JP2011019462A - Selecting and separately culturing medium for enterohemorrhagic escherichia coli o-157, o-26 and o-111 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a selecting and separately culturing medium capable of selecting by identifying and separately culturing enterohemorrhagic Escherichia coli O-157, O-26 and O-111 in test specimens. <P>SOLUTION: This selecting and separately culturing medium for identifying the enterohemorrhagic Escherichia coli O-157, O-26 and O-111 is obtained by adding rhamnose, a pH-indicator and an enzyme chromogenic substrate of a glucuronide derivative as differentiating agents and also adding a tellurite, cefixime, a bile salt and/or lauryl sulfate as selecting agents, to a base medium for detecting the Escherichia coli. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a selective separation medium that can distinguish enterohemorrhagic Escherichia coli serotypes O157, O26, and O111, which are food poisoning bacteria. Further, the present invention provides a method for detecting E. coli of serotypes O157, O26, and O111 from a test sample using the medium, and a method for identifying and isolating from other enteric bacteria. It is related with the isolation method.

  In recent years, food poisoning due to enterohemorrhagic Escherichia coli has been reported in various countries around the world, and occurrence of food poisoning due to enterohemorrhagic Escherichia coli has been observed in Japan. Therefore, food poisoning due to enterohemorrhagic Escherichia coli has become a major social problem, and it is urgent to investigate the cause and establish preventive measures.

  Many enterohemorrhagic E. coli that cause this food poisoning belong to serotype O157, O26, or O111, and serotype O157, O26, or O111 E. coli from test samples obtained from food poisoning patients. It can be said that it is possible to determine whether food poisoning is caused by enterohemorrhagic Escherichia coli based on the presence or absence of detection.

  As a selective separation medium for serotype O157 Escherichia coli, novobiocin, tellurite, and lauryl sulfate are used as selective agents, and 5-bromo-4-chloro-3-indoxyl-β-D-galactopyra is used as a distinguishing agent. A medium containing sugar alcohols such as noside, 4-methylumbelliferyl-β-D-glucuronide, sorbitol, and disaccharides such as cellobiose has been proposed (Patent Document 1).

  This medium suppresses the growth of intestinal gram-negative bacilli other than E. coli by the selective agent. In the above-mentioned discrimination agent, Escherichia coli of serotype O157 cannot decompose sorbitol and cellobiose but decomposes 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside, which is an enzyme substrate, to dark blue. A green colony is formed. On the other hand, general E. coli degrades sorbitol, and general coliforms degrade cellobiose, so that the degradation of the enzyme substrate is delayed. With respect to 4-methylumbelliferyl-β-D-glucuronide, which is a fluorescent substrate, since serotype O157 E. coli does not produce β-glucuronidase, it can be distinguished from other E. coli by ultraviolet irradiation.

  Further, as a selective separation medium for serotype O26 E. coli, cefixime, tellurite, and lauryl sulfate as selective agents, and glucopyranoside derivatives or galactopyranoside derivative chromogenic substrates, pH indicators, and A medium containing rhamnose has been proposed (Patent Document 2).

  This medium suppresses the growth of Gram-positive bacteria such as Bacillus, Escherichia coli other than serotype O26, and Proteus spp. In the discrimination agent, serotype O26 Escherichia coli cannot decompose rhamnose but can decompose glucopyranoside derivatives and galactopyranoside derivatives, which are enzyme color-development substrates, and can determine whether pH changes due to the presence or absence of rhamnose degradation. Confirm by the presence or absence of discoloration of the culture medium, and differentiate from other E. coli.

  Furthermore, as a selective separation medium for Escherichia coli of serotype O111, a medium containing cefixime, tellurite, and bile acids as selective agents, and a pH indicator and L-sorbose as differential agents has been proposed (non-patent document). 1).

  This medium suppresses the growth of bacteria other than serotype O111 by the selective agent. In the discrimination agent, serotype O111 Escherichia coli cannot decompose L-sorbose, so that no pH change occurs, and no color change due to a pH indicator occurs.

  That is, when determining whether food poisoning is caused by enterohemorrhagic Escherichia coli, for example, the above-mentioned three types of selective separation media are prepared, and the test sample is cultured on those media to confirm the presence or absence of proliferation. There is a need to.

JP 2000-342249 A JP 2001-8679 A

Tanaka Hiroshi et al., Japanese Journal of Clinical Microbiology, 1999, Vol. 9, p. 48-50

  In order to determine whether food poisoning is caused by enterohemorrhagic Escherichia coli, it is necessary to prepare selective separation media for serotypes O157, O26, and O111 for E. coli, respectively. Therefore, it is expected that serotypes O157, O26, and O111 E. coli are identified, selected, and cultured by using one type of selective separation medium.

  It is generally known to utilize the ability of sugar metabolism when differentiating bacteria. The serotypes O157, O26, and O111 of E. coli are not uncommon, and are distinguished by using the metabolic ability of sorbitol, rhamnose, and sorbose as an index, respectively.

  Serotypes O157, O26, and O111, and other E. coli were examined in detail with a focus on this sugar metabolic capacity. As a result, by adjusting the amount of rhamnose, which is known to be non-degradable only for serotype O26 E. coli and other E. coli can be degraded, E. coli serotype O157 exhibits the same properties as rhamnose non-degradable. I understood it.

  Further, when various enzyme chromogenic substrates were examined, when a glucuronide derivative was used, Escherichia coli of serotype O157 did not produce its metabolic enzyme, and the amount of metabolic enzyme produced by Escherichia coli of serotype O111 was as follows. It was found that it was low compared to other E. coli production.

  From the knowledge obtained by these studies, by adjusting the amount of rhamnose and glucuronide derivatives added to the medium of the enzyme chromogenic substrate, serotypes O157, O26, and O111, as well as other E. coli, can be obtained using a single medium. I found it possible to identify.

That is, the present invention has the following configuration.
(1) Enterohemorrhagic Escherichia coli O157, O26, and O111, a medium containing rhamnose, pH indicator, enzyme chromogenic substrate, tellurite and cefixime, wherein the enzyme chromogenic substrate is a glucuronide derivative A selective separation medium that can be identified.
(2) The selective separation medium capable of distinguishing enterohemorrhagic Escherichia coli O157, O26, and O111 according to (1), further comprising bile salts and / or sodium lauryl sulfate.
(3) Enterohemorrhagic Escherichia coli according to (1) or (2), wherein the pH indicator is a pH indicator selected from phenol red, neutral red, bromocresol purple, bromothymol blue, bromophenol red, and chlorophenol red A selective separation medium that can distinguish O157, O26, and O111.
(4) The selective separation medium capable of distinguishing enterohemorrhagic Escherichia coli O157, O26, and O111 according to (1) or (2), wherein the pH indicator is neutral red.
(5) The glucuronide derivative is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 3-indolyl-β-D-glucuronide, 4-methyl-umbelliferyl-β-D-glucuronide, It is an enzyme chromogenic substrate selected from p-nitrophenyl-β-D-glucuronide, 6-chloro-3-indolyl-β-D-glucuronide, and 5-bromo-6-chloro-3-indolyl-β-D-glucuronide. A selective separation medium capable of distinguishing enterohemorrhagic E. coli O157, O26, and O111 according to any one of (1) to (4).
(6) Enterohemorrhagic Escherichia coli O157, O26 according to any one of (1) to (4), wherein the glucuronide derivative is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, and A selective separation medium that can identify O111.
(7) Per 1,000 mL of medium, 5-15 g of peptone, 2-5 g of yeast extract, 0.1-10 g of sodium chloride, 0.1-2 g of bile salt, 5-bromo-4-chloro-3-indolyl-β -D-glucuronide 0.05-0.5 g, rhamnose 0.1-8 g, potassium tellurite 1-5 mg, cefixime 0.005-0.1 mg, neutral red 0.001-0.01 g, agar 10-20 g A selective separation medium capable of distinguishing enterohemorrhagic E. coli O157, O26, and O111.
(8) In the method for distinguishing enterohemorrhagic Escherichia coli O157, O26, and O111 from enterobacteria, at least a step of culturing the sample using the selective separation medium according to any one of (1) to (7) A method for distinguishing enterohemorrhagic E. coli O157, O26, and O111.
(9) In the method for isolating and identifying enterohemorrhagic Escherichia coli O157, O26, and O111 from intestinal bacteria, a step of culturing a sample using the selective separation medium according to any one of (1) to (7) A method for isolating and identifying enterohemorrhagic E. coli O157, O26, and O111.

  The present invention will be described by way of example using neutral red as a pH indicator and 5-bromo-4-chloro-3-indolyl-β-D-glucuronide as a glucuronide derivative, but is not limited to this example.

  The composition of the basic medium for growing enterohemorrhagic E. coli O157, O26, and O111 contains 10 g of peptone, 3 g of yeast extract, 5 g of sodium chloride, and 15 g of agar per liter of the medium, and the pH is 7.2 ± 0. 2 is prepared.

  The amount of rhamnose added to this basal medium is suitably 0.1 to 8 g per liter, and the amount of neutral red is suitably 0.001 to 0.01 g per liter. Further, the appropriate amount of 5-bromo-4-chloro-3-indolyl-β-D-glucuronide added to the medium is 0.05 to 0.5 g per liter.

  Furthermore, the amount of potassium tellurite added to suppress E. coli other than enterohemorrhagic Escherichia coli O157, O26, and O111 is 1 to 5 mg per liter, and cefixime is added to suppress Proteus spp. The amount is suitably 0.005 to 0.1 mg per liter, and 0.1 to 2 g per liter of bile salt added to suppress gram-positive bacteria.

  Since the pH of the medium is adjusted to be near neutral and the amount of neutral red added is very small, the color of the medium is transparent with a slight brown color. The rhamnose-degrading bacteria degrade the rhamnose in the medium to produce an acid, and the pH is lowered to make the colony pink. On the other hand, since rhamnose non-degrading bacteria do not produce acid, the pH remains in the vicinity of neutrality and the colonies do not color.

  Escherichia coli is known to produce glucuronidase, and decomposes the enzyme-active substrate of the glucuronide derivative to cause color development. In this example using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide as the glucuronide derivative, the colonies are green. However, serotype O157 E. coli does not produce glucuronidase exceptionally and colonies do not develop color.

  As described above, Escherichia coli of serotype O111 has a small production amount of glucuronidase and decomposes 5-bromo-4-chloro-3-indolyl-β-D-glucuronide to develop a color, but the colony is weak due to a weak reaction. Presents a light green color.

  The color of colonies of E. coli when rhamnose and neutral red and 5-bromo-4-chloro-3-indolyl-β-D-glucuronide shown in this example are combined and added to the medium is shown below.

  Serotype O157 Escherichia coli shows the same behavior as rhamnose non-degrading bacteria by adjusting the amount of rhamnose added, and does not produce glucuronidase, so the colony is colorless.

  Escherichia coli of serotype O26 is a rhamnose non-degrading bacterium and produces glucuronidase, so that the colony is green.

  Escherichia coli of serotype O111 is a rhamnose-degrading bacterium and produces a weak reaction with little production of glucuronidase.

  The growth of most of the other E. coli is suppressed by adding tellurite, but even if it grows, it is a rhamnose-degrading bacterium and produces glucuronidase, so that the colony is purple.

  Although many bacteria other than E. coli are inhibited from growth by adding tellurite, cefixime, and bile salts and / or lauryl sulfate, colonies are small even if they grow. Since the growing bacteria do not produce glucuronidase, the colonies are colorless or pink depending on the presence or absence of rhamnose resolution.

  As described above, by using the culture medium of this example, serotypes O157, O26, O111, and other E. coli and other bacteria can be identified using one type of culture medium.

  By carrying out the present invention, it is possible to select and separate cultures that identify enterohemorrhagic Escherichia coli O157, O26, and O111, which are important as food poisoning bacteria, in a test sample using a single type of medium. .

  Nutrient and salt concentrations to support bacterial growth, and basal medium containing gelling agent to form a plate medium, rhamnose and pH indicator to differentiate bacteria, and glucuronide derivative of enzyme chromogenic substrate, detection target Identify, select and isolate enterohemorrhagic E. coli O157, O26, and O111 by adding tellurite, cefixime, and bile salts and / or sodium lauryl sulfate, which inhibit the growth of other bacteria I can do it.

Confirmation of drug concentration to identify target bacteria
1. In order to examine the difference in the degradability of rhamnose due to the difference in the concentration of rhamnose due to the difference in the rhamnose concentration, the rhamnose was added to a medium having the following composition, and the rhamnose concentration was 0.1, 4, 8 The medium was prepared so as to be 15 g / L. Using one strain each of serotypes O157, O26, O111, and other E. coli as test bacteria, incubate overnight in a heart-infusion broth medium, inoculate the platinum ears on each medium, and incubate at 37 ° C for 18 hours The pink coloration of the colony after this was confirmed.

Medium composition (per liter of medium)
Peptone 10g
Sodium chloride 5g
Neutral red 0.03g
Agar 15g
pH 7.2 ± 0.2

  Serotype O26 E. coli without rhamnose degradability did not show a pink color at any concentration. On the other hand, Escherichia coli other than serotype O26 is known to have rhamnose-degrading properties, and serotype O111 and other Escherichia coli showed a pink color at any concentration. However, serotype O157 E. coli showed a pink color at 15 g / L, but a light pink color at 8 g / L, and no pink color at 0.1 and 4 g / L.

  From these results, it was confirmed that by setting the rhamnose concentration in the medium to 8 g / L or less, serotype O157 E. coli, serotype O111, and other E. coli, which are known to have rhamnose degradability, were displayed as colonies. It was found that it could be identified by color.

2.5-Bromo-4-chloro-3-indolyl-β-D- glucuronide derivative glucuronidase active enzyme chromogenic substrate 5-bromo-4-chloro-3-indolyl-β In order to examine the difference in glucuronidase activity due to the difference in the concentration of D-glucuronide, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide was added to heart infusion broth medium, and 12.5 and 50 mg Prepare the culture medium so as to be / L, and dispense 100 μL each into a microplate. In this medium, one strain each of serotypes O157, O26, O111, and other E. coli was cultured overnight in a heart-infusion broth medium. After inoculating 0.00015 mL of the preparation prepared in 1 and culturing at 37 ° C. for 18 hours, green color development was confirmed.

  In E. coli serotype O157, no color development was observed at both concentrations. In serotype O26 Escherichia coli, color development was observed at both concentrations. In E. coli serotype O111, color development was observed at 50 mg / L, but no color development was observed at 12.5 mg / L. Other E. coli developed color at both concentrations.

  From this result, it was determined that serotype O157 E. coli did not produce glucuronidase, and serotype O111 E. coli produced glucuronidase, but was lower than the production of glucuronidase of serotype O26 and other E. coli. From this, it was found that serotypes O157, O26, and O111 can be distinguished using the color development of a glucuronide derivative, which is an enzyme color development substrate of glucuronidase, as an index.

Combination of pH indicator and glucuronide derivative that is enzyme coloring substrate In order to examine whether or not enterohemorrhagic Escherichia coli O157, O26, and O111 can be distinguished by combinations of various pH indicators and glucuronide derivatives, 0.005 g is added to a medium having the following composition: A medium was prepared by adding / L pH indicator and 0.1 g / L glucuronide derivative. Using one strain each of serotypes O157, O26, O111, and other E. coli as test bacteria, incubate overnight in a heart-infusion broth medium, inoculate the platinum ears on each medium, and incubate at 37 ° C for 18 hours The coloration of the colonies after the check was confirmed.

  In addition, phenol red (PR), bromocresol purple (BCP), bromothymol blue (BTB), bromophenol red (BPR), and chlorophenol red (CPR) are used as pH indicators, and 5-bromo is used as a glucuronide derivative. -4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-indolyl-β-D-glucuronide (Y-Gluc), 4-methyl-umbelliferyl-β-D-glucuronide (MU) -Gluc), p-nitrophenyl-β-D-glucuronide (PNP-Gluc), 6-chloro-3-indolyl-β-D-glucuronide (salmon-Gluc), and 5-bromo-6-chloro-3- Indolyl-β-D-glucuronide (magenta-Gluc) was used.

Medium composition (per liter of medium)
Peptone 10g
Yeast extract 3g
Sodium chloride 5g
Bile salt 1.2g
Rhamnose 7g
Agar 15g
pH 7.2 ± 0.2

  The coloration of serotypes O157, O26, O111 and other colonies of E. coli in the medium of each combination of pH indicator and glucuronide derivative is shown in Table 1, and whether or not each combination can be identified is summarized in Table 2. . The three combinations (BCP and PNP-Gluc, BTB and salmon-Gluc, and BTB and magenta-Gluc) were slightly indistinguishable, but could be easily identified.

Detection of bacteria when neutral red is used as a pH indicator and 5-bromo-4-chloro-3-indolyl-β-D-glucuronide is used as a glucuronide derivative. Serotypes O157, O26, and O111 are used as test bacteria in a medium having the following composition. , Other E. coli and other bacteria, 10 strains, 12 strains, 9 strains, 6 strains, and 42 strains, respectively, were cultured overnight in a heart-infusion broth medium, and one platinum loop was smeared on each medium. Then, after culturing at 37 ° C. for 18 hours, the presence or absence of growth and the coloration of the colonies were examined.

Medium composition (per liter of medium)
Peptone 10g
Yeast extract 3g
Sodium chloride 5g
Bile salt 1.2g
5-Bromo-4-chloro-3-indolyl-β-D-glucuronide 0.2 g
Rhamnose 7g
Potassium tellurite 0.002g
Cefixime 0.05mg
Neutral red 0.005g
Agar 15g
pH 7.2 ± 0.2

  The results are shown in Table 3. As for the serotypes O157, O26, and O111, the growth of E. coli was observed in all cases, and the colors of the colonies differed depending on the serotype, and were colorless, green, and orange, respectively. On the other hand, in other E. coli, growth was observed in 2 out of 6 strains, and the color of the colony was purple. In other bacteria, growth was observed in 8 out of 42 strains, the colony color was 4 colorless and 4 pink, and the 4 colorless strains were as small as about 0.1 mm in colony size. there were.

  From this result, serotypes O157, O26, and O111 of E. coli show good growth on the medium of the present invention, and the colonies exhibit colorless, green, and orange colors, respectively, so that the identified detection is possible. is there. On the other hand, in other Escherichia coli and other bacteria, there are few examples showing good growth in the medium of the present invention, and even if grown, the coloration of micro or colonies is different, so that serotypes O157, O26, and O111 Detection is possible by identifying E. coli.

Comparison between the Invention of the Present Invention and the Conventional Technology The coloration of the growing colonies of the culture medium of the present invention and the conventionally used medium was compared. The medium composition of the present invention was as described in (Example 3). As the medium to be compared, desoxycholate agar medium (DESO), sorbitol-added MacConkey agar medium (S-MAC), rhamnose-added MacConkey agar medium (R-MAC), and sorbose-added MacConkey agar medium (SB-MAC), The composition is shown below.

  In addition, serotypes O157, O26, O111, other Escherichia coli, and other enteric bacteria were used as test bacteria, respectively, 15 strains, 15 strains, 9 strains, 10 strains, and 9 strains, and in a heart infusion broth medium. After culturing overnight, each platinum loop was smeared on each medium, and after culturing at 37 ° C. for 18 hours, the presence or absence of growth and coloration of the colonies were examined.

DESO composition (per liter of medium)
Peptone 10g
Lactose 10g
Sodium desoxycholate 1g
Sodium chloride 5g
2g dipotassium phosphate
2g iron ammonium citrate
Neutral red 0.033g
Agar 15g
pH 7.2 ± 0.2

Composition of S-MAC (per liter of medium)
Peptone 20g
Sorbitol 10g
Bile salt No2 1.5g
Sodium chloride 5g
Neutral red 0.03g
Crystal Violet 0.001g
Agar 15g
pH 7.2 ± 0.2

R-MAC composition (per liter of medium)
Peptone 20g
Rhamnose 10g
Bile salt No2 1.5g
Sodium chloride 5g
Neutral red 0.03g
Crystal Violet 0.001g
Agar 15g
pH 7.2 ± 0.2

Composition of SB-MAC (per 1L of medium)
Peptone 20g
Sorbose 10g
Bile salt No2 1.5g
Sodium chloride 5g
Neutral red 0.03g
Crystal Violet 0.001g
Agar 15g
pH 7.2 ± 0.2

  The results are shown in Table 4. In the culture medium of the present invention, E. coli of serotypes O157, O26, and O111 all developed, and the colonies were colorless, green, and orange, respectively. There were few cases of other bacteria growing, and the grown colonies were purple or pink.

In the desoxycholate agar medium, growth was observed in all cases, and the color was red except that some other bacterial colonies were colorless.
In sorbitol-added MacConkey agar medium, growth was observed in all cases, and the colonies of E. coli of serotype O157 exhibited colorlessness, but otherwise, red except that some other bacterial colonies were colorless. Was presented.

On the rhamnose-added MacConkey agar medium, growth was observed in all cases, and the colonies of E. coli of serotype O26 exhibited colorlessness, but other than that, red color except that some other bacterial colonies were colorless. Was presented.
On sorbose-added McConkie agar, growth was observed in all cases, and serotype O111 colonies were colorless, while others were red except that some other bacterial colonies were colorless. Was presented.

  From this result, when the conventional medium is used alone, only one serotype of enterohemorrhagic Escherichia coli can be identified and detected, but it is difficult to distinguish from some other bacteria. By using the medium of the present invention alone, it is possible to identify and detect three types of enterohemorrhagic E. coli of serotypes O157, O26, and O111.

  Conventionally, for detection of enterohemorrhagic Escherichia coli O157, O26, and O111, O157 is a sorbitol-added McConkey agar medium, O26 is a rhamnose-added McConkey agar medium, and O111 is a sorbose-added McConkey agar medium and a separation medium for each serotype. 3 plate media were required.

  However, in the culture medium of the present invention, enterohemorrhagic Escherichia coli O157, O26, and O111 can be developed in different colors by one selective separation culture, and enterohemorrhagic Escherichia coli O157, O26, or O111 in the test sample can be developed. Can be easily determined.

  Therefore, in the food industry field, the process for verifying the presence of enterohemorrhagic Escherichia coli in raw materials and products can be shortened, contaminated products can be eliminated and products can be shipped quickly, and food distribution Can greatly contribute to the economics of

  Further, in the medical field, feces and vomits can be used as test samples to enable early diagnosis of food poisoning patients with enterohemorrhagic Escherichia coli O157, O26, or O111. Can contribute.

Claims (9)

  A medium containing rhamnose, pH indicator, enzyme chromogenic substrate, tellurite and cefixime, characterized in that enterohemorrhagic E. coli O157, O26 and O111 are characterized in that the enzyme chromogenic substrate is a glucuronide derivative Selective separation medium.   The selective separation medium capable of distinguishing enterohemorrhagic E. coli O157, O26, and O111 according to claim 1, further comprising bile salts and / or sodium lauryl sulfate.   The enterohemorrhagic E. coli O157, O26 according to claim 1 or 2, wherein the pH indicator is a pH indicator selected from phenol red, neutral red, bromocresol purple, bromothymol blue, bromophenol red, and chlorophenol red. A selective separation medium that can identify O111.   The selective separation medium capable of distinguishing enterohemorrhagic Escherichia coli O157, O26, and O111 according to claim 1 or 2, wherein the pH indicator is neutral red.   The glucuronide derivative is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 3-indolyl-β-D-glucuronide, 4-methyl-umbelliferyl-β-D-glucuronide, p-nitro. An enzyme chromogenic substrate selected from phenyl-β-D-glucuronide, 6-chloro-3-indolyl-β-D-glucuronide, and 5-bromo-6-chloro-3-indolyl-β-D-glucuronide. A selective separation medium capable of distinguishing enterohemorrhagic Escherichia coli O157, O26, and O111 according to any one of 1 to 4.   Selection capable of distinguishing enterohemorrhagic Escherichia coli O157, O26 and O111 according to any one of claims 1 to 4, wherein the glucuronide derivative is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide Separation medium.   Per 1,000 mL of medium, 5-15 g of peptone, 2-5 g of yeast extract, 0.1-10 g of sodium chloride, 0.1-2 g of bile salt, 5-bromo-4-chloro-3-indolyl-β-D- Contains glucuronide 0.05-0.5 g, rhamnose 0.1-8 g, potassium tellurite 1-5 mg, cefixime 0.005-0.1 mg, neutral red 0.001-0.01 g, agar 10-20 g A selective separation medium capable of distinguishing enterohemorrhagic E. coli O157, O26, and O111.   A method for distinguishing enterohemorrhagic E. coli O157, O26, and O111 from enterobacteria, comprising at least a step of culturing a sample using the selective separation medium according to any one of claims 1 to 7, Identification method of O157, O26, and O111.   A method for isolating and identifying enterohemorrhagic Escherichia coli O157, O26, and O111 from intestinal bacteria, comprising at least a step of culturing a sample using the selective separation medium according to any one of claims 1 to 7. A method for isolating and identifying hemorrhagic E. coli O157, O26, and O111.