JP2014121338A - Culture medium and method for detecting enterohemorrhagic escherichia coli - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture medium of a specific composition to isolate enterohemorrhagic Escherichia coli that causes food poisoning, which medium allows a short time, easy and accurate detection without special skills or techniques.SOLUTION: The invention provides a culture medium containing tellurite and penicillin-based antibiotic for detecting enterohemorrhagic Escherichia coli.

Description

  The present invention relates to a medium for detecting enterohemorrhagic Escherichia coli and a detection method.

In the present invention, the following abbreviations may be used.
SMAC medium: Sorbit McConkey medium
RMAC medium: Rhamnose McConkey medium
SBMAC medium: Sorbose McConkey medium CTSMAC medium: Cefixime sorbite tellurite sorbite McConkey medium
CTRMAC medium: cefixime rhamnose tellurite rhamnose macconkey medium
CTSBMAC Medium: Cefixime Tellurite Sorbose MacConkey Medium
CLIG medium: Cellobiose Lactose Indole β-D-Glucuronidase agar

  Escherichia coli belongs to the family Enterobacteriaceae and is a Gram-negative, facultative anaerobic Aspergillus oryzae, and the serotype is distinguished by the cell wall antigen structure on the surface of the cell. Food poisoning caused by enterohemorrhagic Escherichia coli is an infectious food poisoning caused by the growth of bacteria in the intestine when meat, milk, eggs and processed foods contaminated with this bacteria are ingested. It is a dangerous food poisoning that can cause ulcerative colitis and lead to death.

  In food inspection, enterohemorrhagic Escherichia coli (O157, O26, or O111) is generally detected as follows. First, the sample is enriched using a medium for enrichment such as modified EC (novobiocin-added mEC) medium with novobiocin and then inoculated into a selective separation medium such as SMAC (RMAC, SBMAC) medium or CTSMAC (CTRMAC, CTSBMAC) medium. Then, separate and culture, and observe colonies on the medium. In addition, from the colonies of sorbite (rhamnose, sorbose, etc., non-degradable or slow-degrading sugar) non-degraded colonies on CTSMAC plate medium, O157 (O26, O111, enterohemorrhagic Escherichia coli) and colonies with suspected properties were caught, After inoculating in a confirmation medium such as CLIG and culturing at 37 ° C. for 18-24 hours, an immunological test using antiserum is conducted to confirm the serotype of enterohemorrhagic Escherichia coli. Some selective separation media have improved visibility using a chromogenic substrate, but they are still expensive because of the high cost, such as SMAC (RMAC, SBMAC) medium and CTSMAC (CTRMAC, CTSBMAC) medium. A culture medium that separates by using is widely used.

  In a stool test, a stool sample is inoculated into a selective separation medium and separated and cultured, and thereafter, in the same manner as in a food test, a check medium is inoculated and an immunological test using antiserum is performed.

  Thus, in addition to complicated processes, the conventional method for detecting enterohemorrhagic Escherichia coli is selected from a non-sugar-decomposing colony to select a colony that seems to be enterohemorrhagic Escherichia coli. There was a problem that a long time was required. In addition, in various selective separation media to be used, many intestinal bacteria may grow and form colonies similar to the bacteria to be detected. Therefore, skill and experience are required to distinguish strains suspected of enterohemorrhagic Escherichia coli from such a selective separation medium, and in order to conduct a confirmation test and subsequent serum test on the colony where the fungus was conducted at this time, It is also related to the cost of the entire inspection. The materials that are usually tested often contain both enterohaemorrhagic Escherichia coli that causes food poisoning, as well as normal Escherichia coli and bacteria that form similar colonies. It is necessary to identify the causative bacteria.

  Among the food and beverage workers, sushi restaurants, caterers including bento restaurants, soba restaurants, seafood sales, tofu manufacturing, group food facility cooking workers, water facility workers, Legal check-up flights (carrier detection business) are carried out once a month between May and September with seasonal inns designated as priority industries. In addition, stool tests are recommended throughout the year, even in other industries and outside the legal period. For this reason, a vast number of stool examinations are carried out in facilities such as inspection centers and health centers. However, since fecal specimens contain a large amount of bacteria, it is difficult to separate enterohemorrhagic Escherichia coli due to the growth of resident bacteria in the above-mentioned conventional culture media and detection methods. Was necessary.

JP 2002-281959 A JP 2003-235545 A Patent No. 3026005 Patent No. 3145125 Patent No. 3711563

  An object of the present invention is to provide a specific medium composition capable of easily and accurately detecting enterohemorrhagic Escherichia coli in a sample in which a plurality of bacteria are mixed, without requiring special skills and techniques, and in a short time. It is to provide a method for detecting enterohemorrhagic Escherichia coli.

Samples for stool examination of enterohemorrhagic Escherichia coli contain large amounts of E. coli other than enterohemorrhagic Escherichia coli or other intestinal bacteria even if they contain enterohemorrhagic Escherichia coli. It was not easy to detect enterohemorrhagic E. coli. For example, O157, one of typical enterohemorrhagic Escherichia coli, is a bacterium that does not degrade sorbit. For example, on CTSMAC, the central brown area forms a transparent or translucent settlement. In contrast, most E. coli or enterobacterial colonies that degrade sorbitol have a pH lowering due to the effects of the organic acid produced, and the neutral red pH indicator is pink. Distinguished from villages. However, in the vicinity of the densely populated cells, the presence of a large amount of Escherichia coli / intestinal bacteria that decompose sorbitol tended to weaken the pink coloration. Therefore, when distinguishing O157, which is non-degradable or slow degrading sorbite, in the vicinity of densely populated villages, there is a tendency that it is difficult to distinguish because there is a mixture of E. coli / intestinal bacteria that degrade sorbite whose pink color has weakened. It was. Similarly, O26 is rhamnose nondegradable or slow degrading, O111 is sorbose nondegradable or slow degrading, and O26 and O111 can be discriminated similarly to O157 using the pH indicator neutral red. However, in the densely populated areas, E. coli / intestinal bacteria that decompose sorbite, which has weakened pink coloration, coexist, and it was difficult to distinguish.

The present inventor has obtained 2,3,5-triphenyltetrazolium chloride (TTC) and the like in a medium for detecting enterohemorrhagic Escherichia coli in which a sugar for improving selectivity is added to a medium for inoculating a specimen, particularly a stool specimen. Such a redox dye was added. As a result, the difference in color and / or contrast between non-degrading colons of enterohemorrhagic Escherichia coli, etc., and bacteria that decomposes sugar, such as in the enterohaemorrhagic Escherichia coli, can be distinguished more clearly in densely populated areas. Found that it can be made easier. More preferably, the present invention has been completed by enhancing the ability of selective growth to enterohemorrhagic Escherichia coli by adding tellurite and cephem and penicillin antibiotics.

  Coloring of coexisting bacteria based on pH reduction due to degradation of sugar using pH indicator, coloring with redox dye of enterohemorrhagic Escherichia coli that does not degrade sugar, and selective inhibitory power against non-degrading bacteria other than enterohemorrhagic Escherichia coli Since it is high, it is the greatest feature of the present invention that it can be easily distinguished even in densely populated areas.

The present invention provides the following medium for detecting enterohemorrhagic Escherichia coli and a method for detecting enterohemorrhagic Escherichia coli.
Item 1. A medium for detecting enterohemorrhagic Escherichia coli containing redox dye and sugar.
Item 2. Item 2. The medium for detecting enterohemorrhagic Escherichia coli according to Item 1, wherein the sugar is non-degradable or slow-degrading in the detection target bacterium.
Item 3. Item 3. The medium for detecting enterohemorrhagic Escherichia coli according to Item 1 or 2, wherein the sugar is selected from sorbitol, rhamnose, and sorbose.
Item 4. Item 4. The medium for detecting enterohemorrhagic Escherichia coli according to any one of Items 1 to 3, further comprising a pH indicator.
Item 5. The medium according to any one of claims 1 to 4, wherein rhamnose is used as a sugar, and enterohemorrhagic Escherichia coli O157, O26, O111 can be discriminated by adjusting the amount thereof.
Item 6. The medium according to claim 5, wherein the amount of rhamnose is 2 to 23 g.
Item 7. Item 7. The medium for detecting enterohemorrhagic Escherichia coli according to any one of Items 1 to 6, further comprising at least one selection agent selected from the group consisting of tellurite and antibiotics.
Item 8. Item 8. The medium for detecting enterohemorrhagic Escherichia coli according to Item 7, wherein the antibiotic is at least one selected from the group consisting of cephem antibiotics and penicillin antibiotics.
Item 9. Item 8. The medium for detecting enterohemorrhagic Escherichia coli according to Item 7, further comprising tellurite, a cephem antibiotic, and a penicillin antibiotic.
Item 10. Item 10. The medium for detecting enterohemorrhagic Escherichia coli according to any one of Items 1 to 9, wherein the enterohemorrhagic Escherichia coli is selected from the group consisting of O157, O26, and O111.
Item 11. Item 11. The medium for detecting enterohemorrhagic Escherichia coli according to any one of Items 1 to 10, wherein the redox dye develops in any color selected from the group consisting of red, blue, purple, orange, yellow, and green.
Item 12. Item 5. The culture medium for detecting enterohemorrhagic Escherichia coli according to Item 4, wherein the acidic side color of the pH indicator is any color selected from the group consisting of red, blue, yellow, orange, green, and purple.
Item 13. Any one of Items 1 to 12, wherein the sugar is non-degradable or slow-degrading in the detection target bacterium, and the color on the acidic side of the pH indicator and the color of the redox pigment developed by the enterohemorrhagic Escherichia coli are distinguishable A medium for detecting enterohemorrhagic Escherichia coli according to any one of the above.
Item 14. The intestinal tract according to Item 13, wherein the acidic side color of the pH indicator is yellow, blue or red, and the redox dye is purple, red or blue (however, the acidic side color of the pH indicator is a different color). Culture medium for detection of hemorrhagic E. coli.
Item 15. Item 15. The medium for detecting enterohemorrhagic Escherichia coli according to Item 13 or 14, wherein the pH indicator and the redox dye are any combination of the following:
(1) The pH indicator is phenol red, and the redox dye is tetrazolium violet (TTV), 2,3,5-triphenyltetrazolium chloride (TTC) or nitrotetrazolium blue (NTB);
(2) The pH indicator is bromthymol blue (BTB) and the redox dye is tetrazolium violet (TTV), 2,3,5-triphenyltetrazolium chloride (TTC) or nitrotetrazolium blue (NTB);
(3) pH indicator is water blue and redox dye is 2,3,5-triphenyltetrazolium chloride (TTC);
(4) The pH indicator is neutral red and the redox dye is tetrazolium violet (TTV), 2,3,5-triphenyltetrazolium chloride (TTC) or nitrotetrazolium blue (NTB).
Item 16. Item 16. The medium for detecting enterohemorrhagic Escherichia coli according to any one of Items 1 to 15, for examining a sample derived from stool, food, or water.
Item 17. Item 17. A method for detecting enterohemorrhagic Escherichia coli in a sample, comprising culturing the sample in a medium according to any one of Items 1 to 16, and detecting the presence or absence of enterohemorrhagic E. coli.

  According to the present invention, a small amount of enterohemorrhagic Escherichia coli can be clearly discriminated from an intestinal bacterium such as a stool sample or a sample containing a large amount of Escherichia coli other than enterohemorrhagic Escherichia coli.

In addition, O157, O26, and O111, which are typical enterohemorrhagic Escherichia coli, can be discriminated with one medium.

The result of Example 2 is shown. The result of Example 3 is shown. The result of Example 4 is shown. The result of Example 5 is shown.

(I) Enterohemorrhagic Escherichia coli Enterohemorrhagic Escherichia coli to be measured in the present invention is not particularly limited as long as it is Escherichia coli having enterohemorrhagic properties, for example, O157, O26, O111, O121, O126, O103, O108, For example, O145. Since these enterohemorrhagic E. coli are different from E. coli and other coexisting bacteria other than the bacteria to be detected in terms of the use of sugar, the presence of enterohemorrhagic E. coli is determined by adding such sugar to the medium. Can help you.
(II) Sugar The sugar used in the present invention is non-degradable or slow-degrading in the enterohemorrhagic E. coli to be detected, and is degradable by other bacteria coexisting with the enterohemorrhagic E. coli in the sample. However, the amount of acid produced by glycolysis differs due to the difference in sugar degradability. For example, enterohemorrhagic Escherichia coli is differentiated from other coexisting bacteria based on the change in pH due to acid production. It is useful to do.

  In the present specification, “non-degradation” means that the detection target bacterium does not decompose sugar, and “slow decomposition” means that the sugar decomposition rate of the detection target bacterium is slow. Inoculated and cultured in a medium, even after observation, the bacterium shows a state that does not permanently decompose the sugar contained in the medium, while slow degradation is inoculated, cultured, In a general culture time (18 to 24 hours for enterobacteria such as O157) by those skilled in the art, it shows a state in which glycolysis is not recognized by the bacterium and is subsequently degraded over time. .

Examples of sugars that are non-degradable or slow-degrading enterohemorrhagic Escherichia coli used in the present invention and other bacteria that coexist with enterohemorrhagic Escherichia coli in the specimen are degradable include sorbitol (sorbitol) , Rhamnose, sorbose, inositol, adonitol, raffinose, rhamnose, salicin and the like. Examples of such a combination of sugar and enterohemorrhagic E. coli are as follows.

  In order to distinguish enterohemorrhagic Escherichia coli such as O157, O26, and O111 with one medium, it is preferable to use rhamnose as a sugar, and a medium based on CT-RMAC and supplemented with a redox dye is particularly preferable. .

(III) Redox dye The redox dye is colored in all the villages without color selectivity. However, when the color is already developed by a pH indicator or the like, the influence is small, and in SMAC, for example, the effect is strongly expressed on a village such as O157 that does not decompose sorbit. Until now, in SMAC, the colonies other than the detected bacteria were colored only, and in the densely populated parts of the villages, pH indicator fading occurred, so there were many cases where it was difficult to distinguish the colonies of O157 as the detection target. It was happening. Surprisingly, the redox dyes highlight the coloration by the pH indicator in the dense part and prevent the fading, so that the colonies of the saccharide-decomposable bacteria are also colored in a brighter and darker color in the dense part. Coloring non-sugar-decomposable colonies such as hemorrhagic Escherichia coli with a dark dark color, and even if the two villages overlap mostly, non-sugar-decomposable colonies such as enterohemorrhagic Escherichia coli Can be identified. In the present invention, by adding a redox dye together with sugar, it becomes easy to discriminate even a small colony found in a dense part, and by clarifying the color difference from the pH indicator, further enterohemorrhagic Escherichia coli The visibility can be improved.

  Conventional redox dyes have been used in combination with β-galactosidase substrate to color the whole E. coli (for example, JP 2002-360296, JP 2004-187588). There is no example used as a culture medium. This seems to be because, when combined with sugar, it was thought that the use of a redox dye was not necessary because the sugar-degrading bacteria can be strongly colored by the pH indicator. In fact, if each colony is well separated, enterohemorrhagic Escherichia coli can be differentiated with a conventional medium using sugar and pH indicator, but it is like a stool specimen in which other coexisting bacteria coexist. In the case of a small sample, the acid side coloring of the pH indicator in a densely populated area was weakened, making differential diagnosis difficult. On the other hand, when the redox dye used in the present invention is used in combination with a suitable pH indicator, the coloring of the redox dye is added to the coloring of the saccharide-degrading bacteria by the pH indicator even in such a densely populated area. The non-sugar-degrading enterohemorrhagic Escherichia coli forms a colony with a color and / or contrast that is different from that of glycolytic bacteria. Clearly, the difference can be clearly shown even in the densely populated areas, which was a surprising discovery for the inventor. A medium for detecting enterohemorrhagic Escherichia coli using a synergistic effect of improving visibility by using such a sugar, a redox dye, and a pH indicator in combination has not been known at all. In addition, in the present invention, it is possible to detect enterohemorrhagic Escherichia coli even in a densely populated area by further adding an antibiotic having a growth-inhibiting effect against non-degrading bacteria other than the intestinal hemorrhagic Escherichia coli that grows. did.

  In the medium of the present invention, a redox dye is used in addition to sugar. This redox dye is a reagent that is non-selectively oxidized / reduced in all bacteria. However, enterohemorrhagic Escherichia coli and other coexisting bacteria have different sugar degradability, so the colony coloration in the medium is different. Preferably, enterohemorrhagic Escherichia coli colonies are transparent in the absence of redox dyes. The coexisting bacteria are strongly colored. In this state, when enterohemorrhagic Escherichia coli oxidizes / reduces the redox dye, this dye colors the colonies of non-sugar-degrading bacteria including enterohemorrhagic Escherichia coli.

  Enterohemorrhagic Escherichia coli is weakly colored due to sugar. For example, there is a pH indicator in the medium. This pH indicator does not lower the pH of the medium in enterohemorrhagic Escherichia coli whose sugar is non-degradable / slowly degrading. Therefore, in the coexisting bacteria that decompose sugars, the sugars are rapidly decomposed to produce organic acids and lower the pH. Therefore, when they are strongly colored, or enterohemorrhagic Escherichia coli is selective for sugars. If many of the coexisting bacteria do not degrade the sugar, or decompose slowly, the pH indicator should be strongly colored near neutrality, and it should be selected to be transparent / colorless or weakly colored when the pH decreases. .

  As redox dyes, methylene blue, indigosulfonic acid, safranin T, phenosafranine, N-phenylanthranilic acid, malachite green, rhodamine B, diphenylamine, diphenylbenzidine, diphenylaminesulfonic acid, 5,6-dimethylferroin, erioglaucine A , 5-methylferroin, ferroin, nitroferroin, tetrazolium salt and the like. Preferred are tetrazolium salts, specifically 2,3,5-triphenyltetrazolium bromide, 2,3,5-triphenyltetrazolium chloride, 2,3,5-triphenyltetrazolium iodide, 2,3,5- Tris (p-tolyl) tetrazolium chloride, 2,3-bis (3-chlorophenyl) -5-phenyltetrazolium chloride, 2,3-bis (3-fluorophenyl) -5-phenyltetrazolium chloride, 2,3-bis ( 3-methylphenyl) -5-phenyltetrazolium chloride, 2,3-bis (4-chlorophenyl) -5-phenyltetrazolium chloride, 2,3-bis (4-ethylphenyl) -5-phenyltetrazolium chloride, 2,3 -Bis (4-fluorophenyl) -5-phenyltetrazolium chloride, 2,3-bis (4-methoxyphenyl) -5- (4-cyanophenyl) tetrazolium chloride, 2,3-bis (4-methoxyphenyl) Nyl) -5-phenyltetrazolium chloride, 2,3-bis (4-methylphenyl) -5- (4-cyanophenyl) tetrazolium chloride, 2,3-bis (4-nitrophenyl) -5-phenyltetrazolium chloride 2,3-di (p-tolyl) -5-phenyltetrazolium chloride, 2,3-diphenyl-5- (2-thienyl) tetrazolium chloride, 2,3-diphenyl-5- (4-chlorophenyl) tetrazolium chloride 2,3-diphenyl-5- (4-methoxyphenyl) tetrazolium chloride, 2,3-diphenyl-5- (4-tolyl) tetrazolium chloride, 2,3-diphenyl-5-aminotetrazolium chloride, 2,3- Diphenyl-5-aminotetrazolium chloride, 2,3-diphenyl-5-carboxytetrazolium chloride, 2,3-diphenyl-5-ethyltetrazolium chloride, 2,5-di (p-tolyl) -3-phenylteto Zorium chloride, 2,5-diphenyl-3- (4-styrylphenyl) tetrazolium chloride, 2- (2-benzothiazolyl) -3,5-diphenyltetrazolium bromide, 2- (2-benzothiazolyl) -3- (4- Nitrophenyl) -5-phenyltetrazolium bromide, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyltetrazolium chloride, 2-phenyl-3- (4-biphenylyl) -5-methyl Tetrazolium chloride, 2-phenyl-3- (4-carboxyphenyl) -5-methyltetrazolium chloride, 3,3 '-(3,3'-dimethoxy-4,4'-diphenylene) bis (2-phenyl-5- Belatryltetrazolium chloride), 3- (3-nitrophenyl) -5-methyl-2-phenyltetrazolium chloride, 3- (4-nitrophenyl) -5-methyl-2-phenyltetrazolium chloride, blue tetrazolium, m- Triltetrazoli Mured, nitroblue tetrazolium, o-tolyltetrazolium red, p-tolyltetrazolium red, tetranitroblue tetrazolium, tetrazolium violet, thiocarbamyl nitroblue tetrazolium chloride, WST-1, WST-8, 3- [4,5-dimethyl Thiazol-2-yl] -2,5-diphenyltetrazolium bromide, Alamar Blue (Alamar Biosciences, USA), 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide (MTT) ), 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxyanilide (XTT), and the like.

For example, when bromthymol blue (BTB) is used as a pH indicator, the acidic side is colored yellow, so avoid yellow pigments of similar colors. In this case, by adding 2,3,5-triphenyltetrazolium chloride (TTC) to the sorbite-containing medium, both the reddish brown color of the fungal colonies that do not decompose O157 and the yellow colony of the fungus that decomposes sorbitol Since the coloring is enhanced at the same time, as a result, the visibility in the dense part is greatly improved, and the determination of O157 becomes very easy.
(IV) pH indicator The coloration of the coexisting bacteria accompanying the decomposition of sugar is based on the decrease in pH accompanying the production of organic acid, and is greatly affected by the pH indicator. The medium of the present invention preferably contains a pH indicator.

  As pH indicators, benzoperpurine 4B, 2,6-dinitrophenol, 2,4-dinitrophenol, methyl yellow, bromophenol blue, methyl orange, ethyl orange, congo red, alizarin red, bromocresol green, chlorocresol green, 2,5-dinitrophenol, methyl red, lactoid, TBPE, litmus, methyl purple, azolithamine, chlorophenol red, o-nitrophenol, p-nitrophenol, Andrade reagent, resazurin, bromocresol purple, bromophenol red, bromo Thymol blue, pararosolic acid, 3 ', 3' ', 5', 5 ''-tetraiodophenol sulfonephthalein, neutral red, phenol red, aurine, 3-nitrophenol, water blue (aniline blue), chi Inablue, cyanine, cresol red, α-naphtholphthalein, curcumin, metacresol purple, thymol blue, p-xylenol blue, o-cresolphthalein, phenolphthalein, α-naphtholbenzyne, thymolphthalein, etc. Congo red, alizarin red, bromocresol green, chlorocresol green, 2,5-dinitrophenol, methyl red, lactoid, TBPE, litmus, methyl purple, azolithamine, chlorophenol red, o-nitrophenol, p-nitro Phenol, Andrade reagent, resazurin, bromocresol purple, bromophenol red, bromothymol blue, pararozolic acid, 3 ', 3' ', 5', 5 ''-tetraiodophenol sulfonephthalein, new Ralred, phenol red, aurin, 3-nitrophenol, water blue (aniline blue), China blue, cyanine, cresol red, α-naphtholphthalein, curcumin, metacresol purple, thymol blue, p-xylenol blue, o-cresol Phthalein, phenolphthalein, α-naphthol benzyne, more preferably methyl red, lactoid, TBPE, litmus, methyl purple, azolithamine, chlorophenol red, o-nitrophenol, p-nitrophenol, Andrade reagent, resazurin, bromo Cresol purple, bromophenol red, bromothymol blue, pararosoleic acid, 3 ', 3``, 5', 5 ''-tetraiodophenol sulfonephthalein, neutral red, phenol red, aurin 3-nitrophenol, water blue (aniline blue), China blue, cyanine, cresol red, α-naphtholphthalein, curcumin, metacresol purple, thymol blue, p-xylenol blue, o-cresolphthalein, phenolphthalein, α-Naphthol benzyne, thymolphthalein, etc. are mentioned, preferably Congo Red, Alizarin Red, Bromocresol Green, Chlorocresol Green, 2,5-Dinitrophenol, Methyl Red, Lactoid, TBPE, Litmus, Methyl Purple, Azolitmine , Chlorophenol red, o-nitrophenol, p-nitrophenol, Andrade reagent, resazurin, bromocresol purple, bromophenol red, bromothymol blue, pararosoleic acid, 3 ', 3' ', 5', 5 ''-tetraiodophenol sulfonephthalein, neutral red, phenol red, aurine, 3-nitrophenol, water blue (aniline blue), China blue, cyanine, cresol red, α -Naphtholphthalein, curcumin, metacresol purple, thymol blue, p-xylenol blue.

  For example, when the sugar is non-degradable or slow-degrading in the bacteria to be detected, BTB has a contrast between the colony color of enterohemorrhagic Escherichia coli from blue to green and the yellow colony of sugar-degrading bacteria. It can be strengthened to facilitate identification of enterohemorrhagic E. coli.

  A preferable combination of the color on the acidic side of the redox dye and the pH indicator is described below.

  In the present invention, preferred combinations of pH indicators and redox dyes are shown below.

(V) Other components In the medium for detecting enterohemorrhagic Escherichia coli of the present invention, in addition to sugar, a selective agent that suppresses the growth of bacteria in specimens other than enterohemorrhagic Escherichia coli can be preferably used. Examples of such a selective agent include tellurite (preferably potassium tellurite), antibiotics such as cephem antibiotics such as cefixime, penicillin antibacterial agents, and the like. By using at least one of these selective agents, it is possible to suppress the growth of bacteria that affect the discrimination of enterohemorrhagic Escherichia coli containing O157. In particular, by using a combination of cephem antibiotics and penicillin antibacterial agents, Morganella, which is often caught as false positives among non-degrading saccharides (e.g., sorbite) that show colony color similar to enterohemorrhagic Escherichia coli (e.g., O157) And the growth of Proteus and Providencia can be prevented, false positives can be suppressed, and the number of fishing bacteria used in the secondary test can be reduced. When a tellurite such as potassium tellurite is added as a selective agent, enterohemorrhagic E. coli (eg, O157) forms a brown colony.

  Penicillin antibiotics include benzylpenicillin, benzylpenicillin benzathine, pheneticillin, aspoxicillin, cyclacillin, renaipicillin, sulbenicillin, methicillin, oxacillin, penicillin N, penicillin O, penicillin V, penicillin G, cloxacillin, dicloxaline, dicloxaline, , Ampicillin, amoxicillin, bacampicillin, tarampicillin, carbenicillin, ticarcillin, mezlocillin, abalacillin, sultamicillin, pibmesilinum and the like. Wide area penicillins such as ampicillin, amoxicillin, bacampicillin, tarampicillin, carbenicillin, ticarcillin, mezlocillin, abalacillin, piperacillin, sultamicillin, azocillin, pibmesililin. More preferred is carbenicillin.

  The cephem antibacterial agents include cephalothin, cefazoline, ceftezol, cephaloridine, cephalexin, cephatrizine propylene glycol, cefadroxyl, cefradine, cefloxazine, cefaclor, cefamandole, cefotiam, cefothiam hexetyl, cefuroxime, cefuroxime axetil, Cefminox, cefotaxime, ceftizoxime, cefmenoxime, cefoperazone, ceftriaxone, ceftazidime, cefpyramide, cefthrozine, cefodizime, cefpirom, cefozopran, cefepime, cefoselas, cefterampiboxime, cefetamethifepoxicefide , Ceftibbutene, cefditoren pivoxil, cefcapene pivo Sill, Sefuperazon, cefotetan, latamoxef, and the like flomoxef. Preferably cefotaxime, ceftizoxime, cefmenoxime, cefoperazone, ceftriaxone, ceftazidime, cefpyramide, cefthrozine, cefodizime, cefpyrom, cefozopran, cefepime, cefoselas, cefterampiboxime, cefetamethoxipifem cepoxime cepoxime cepoxime , 3rd generation cephem such as ceftibene, cefditoren pivoxil, cefcapene pivoxil, cefperazone, cefotetan, latamoxef, flomoxef. More preferably, cefotaxime, ceftizoxime, cefmenoxime, cefoperazone, ceftriaxone, ceftazidime, cefpyramide, cefthrosin, cefodizime, cefpyrom, cefozopran, cefepime, cefoselas, cefterampiboxime, cefetamethoxipicepoxime cepoxime cepoxime Cephalosporin-based cephem such as cetyl, ceftibbutene, cefditoren pivoxil, cefcapene pivoxil. Most preferred is cefixime.

  In the present specification, the stool subject to detection of enterohemorrhagic Escherichia coli includes stool specimens, in particular, specimens of legal stool specimens of food and beverage workers, and foods that are suspected of food poisoning, such as sushi, lunch boxes, Examples include noodles, seafood, tofu, school lunch, and meat. Examples of water include rivers, lakes, well water, ground water, and waterworks.

  The pH of the medium of the present invention is usually about 4 to 10, preferably about 5 to 9, although it depends on the type of pH indicator used. For example, the preferred pH of the medium when bromthymol blue (BTB) is used as the pH indicator is about 6.0 to 8.0.

The amount of each component per 1000 mL of medium used in the present invention is as follows:
2,3,5-triphenyltetrazolium chloride (TTC) about 0.1 to 500 mg;
Sorbit about 1-50g;
Rhamnose about 1-50g;
Bromothymol blue (BTB) about 1 to 500 mg;
About 0.1 to 100 mg of potassium tellurite;
One of sorbit and rhamnose is usually used, but both may be used in combination.

A person skilled in the art can easily determine the amount of other redox dye used with reference to the above description of the amount of TTC used. Similarly, the amount of sugar other than sorbitol and rhamnose, pH indicator other than BTB, and tellurite other than potassium tellurite can be easily determined by those skilled in the art with reference to these amounts.
Cefixime 0.005 to about 1 mg;
Carbenicillin about 0.01-100 mg.

  Antibiotics such as cefixime and carbenicillin are preferably used in the above range in order to prevent the growth of Morganella, Proteus and Providencia, which are often captured as false positives, and to suppress false positives. Cephem antibiotics other than cefixime and penicillin antibacterials other than carbenicillin allow colonization of enterohemorrhagic Escherichia coli, but allow the amount used to prevent the growth of Morganella, Proteus and Providencia. A person skilled in the art can easily determine the amount of cefixime and carbenicillin used as a reference.

  The medium of the present invention may contain the above components. Other components are not particularly limited as long as they are components of a medium usually used for detection of E. coli. Examples of such a medium include SMAC medium, RMAC medium, SBMAC medium, CT-SMAC medium, CTRMAC medium, and CTSBMAC medium. Since sugars such as sorbitol, rhamnose, and sorbose are contained in these known media, the sugar as a component of the media can be used as it is. However, when the enterohemorrhagic Escherichia coli O157, O26, and O111 are discriminated simultaneously, it is necessary to appropriately set the type and amount of sugar. In the present invention, by using 2-23 g / L rhamnose for sugar, it becomes possible to discriminate O157, O26, and O111 grown on one medium. On the other hand, since the redox dye is not contained in these known media, a necessary amount is added. In addition, as the pH indicator, one contained in a known medium for Escherichia coli such as neutral red may be used as it is, and it is changed to a preferable pH indicator such as BTB instead of the pH indicator contained in advance. Also good. Moreover, since cefixime and potassium tellurite are contained in the CT-SMAC medium, they may be used as they are, and the blending amount may be adjusted. For example, when SMAC medium is used, cefixime and potassium tellurite may be added as optional components in the amounts exemplified above. Furthermore, it is not particularly limited as long as it is an amount that can suppress the growth of non-degrading sorbite such as Morganella, Proteus and Providencia by combining penicillin-based antibacterial agents, and can be appropriately selected within the concentration range exemplified above. . Addition of cefixime and tellurite (potassium tellurite, etc.) selective agents can suppress the growth of bacteria that cause false positives, but penicillin antibacterial agents can be used in combination with cefixime to prevent false positives. Has a synergistic effect on inhibition.

  Moreover, this culture medium can be made into a liquid culture medium, a semi-fluid culture medium, and a solid culture medium (plate) by adjusting the amount of agar in a composition. In other words, when the agar concentration is 0 to 0.04% (0 to 0.4 g / L), the medium becomes a liquid medium, and the agar concentration is 0.05 to 0.6% (0.5 to 6.0 g / L). In this case, the medium is a semi-fluid medium, and when the agar concentration is 1-2% (10.0-20.0 g / L), the medium is a solid medium. The liquid medium is useful for micro liquid dilution methods using microplates and the like, and the semi-fluid medium is suitable for puncture culture, so it is useful as a medium for storing bacteria and transporting specimens. The solid medium (agar plate) It is useful for general bacterial culture and plate dilution method.

  In the method for detecting enterohemorrhagic Escherichia coli of the present invention, a stool specimen (stool specimen) or a stool specimen suspension (dilution) is smeared on a solid medium such as an agar plate medium using a sterilized plastic stick (stool collection stick) or the like. Incubating at a temperature of about 35 to 37 ° C. for about 12 to 48 hours, preferably about 16 to 24 hours, and detecting colony of enterohemorrhagic E. coli visually or mechanically, Whether or not E. coli is contained can be detected. Since the detection method of the present invention has very high sensitivity (selectivity) and has a lower false positive ratio than the conventional method using a culture medium, a large amount of stool specimens can be efficiently examined.

  Hereinafter, the present invention will be described in more detail based on examples. It should be noted that the following examples are merely illustrative and do not limit the present invention.

Example 1
Regarding the commercially available CT-SMAC agar medium and the sorbitol / Macconkey medium of the present invention (the medium of the present invention) containing the following components, the growth of each strain shown in Table 4 was determined based on the Misra method.
Medium of the present invention: In the sorbitol / Macconkey medium shown in “Composition 10” of Example 1 of JP-A-2002-281959, the pH indicator neutral red was changed to bromthymol blue, and 2,3,5-tri A medium containing phenyltetrazolium chloride (TTC), carbenicillin in an amount in the range indicated in the specification.

Specifically, inoculate the culture medium of the present invention (agar plate medium) and commercially available CT-SMAC agar medium (control medium) with the bacterial species described in Table 4, and culture at 37 ° C. for 20 hours. Investigated by the Misra method. That is, a McFarland No. 1 bacterial suspension was prepared from a colony pre-cultured on a blood agar medium, and diluted in a 10-fold serial dilution series. Each medium was inoculated with 10 μL of each diluted bacterial solution of 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ , and 10 ⁻⁶ .

As shown in Table 4, Morganella (M. morganii), which is a non-degrading sorbite bacterium, has the same growth of O157 as the commercially available CT-SMAC medium, but false positives are problematic. And Proteus (P. vulgaris) and Providencia (P. rettgeri) growth can be suppressed.
Example 2
A mixed solution of O157 and K. pneumoniae, which is a sorbite-degrading bacterium, was inoculated into the medium of the present invention and commercially available CT-SMAC, and cultured at 37 ° C. for 24 hours. The results are shown in FIG.

As shown in FIG. 1, the media of the present invention clearly showed distinction between O157 (reddish brown) and K. pneumoniae (yellow), and the visibility was improved.
Example 3
A mixed solution of O26 and rhamnose-degrading bacterium K. pneumoniae was inoculated into the medium of the present invention and cultured at 37 ° C. for 24 hours. The results are shown in FIG.

  As shown in FIG. 2, the media of the present invention clearly showed distinction between O26 (reddish brown) and K. pneumoniae (yellow), and the visibility was improved.

Example 4
A mixed solution of O157, O26, and O111 was inoculated into the following medium of the present invention and a conventional medium (neutral red medium) and cultured at 37 ° C. for 24 hours. The results are shown in FIG.

As shown in FIG. 3, in the culture medium of the present invention, the distinction between O157 (yellowish brown), O26 (reddish brown), and O111 (yellow) was clear, and it became clear that the visibility was improved. On the other hand, it became clear that it was difficult to distinguish between O157 and O26 in the conventional medium.
Medium of the present invention: In the sorbitol / Macconkey medium shown in “Composition 10” of Example 1 of JP-A-2002-281959, rhamnose (9 g / L) is used instead of sorbitol, and neutral red, which is a pH indicator, is bromthymol A medium changed to blue and containing 2,3,5-triphenyltetrazolium chloride (TTC), carbenicillin in an amount in the range indicated in the specification.
Conventional medium: A medium in which rhamnose (9 g / L) is used instead of sorbitol in the sorbitol-Macconkey medium shown in “Composition 10” of Example 1 of JP-A-2002-281959.
Example 5
In the formulation of the present invention of Example 4, the same experiment was conducted by changing the amount of rhamnose to 2 g / L, 3 g / L, and 23 g / L. The results are shown in FIG. It was revealed that O157, O26, and O111 can be differentiated when rhamnose is in the range of 3 to 23 g / L. With rhamnose 2g / L, it was difficult to distinguish three types of enterohemorrhagic E. coli, indicating a critical significance over rhamnose 3g / L.
Example 6
In the medium of Example 1, the medium in which the pH indicator and the redox dye were changed as follows was tested whether O157 and K. pneumoniae could be distinguished with the naked eye. The results are shown in Table 5 below. In Table 5,
Phenol red: PR
Bromothymol Blue: BTB
Neutral red: NR
Water blue: WB
o-Nitrophenyl-β-D-galactoside: ONPG
Means.

  From the above results, excellent visibility of the culture medium of the present invention in which a pH indicator and a redox dye were combined was confirmed.

Claims (3)

  A medium for detecting enterohemorrhagic Escherichia coli containing tellurite and penicillin antibiotics.   The medium for detecting enterohemorrhagic Escherichia coli according to claim 1, further comprising cephem antibiotics in addition to tellurite and penicillin antibiotics.   The medium for detecting enterohemorrhagic Escherichia coli according to claim 1 or 2, which suppresses the growth of Morganella spp., Providencia spp. And / or Proteus spp. Present together with enterohemorrhagic Escherichia coli in a test sample .