JP2002335957A - New bacteriophage, method for screening the same, new biobactericidal material prepared by using the same and reagent for detecting the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain both a bacteriophage having a high specificity for enterohemorrhagic Escherichia coli and a biobactericidal material prepared by using the phage, to provide a method for simply screening a phage and to obtain both a reagent for simply detecting a pathogenic bacterium in a short time by using the phage and a reagent kit. SOLUTION: The bacteriophage has a DNA containing a fragment with a base sequence represented by sequence number 1-1 to 1-4, sequence number 2-1 to 2-10, sequence number 3-1 to 3-5 or sequence number 4-1 to 4-5 (referred to the specification), respectively. The bacteriophage is infected with a pathogenic bacterium in a restriction enzyme minus and modified enzyme positive state, amplified, treated at a high temperature, consequently only a bacteriophage containing a deleted DNA is selected to screen the bacteriophage. The biobactericidal material may contain a cocktail of two or more kinds of bacteriophages different in properties so as to sterilize two or more kinds of pathogenic bacteria at the same time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] 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 bacteriophage having high specificity for various pathogenic bacteria including pathogenic Escherichia coli, a screening method thereof, and a novel bio-sterilizing material using such a bacteriophage alone or as a cocktail, The present invention relates to a reagent or reagent kit for detecting pathogenic bacteria using the stabilizer and bacteriophage.

[0002]

2. Description of the Related Art Various types of bacteria are distributed in nature, and many of them carry out important life phenomena such as spoilage and fermentation. Among these bacteria, so-called pathogenic bacteria exist, and the pathogenic bacteria infect animals such as humans, pets such as dogs and cats, as well as various plants, causing disease. Many bacteria which cause disease such as pathogens or pathological symptoms, and which invade and proliferate in the body tissues of such animals and plants to cause disease are also known.
Escherichia coli, which is one of such pathogenic bacteria, is ubiquitous in nature and also a bacterium resident in the intestinal tract of humans and mammals. Almost all of such Escherichia coli usually have no harm to humans and mammals. However, among Escherichia coli, so-called pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli is present, and this pathogenic Escherichia coli may infect humans and mammals and cause serious results. Many types of fungicides are conventionally used to prevent infection from pathogenic bacteria such as pathogenic Escherichia coli or for treatment when infected with bacteria such as Escherichia coli. For example, iodine-containing fungicides such as iodine tincture and chlorhexidine-containing maquilon are often used for disinfecting wounds. Furthermore, benzoic acid and the like are often used as preservatives that can be added to foods. Various effective antibiotics have been used for the prevention or treatment of bacterial infections.

[0003] When there is a possibility that enterohaemorrhagic Escherichia coli is transmitted through vegetables such as raw cow liver, cayenne radish, lettuce, etc., as in the case of infection recently caused by pathogenic Escherichia coli, ie enterohemorrhagic Escherichia coli. In some cases, infections such as sashimi infections and rare steaks may be suspected. However, when it comes to fungicides that can be used to prevent infections from foodstuffs such as fresh foods, iodine tinctures and macirons cannot be used at all for such purposes due to toxicity problems. Furthermore, antibiotics used for the prevention or treatment of bacterial infections cannot be used at all for the purpose of protecting foodstuffs such as pathogenic Escherichia coli. Further, even if a safe bactericide is present, if it is used in fresh food, it is difficult to spray it evenly, so that a large concentration difference is produced, and local unevenness of the bactericidal effect occurs. This is significant because it implies a germicidal effect. Further, a substance having a so-called bacteriostatic action, such as polylysine, which can suppress the growth of bacteria without having a bactericidal action, is also known. In the case of polylysine, 0.5-1% It must be used at a high concentration, and when used in foodstuffs such as fresh food, a change in taste is expected, it is expensive, and uneven bacteriostatic action is also expected. As described above, there is no sterilizing agent that is harmless to humans and can be used for foods such as fresh foods.Therefore, sterilization of foods such as raw vegetables in school lunches requires disinfection with boiling water after all. It is currently being done. Therefore, there is a demand for the development of a sterilizing material that can be used for foodstuffs such as fresh foods and is safe for humans.

[0004] A bacteriophage (hereinafter, sometimes abbreviated simply as "phage") is composed of only a protein and a nucleic acid, each of which can be propagated only in a specific bacterium using a specific bacterium as a host and an inanimate organism. And is very small and can only be observed with an electron microscope. Bacteriophages are known to infect only bacteria using the bacterium as a host, and to increase the progeny by exhausting the infected bacterium. At present, a large number of bacteriophages using various bacteria as hosts are known. For example, Salmonella specific phages using Salmonella as a host and Vibrio specific phages using Vibrio as a host are known. Also, many phages that destroy phagocytosis using Escherichia coli as a host are known, and about 300 species are known so far, and many exist in the air. Both of these bacteriophages infect E. coli but are completely harmless to humans and other mammals. Similarly, phages using bacteria other than Escherichia coli as hosts are harmless because they do not infect humans or other mammals at all. However, such bacteriophages act only on bacteria, and further, have high specificity for host bacteria, and act only on one particular bacterium or only a small number of a plurality of particular bacteria. Are known. For example, phages that act on E. coli are no exception, and are not able to act on all or many different types of E. coli, and it is also known that the well-known T2 phage cannot act on C-type E. coli.
Thus, bacteriophages have been used only as research targets and have made great progress in genetic research, but their specificity is too high to use bacteriophages in industrial applications. Not to be noticed much. Therefore, it has never been considered to use such a bacteriophage as a bactericide. As described above, many bacteriophages using various bacteria as hosts are known, but no bacteriophage using pathogenic bacteria such as enterohemorrhagic Escherichia coli as a host has been known so far.

[0005] On the other hand, among bacteria which serve as hosts for bacteriophages, Escherichia coli, in particular, has an extremely strong growth ability, and
Divide once every 30 minutes to 30 minutes
E. coli grows to 1 gram per day. As described above, Escherichia coli is a bacterium that is ubiquitous in the natural world and also resident in the intestinal tract of humans and mammals, and usually has no harm to humans or mammals. Most of the so-called biopharmaceuticals are produced by culturing Escherichia coli by utilizing such properties of Escherichia coli. As described above, a bacteriophage using a pathogenic bacterium such as enterohemorrhagic Escherichia coli as a host has not been known, but screening can be performed by a conventional bacteriophage screening method. It can be said that an efficient and reliable screening method has not yet been established. Therefore, there is also a need to develop a screening method for efficiently and reliably screening such bacteriophages.

Further, it can be said that there is no effective bactericide which can be used directly for foodstuffs such as fresh foods. Therefore, there is a need to develop products that can be directly used for foodstuffs such as fresh foods. In developing such a bactericide, the bactericide may be used as a stabilizing agent so that it can be stored for a long period of time, particularly when used directly in foodstuffs such as fresh foods, while ensuring its safety. Alternatively, development of preservatives is also required. Hitherto, as a phage preservation solution, Tris-HCl buffer, phosphate buffer, and the like are well known, but these have been used only for research so far, and are directly used for fresh foods and other foods. Can not be used. In addition, no phage preservation solution that can be directly sprayed on fresh food and drinkable has been known.

In the past, when food poisoning or the like due to infection with a pathogenic bacterium occurs, it takes a certain number of days to identify the infectious pathogenic bacterium that causes the food poisoning. Therefore, in the case of food poisoning caused by infection with pathogenic bacteria such as enterohemorrhagic Escherichia coli, it is necessary to quickly identify the infecting bacteria in order to perform prompt treatment. Therefore, development of a detection reagent and a reagent kit that can detect pathogenic bacteria such as enterohemorrhagic Escherichia coli in a short time and reliably is also required.

[0008]

In order to meet the above-mentioned demands, the present inventors have made intensive efforts focusing on the specific action of bacteriophage as described above. In addition to finding a specific sterilizer, even when the sterilizing material containing the bacteriophage is used for food such as fresh food, such pathogenic Escherichia coli can be sterilized, and food such as fresh food can be used. Has been found to be safe.

Accordingly, an object of the present invention is to provide a bacteriophage exhibiting extremely high specificity only for pathogenic bacteria. Another object of the present invention is to provide a screening method capable of efficiently and reliably screening for bacteriophages having such high specificity.

Further, the present invention provides a bio-sterilizer containing a bacteriophage which can stabilize phage for a long period of time while ensuring extremely high safety even when the bacteriophage is directly used for foodstuffs such as fresh food. It is intended to provide materials. Furthermore, the present invention can stabilize the phage for a long period of time in such a bio-sterilized material containing a bacteriophage while ensuring extremely high safety even when the phage is used for foodstuffs such as fresh food. It is intended to provide a phage preservative.

[0011] In addition, the present invention provides a bacteriophage detection reagent or a bacteriophage detection reagent capable of efficiently, quickly and reliably detecting a host pathogenic bacterium by utilizing the phagocytic action of a bacteriophage. It is intended to provide a reagent kit.

The term "pathogenic bacterium" as used herein is a bacterium that infects animals such as humans and pets such as dogs and cats and gives the origin of diseases and pathological symptoms. In addition, it means a bacterium that is a host of a bacteriophage and is phagocytosed by the bacteriophage, and is to be understood as a collective term for pathogenic bacteria including, for example, pathogenic Escherichia coli including enterohemorrhagic Escherichia coli. It is. In this specification, Escherichia coli, which is a representative bacterium, will be described as a pathogenic bacterium. However, Escherichia coli is merely a representative of a pathogenic bacterium and is described as an example of a pathogenic bacterium. It should also be understood that the present invention is not limited to such an example.

[0013]

In order to achieve the above-mentioned object, the present invention provides a bacteriophage having high specificity only for pathogenic bacteria. The present invention also provides, in a preferred embodiment thereof, a bacteriophage having high specificity only for a certain kind of pathogenic Escherichia coli. Further, the present invention provides a novel bacteriophage having high specificity only for a certain kind of pathogenic Escherichia coli, thereby providing a bacterium useful for pathogenic bacteria including various pathogenic Escherichia coli. It is possible to respond to a wide range of requests using phage.

Furthermore, the present invention provides a screening method for efficiently and reliably screening for bacteriophages having high specificity only for pathogenic bacteria.

The present invention provides a novel biocidal material containing bacteriophages having high specificity only for pathogenic bacteria. Such a new bio-sterilizing material is
Utilizing the fact that the bacteriophage can phagolytically destroy potentially infectious pathogenic bacteria without causing any adverse effects on the human body, etc., it can be used directly on food products such as fresh foods. it can. In addition, the novel bio-sterilizing material provided by the present invention allows such a bacteriophage to consist of a cocktail of two or more different bacteriophages and to simultaneously address a plurality of different pathogenic bacteria. ing. Further, the novel bio-sterilizing material contains a stabilizer or preservative of a culture solution of bacteriophage which can stably store the bacteriophage for a long period of time. It can be stored for a long time while ensuring its safety and stability.

The present invention also provides a method for producing a culture solution of bacteriophage, which comprises obtaining a culture solution of bacteriophage by infecting and growing a bacterium which is a host in which the bacteriophage is cultured. Is what you do. As a preferred embodiment of the present invention, a method for producing a bacteriophage having high specificity for pathogenic bacteria by providing calcium ions in a medium for culturing pathogenic bacteria is provided.

The present invention further provides a bacteriophage culture stabilizer or preservative which can stably store a bacteriophage culture for a long period of time.

According to another aspect of the present invention, there is provided a pathogen capable of detecting a pathogenic bacterium using a bacteriophage having specificity with certainty, easily and in a short time. We provide a kit for detecting bacteria.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION (Bacteriophage) Bacteriophages are excreted from many animals such as domestic animals such as cattle, pets such as dogs and cats, birds such as pigeons and crows, and poultry such as chickens. It is widely known that these substances are present in such wastes, and it is also known that they can be separated from such wastes and sewage. Regardless of the origin, 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. And is not limited to a particular type of phage. That is,
As such a bacteriophage, any bacteriophage capable of adsorbing on a pathogenic bacterium serving as a host and specifically lysing and destroying the bacterium to achieve the object of the present invention can be used. it can. Examples of such bacteriophages include, for example, T-type phage (T1, T2, T3, T4, T4
5, T6, T7, etc.), φX-174, λ, φX80,
In addition to Qβ, P1, etc., naturally, a novel bacteriophage screened by the screening method according to the present invention can also be mentioned. In addition to Escherichia coli, SZP01, SP
Bacteriophage such as 02. Among such bacteriophages, in particular, SEQ ID NOs: 1-1 to 1-4 and SEQ ID NOs: 2-1 to 2 shown in the following sequence listing
-10, SEQ ID NOs: 3-1 to 3-5 or SEQ ID NO: 4
Bacteriophages having DNAs containing fragments having the nucleotide sequences represented by -1 to 4-5, respectively, are enterohemorrhagic Escherichia coli O157
Has high specificity for The DNA sequence of each of these bacteriophages was identified by the method described below.

(Bacteriophage Screening Method) Many bacteriophages which have been conventionally used for the study of molecular biology lyse pathogenic bacteria which are the subject of the present invention, particularly pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli. Turned out to be impossible. It is also possible to screen for bacteriophages with high specificity according to the present invention by conventional methods for screening such bacteriophages. However, a screening method capable of screening bacteriophages having high specificity against pathogenic bacteria, particularly pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli has not been established yet. Therefore, in order to enhance the efficiency and certainty of the screening, the following bacteriophage screening method was devised, and it was lysed with high specificity from nature, especially to pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli. Bacteriophages that can be screened. In the present invention, samples of various animal excrement and sewage are collected to screen for bacteriophages having high specificity for pathogenic bacteria considered to be present in the samples. First, sample
For example, E. coli K strains, B strains and C strains are infected with a modified enzyme positive (m ⁺ ) host with a restriction enzyme minus (r ⁻ ) to amplify the phage present in the sample.
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 high titer stock. Of these, only clear plaques, which were almost completely lysed, were selected. In the next step, only phages containing the naturally occurring deletion DNA were exposed to EDTA at a high temperature (55 ° C.-65 ° C.) for several hours. select. By repeating this operation several times, stable phages having only essential genes can be selected.

(Cultivation of bacteriophage) The novel method of culturing bacteriophage according to the present invention will be described by taking as an example the case of culturing bacteriophage using Escherichia coli as a host. First, Escherichia coli is cultured in a medium containing polypeptone, yeast extract, or the like at room temperature or by heating, using an incubator such as a jar fermenter until the number of Escherichia coli cells reaches a predetermined number. When the number of E. coli cells is a predetermined number, for example, 2-3 × 10 ⁸ / ml,
At this stage (eg, OD = 0.2), bacteriophage is added to the medium. When the cultivation is further continued under the same conditions as before after adding the bacteriophage, the bacteriophage infects E. coli, and the bacteriophage grows in the E. coli cells and completely destroys the E. coli. As a result, if the cultivation is continued for a predetermined time, Escherichia coli is no longer alive in the culture solution, and a culture solution of bacteriophage is obtained. This culture solution is purified according to a standard method,
Escherichia coli residues and the like can be removed by a conventional method such as centrifugation to obtain a bacteriophage culture solution. Further, the bacteriophage according to the present invention can also be cultured using an agar medium containing, for example, polypeptone, yeast extract and the like. In this case, cultivation can also be performed by using double agar medium having different concentrations, adding Escherichia coli and phage to the upper agar medium, and culturing at room temperature or a heated temperature. The phage cultured in this manner is dissolved, for example, by adding the agar medium to the phage stock solution or the like, dissolving the mixture, separating the mixture from solids such as culture residue by centrifugation, and separating the supernatant. Thus, the supernatant can be used as a stock solution. In other words, the surface of Escherichia coli has a receptor structure to which bacteriophage is adsorbed. inject. When such DNA or RNA is injected into the cells, the bacteriophage phagocytoses Escherichia coli and grows in the cells within a short time. When bacteriophages grow in the cells,
The E. coli is completely destroyed, so that the bacteriophage grows in the culture and becomes a bacteriophage culture. This mechanism is the same when using the bio-sterilizing material of the present invention containing bacteriophage to kill pathogenic bacteria. In this bacteriophage culture method, when culturing a bacteriophage using Escherichia coli as a host with pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli, a trace amount of metal such as magnesium, manganese, or calcium is added to the medium for culturing the Escherichia coli. Is preferably added, and it may be necessary to make calcium ions particularly present in the medium. As the agent to be used, any agent that releases calcium ions in a medium for culturing Escherichia coli or the like and does not adversely affect Escherichia coli or bacteriophage to be cultured can be added. Examples thereof include calcium chloride.

(Stabilization of the phage stock solution) In order to stably store the bacteriophage according to the present invention cultured as described above for a long period of time, a buffer solution containing salts, Mg, A trace amount of metal such as Mn and Ca can be added. In order to further enhance stabilization, glycerol is added in an amount of about 0.001 to 5%, preferably 0.1%.
It can be added to about -1%. Furthermore, sugars,
For example, polysaccharides such as maltose and glucose, amino acids such as glycine, arginine and lysine, ethylparaben and polylysine can be added. The novel phage stock according to the present invention uses amino acids such as glycine, arginine and lysine, preferably glycine, in order to stabilize the bacteriophage contained therein while ensuring high safety for food. Is good. In this case, the amino acid is a buffer containing 10 mM to 1 M, preferably 50 mM to 500 mM, at pH6.
-8, preferably pH 6.5-7.5, to which 5% sodium chloride, preferably 0.1%, if necessary.
It is also possible to add calcium chloride in the range of 03% -1%, 10 mM, preferably 0.1-1 mM.
As described above, the novel phage preservation solution according to the present invention comprises:
The phage is not inactivated even when diluted up to about 100 times with ordinary tap water, and can exert a sufficiently powerful phagocytosis-destroying effect. The same is true even when diluted with household alkaline water or acidic water, and the phage contained therein is not deactivated, and a sufficient phagocytosis-destroying effect is exhibited.

(Novel Bio-sterilizing Material) A novel bio-sterilizing material using the bacteriophage according to the present invention can be prepared as follows. That is, the culture solution of the bacteriophage cultured as described above is separated from the culture residue by a conventional method such as centrifugation, and the culture solution is prepared as a stock solution of the novel biosterile material according to the present invention. In the novel bio-sterilized foodstuff according to the present invention,
Bacteriophage, for example, 10 ² cells / ml to ^{10 12} cells / ml, and preferably contains 10 ³ cells / ml 1
0 may be a any concentration in the range of ⁸ / ml. In addition, the novel bio-sterilizing material according to the present invention can contain such bacteriophage alone or in a cocktail of two or more kinds,
The novel bio-sterilizing material according to the present invention can be used by appropriately changing the combination according to the purpose of use. In this case, of course, the pathogenic bacteria hosting each of the combined cocktails of bacteriophages can be simultaneously phagocytosed. Since the concentration of the phage is usually too high, the stock solution of the novel biosterile material thus obtained is used after diluting the stock solution to an appropriate ratio. In this case, it is preferable to dilute with the phage preservation solution described above, but it is also possible to dilute with tap water or the like. Further, in the present invention, by using as a cocktail of two or more kinds of bacteriophages having different properties, it is also possible to suppress the emergence of resistant bacteria due to high frequency use. For example, in the present invention, in order to suppress the emergence of resistant bacteria at the time of frequent use in the sterilization of pathogenic Escherichia coli, for example, T
When a cocktail in which equal amounts of 2 phage and λ phage were mixed was used as a bactericidal material, the frequency of emergence of each resistant bacterium was 10 ⁻⁶ , whereas the frequency of emergence of resistant bacterium when the above cocktail was used. Is 10 ⁻¹² , which makes it possible to virtually eliminate the appearance of resistant bacteria theoretically and experimentally. Further, in order to stabilize the novel bio-sterilizing material according to the present invention, a fungicide for food, for example, benzoic acid is added to 0.1%.
It can be added at about 002% -2%, preferably about 0.1% -0.3%. Furthermore, since the novel bio-sterilizing material according to the present invention has no odor or taste, a flavor such as lemon may be added when used with food.

The mechanism of action of the bactericidal material according to the present invention will be described with reference to pathogenic Escherichia coli as an example. When bacteriophages are cultured, the mechanism is the same as when phages grow while phagocytosing Escherichia coli. . That is, the surface of Escherichia coli has a receptor structure to which bacteriophage is adsorbed, and through this, the bacteriophage is adsorbed to the bacteria and its own gene, DNA or RN
A or the like is injected into the cells of Escherichia coli. Such DNA or R
When NA or the like is injected into the cells, the bacteriophages phagocytose E. coli within a short period of time and grow in the cells. When the bacteriophage multiplies in the cells, E. coli is completely destroyed and, as a result, is killed. Therefore,
The germicidal material according to the present invention has a completely different mechanism of action from other conventionally used germicides. The bacteriophage used in the present invention is relatively strong in the air, but is weak against gastric acid and the like because it does not have a cell membrane unlike Escherichia coli, and when ingested orally, it is inactivated in gastric juice. , Followed by digestion and absorption. Therefore, the bactericide according to the present invention containing the bacteriophage can be said to be a germicidal material or bactericidal material 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 new bio-sterilizing material containing bacteriophage.

The novel bio-sterilizing material of the present invention is bactericidal and proliferates because of its bactericidal action by the above-mentioned action mechanism. Therefore, it is not affected by the unevenness in concentration. Can be sterilized by phagocytosis over time. Therefore, there is an advantage that the substance can be used at a very low concentration.

(Reagent for Bacteriophage Detection and Reagent Kit Thereof) The bacteriophage according to the present invention can be used as a reagent for detecting pathogenic bacteria serving as its host. Since this phage can phagocytose and destroy the host pathogenic bacteria, a reagent for detecting the host pathogenic bacteria is prepared using the phagocytosis of this phage. can do. As a reagent kit using this phage,
There are various types, and examples thereof include a reagent kit in which an agar medium on which phage can be cultured is injected into a container such as a petri dish. When a reagent kit of this type is used, a sample containing a specimen that may contain the pathogenic bacteria to be detected is applied to the surface of the agar medium, and cultured at an appropriate temperature. When the pathogenic bacteria which host the contained phage are present, the phage will be phagocytosed. The traces of this phagocytosis cause the medium to be lysed and become transparent or translucent. When the agar medium is in such a state, the sample examined contains pathogenic bacteria corresponding to the phage included as a detection reagent in the agar medium, and the corresponding pathogenic bacteria are detected. It will be. For example, when the pathogenic bacterium to be detected is pathogenic Escherichia coli, the Escherichia coli is extremely short in time to divide and proliferate. Is applied to an agar medium serving as the above detection reagent kit, and if about 30 minutes have passed, it can be determined whether or not Escherichia coli to be detected is present. That is, using the phage according to the present invention, for example, when detecting Escherichia coli, the detection can be performed in a short time of about 30 minutes, which is extremely convenient. In addition, the use of this detection reagent or its reagent kit makes it possible to reliably detect phagocytosis of phage even if the pathological bacteria to be detected are extremely small in the sample to be tested. It is very useful. By using phage having two or more different properties as such a detection reagent or a reagent kit thereof, different types of pathogenic bacteria can be simultaneously detected, which is extremely useful and convenient. Therefore, the reagent for detecting pathogenic bacteria or the reagent kit thereof according to the present invention is brought into contact with a specimen to be tested, for example, in a petri dish containing an agar medium containing phage, at a temperature at which the pathogenic bacteria can grow. Since the presence or absence of the pathogenic bacterium can be determined simply by leaving it in an appropriate place, no other measuring instruments or devices for detection are required, and the method of use is extremely simple. Therefore, the reagent for detecting pathogenic bacteria and the reagent kit therefor according to the present invention can be easily used, for example, for the primary screening for detecting pathogenic bacteria.

The novel bio-sterilizing material according to the invention can also be applied in various ways of use. The novel bio-sterilizing material according to the present invention can be used for any place where pathogenic bacteria can be present, or any article that can be infected with pathogenic bacteria, and those articles are pathogenic bacteria. It can protect against infection or test for contamination by such pathogenic bacteria. That is, the novel bio-sterilizing material according to the present invention is:
For example, it can be directly used by spraying on foodstuffs such as fresh foods, dipping the foodstuffs therein, or washing it with it. Therefore, the novel bio-sterilizing material can be used at any stage such as preservation and cooking of foods such as fresh foods so that the foods and the like are not contaminated by pathogenic bacteria. In addition, the novel bio-sterilizing material according to the present invention must be kept clean, for example, in a kitchen, kitchen, storage room, etc., in a place such as a hospital where cleanliness should be maintained, or in aprons, white coats and other clothes. By spraying directly onto an article or the like, the place or the article can be prevented from being contaminated by pathogenic bacteria. Furthermore, the novel bio-sterilizing material can be used as a washing liquid to prevent pathogenic bacteria from entering the body or infecting others through the body such as hands.
Furthermore, the novel bio-sterilizing material can be used, for example, in the cultivation or production process of fresh foods and the like. For example, spraying on foodstuffs such as fresh food, mixing and adding to the cultivation medium of cultivated vegetables such as kai radish, spraying on cultivated vegetables such as kai radish, or especially in hydroponics It can be used by putting a predetermined concentration in water. The novel bio-sterilizing material can also be used for the purpose of sterilization, for example, in a fish market, a place where fresh food is processed such as a meat processing plant, or a breeding place for livestock such as a barn.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are described only for illustrative purposes of the present invention,
It is not intended to limit the invention in any way, and all modifications and alterations that fall within the scope and spirit of the invention should be construed as being included within the scope of the invention.

[0029]

EXAMPLES (Example 1) Screening of bacteriophage Samples were collected from feces of animals such as pigeons, crows, cattle, dogs, cats, poultry, and sewage. About 1g of each sample
In the case of sewage, 5 to 10 ml was dissolved in an LB culture solution, and the residue was precipitated by centrifugation to obtain a supernatant. First, a phage presumed to be present in a small number in this supernatant was infected with a modified enzyme positive (m ⁺ ) host using Escherichia coli K, B, and C strains with a restriction enzyme minus (r ⁻ ) to amplify the phage. . The lysate was plated on O157 to separate single plaques. Thereafter, several rounds of amplification were performed to obtain a high titer stock. Of these, only clear plaques that were almost completely lysed were selected and, as the next step, were exposed to 5-10 mM EDTA at high temperature (55 ° C.-65 ° C.) for several hours, and the naturally occurring deletion DNA was removed.
Only phages containing were selected. By repeating this operation several times, stable phages having only essential genes could be selected. A large number of phages were isolated by screening in this manner. As a result of investigating the effects of these phages on a plurality of types of enterohemorrhagic O157, about four types of phages having strong effects were selected and bacteriophages # 1, # 2, and # 3 were selected.
And # 4. DN for these phages
The A sequence was determined as follows. (Example 2) Bacteriophage preparation method Escherichia coli was cultured in a 1-liter jar fermenter using an E. coli culture medium (Elbros) containing polypeptone, yeast extract, and the like, and the absorbance was 0.2 (the number of E. coli cells 2). (-3 × 10 ⁸ / ml), a phage that specifically destroys only pathogenic Escherichia coli O157 obtained by screening in Example 1 was added so as to have a Moi of 20 (MOI = 20). 8-261132), 37 ° C
For 4 hours or more. When the culture broth became slightly clear, add a few drops of chloroform and further at 37 ° C for 1 hour.
After culturing for 0 minutes, the mixture was centrifuged at 8,000 rpm for 30 minutes to obtain a supernatant. The obtained supernatant was subjected to aseptic filtration with a 0.45 micron Millipore filter to obtain a phage which specifically destroys only pathogenic Escherichia coli O157 in phagocytosis, and stored at 4 ° C. (Example 3) (Preparation of a new phage stock solution) (1) 15 g of glycine and 0.5 g of CaCl ₂ .H ₂ O
mM, 0.5% NaCl, 0.02% glycerol
And pH is adjusted to 6.8-7.0 with NaOH.
Was adjusted. Thereafter, the volume was adjusted to 1,000 ml with pure water, and sterilized in an autoclave at 121 ° C. for 15 minutes to prepare a new phage stock solution. (2) After adding 15 g of glycine, 0.5% of NaCl and 0.02% of glycerol, NaOH was added.
Adjust to pH 6.8-7.0 with 1,000 ml with pure water
After that, the mixture was sterilized in an autoclave at 121 ° C. for 15 minutes to prepare a new phage stock solution. (3) 15 g of glycine and 0.5 of CaCl ₂ .H ₂ O
After adjusting the pH to 6.8-7.0 with NaOH, the mixture was adjusted to 1,000 ml with pure water. Then, in an autoclave,
Sterilization was performed at 21 ° C. for 15 minutes to prepare a new phage stock solution. (4) 15 g of glycine and 0.5 of CaCl ₂ .H ₂ O
mM and NaCl to 0.5%.
After adjusting the pH to 6.8-7.0 with OH, the solution was adjusted to 1.0 with pure water.
It was made up to 00 ml. Then, 121 ° C. in an autoclave,
A new phage stock solution was prepared by performing a sterilization treatment for 15 minutes. (5) CaCl ₂ .H ₂ O was added to 0.5 mM, NaCl to 0.5%, and glycerol to 0.02%, and adjusted to pH 6.8-7.0 with NaOH. Make up to 1,000 ml with water. Thereafter, the mixture was sterilized in an autoclave at 121 ° C. for 15 minutes to prepare a new phage stock solution. (6) 7.5 g of glycine and 0.1 g of CaCl ₂ .H ₂ O
5 mM, 0.5% NaCl, 0.02 glycerol
%, And pH 6.8-7.
Adjusted to zero. Then, make it 1,000 ml with pure water,
A new phage stock solution was prepared by performing a sterilization treatment at 121 ° C. for 15 minutes in an autoclave. (7) 0.2M arginine and 0% CaCl ₂ .H ₂ O
5 mM, 0.5% NaCl, 0.02 glycerol
%, And pH 6.8-7.
Adjusted to zero. Then, make it 1,000 ml with pure water,
A new phage stock solution was prepared by performing a sterilization treatment at 121 ° C. for 15 minutes in an autoclave. (8) Lysine 0.2M, CaCl ₂ .H ₂ O 0.5m
M, 0.5% of NaCl and 0.02% of glycerol were added, and the pH was adjusted to 6.8 to 7.0 with NaOH. Thereafter, the volume was adjusted to 1,000 ml with pure water, and sterilized in an autoclave at 121 ° C. for 15 minutes to prepare a new phage stock solution. Example 4 Method for Preparing New Bio-Sterilized Material The bacteriophage prepared in Example 1 was multiplied by a known method using host Escherichia coli.
A 0-fold increase was measured. In other words, E. coli that has reached the logarithmic growth phase is subjected to MOI (Multiplicity of In).
A bacteriophage was infected with the F.Fection 10 and chloroform was added when the cells were lysed about 100 minutes later. Then, bacterial debris (DNA, RNA, protein, cell membrane, etc.) was removed by centrifugation to prepare a crude bacteriophage product. The crude product obtained is about 10
It had an infectivity of ⁹ / ml. 0.1-0.01% of glycerol, a stabilizer such as lauroyl sarcosine salt and salts such as benzoate are added to the obtained bacteriophage sample, and Mg, Mn or Ca is used as a metal.
Was added to prepare a new biosterile material. When this sterilized material was stored in a cold room (4 ± 1 ° C.), the bacteriophage titer could be stored stably without inactivation for 6 months. In preparing a new bio-sterilizing material, bacteriophage was prepared basically using two different species so that no residual bacteria appeared. For example, when T2 phage and λ phage were used, the appearance frequency of resistant bacteria was 10 ⁻⁶ × 10 ⁻⁶ = 10 ⁻¹² , which was no problem theoretically or experimentally. As a result, by using the bio-sterilizing material of this example, harmful bacteria including pathogenic Escherichia coli could be completely eliminated.
In addition, it was possible to eliminate the reduction of the germicidal effect due to frequent use. Example 5 Method for Preparing New Bio-Bacterial Material Bacteriophages were grown in a known manner using host Escherichia coli, and an approximately 100-fold increase was measured in each step. That is, MOI (Mul) was added to E. coli that reached the logarithmic growth phase.
Tipicity of Infection) 10
Then, bacteriophage was infected, and after lysis about 100 minutes, chloroform was added. Then, bacterial debris (DNA, RNA, protein, cell membrane, etc.) was removed by centrifugation, and the supernatant bacteriophage was purified.
Purification was performed by precipitation with polyethylene glycol and CsCI.
About 10 ¹² / ml by density gradient centrifugation
Infectious preparation was obtained. 0.1-0.01% of the obtained bacteriophage sample is added with a stabilizer such as salts such as lauroyl sarcosine salt and benzoate, and Mg, Mn or Ca is added as a metal to produce a new biosterilizer. Materials were prepared. This sterilizing material is placed in a cold room (4
(± 1 ° C.), the bacteriophage titer could be stably stored without inactivation for 6 months. The biosterile material was prepared in the same manner as in Example 1. As a result, the same functions and effects as those of the first embodiment were obtained. Depending on the purpose of use, the concentration was adjusted so that the bacteriophage became MOI: 10 with respect to the bacteria. These operations and storage were performed in a low-temperature room (4 ° C. ± 1 ° C.) in principle. (Example 6) Preparation method of new bio-sterilized material Escherichia coli was added to an L-broth medium composed of 10 g of polypeptone, 3 g of yeast extract, and 2.5 g of NaCl, and cultured at 37 ° C in a jar fermenter. . Bacteriophage was added when the OD reached 0.2 in the spectrophotometer (MOI: 20). In addition,
At this stage, the number of E. coli cells is about 2-3 × 10 ⁸ / ml.
Met. After adding the bacteriophage, the cells were cultured in a jar fermenter at 37 ° C. for 4 hours with aeration, and several drops of chloroform were dropped when the culture solution became slightly transparent. Thereafter, the residue was removed by centrifugation at 8,000 rpm for 30 minutes to obtain a supernatant. This supernatant was used as a stock solution for the preparation of a sterilizing material. (Example 7) (1) Kit for detection of pathogenic Escherichia coli A 10% agar medium having the following composition was heated to 45 ° C and completely dissolved, uniformly poured as a lower layer medium on a petri dish, and left at room temperature. And solidified. 10% polypeptone 3% Yeast extract 2.5% NaCl 0.1% Glucose 5Mm CaCl ₂ 3 ml of 5% agar medium having the same composition separately prepared on the surface of this 10% agar underlayer medium was added to E. coli. coli O15
7: The mixture was added to 0.1 ml of a solution containing the H7 strain (the number of cells: 4 × 10 ⁷ /0.2 ml), and the mixture was diluted with 45 ml.
The mixture was completely dissolved by heating to ℃, uniformly poured as an upper layer medium, allowed to stand at room temperature and solidified to prepare a double agar medium.
Phage-containing solution 0.1 m on the surface of this double agar medium
l, and cultured at 37 ° C. for 7 hours to obtain enterohemorrhagic E. coli E. coli. E. coli O157: H7 was grown and the number of holes (plaques) due to the phagocytosis of the phage was counted and the phage number was calculated accordingly. As a result, when the target phage was present in the test sample, plaques were generated on the agar medium in accordance with the number of phages, and the target phage was detected. (2) Kit for detecting pathogenic Escherichia coli An agar medium having the following composition was prepared. 10% polypeptone 3% yeast extract 2.5% NaCl 0.1% glucose 5Mm CaCl ₂ enterohemorrhagic E. coli E. coli O157: H7 strain (number of cells: 4 × 10 ⁷ /0.2 ml) and phage of each concentration were mixed in 0.1 ml of a solution, and 3 ml of a 5% agar medium prepared separately and having the same composition was added. The mixture was heated to 45 ° C., completely dissolved, uniformly injected as an upper layer medium, and immediately cultured at 37 ° C. for 7 hours. E. coli O157: H7 was grown, and the growth inhibition rate of bacterial cells due to the phagocytosis of phage was observed.
When a low concentration phage was inoculated, the number of holes (plaques) due to the phagocytosis of the phage was counted, and the phage number was calculated accordingly. As a result of the above, enterohemorrhagic E. coli E. coli. For E. coli O157: H7, the Escherichia coli could be detected by utilizing the phagocytosis of a phage specific thereto. In addition, the phage number (titer) can be assayed by this method. The results obtained above are shown in the table below,
The titer of the stock solution in this case was 4.0 × ^{10 10} / ml. (Phage titer assay) Escherichia coli was grown at an absorbance = 0.
2 and the diluted sample of phage are mixed,
Phage titers were assayed by overlaying on agar medium as described above.

The effects of the bacteriophage according to the present invention and the bio-sterilizing material using the same will be further described. (Bactericidal Effect on Solid Medium) The bactericidal effect of the present bactericidal material was examined in consideration of the case where pathogenic bacteria, which are harmful bacteria, adhered or proliferated on solids. As shown in FIG. 1, E. coli was spread on one side. Before or after this application, the bio-sterilized material prepared in Example 1 was used for 10 ⁻¹ , 10 ⁻² ,
The solution diluted to 10 ⁻³ was applied vertically (this method is called cross streak method). After overnight cultivation at 37 ° C., all bacteria that contacted the bacteriophage were completely lysed. No colony formation was observed at all, indicating a strong effect of the present sterilizing material.

(Bactericidal effect in liquid medium) In the liquid of food (juice, soup), sprouts or radish radishes by hydroponics, bacteria causing food poisoning often grow. In such a case, it is effective to give this agent when the concentration of the bacterium is low with respect to the unit volume. Therefore, the following experiment was conducted to confirm the effect. About 1/10 of a bouillon medium was added in order to inoculate Escherichia coli into a commonly used commercially available medium for hydroponics and measure proliferation. After several hours, culturing was started at 37 ° C., and the bactericidal material was added. After 12 hours, the bacteria were completely killed. Even if harmful bacteria grow, MO
I: When this sterilizing material was used at 10-50, the harmful bacteria that had grown could be completely eliminated.

(Bactericidal Effect by Spray) The bactericidal effect of this agent was examined using a chopping board used at home for about one year. First, 10,000 Escherichia coli are dissolved in about 10 ml of a medium (bouillon medium), and this solution is applied to both sides of a cutting board.
After 30 minutes, excess water was removed. Thereafter, the cutting board was divided into two equal parts, one of which was used as a test for applying the agent 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 overnight storage at 37 ° C, the number of remaining bacteria adhering to both
The cells were washed with ml of the medium and counted to determine the number of bacteria. As a result, no E. coli was detected from the experimental cutting board, but about 1 to 2 million E. coli was detected from the control cutting board.

(Effect on Escherichia coli adhering to meat) 10 pieces of beef for steak were used, and an average of about 1
The number of E. coli that gave out 00 colonies was sprayed. These were divided into two groups, an experimental group and a control group. The experimental group was sprayed with a spray containing the drug and the control group was not sprayed. 12 at 37 ° C
After a certain period of time, a part of this steak was randomly cut (about 10 cuts), replicated on an agar medium, and further cultured overnight, and the number of colonies formed in both samples was counted. As a result, the number of colonies generated in the steak of the experimental group to which the drug was sprayed was zero, whereas that of the control group in which the drug was not sprayed was 100.
± 10 colonies were found. This result indicated that the bactericide according to the present invention exhibited an excellent bactericidal effect.

(Method of determining base sequence of bacteriophage)
Among the bacteriophages according to the present invention, bacteriophages # 1, # isolated by the above screening
The nucleotide sequence of each of the DNAs # 2, # 3 and # 4 was determined according to a conventional method. (1) Propagation of bacteriophage L-liquid medium was infected with Escherichia coli N60, cultured to a OD of 0.2 with a spectrophotometer, bacteriophage was added so that the MOI became theoretically 10, and left on ice for 30 minutes. Thereafter, it was shaken at 37 ° C. for about 20 hours. Thereafter, one drop of chloroform was added and shaken for 5 minutes. The culture was centrifuged at 4,500 rpm at 4 ° C. for 20 minutes, and only the supernatant was transferred to a sterilized bottle, and the resultant was spread on an L-agar medium to measure the concentration. As a result, 1x10
500 ml of bacteriophage at a concentration of ¹⁰ / ml were obtained. (2) Concentration of bacteriophage 20% PEG · 2M was added to the phage culture obtained above.
NaCl was added and left on ice for 1 hour. 12,000
The mixture was centrifuged at 0 rpm at 4 ° C. for 20 minutes to remove water, and 1 ml of SM solution was added to the precipitate, suspended well, and transferred to a microcentrifuge tube. To check the concentration,
As a result of measuring the concentration by spreading on an L-agar medium, 1.3 ×
It became 10 ²⁰ / ml. (3) Preparation of phage DNA 10% SDS 0.5M EDTA was added to the concentrated culture solution to 0.1% and 5 mM, respectively (1/1 each).
), And heated at 68 ° C for 15 minutes. After extracting with phenol and then with phenol / chloroform,
Extracted again with chloroform. An equal amount of isopropanol was added to the obtained aqueous layer, cooled to -70 ° C, and left for 10 minutes. Then, at 15,000 rpm, 4 ° C, 15
The supernatant was removed by centrifugation under the conditions of minutes, and the precipitate was washed with ethanol, suction-dried, and dissolved in 1 ml of a TE solution. (4) Confirmation of cleavage by electrophoresis To 4 µl of this DNA aqueous solution, 2 µl of M buffer, 13 µl of sterilized water, and 1 µl of restriction enzyme Hind III were added.
Shake at 37 ° C. for 1 hour. One tenth of the sample overlay reagent was added, and electrophoresis was performed on a 0.7% agarose gel at 100 V for 2 hours. The agarose gel was left in a solution in which 20 μl of ethidium bromide was dissolved in 400 ml of TAE solution for 2 hours, and the band was confirmed by a UV lamp. (5) Dephosphorylation of vector plasmid DNA Next, 3 μl of plasmid (pkk223), 2 μl of M buffer, 14 μl of sterilized water, restriction enzyme Hind III
1 μl was added and shaken at 37 ° C. for 1 hour. Hin
It was left in a hot water bath at 70 ° C. for 15 minutes to inactivate d III. In order to perform dephosphorylation, this vector
1, buffer 2 μl, BAP 0.5 μl, sterile water 2
6.5 μl was added and shaken at 37 ° C. for 2 hours. Thereafter, extraction was performed twice with phenol / chloroform, and ethanol precipitation was performed, followed by dissolving in 80 μl of a TE solution. In order to confirm the presence or absence of the vector, 1/10 volume of a reagent for superimposing a sample was added, and electrophoresis was performed at 100 V for 2 hours on a 0.7% agarose gel. (6) Deactivation of enzyme 4 μl of aqueous phage DNA solution for ligase reaction
Buffer 2 μl, sterile water 13 μl, restriction enzyme Hi
1 μl of nd III was added, shaken at 37 ° C. for 1 hour, and then left in a water bath at 70 ° C. for 15 minutes to inactivate Hind III. (7) Ligase reaction The aqueous phage DNA solution and the vector that had been dephosphorylated as described above were allowed to stand in a 60 ° C water bath for 10 minutes. (1) Phage DN digested with Hind III
A, (2) Phage DNA cut with Hind III
And (3) a vector dephosphorylated, (4) a vector cut with HindIII, 10 μl of 10 × buffer, DN
A solution (10 μl) and sterilized water were added so that the total amount became 100 μl. coli DNA ligase2
μl was added and left at 16 ° C. overnight. (8) Transformation Competent Cell (HB101) 50 μl
Then, 5 μl of each of the above (1), (2), (3) and (4) was added, left on ice for 30 minutes, then left at 42 ° C. for 45 seconds, and left on ice for another 2 minutes. Then
Add L-liquid medium to 0.5 ml and add 1 hour,
After shaking at 37 ° C, the cells were spread on an L-agar medium supplemented with ampicillin. (9) Culture of recombinant clone The colony of the above (2) was placed in 500 ml of L-liquid medium supplemented with ampicillin, and cultured for about 24 hours. The culture was centrifuged at 6,000 rpm at 4 ° C. for 15 minutes, and the obtained supernatant was removed. (10) Purification of plasmid The plasmid containing the phage DNA was purified using a plasmid purification kit (manufactured by Funakoshi). First, 10 ml of P1 and 10 ml of P2 were added to the pellet, and the mixture was slowly stirred. After standing at room temperature for 5 minutes, the cooled P3
After adding 0 ml and stirring slowly, the mixture was left on ice for 20 minutes. Thereafter, the mixture was centrifuged at 20,000 g at 4 ° C. for 30 minutes, and the supernatant was collected.
Centrifugation was performed at 000 g, 4 ° C., for 30 minutes. The column was washed by adding 10 ml of QBT. The supernatant was added to the column, and the column was washed twice with 30 ml of QC. Thereafter, purification was performed using 15 ml of QF, and 0.7 vo
lume of isopropanol was added. This mixture is
The mixture was centrifuged at 5,000 g and 4 ° C. 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 is called Hin
After cutting with dIII, electrophoresis was performed. Analysis of the nucleotide sequence of the thus obtained fragment according to a conventional method revealed that the bacteriophage #
1, # 2, # 3 and # 4 have the following SEQ ID Nos. 1-1 to 1-4, SEQ ID Nos. 2-1 to 2-10, respectively.
It was confirmed that they had DNAs containing fragments having partial sequences as shown in SEQ ID NOs: 3-1 to 3-5 and SEQ ID NOs: 4-1 to 4-5, respectively.

[0035]

According to the present invention, a bacteriophage having a high specificity for a certain kind of pathogenic bacteria is provided, and the bacteriophage can be used to produce foodstuffs such as fresh foods. It can be used to store, cook, and process foods and other foods, and to prevent humans who handle the foods from being infected with the pathogenic bacteria. Very useful. In addition, the use of the phage according to the present invention disinfects not only the infected place but also the place or the article which is expected to be infected, thereby easily and surely sterilizing the infectious bacteria. It can be performed in a short time and is extremely useful.
At the same time, by using such a phage, the pathogenic bacterium serving as the host can be detected easily, reliably, and in a very short time. Therefore, there is a great advantage that infection prevention and treatment can be performed promptly. In addition, by providing a bacteriophage having high specificity for pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli among certain types of pathogenic bacteria according to the present invention, such as foodstuffs such as fresh foods, Places and utensils that store, cook, and process these foods, as well as humans who handle the foods, can reliably prevent infection with pathogenic Escherichia coli, particularly enterohemorrhagic Escherichia coli. In addition, even in the event of infection or in the event of infection, the pathogenic Escherichia coli can be easily, easily and quickly disinfected by disinfecting the infected items or places or the suspected infection. It can be sterilized in time and is very useful. In addition, since the corresponding pathogenic Escherichia coli can be detected in a short time and reliably by the phage, the presence or absence of infection and identification of the source of infection can be performed very easily, in a short time, reliably, and extremely useful. It is. Furthermore, by providing a novel bacteriophage according to the present invention, the type of pathogenic bacteria to be its host will be increased, and it will be possible to cope with more types of pathogenic bacteria,
A wide range of measures can be taken in the prevention and treatment of infection. Naturally, bacteriophages having high specificity for specific pathogenic bacteria, particularly pathogenic Escherichia coli as described above can be used alone, but two or more phages having different properties can be used. The use of a mixed cocktail is extremely convenient and advantageous because it can simultaneously cope with other types of pathogenic bacteria.

The present invention also provides such a novel bacteriophage screening method. The screening method has an effect that a novel phage having extremely high specificity for pathogenic bacteria can be reliably and easily screened.

By providing a novel biocidal material containing such a novel bacteriophage according to the present invention, the effects achieved by providing the phage as described above can be put to practical use. In other words, by including such phage in the novel bio-sterilizing material, for example, food products such as fresh food, such food products and the like, storage, cooking, processing,
It can be used for instruments, humans who handle such foodstuffs, etc., and can surely prevent infection with pathogenic bacteria including pathogenic Escherichia coli, such as enterohemorrhagic Escherichia coli. Even if infected or suspected to be infected, disinfect the infected item or place, or the suspected infection, so that the pathogenic bacteria can be disinfected easily, easily and in a short time. There is a great effect that you can. By adding the preservative or stabilizing agent according to the present invention to the novel bio-sterilizing material, the novel bio-sterilizing material can be stably stored for a long period of time while maintaining the action of the phage, and is useful. is there. In addition, by including a plurality of types of the novel bacteriophage according to the present invention in the novel bio-sterilizing material, there is a great effect that the bio-sterilizing material can simultaneously cope with a plurality of types of pathogenic bacteria.

By providing a method for producing such a novel bacteriophage according to the present invention, the phage can be produced in a large amount and at low cost, and in particular, the novel bacteriophage can be produced according to the present invention. It is extremely advantageous in putting the new bio-sterilizing material included into practical use. Further, in this production method, by adding metal ions such as calcium ions to a medium for culturing the phage,
It has the effect of promoting the growth of phage requiring such metal ions and accelerating the growth of phage.

In addition, the present invention provides a stabilizing agent or a preservative capable of stably storing such a novel bacteriophage, whereby the phage can be stably stored for a long period of time. The novel bacteriophage 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 period of time.

Furthermore, the present invention provides a method for detecting a pathogenic bacterium and a reagent kit for detecting the pathogenic bacterium, whereby a pathogenic bacterium serving as a host thereof can be easily and quickly obtained by using the phage according to the present invention. It is very useful because it can be reliably detected. By using this detection method and its detection reagent kit, even if urgent action is required, such as when infection by pathogenic bacteria is suspected, suspected pathogenic bacteria are present in the sample at the site where the sample was collected There is a great advantage that the assay can be reliably performed in a short time using only the detection reagent kit, that is, without using any other measuring device for detection. Furthermore, this method for detecting pathogenic bacteria can be used as a host by the proliferation of bacteria, even if the amount of the specimen is very small, and at least one pathogenic bacteria to be detected is present in the specimen. It is an extremely sensitive method that can detect the bacterium because the phage acts. In addition, a reagent kit for detecting a pathogenic bacterium using this method for detecting a pathogenic bacterium may have an extremely simple configuration, such as a normal container such as a petri dish containing an agar medium containing phage. It is also a great effect that the phage only needs to be left in an environment where bacteria can easily proliferate, and only for a short period of time, that is, for the phage phagocytosis of the bacterium.

[Brief description of the drawings]

FIG. 1 is a diagram showing the bactericidal effect of a cross streak method on a plate, in which it is shown that the bacteria that have come into contact with the test bacteriophage are completely killed.

FIG. 2 is a view showing the effect of the present agent in a liquid medium,
In the figure, (-) shows the growth curve of the bacteria when this drug was not added, and (+) shows that the growth of the bacteria was suppressed when this drug was added at MOI = 10.

FIG. 3 is a diagram showing a culture state of bacteriophage. In FIG. 3, a) shows a petri dish in which only an agar medium was cultured, and a) shows an enterohemorrhagic O15 on an agar medium.
7 shows a petri dish in which No. 7 was cultured. In this figure, it is found that the petri dish became white due to the proliferation of enterohemorrhagic O157. C) Enterohemorrhagic O157 on agar medium
In addition, a phage stock solution (2 × ^{10 10} / ml) that phagocytosically destroys only pathogenic Escherichia coli O157 was added and cultured.
It turns out that all 57 have been phagocytosed. D) shows a culture obtained by adding a liquid (2 × 10 ⁷ / ml) obtained by diluting the phage 1,000-fold with a new phage stock on an agar medium together with enterohemorrhagic O157. , Enterohemorrhagic O1
57 are all shown to be phagocytosed.
E) on an agar medium together with enterohemorrhagic O157,
A solution obtained by diluting the phage 1,000-fold with a new phage stock solution (2 × 10 ⁷ / ml) and further adding a 20-fold dilution with tap water and culturing it is shown. Enterohemorrhagic O157 has all been shown to be phagocytosed. (F) A solution obtained by diluting the phage 1,000-fold with a new phage stock solution together with enterohemorrhagic O157 on an agar medium (2 × 10
⁷ / ml) was further added and diluted 100 times with tap water and cultured. In this figure,
Enterohemorrhagic O157 has all been shown to be phagocytosed.

[Sequence list]

SEQ ID NO: 1-1 Sequence length: 628 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 1-2 Sequence length: 780 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type; Genomic DNA Sequence origin: Phage sequence SEQ ID NOs: 1-3 Sequence length: 518 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 1-4 Sequence length: 599 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-1 Sequence length: 585 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-2 Sequence length: 578 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-3 Sequence length; 598 Sequence type: Nucleic acid strand number; Double stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-4 Sequence length: 600 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-5 Sequence length: 603 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-6 Sequence length: 601 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-7 Sequence length: 607 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-8 Sequence length: 585 Sequence type; number of nucleic acid strands: double-stranded Topology: linear Sequence type: Genomic DNA Sequence origin: phage sequence SEQ ID NO: 2-9 Sequence length; 607 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 2-10 Sequence length: 563 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 3-1 Sequence length: 528 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 3-2 Sequence length: 543 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 3-3 Sequence length: 557 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 3-4 Sequence length: 528 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 3-5 Sequence length: 528 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 4-1 Sequence length: 517 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 4-2 Sequence length: 353 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 4-3 Sequence length: 416 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 4-4 Sequence length: 517 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence SEQ ID NO: 4-5 Sequence length: 528 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence origin: Phage sequence

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) A23L 3/3463 A23B 4/00 J F term (reference) 4B021 MC01 MK06 MK30 MP01 MP05 4B065 AA26X AA98X AA98Y AC20 BA21 CA46 CA60 4B069 AA04 HA01 HA11 KA01 KB03 KC19 4H011 AA01 BA04 BB21 BC06

Claims (16)

[Claims] 1. A novel bacteriophage characterized by having a high specificity only for a particular pathogenic bacterium. 2. The bacteriophage according to claim 1, wherein the pathogenic bacterium is enterohemorrhagic Escherichia coli. 3. The bacteriophage according to claim 1, wherein the bacteriophage is SEQ ID NO: 1.
-1 to 1-4, SEQ ID NOs: 2-1 to 2-10, SEQ ID NOs: 3-1 to 3-5, and DNAs containing fragments having the base sequences represented by SEQ ID NOs: 4-1 to 4-5, respectively. It is characterized by having. 4. A bacteriophage is screened by infecting a pathogenic bacterium with a restriction enzyme minus and a modification enzyme positive, amplifying the bacteriophage, and treating at high temperature to select only the bacteriophage containing the deleted DNA. A method for screening bacteriophages, which comprises: 5. A novel bio-sterilizing material comprising the bacteriophage according to claim 1. Description: 6. The novel bio-sterilizing material according to claim 5, wherein a stabilizing agent or a preservative for stabilizing the bacteriophage is contained. 7. The novel biocidal material according to claim 5, wherein the bacteriophage comprises a single or a cocktail of bacteriophages having two or more different properties. 8. A novel bio-sterilizing material comprising the bacteriophage according to any one of claims 1 to 3 or the bacteriophage according to any one of claims 5 to 6, for killing pathogenic bacteria. Use of a bacteriophage for killing a pathogenic bacterium, wherein the bacteriophage is used for sterilization. 9. Use according to claim 8, wherein the bacteriophage or the biocidal biomaterial is applied to a subject infected or suspected of being infected by the pathogenic bacterium. do it,
It is characterized by being used for killing the pathogenic bacteria or preventing the pathogenic bacteria from being infected. 10. The use for bacteriophage killing according to claim 8 or 9, characterized in that the bacteriophage consists of a single or a cocktail of two or more different bacteriophages. 11. When a pathogenic bacterium is cultured in a medium and reaches a predetermined number of cells, a bacteriophage is added to the medium to infect the bacteriophage with the pathogenic bacterium. A method for producing a culture solution of bacteriophage, wherein a culture solution of the bacteriophage is obtained by removing bacterium. 12. The method for producing a bacteriophage culture solution according to claim 11, wherein the medium contains metal ions such as calcium ions. 13. A pH using a food material such as an amino acid.
Is adjusted to 6.5 to 7.5. 14. The stabilizer for bacteriophage according to claim 13, wherein the amino acid is glycine, arginine or lysine. 15. A method for detecting a pathogenic bacterium, comprising using the bacteriophage according to any one of claims 1 to 3 for detecting a pathogenic bacterium. 16. A reagent or kit for detecting a pathogenic bacterium using the method for detecting a pathogenic bacterium according to claim 15.