).
  • Aeromonas hydrophila AH16 cultures Bacterial lysis activity of PAH1-C and PAH6-C was tested in Aeromonas hydrophila AH16 cultures (FIG. 6). When the Aeromonas hydrophila AH16 culture was not infected with PAH1-C and PAH6-C (control), OD 600 values continued to increase during incubation. However, 24 hours after PAH1-C and PAH6-C infection, survival of Aeromonas hydrophila AH16 was significantly inhibited at MOI 0.01, 1 and 100.
  • Aeromonas hydrophila AH16 only died within 3 days, and delayed to reduced mortality was observed in the group treated with PAH1-C and PAH6-C. In addition, it was found that PAH6-C was superior to PAH1-C in inhibiting aeromonas hydrophilic infectious disease (FIG. 7).
  • Using the phages according to the present invention is expected to contribute to improving the competitiveness and public health of the aquaculture industry.

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Abstract

La présente invention concerne un nouveau phage qui est nouvellement isolé et identifié, et une composition pour l'inhibition de la prolifération de bactéries pathogènes ou pour tuer des bactéries pathogènes, le contenant en tant que principe actif. La présente invention peut être utilisée en variante dans une composition pour la prévention ou le traitement de maladies infectieuses bactériennes, une composition pour le traitement de l'eau de ballast, des antibiotiques, un désinfectant, un additif alimentaire et similaires.
PCT/KR2013/003334 2012-06-22 2013-04-19 Nouveau bactériophage et son utilisation pour l'inhibition de la prolifération de bactéries pathogènes WO2013191363A1 (fr)

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KR1020120067606A KR101466620B1 (ko) 2012-06-22 2012-06-22 신규한 박테리오파지 및 이의 아에로모나스 히드로필라 증식 억제 용도
KR10-2012-0067606 2012-06-22
KR1020120067811A KR101381336B1 (ko) 2012-06-25 2012-06-25 신규한 박테리오파지 ｐｖｐ-1 및 이의 비브리오 파라헤몰리티쿠스 증식 억제 용도
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CN108699532A (zh) * 2015-12-21 2018-10-23 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体Vib-PAP-2及其用于抑制副溶血弧菌增殖的用途
CN110214181A (zh) * 2016-12-21 2019-09-06 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体vib-pap-4及其用于抑制副溶血弧菌细菌增殖的用途
CN115181731A (zh) * 2021-04-02 2022-10-14 青岛诺安百特生物技术有限公司 一株坎贝氏弧菌噬菌体、制备方法及其应用

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WO2007130655A2 (fr) * 2006-05-05 2007-11-15 Gangagen, Inc. Activités antimicrobiennes dérivées de bactériophages
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Publication number Priority date Publication date Assignee Title
CN108699532A (zh) * 2015-12-21 2018-10-23 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体Vib-PAP-2及其用于抑制副溶血弧菌增殖的用途
CN108699532B (zh) * 2015-12-21 2022-08-23 尹特荣生物科技株式会社 副溶血弧菌噬菌体Vib-PAP-2及其用于抑制副溶血弧菌增殖的用途
CN110214181A (zh) * 2016-12-21 2019-09-06 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体vib-pap-4及其用于抑制副溶血弧菌细菌增殖的用途
CN115181731A (zh) * 2021-04-02 2022-10-14 青岛诺安百特生物技术有限公司 一株坎贝氏弧菌噬菌体、制备方法及其应用
CN115181731B (zh) * 2021-04-02 2023-12-15 青岛诺安百特生物技术有限公司 一株坎贝氏弧菌噬菌体、制备方法及其应用

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