+

WO1998008944A1 - Nouveau bacteriophage et procede de criblage associe, nouvelles matieres biobactericides preparees avec ledit bacteriophage et reactif utilise pour detecter celui-ci - Google Patents

Nouveau bacteriophage et procede de criblage associe, nouvelles matieres biobactericides preparees avec ledit bacteriophage et reactif utilise pour detecter celui-ci Download PDF

Info

Publication number
WO1998008944A1
WO1998008944A1 PCT/JP1997/002957 JP9702957W WO9808944A1 WO 1998008944 A1 WO1998008944 A1 WO 1998008944A1 JP 9702957 W JP9702957 W JP 9702957W WO 9808944 A1 WO9808944 A1 WO 9808944A1
Authority
WO
WIPO (PCT)
Prior art keywords
phage
pathogenic
bacteriophage
bacterium
bacteria
Prior art date
Application number
PCT/JP1997/002957
Other languages
English (en)
Japanese (ja)
Inventor
Seishi Takahashi
Koji Nishikori
Original Assignee
Bio Venture Bank Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP13023697A external-priority patent/JP2002335956A/ja
Application filed by Bio Venture Bank Co., Ltd. filed Critical Bio Venture Bank Co., Ltd.
Priority to US09/242,901 priority Critical patent/US6322783B1/en
Priority to AU38692/97A priority patent/AU3869297A/en
Publication of WO1998008944A1 publication Critical patent/WO1998008944A1/fr

Links

Classifications

    • 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
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A23B2/783—Microorganisms; Enzymes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention relates to a novel bacteriophage, a screening method thereof, a novel bio-sterilizing material using the same, and a detection reagent. More specifically, the present invention relates to a novel pacteriophage having ⁇ ⁇ specificity to various pathogenic bacteria including pathogenic Escherichia coli, a method for screening the same, and the use of such a pacteriophage alone or as a mixed force vector. The present invention relates to a novel biocidal material, a stabilizing agent and a reagent or test kit for detecting pathogenic bacteria using a bacteriophage. Background technology
  • Escherichia coli is ubiquitous in nature and is also a resident bacterium in the intestine of humans and mammals. Almost all such Escherichia coli usually do no harm to humans and babies. However, so-called pathogenic Escherichia coli, such as enterohemorrhagic Escherichia coli, exists in Escherichia coli, and this pathogenic Escherichia coli can infect humans and mammals and have serious consequences.
  • fungicides To prevent infection from pathogenic bacteria such as pathogenic Escherichia coli or to treat when infected with bacteria such as Escherichia coli, more types of fungicides are used than before. It is used. For example, iodine-containing germicides such as eodo tincture and chlorhexidine-containing maquilon are used to disinfect wounds. In addition, benzoic acid and the like are often used as preservatives that can be added to foods. Various effective antibiotics are used to prevent or treat bacterial infections.
  • a bacteriophage (hereinafter sometimes simply referred to as “phage”) is composed of only proteins and nucleic acids, each of which is a medium between an organism and an inanimate organism that can grow only in a specific bacterium using a specific bacterium as a host. It is very small and can only be observed with an electron microscope.
  • Pacteriophages are bacteria It is known that the disease spreads only to infected bacteria, and that the number of progeny is increased by eating out the infected bacteria. At present, a number of pacteriophages using each cell as a host are known. For example, Salmonella has a specific phage using Salmonella as a host, and Vibrio has a specific phage using Vibrio as a host.
  • pacteriophages act only on bacteria, and have high specificity for host bacteria, and act only on one particular bacterium or only a small number of a plurality of particular bacterium. It is known. For example, phage acting on Escherichia coli are no exception, they cannot act on all or many different types of Escherichia coli, and it is also known that the well-known T2 phage cannot act on Escherichia coli C . Thus, although pacteriophages have been used only as research targets and have made great progress in gene research, on the other hand, their specificity is too high, so that pacteriophages are used industrially. It has not received much attention so far. Therefore, there has never been any idea of using karoku pacteriophage as a bactericide.
  • pacteriophages using various bacteria as hosts are known, but no pacteriophage using pathogenic bacteria such as enterohemorrhagic Escherichia coli as a host has been known so far.
  • Escherichia coli among the bacteria hosting the pacteriophage, Escherichia coli, in particular, has a very strong growth potential, and divides once every 20 to 30 minutes. Grows to 1 gram per day.
  • Escherichia coli is a bacterium that is ubiquitous in nature and also in the human intestine of mammals, and usually has no harm to humans or human milk excretions. Has no effect. Utilizing such a property of Escherichia coli, many so-called biomedical products are produced by culturing Escherichia coli.
  • a stabilizer is required so that it can be stored for a long time while ensuring its safety.
  • development of preservatives is also required.
  • Tris-HCl buffer, phosphate buffer, and the like are well known, but these have been used only for research so far, and are used for fresh foods and other foods. It cannot be used directly.
  • no phage preservation solution that can be sprayed on fresh food and drinkable has been known at all.
  • the present inventors have made intensive efforts focusing on the above-mentioned specific effects of pacteriophages, and as a result, a specific pacteriophage specifically kills pathogenic Escherichia coli. And the use of the bactericidal material containing the pacteriophage in fresh foods and other foodstuffs to kill such pathogenic E. coli. It has been found that bacteria can be germ-free and the safety of food products such as fresh food can be ensured.
  • an object of the present invention is to provide a bacterium phage exhibiting extremely high specificity only for pathogenic bacteria.
  • Another object of the present invention is to provide a screening method that can efficiently and reliably screen for such highly specific pateriophages.
  • the present invention relates to a biocidal material containing a pacteriophage that can stabilize a phage for a long period of time while ensuring extremely high safety even when the pacteriophage is used directly in foodstuffs such as fresh food. It is intended to provide
  • the present invention provides a method for stabilizing phage for a long period of time in such a biocidal material containing bacteriophage while ensuring extremely high safety even when the phage is used for foodstuffs such as fresh food.
  • the purpose of the present invention is to provide a preservative for phage that can be used.
  • the present invention provides a reagent for detecting a bacterial phage capable of efficiently, quickly and reliably detecting a host pathogenic bacterium by utilizing the phagocytic action of pateriophage.
  • the purpose is to provide the reagent kit.
  • pathogenic bacteria refers to bacteria that infect animals such as pets such as humans, dogs, and cats and give them a source of disease or pathological symptoms.
  • pathogenic bacteria means a bacterium that becomes a host of a bacteriophage and is phagocytosed and destroyed by the bacteriophage, and should be understood as a generic term for pathogenic bacteria including pathogenic E. coli including enterohemorrhagic E. coli. It is.
  • Escherichia coli which is a representative bacterium, will be described as a pathogenic bacterium, but Escherichia coli is merely a representative of a pathogenic bacterium and is described as an example of a pathogenic bacterium. It should be understood that the present invention is not limited to such an example.
  • the present invention provides pacteriophages having high specificity only against pathogenic bacteria.
  • the present invention also provides, as a preferred embodiment thereof, a Pacteriophage having high specificity only against a specific pathogenic Escherichia coli.
  • the present invention provides a novel bacteriophage having high specificity only for a specific type of Escherichia coli, and thereby provides a novel bacteriophage containing various pathogenic Escherichia coli. It will be possible to respond to a wide range of demands using bacterium phage useful for bacteria.
  • the present invention provides a screening method for efficiently and reliably screening for pateriophages having ⁇ ⁇ specificity only for pathogenic bacteria.
  • the present invention provides a novel bio-sterilizing material containing pateriophage having high specificity only for pathogenic bacteria.
  • a novel bio-sterilizing material has the advantage that its bacteriophages can destroy pathogenic microbes that can be infected without causing any adverse effects on the human body. It can also be used directly on products.
  • novel biocidal material provided by the present invention includes such a bacteriophage consisting of a cocktail of bacteriophages having two or more different properties so that it can simultaneously cope with a plurality of different pathogenic bacteria. ing.
  • novel bio-sterilized material contains a stabilizing agent or preservative for a culture solution of bacteriophage which can stably store the paterio phage for a long period of time. Disinfecting materials can be stored for long periods of time, while ensuring their safety and stability.
  • the present invention also provides a method for producing a culture medium of Pacteriophage, which comprises obtaining a culture medium of Pacteriophage by infecting and growing a bacterium, which is a host, in which Pacteriophage is cultured. Is what you do.
  • a medium for culturing pathogenic bacteria is made to contain calcium ions, so that a pacteriopha having high specificity for pathogenic bacteria is obtained.
  • This ⁇ ijlj provides L1I with a stabilizer or preservative for the culture of bacteriopacteriophage, which can save bacteriophage solution in 7iZ and store it.
  • Another aspect of the present invention is a pathogen capable of detecting the presence of a pathogenic bacterium easily and in a short time by using a specific pateriophage.
  • FIG. 1 is a diagram showing the bactericidal effect of the cross streak method on the plate, and shows that the bacteria that have come into contact with the test bacterium are completely killed.
  • Figure 2 shows the effect of this drug in a liquid medium.
  • (1) shows the growth curve of bacteria without this drug
  • FIG. 3 is a diagram showing a culture state of bacteriophage.
  • a) shows a petri dish in which only the agar medium was cultured
  • a) shows a petri dish in which enterohemorrhagic 0157 was cultivated on the agar medium. It turns out that the petri dish is white.
  • (Ii) shows a culture obtained by adding an undiluted phage solution (2 ⁇ 10 1 D / ml) on an agar medium, which phagocytoses and destroys only pathogenic adenobacterium 0157 together with enterohemorrhagic 0157. In this figure, it is clear that all enterohemorrhagic 0157 has been destroyed by phagocytosis.
  • E) is a solution obtained by diluting the phage by a factor of 1,000 with a new phage stock solution (2 x 10 7 / ml) and a 20-fold dilution with tap water together with enterohemorrhagic 0157 on an agar medium.
  • Bacteriophages are present in the excrement of many animals, such as domestic animals such as sea lions, pets such as dogs and cats, birds such as cattle and crows, and poultry such as chickens, and sewage. It is widely known that it can be separated from such excrement and sewage.
  • any bacteriophage that can be used in the present invention achieves the object of the present invention as long as it has high specificity against pathogenic bacteria such as pathogenic Escherichia coli including enterohemorrhagic Escherichia coli. It is not limited to a particular type of phage. In other words, as the pateriophage, any pacteriophage capable of adsorbing on the host pathogenic bacterium and specifically lysing and destroying the bacterium to achieve the object of the present invention can be used. can do. Examples of such bacteriophages include, for E.
  • bacteriophages such as SZP01 and SP02 are also available against Bacillus subtilis.
  • SEQ ID Nos. 1-1 to 1 to 4 SEQ ID Nos. 2-1 to 2-10, SEQ ID Nos. 3-1 to 3-5 or SEQ ID No. 4- shown in the following sequence listing
  • Each of the Pacteriophages having DNA containing a fragment having the nucleotide sequence represented by each of 1 to 4-5 has specificity against enterohemorrhagic Escherichia coli 0157.
  • Each DNA sequence was identified by the method described below.
  • pacteriophage it is also possible to screen for the highly specific pacteriophage according to the present invention by the conventional method for screening pacteriophage.
  • a screening method that can screen for pacteriophage having ⁇ ⁇ specificity against pathogenic bacteria, particularly pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli, has not been established yet. Therefore, in order to increase the efficiency and certainty of the screening, we devised the following bacteriophage screening method, and have a high specificity from nature, especially for pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli. It has been found that pacteriophage that can be lysed sexually can be screened.
  • the samples are collected.
  • the sample is infected with, for example, Escherichia coli ⁇ , ⁇ , and C strains, and the positive enzyme (m + ) is modified with the restriction enzyme minus ( ⁇ ⁇ —) to amplify the phage present in the sampzole. I do.
  • This lysate is plated on pathogenic bacteria, a single plaque is separated, and such an operation is repeated several times to perform several rounds of amplification to obtain a Takata ita stock.
  • the method for culturing a novel pateriophage according to the present invention will be described by taking, as an example, the case of cultivating pateriophage using Escherichia coli as a host.
  • the cultivation is continued under the same conditions as before, after adding the pacteriophage, the pacteriophage will infect the large intestine, and the pacteriophage will grow in the E. coli cells and completely destroy the E. coli. If the ligation and cultivation are continued for a predetermined time, Escherichia coli is no longer alive in the culture solution, and a culture solution of pacteriophage is obtained.
  • This culture solution is purified according to a conventional method, and a residue of Escherichia coli is removed according to a conventional method such as centrifugation to obtain a culture solution of bacteriophage.
  • the pacteriophage according to the present invention can be cultured using an agar medium containing, for example, polypeptone, yeast extract and the like.
  • agar medium containing, for example, polypeptone, yeast extract and the like.
  • the phage cultivated in this manner is dissolved, for example, by adding the natural medium to the phage preservation solution described above and dissolving the mixture, and separating the mixture from solids such as culture residue by centrifugation or the like. And the supernatant can be used as a stock solution.
  • RNA etc., into E. coli cells.
  • DNA or RNA is injected into the cells, the pacteriophages phagocytose the colon in a short time and grow in the cells.
  • the Escherichia coli is completely destroyed, and as a result, the bacterium is propagated in the culture solution to form the bacterium solution.
  • This mechanism can be said to be the same when killing pathogenic bacteria using the bio-sterilizing material of the present invention including pateriophage.
  • a buffer containing salts in the culture and a trace amount of metal such as Mg, Mn, or Ca.
  • metal such as Mg, Mn, or Ca.
  • glycerol can be added at about 0.001-5%, preferably about 0.1-1%.
  • sugars such as polysaccharides such as maltose and glucose, amino acids such as glycine, arginine and lysine, ethylparaben and polylysine can also be added.
  • the novel phage preservation solution according to the present invention uses an amino acid such as glycine, arginine, lysine, or the like, and preferably glycine, in order to stabilize bacteriophage contained therein while ensuring high safety for food. Good to do.
  • the amino acid is prepared by adjusting a buffer containing 10 mM to ⁇ M, preferably 50 mM to 500 mM to pH 6 to 8, preferably pH 6.5 to 7.5, and adding 5% of sodium chloride as necessary.
  • calcium chloride can be added in the range of 0.03% -1%, and calcium chloride in the range of 10 mM, preferably 0.1-lniM.
  • novel phage preservation solution according to the present invention does not inactivate the phage even when diluted up to about 100-fold with ordinary tap water, and can exert a sufficiently strong phagocytic-destroying effect. The same applies when diluted with household alkaline water or acidic water. The phage contained is not inactivated, and a sufficient phagocytic destruction effect is exhibited.
  • the novel biocidal material using the pacteriophage according to the present invention can be prepared as follows. That is, the culture solution of Pacteriophage cultivated as described above is separated from the culture residue by a conventional method such as centrifugation, and the culture solution is used as the novel bio-orchid material of the present invention. Prepare as stock solution. In the novel bio sterilizing food according this 3 ⁇ 4fjm this, Park bacteriophage, for example, 10 2 cells / ml to 10 12 cells / ml, at any concentration preferably in the range of 10 3 cells / ml to 10 8 cells / ml I just need.
  • novel bio-sterilizing material according to the present invention can contain such a bacterium phage alone or in a cocktail of two or more kinds.
  • the combination can be appropriately changed according to the purpose of use.
  • the pathogenic bacteria hosting each of the combined bacteriophage cocktails can be simultaneously phagocytosed.
  • the stock solution of the novel biocidal Si material obtained in this manner is used after diluting the stock solution to an appropriate ratio because the concentration of the phage is usually too high.
  • dilution with the phage preservation solution described above is preferable, but dilution with tap water or the like is also possible.
  • the use of a cocktail of two or more bacteriophages having different properties can suppress the emergence of resistant bacteria due to high frequency use.
  • a cocktail in which equal amounts of T2 phage and ⁇ phage are mixed is used as a sterilizing material.
  • the each of the resistant bacteria frequency is 10 6
  • resistant bacteria frequency in the case of using the above cocktail 10- 12 becomes also the emergence of resistant bacteria in theory experimentally also Almost nothing can be achieved.
  • a bactericide for food for example, benzoic acid is added in an amount of 0.002%. It can be added at a rate of about 2%, preferably about 0.1% to 0.3%.
  • novel bio-sterilizing material according to the present invention has no smell or taste, when used with foods, a flavor such as lemon may be added.
  • the mechanism of the production of the killing material according to the present invention will be described by taking, as an example, the origin of bacterial Escherichia coli. In the case of culturing bacteriophage, the mechanism is the same as that when the phage grows while eating Escherichia coli. is there.
  • the germicidal material according to the present invention has a completely different mechanism of action from other conventionally used germicides.
  • the pacteriophage used in the present invention is relatively strong in air, but is weak against stomach acid, etc. because it does not have a cell membrane unlike E. coli, and is lost in gastric juice when it enters the body orally. It is alive and subsequently digested and absorbed. Therefore, the bactericide according to the present invention containing the pacteriophage can be said to be a germicidal material or bactericidal food having a bactericidal action due to phagocytosis, unlike the conventional bactericide. Based on this property, the sterilizing material according to the present invention was named as a novel bio-sterilizing material containing pacteriophage.
  • novel biocidal material of the present invention is bactericidal by the action mechanism of flii, it is phagocytosed and proliferates, so it is not affected by concentration unevenness. Can be sterilized by phagocytosis over time. Therefore, there is an advantage that the material can be used at an extremely low concentration. (Reagents for detecting pacteriophage and their kits)
  • the pacteriophage according to the present invention can be used as a reagent for detecting a pathogenic bacterium serving as its host. Since this phage can phagocytose and destroy the host pathogenic bacterium, a reagent for detecting the host pathogenic bacterium using the phagocytic action of the phage is used. Can be prepared. There are various types of reagent kits using this phage.For example, an agar medium capable of culturing a phage is injected into a container such as a petri dish. Reagent kits in different formats.
  • a sample containing a test substance that may contain the pathogenic cell to be detected is applied to the surface of the culture medium, and an appropriate temperature ⁇ culture is performed. Then, if the phage contained in the agar medium is present in the presence of the iogenic force of the host, the phage will be phagocytosed. The traces of this phagocytosis cause the medium to be lysed and become transparent or translucent. If the agar medium is in such a state, the sample examined contains pathogenic bacteria corresponding to the phage included in the agar medium as a detection reagent, and the corresponding pathogenic bacteria are not detected. It has been detected.
  • the pathogenic bacterium to be detected is pathogenic Escherichia coli
  • the Escherichia coli divides and proliferates in a very short time.
  • About 30 minutes after the sample is applied to the agar medium serving as the detection reagent kit it can be determined whether or not Escherichia coli to be detected is present.
  • using the phage according to the present invention for example, when detecting Escherichia coli, it can be detected in a short time of about 30 minutes, which is extremely convenient.
  • the use of this detection kit or its reagent kit makes use of the phagocytosis of phage, even if the pathogenic bacteria to be detected are not extremely profitable in the sample to be tested. This is very useful because it can be reliably detected.
  • phage having two or more different properties as a test vein or a reagent kit for such detection different types of pathogenic bacteria can be simultaneously detected, which is extremely useful and convenient. .
  • the reagent for detecting a protozoan bacterium or the reagent kit thereof according to the present invention has such an extent that a specimen to be tested is brought into contact with a petri dish containing an agar medium containing a phage and the pathogenic bacterium can grow. Since the presence or absence of the pathogenic bacterium can be determined simply by leaving it in an appropriate place at a temperature, no other measuring instruments or devices for detection are required, so the method of use is extremely simple. Therefore, the reagent for detecting a pathogenic bacterium and the reagent kit thereof according to the present invention can be easily used, for example, for primary screening for detecting a pathogenic bacterium f.
  • novel biosterile material according to the present invention can also be applied in various ways.
  • the novel biocidal material according to the present invention can be used for any place, such as a place where the immature microbial iMi can exist, or an article that can be infected by pathogenic bacteria. Can be protected from infection with pathogenic bacteria, or can be tested for contamination by such pathogenic bacteria.
  • the novel bio-sterilizing material according to the present invention can be directly used, for example, by spraying food products such as fresh foods, dipping the food products therein, or washing it. Therefore, this novel bio-sterilizing material can be used at any stage of preservation and cooking of foods such as fresh foods so that the foods and the like are not contaminated by pathogenic bacteria.
  • novel bio-sterilizing material according to the present invention must be kept clean, for example, in places where cleanliness should be maintained, such as kitchens, kitchens, storage rooms, hospitals, and clothing such as aprons and white coats. By spraying directly on items that should not be used, it is possible to prevent contamination of the place or items by pathogenic bacteria.
  • the novel bio-sterilizing material can be used as a washing solution to prevent pathogenic bacteria from entering the body or infecting others through the body such as hands.
  • this novel bio-sterilizing material can be used, for example, in the cultivation or production process of fresh foods and the like.
  • spraying on foodstuffs such as fresh food mixing and adding to cultivation vegetables for cultivated vegetables such as kale radish, spraying on cultivated vegetables such as kale radish, or especially in hydroponics It can be used by putting a certain concentration in the water.
  • the new bio-sterilizing material can also be used for sterilization purposes, for example, in fish markets, meat processing plants and other places where fresh food is processed, and cattle barns and other livestock breeding grounds.
  • Samples were collected from animals such as pigeons, crows, sea lions, dogs, cats and poultry, and from sewage. Approximately 1 g of each sample or 5 to ⁇ 0 ml in the case of sewage was dissolved in B medium, and the residue was precipitated by centrifugation to obtain a supernatant. Phage, which is presumed to be present in a small amount in this supernatant, was amplified by infecting E. coli K strains, B strains and C strains with the restriction enzyme minus () to the modified enzyme positive (m + ) host. . This lysate was plated on 01 (37, and plaques were separated.
  • a high-yield Yuichi stock was obtained.
  • phages containing naturally-occurring deleted DNA were selected by exposing them to 5-10 mM EDTA for several hours at high temperature (55 ⁇ 65 ⁇ :). By repeating this operation several times, it was possible to select a phage that is stable and has only essential genes.
  • Escherichia coli is cultivated in a 1-liter jar fermenter using an E. coli culture medium (Elbros) containing polypeptone, a list extract, etc., and the absorbance becomes 0.2 (the number of E. coli cells 2-3 X lOVml).
  • E. coli culture medium Elbros
  • Lysine 0.2M, CaCl 2 ⁇ H 20 were added so that ⁇ . ⁇ . NaCl was 0.5% and glycerol was 0.02%, and ⁇ 6.8-7.0 was adjusted with NaOH. Then, the volume was adjusted to 1,000 ml with pure water, and sterilized by autoclaving for 121 and 15 minutes to prepare a new phage stock solution.
  • the pacteriophage prepared in Example 1 above was propagated by a known method using host Escherichia coli, and an increase of about 100-fold was measured in each step. That is, Escherichia coli that reached the logarithmic growth phase was infected with bacterio phage by MOI (Multiplicity of Infection) 10 and lysed about 100 minutes later, and then clonal form was added. Then, bacterial debris (DNA, RNA, protein, cell membrane, etc.) was removed by centrifugation to prepare a crude product of Pacteriophage. The resulting crude preparation had an infectivity of about 10 V ml.
  • MOI Multiplicity of Infection
  • Glycerol is added to the obtained bacteriophage sample in a concentration of 0.1 to 0.01%, and stabilizers such as lauroyl sarcosine salt and benzoate are added, and Mg, Mn, or Ca is added as gold to add new biotin.
  • a sterilizing material was prepared. When this sterilized material was stored in a low-temperature room (4 ⁇ 1), the bacterium of Pacteriophage could be stored stably without inactivation for 6 months. In preparing the new bio-sterilized material, the bacterio phage was basically prepared using two different species so that no residual bacteria appeared.
  • Bacteriophages were propagated in a known manner using host E. coli and measured at about a 100-fold increase at each step. That is, Escherichia coli that reached the logarithmic growth phase was infected with bacteriophage by MOI (Multiplicity of Infection UO), and after about 100 minutes of lysis, clostic form was added. Then, bacterial debris (DNA, RNA, protein, (Cell membranes etc.) were removed by centrifugation, and the supernatant was purified for pateriophage.Purification was performed by precipitation with polyethylene glycol and density gradient centrifugation with CsCI to give an infectious sample of about 10 12 / ml. I got
  • the bacterium was used after adjusting the concentration of bacterio phage to a MOI of 10. These operations and preservation were performed in a cold room (4 in soil 1) in principle.
  • Escherichia coli was added to an L-broth medium composed of 10 g of polypeptone, 3 g of extract extract, and 2.5 g of NaC1 for s and cultured at 37 ⁇ in a jar armen overnight.
  • pacteriophage was added (MOI: 20).
  • the number of E. coli cells was about 2-3 x 10 V ml.
  • the cells were cultured for 4 hours at 37 ° C. in a jar amen while aeration was performed, and a few drops of black-mouthed form were dropped when the culture solution became slightly transparent. Then, the mixture was centrifuged at 8,000 n) m for 30 minutes to remove the residue, and the supernatant was obtained. This supernatant was used as a stock solution for the preparation of a sterilizing material. (Example 7)
  • a 10% agar medium having the following composition was heated to 451: and completely dissolved, uniformly poured as a lower layer medium on a petri dish, and allowed to stand at room temperature to solidify.
  • a solution containing 0.1 ⁇ m 1 of phage was coated on the surface of this double agar medium in a strip shape, and cultured at 37 for 7 hours to grow enterohemorrhagic Escherichia coli E. coli 0157: H7.
  • the number of holes (plaques) due to phagocytosis was counted and the phage number was calculated accordingly.
  • plaques were formed on the agar medium in accordance with the number of phages, and the target phage was detected.
  • An agar medium having the following composition was prepared.
  • E. coli 0157: H7 strain (number of bacteria: 4 ⁇ 10 7 /0.2 ml) and phage of each degree were mixed in 0.1 ml of a 5% cold solution having the same composition prepared separately. Add 3 ml of natural medium, warm this mixture to 45, dissolve completely, inject uniformly as the upper medium, and immediately incubate at 37: 7 for 7 hours.
  • E. coli 0157: H7 was grown, and the rate of cell growth inhibition by the phagocytosis of phage was observed. When a low concentration of phage was inoculated, the number of holes (plaques) due to the phagocytosis of the phage was counted, and the phage number was calculated accordingly.
  • the growth of Escherichia coli having reached an absorbance of 0.2 and the diluted sample of the phage were mixed, layered on an agar medium as described above, and the phage titer was assayed.
  • the bactericidal effect of the germicidal material was examined in consideration of the case where pathogenic bacteria, which are harmful bacteria, adhered or bleed on the 151-shaped object.
  • Bacteria causing food poisoning often proliferate in food liquids (juices, soups) or in hydroponic sprouts or radish. In such a case, it is effective to give this drug when the bacterial concentration is low per unit volume, so the following experiment was conducted to confirm the effect.
  • Escherichia coli was planted in a commonly used commercially available medium for hydroponic culture, and about 1/10 of a bouillon medium was added to measure proliferation. A few hours later, the culture was started at 37, and this fungicide was added. After 12 hours, ⁇ completely died.
  • the bactericidal effect of this drug was examined using chopping boards used at home for about one year.
  • 10,000 Escherichia coli were dissolved in about 10 ml of a medium (bouillon medium), and this solution was applied to both sides of a cutting board, and after 30 minutes, excess water was removed.
  • the chopping board was then divided into two equal parts, one of which was used as a test for the application of this drug and the other was used as a control.
  • the agent was sprayed all over the experimental chopping board. On the other hand, the same amount of water was sprayed on the control cutting board. After storage overnight 37, the number of remaining bacteria adhering to both cutting boards was examined by washing the cutting boards with 500 ml of medium and counting the number of bacteria. As a result, although no large intestine was detected from the experimental cutting board, about 1 to 2 million E. coli cells were detected from the control ffl cutting board. (Effect on Escherichia coli attached to meat *)
  • the nucleotide sequences of the DNAs of bacteriophages # 1, # 2, # 3 and # 4 isolated by the above screening were determined in accordance with a conventional method.
  • aqueous DNA solution 41 was added 2 U of M buffer, sterile water 13 I restriction enzyme Hind 1 / zl, and the mixture was shaken at 37'C for 1 hour.
  • One-tenth volume of the sample overlay was added, and one electrophoresis was performed on a 0.7% agarose gel at 100 V for 2 hours.
  • the agar-mouth gel was left in a solution of 20 a1 of ethidium ore in 400 ml of TAE solution for 2 hours, and the band was confirmed with a UV lamp.
  • plasmid 21 (pkk223) 3 iU buffer 21, sterile water 14 n 1 and restriction enzyme Hind ffl 11 were added, and the mixture was shaken at 37 for 1 hour.
  • Hind 111 was deactivated for 15 minutes in a water bath at 70 to deactivate.
  • sterilized water 26.51 was added, and shaken at 37 for 2 hours. After that, extraction was performed twice with phenol-cloth form, ethanol precipitation was performed, and the resultant was dissolved in TE solution 80H1.
  • 1/10 volume of the reagent for superimposing the sample was added, and electrophoresis was performed at V0.7% agarose gel at 100 V for 2 hours. Contamination and the presence of the vector were confirmed.
  • the colony of the above (2) was taken in 500 ml of L-liquid medium supplemented with ampicillin, and cultured for about 24 hours. The supernatant was removed by centrifugation of the medium at 6,000 rpm for 4 and 15 minutes.
  • the plasmid containing the phage DNA was purified using a plasmid purification kit (Funakoshi). First, 10 ml of P1 and 10 ml of P2 were added to the pellet and stirred slowly. After standing at room temperature for 5 minutes, 10 ml of cooled P3 was added, and the mixture was stirred slowly, and then left on ice for 20 minutes. After that, centrifugation was performed at 2,000 g for 4 and 30 minutes, and the supernatant was collected. This supernatant alone was further centrifuged at 20,000 g for 4 ⁇ for 30 minutes. The column was washed by adding 10 ml of QB.
  • the supernatant was added to the column, and the column was washed twice with 30 ml of QC. Then, it was purified with 15 ml of QF, and 0.7 volume of isopropanol was added to the purified solution. This mixture was centrifuged at 15,000 g and 4 at 30 minutes for 30 minutes, and the obtained precipitate was washed with 70% ethanol, dried for 5 minutes and dissolved in 1 ml of sterilized water. This plasmid was cut with HindIII and subjected to electrophoresis. The nucleotide sequence of the thus obtained fragment was analyzed according to a conventional method.
  • the bacteriophages # 1, # 2, # 3 and # 4 according to the present invention had the following sequences, respectively.
  • a fragment having a partial sequence as shown in SEQ ID NOs: 1-1 to 1-4, SEQ ID NOs: 2-1 to 2-10, SEQ ID NOs: 3-1 to 3-5 and SEQ ID NOs: 4-1 to 4-5 It was confirmed that each had the contained DNA.
  • the present invention provides bacteriophages having high specificity for a specific kind of pathogenic bacteria, and uses such bacteriophages to produce foodstuffs such as foodstuffs such as fresh foods.
  • foodstuffs such as foodstuffs such as fresh foods.
  • the place where food is stored, cooked, processed, and the like, as well as the birds handling such foods can be reliably prevented from being infected with the pathogenic bacteria, and can be easily used for a wide range of applications. Extremely useful.
  • the use of the phage according to the present invention disinfects not only the infected place but also the place and the article which are expected to be infected, thereby easily and surely sterilizing the infectious bacteria. It can be performed in a short time and is extremely useful.
  • the present invention provides bacterio phages having high specificity for pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli among pathogenic bacteria of a specific kind, thereby providing food products such as fresh foods.
  • pathogenic Escherichia coli especially intestinal bleeding Escherichia coli, from preserving, cooking, and processing such foods, etc., and humans handling such foods.
  • the pathogenic Escherichia coli can be removed by disinfecting infected items and places or suspected infection. It is surely easy to kill in a short time and is extremely useful.
  • the phage can be used to quickly and reliably reconstitute the corresponding pathogenic colon II in a short time. Since it can be detected in a short time, it is very easy and reliable to determine the presence or absence of the infection and the source of the infection.
  • the present invention provides a novel pacteriophage, thereby increasing the number of pathogenic bacterial rice plants to be used as host cells by 3%, and can cope with more rice-producing bacterial strains. As a result, a wide range of measures can be taken in infection prevention and treatment.
  • Pacteriophages having high specificity for specific pathogenic bacteria, especially pathogenic Escherichia coli, as described above, can be used alone, but phage with different properties are used.
  • the use of a cocktail in which two or more types are mixed makes it possible to simultaneously cope with other types of pathogenic cells, which is extremely convenient and advantageous.
  • the present invention also provides such a novel method for screening pateriophages.
  • the screening method has an effect that a novel phage having extremely high specificity for pathogenic bacteria can be reliably and easily screened.
  • the present invention provides a novel bactericidal material containing such a novel bacterium phage, and the effect achieved by providing the phage as described above can be practically used.
  • a novel bio-sterilized material for example, food products such as fresh foods, places where such food products are stored, prepared, processed, equipment, etc., and those food products, etc. It can be used for handling humans, etc., and can reliably prevent infection with pathogenic bacteria including pathogenic colon bacilli such as enterohemorrhagic colon ⁇ . Even if infection is expected, the pathogenic bacteria can be sterilized easily, in a short time, by disinfecting the infected article or place or the suspected article or place. There is a big effect that it can be done.
  • the novel biocidal material can be stably stored for a long period of time while maintaining the action of the phage. It is.
  • the novel biocidal IS material to contain a plurality of novel bacteriophages according to the present invention, the biocidal material can simultaneously cope with multiple types of pathogenic bacteria. There is a big effect that.
  • the phage can be produced in a large amount and at low cost.
  • the novel bacteriophage according to the present invention can be produced. This is extremely advantageous in commercializing a new bio-sterilizing material containing.
  • the addition of gold ions such as calcium ions to the medium in which the phages are cultured has the effect of promoting the growth of phages requiring such metal ions and hastening the increase of the phages. .
  • the present invention provides a stabilizing agent or a preservative that enables stable storage of the novel Bataterio phage, whereby the phage can be stored stably for a long period of time.
  • the novel bacterio phage according to the present invention which is prepared by adding the phage stabilizing solution, also has a great effect that it can be stably stored for a long time.
  • the present invention provides a method for detecting a pathogenic bacterium and a kit for detecting the pathogenic bacterium, whereby a pathogenic bacterium serving as a host can be easily and quickly obtained using the phage according to the present invention. It is extremely useful because it can be detected in a collision. By using this detection method and the reagent kit for detection, even if urgent measures such as suspected infection by pathogenic bacteria are required, the suspected pathogenic bacteria can be collected at the site where the sample was collected.
  • the use of only the detection reagent kit to determine whether or not the sample is present means that the assay can be reliably performed in a short period of time without using any other measurement equipment for detection. There are advantages.
  • this method for detecting pathogenic bacteria can be performed by increasing the amount of at least one pathogenic bacterium to be detected, even if the amount of the specimen is very small. Since phagocytic bacteria act as a host, it is an extremely sensitive method that can detect the bacteria.
  • the reagent kit for detecting pathogenic bacteria using this method for detecting pathogenic bacteria is extremely limited to a normal container such as a petri dish containing an agar medium containing phage.
  • the kit can be used with a simple configuration.For detection, the kit needs to be released in an environment where bacteria grow and spread for a short period of time, that is, only for the time required for phage to phagocytize the fine sprout. It is also a great advantage that it is not necessary at all.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • GACTGGGAGC 300 CAGTNAAAAC AGT AAACTG TGGAAGTACT ATTACAAGCA AGGTGNTTAC 350 CACTTITGAG CTTTGAGTCA AGTCGCTACA CAAGGATTGA TTGATGCANC 400 ATATACACCA ATTGAGTTTG AAGTTTCNCC GTATGACTCA NGTAGCAATT 450 GTTAAAGACT ACTTGAAATC AGTTGGGTGG ATTCCAGATG ACTGGAACTA 500 CAAGAAAGAT TCAGACGGTC GCCCTGTC 528 SEQ ID NO: 3-5
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Virology (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un bactériophage qui présente une très grande spécificité envers des bactéries pathogènes spécifiques et qui peut donc exterminer efficacement par phagocytage les bactéries hôtes. L'invention se rapporte en outre à des matières biobactéricides nouvelles, qui sont préparées avec ledit phage et qui sont utilisées pour stériliser tout ce qui doit être protégé d'une infection par bactéries pathogènes, par exemple des denrées alimentaires, telles que des produits frais, et des cuisines de restaurants ou d'écoles, afin d'exterminer lesdites bactéries. Ces matières biobactéricides peuvent contenir un mélange de deux ou plusieurs bactériophages aux propriétés différentes. De telles matirères sont très utiles, étant donné que deux ou plusieurs bactéries pathogènes peuvent être exterminées au même temps. Les bactéries pathogènes non humaines hôtes sont exclusivement infectées par ledit phage, qui est donc tout à fait sans danger. Il est en outre puissamment efficace. Le bactériophage présentant une spécificité exclusivement envers des bactéries pathogènes spécifiques constitue par conséquent une matière biobactéricide extrêmement sûre, qui est utilisée pour protéger par exemple des denrées alimentaires, telles que des produits frais, et des cuisines de restaurants ou d'écoles d'une infection par des bactéries pathogènes. L'invention concerne également un procédé de criblage facile dudit phage, un procédé de fabrication dudit phage, un stabilisateur servant à stabiliser ledit phage ou un agent conservateur dudit phage ainsi qu'un réactif et un kit de réactif permettant de détecter aisément les bactéries pathogènes en peu de temps au moyen du phage.
PCT/JP1997/002957 1996-08-26 1997-08-26 Nouveau bacteriophage et procede de criblage associe, nouvelles matieres biobactericides preparees avec ledit bacteriophage et reactif utilise pour detecter celui-ci WO1998008944A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US09/242,901 US6322783B1 (en) 1996-08-26 1997-08-26 Bacteriophages, method for screening same and bactericidal compositions using same, and detection kits using same
AU38692/97A AU3869297A (en) 1996-08-26 1997-08-26 Novel bacteriophage, method for screening the same, novel biobactericidal materials prepared with the use of the same, and reagent for detecting the same

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP8/261132 1996-08-26
JP26113296 1996-08-26
JP9/130236 1997-04-14
JP13023697A JP2002335956A (ja) 1997-04-14 1997-04-14 新規ファージ保存液
JP13571697 1997-04-19
JP9/135716 1997-04-19

Publications (1)

Publication Number Publication Date
WO1998008944A1 true WO1998008944A1 (fr) 1998-03-05

Family

ID=27316080

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1997/002957 WO1998008944A1 (fr) 1996-08-26 1997-08-26 Nouveau bacteriophage et procede de criblage associe, nouvelles matieres biobactericides preparees avec ledit bacteriophage et reactif utilise pour detecter celui-ci

Country Status (2)

Country Link
AU (1) AU3869297A (fr)
WO (1) WO1998008944A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998047521A1 (fr) * 1997-04-24 1998-10-29 Idaho Research Foundation, Inc. Phages, procedes de culture et de detection de phages, et utilisation de phages
US6656463B2 (en) * 2000-11-13 2003-12-02 Iowa State University Research Foundation, Inc. Compositions and methods for reducing the amount of salmonella in livestock
US7232564B2 (en) 2001-07-18 2007-06-19 Instytut Immunologii I Terapii Doswiadczal-Nej Pan Methods of polyvalent bacteriophage preparation for the treatment of bacterial infections
WO2010036132A1 (fr) 2008-09-29 2010-04-01 Instytut Immunologii i Terapii Doświadczalnej PAN Nouvelles souches de bactériophage pour le traitement d’infections bactériennes, en particulier des souches pharmacorésistantes du genre enterococcus
US7972773B2 (en) 2002-04-12 2011-07-05 Colorado School Of Mines Method for detecting concentrations of a target bacterium that uses phages to infect target bacterial cells
WO2011162626A1 (fr) 2010-06-20 2011-12-29 Instytut Immunologii i Terapii Doświadczalnej PAN Nouvelles souches de bactériophages pour le traitement d'infections bactériennes, en particulier par des souches de bactéries pharmacorésistantes du genre stenotrophomonas
US8092990B2 (en) 2005-03-31 2012-01-10 Colorado School Of Mines Apparatus and method for detecting microscopic organisms using bacteriophage
US8216780B2 (en) * 2002-04-12 2012-07-10 Microphage (Tm) Incorporated Method for enhanced sensitivity in bacteriophage-based diagnostic assays
CN103271411A (zh) * 2013-06-19 2013-09-04 太仓市荣德生物技术研究所 一种高效防腐剂
US8821855B2 (en) 2005-01-10 2014-09-02 Omnilytics, Inc Methods for isolating phage and for controlling microorganism populations with the phage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J. FOOD PROT., Vol. 53, No. 11, (1990), A.B. RONNER et al., "Isolation and Characterization of a Coliphage Specific for Escherichia Coli o157:H7", pages 944-947. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998047521A1 (fr) * 1997-04-24 1998-10-29 Idaho Research Foundation, Inc. Phages, procedes de culture et de detection de phages, et utilisation de phages
US6656463B2 (en) * 2000-11-13 2003-12-02 Iowa State University Research Foundation, Inc. Compositions and methods for reducing the amount of salmonella in livestock
US7232564B2 (en) 2001-07-18 2007-06-19 Instytut Immunologii I Terapii Doswiadczal-Nej Pan Methods of polyvalent bacteriophage preparation for the treatment of bacterial infections
US7972773B2 (en) 2002-04-12 2011-07-05 Colorado School Of Mines Method for detecting concentrations of a target bacterium that uses phages to infect target bacterial cells
US8216780B2 (en) * 2002-04-12 2012-07-10 Microphage (Tm) Incorporated Method for enhanced sensitivity in bacteriophage-based diagnostic assays
US8821855B2 (en) 2005-01-10 2014-09-02 Omnilytics, Inc Methods for isolating phage and for controlling microorganism populations with the phage
US8092990B2 (en) 2005-03-31 2012-01-10 Colorado School Of Mines Apparatus and method for detecting microscopic organisms using bacteriophage
WO2010036132A1 (fr) 2008-09-29 2010-04-01 Instytut Immunologii i Terapii Doświadczalnej PAN Nouvelles souches de bactériophage pour le traitement d’infections bactériennes, en particulier des souches pharmacorésistantes du genre enterococcus
WO2011162626A1 (fr) 2010-06-20 2011-12-29 Instytut Immunologii i Terapii Doświadczalnej PAN Nouvelles souches de bactériophages pour le traitement d'infections bactériennes, en particulier par des souches de bactéries pharmacorésistantes du genre stenotrophomonas
CN103271411A (zh) * 2013-06-19 2013-09-04 太仓市荣德生物技术研究所 一种高效防腐剂

Also Published As

Publication number Publication date
AU3869297A (en) 1998-03-19

Similar Documents

Publication Publication Date Title
US6322783B1 (en) Bacteriophages, method for screening same and bactericidal compositions using same, and detection kits using same
Nakayinga et al. Xanthomonas bacteriophages: a review of their biology and biocontrol applications in agriculture
Baert et al. The efficacy of preservation methods to inactivate foodborne viruses
Hudson et al. Bacteriophages as biocontrol agents in food
Cooper A review of current methods using bacteriophages in live animals, food and animal products intended for human consumption
Ye et al. Control of Salmonella on sprouting mung bean and alfalfa seeds by using a biocontrol preparation based on antagonistic bacteria and lytic bacteriophages
CN105209610B (zh) 噬菌体、包括其的组成物及其应用
CN105008525A (zh) 新颖噬菌体和包括其的抗细菌组合物
CN113583971B (zh) 一株可同时裂解大肠杆菌的沙门氏菌噬菌体及其应用
US10849942B2 (en) Treatment of bacterial infections in aquaculture
US8685697B1 (en) Listeria monocytogenes bacteriophages and uses thereof
Dhowlaghar et al. Biofilm formation by Salmonella spp. in catfish mucus extract under industrial conditions
Ngene et al. Bacteriophages as Bio-control agent against Food-Borne Pathogen E. coli O157: H7
CN101115393A (zh) 处理食品的方法
WO1998008944A1 (fr) Nouveau bacteriophage et procede de criblage associe, nouvelles matieres biobactericides preparees avec ledit bacteriophage et reactif utilise pour detecter celui-ci
KR102193499B1 (ko) 신규한 바실러스 세레우스 균 특이 박테리오파지 bc13 및 이를 포함하는 항균 조성물
Ahmed et al. Bacteriophage therapy revisited
KR101830900B1 (ko) 신규한 조류 병원성 대장균 특이 박테리오파지 ec13 및 이를 포함하는 항균 조성물
KR102003773B1 (ko) 신규한 비브리오균 특이 박테리오파지 vp4 및 이를 포함하는 항균 조성물
KR102037383B1 (ko) 신규한 비브리오균 특이 박테리오파지 vp2 및 이를 포함하는 항균 조성물
JP2002335957A (ja) 新規なバクテリオファージならびにそのスクリーニング法 およびそれを使用した新規バイオ殺菌材料ならびに検出用 試薬
KR102037398B1 (ko) 신규한 살모넬라균 특이 박테리오파지 sg102 및 이를 포함하는 항균 조성물
KR101842668B1 (ko) 신규한 살모넬라균 특이 박테리오파지 sg2 및 이를 포함하는 항균 조성물
KR20190069901A (ko) 신규한 비브리오균 특이 박테리오파지 vp2 및 이를 포함하는 항균 조성물
Islam et al. Determination of optimum survivability factors of highly pathogenic Vibrio cholerae 01 serogroup specific bacteriophage JSF4ϕ

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA KR NZ US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

WR Later publication of a revised version of an international search report
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 09242901

Country of ref document: US

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载