JP3026005B1 - Development of rhamno-smacconkey (RMAC) medium and rhamno-smacconkey (CT-RMAC) medium supplemented with cefixime potassium potassium tellurite, which are selective separation media for enterohemorrhagic Escherichia coli O26 - Google Patents

Abstract

The present invention provides a selective separation medium capable of selectively separating enterohemorrhagic Escherichia coli O26 by culturing a sample. SOLUTION: The selective separation medium for enterohemorrhagic Escherichia coli O26 according to the present invention comprises a basal medium for detecting Escherichia coli,
Rhamnose is added as a sugar component, and tellurite and cefixime are added to inhibit the growth of other bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a selective culture medium for enterohemorrhagic Escherichia coli for culturing a sample to selectively isolate enterohemorrhagic Escherichia coli O26. The present invention also relates to an isolation method for isolating enterohemorrhagic Escherichia coli O26 from other intestinal bacteria using the above-mentioned medium, and to an identification method for identification.

[0002]

2. Description of the Related Art In recent years, food poisoning due to enterohemorrhagic Escherichia coli (EHEC) has been reported from all over the world. The first report was made in Japan in 1984, and in July 1996.
O15, a type of enterohemorrhagic Escherichia coli in Sakai City, Osaka
7 has become a major social problem since the outbreak of food poisoning, and it is urgently necessary to investigate the cause and establish preventive measures.

[0003] Here, as for O157, it is known that O157 has a slow decomposition property to sorbitol, and a selective separation medium of O157 has been developed using this as an index. Therefore, by culturing a sample using this, O157 can be accurately detected, and a rapid and accurate test method for infectious diarrhea has been successfully established.

[0004]

Since 1996, cases of infectious diarrhea caused by enterohemorrhagic Escherichia coli other than O157, such as O26 and O111, have been increasing. In particular, O26 is currently known as enterohemorrhagic E. coli.
It is detected as the causative bacterium at a frequency after 157.

For enterohemorrhagic Escherichia coli other than O157, a suitable selective separation medium has not been developed yet.
In order to detect enterohemorrhagic Escherichia coli other than 57, DHL (Desoxycholate Hydrogen) which is a selective separation medium for detecting common pathogenic Escherichia coli (including enterohemorrhagic Escherichia coli) is used.
Bacteria had to be grown using sulfide lactose) media.

However, when this DHL medium is used, pathogenic Escherichia coli and many other nonpathogenic Escherichia coli present in the living body of animals including humans and in the natural world decompose lactose in the medium to red. They grow by forming the indicated communities. For this reason, in order to detect O26, it was necessary to extract all Escherichia coli (take the bacteria out of the culture medium) and conduct biochemical and serologic tests on all of them. Such a detection method required a great deal of time and labor due to inefficiency.

Accordingly, the present invention has been made in view of the above-mentioned circumstances, and has as its object to provide a selective isolation medium capable of selectively isolating only enterohemorrhagic Escherichia coli O26 by culturing a sample. I have.

Another object of the present invention is to provide a method for isolating enterohemorrhagic Escherichia coli O26 using the above-mentioned medium, and a method for identifying this enterohemorrhagic Escherichia coli O26.

[0009]

Means for Solving the Problems Here, in order to solve the above-mentioned problems, the selective separation medium for enterohemorrhagic Escherichia coli O26 according to the present invention is used as a basal medium for detecting Escherichia coli.
It is characterized by adding rhamnose as a sugar component. This rhamnose is added in an amount of 0.1 per 1000 ml of the culture medium.
g to 50 g.

[0010] It is preferable that the above-mentioned basal medium is added to an intestinal bacterial isolation medium using bile salts.

Further, it is preferable that tellurite is added to the selective separation medium. As the tellurite, potassium tellurite is preferable.
It is added in an amount of 0.1 mg to 25 mg per 0 ml.

It is preferable that an antibiotic that inhibits the growth of the intestinal flora be added. Cefixime is preferred as the antibiotic, and 0.1 mg / 1000 ml of medium is used.
It is added in an amount of from 01 mg to 1.0 mg.

The enterohemorrhagic Escherichia coli O2 according to the present invention is also provided.
The isolation method according to 6 is characterized by including at least a step of culturing the sample using any one of the selective separation media described above.

The enterohemorrhagic Escherichia coli O2 according to the present invention is also used.
The identification method of No. 6 is characterized by including at least a step of culturing a sample using any of the selective separation media described above.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below.

The present inventors have proposed enterohemorrhagic Escherichia coli O15.
7, O26 (hereinafter simply referred to as “O157”, “O2
As a result of examining the biochemical properties of many Escherichia coli such as O.6, it is found that unlike other Escherichia coli, O26 cannot degrade the sugar rhamnose (6-deoxymannose). It was found to be resistant to potassium tellurite.

From the findings obtained here, a basal medium for detecting Escherichia coli, for example, a medium using bile salts which is widely known as a medium for separating intestinal bacteria, for example, a medium developed by MacConkey et al. The inventors have found that O26 can be detected by replacing lactose, which is a sugar component of (MAC medium), with rhamnose, and have completed the present invention.

The selective isolation medium for enterohemorrhagic Escherichia coli O26 according to the present invention is obtained by adding rhamnose as a sugar component to a basal medium for detecting Escherichia coli.

Here, the basal medium includes MacConkey's medium, which is a medium for separating intestinal bacteria using bile salts. Table 1 shows a composition example of a typical MacConkey medium and a composition example of a selective separation medium (rhamnose MAC medium: RMAC medium) for enterohemorrhagic Escherichia coli O26 according to the present invention.

[0020]

[Table 1]

In the RMAC medium as shown in Table 1, intestinal bacteria other than O26 form red colonies and grow. This is because other intestinal bacteria have rhamnose degradability.

That is, when intestinal bacteria such as Escherichia coli grow in a medium and decompose sugar components, an acid is generated. This acid
At the same time, the yellowish brown color of neutral red added to the medium as an indicator is changed to red, and at the same time, insoluble bile acids are precipitated from bile salts. Since this bile acid originally has a property of strongly binding to a dye such as neutral red or methylene blue, the precipitated bile acid binds to reddened neutral red, so that the colony exhibits a clear brick red color. Therefore, a red settlement is formed.

On the other hand, Escherichia coli which does not decompose the sugar component does not generate acid during the growth process in the medium, so that neutral red does not turn red. Therefore, the red settlements are not formed as described above.

In the present invention, O26, unlike other intestinal bacteria, does not degrade rhamnose, which is a sugar component.
It does not turn neutral red and therefore does not form red colonies, but forms white colonies and develops. By culturing bacteria from a specimen using the RMAC medium based on such a principle, O26 in the specimen can be selectively separated.

In the medium composed of the components shown in Table 1, the rhamnose content is used in an amount of 0.1 g to 50 g, preferably 5 g to 15 g, particularly preferably 8 g to 12 g per 1000 ml of distilled water. Can be

If a large amount of rhamnose is used, the osmotic pressure may change and cell growth may be inhibited, and if it is a small amount, the nature of rhamnose degradation exhibited by bacteria becomes difficult to see.

As the bile salt, for example, sodium taurocholate is used, which is mainly used for inhibiting the growth of Gram-positive bacteria.

The bile salt must prevent the growth of Gram-positive bacteria and must have an amount that does not affect E. coli. For example, Bacto Bile sal
In ts No.3 (difco), per 1000ml of distilled water,
It is used in an amount of 0.1 g to 15 g, preferably 0.5 g to 5 g, particularly preferably 1.0 g to 2.0 g.

Crystal violet inhibits the growth of Gram-positive bacteria and interacts with bile salts.

Although the selective separation medium of the present invention has been described by way of example with the composition shown in Table 1 above (RMAC medium), it is needless to say that the present invention is not limited to this. In addition, an example using neutral red as an indicator is given, but the present invention is not limited to this, and a specific color is exhibited by reacting with an acid generated by decomposing a sugar component in a medium during the growth process of Escherichia coli. At the same time, any indicator may be used as long as the reactant further reacts with bile acid to give a predetermined color.

The selective culture medium for enterohemorrhagic Escherichia coli O26 according to the present invention preferably further contains tellurite which mainly inhibits the growth of Gram-negative bacteria. Examples of the tellurite include potassium tellurite, sodium tellurite and the like, and potassium tellurite is particularly preferred.

[0032] Potassium tellurite is distilled water 1000m
0.1 mg to 25 mg, preferably 1 mg
-7 mg, particularly preferably 1.5 mg-5.0 mg.

Here, tellurite is a drug that mainly suppresses the growth of Gram-negative bacteria, but enterohemorrhagic Escherichia coli generally shows resistance to this tellurite, Most enterobacteria are susceptible,
Its growth is inhibited.

Therefore, the growth of intestinal bacteria other than enterohemorrhagic Escherichia coli is inhibited by using a medium obtained by adding tellurite to the RMAC medium. Further, in this medium, enterohemorrhagic Escherichia coli other than O26 grows by forming red colonies, and O26 grows by forming white colonies.

As described above, when a medium containing tellurite is used, even if the enterohemorrhagic Escherichia coli O26 is a sample in which many other enterobacteria are mixed, the enterohemorrhagic hemorrhage can be obtained by simply culturing the sample. Escherichia coli is selectively cultured, especially O.
Only 26 form white colonies and are cultured.

When this medium to which tellurite is added is used, it is preferable to add an antibiotic which inhibits the growth of Proteus which is a part of the intestinal flora. Examples of the antibiotic include cefixime, cefuroxime, cephalexin, etc., with cefixime being particularly preferred.

Cefixime is generally used in an amount of 0.01 mg to 1.0 mg, preferably 0.0 mg to 1000 ml of distilled water.
1 mg to 0.2 mg, particularly preferably 0.01 mg to
Used in an amount of 0.08 mg.

The enterohemorrhagic Escherichia coli O2 according to the present invention is also used.
The identification method of No. 6 is a method including at least a step of culturing a sample using the selective separation medium described above.

As described above, when the selective separation medium of the present invention is used without the addition of tellurite, many intestinal bacteria including gram-negative bacteria form red colonies and grow. In contrast, O26 forms white colonies and grows. On the other hand, when a medium supplemented with tellurite is used, only enterohemorrhagic E. coli resistant to this drug grows, and enterohemorrhagic E. coli other than O26 forms a red colony. Therefore, O26 can be identified because O26 forms a white settlement.

The enterohemorrhagic Escherichia coli O2 according to the present invention is also used.
The isolation / identification method of No. 6 is a method including at least a step of culturing a sample using the selective separation medium described above.

As described above, no matter which selective separation medium of the present invention is used, the sample is cultured as it is, and the presence or absence of the formation of white colonies is visually identified. Whether the body is due to O26 or not can be confirmed by examining biochemical and serological properties. The biochemical properties are properties of Escherichia coli and the presence or absence of the production of verotoxin characteristic of enterohemorrhagic Escherichia coli. The serological properties are detection of bacterial antigen (O26).

As described above, since the selective separation medium for enterohemorrhagic Escherichia coli O26 according to the present invention is obtained by adding rhamnose as a saccharide component of a basal medium for detecting Escherichia coli, a general method for decomposing rhamnose is used. Intestinal bacteria form red colonies and grow, whereas enterohemorrhagic Escherichia coli O26, which does not degrade rhamnose, forms white colonies and grows. Therefore, by culturing a sample using this medium, O26 can be selectively separated.

Further, by adding tellurite to the RMAC medium, the growth of intestinal bacteria other than enterohemorrhagic Escherichia coli is inhibited. Enterohemorrhagic Escherichia coli other than O26 grows by forming red colonies, and O26 grows by forming white colonies. Therefore, by simply culturing bacteria in a sample using this selective separation medium, enterohemorrhagic Escherichia coli can be selectively cultured even in a sample in which a large number of various intestinal bacteria are mixed. Only develop white colonies.

The enterohemorrhagic Escherichia coli O2 according to the present invention is also used.
According to the identification method and the isolation / identification method of No. 6, intestinal bleeding can be performed simply by culturing from a sample using the selective separation medium and visually confirming whether or not white colonies are formed. Identification, isolation and identification of sex E. coli O26 can be facilitated.

Therefore, when discriminating the presence or absence of O26 in a sample, fishing bacteria for each colony of all Escherichia coli, which were conventionally performed after culturing intestinal bacteria using a general medium, There is no need to test for biochemical and serological properties, resulting in a very large reduction in labor and time involved in each operation. From the foregoing, it is possible to provide a method for quickly and reliably testing for infectious diarrhea derived from enterohemorrhagic Escherichia coli O26.

[0046]

According to the present invention, it is possible to provide a selective isolation medium for selectively isolating enterohemorrhagic Escherichia coli O26 in bacterial culture from a sample.

Further, by using the above selective separation medium,
Enterohemorrhagic Escherichia coli O26 can be easily isolated. Further, the presence / absence of O26 can be quickly, easily, and reliably determined without spending much labor and time.

[0048]

EXAMPLES Hereinafter, preferred embodiments of the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[0049]

Examples 1 to 12 Table 2 below shows the selective separation medium.
The composition shown in Table 2 was used.

[0050]

[Table 2]

Using the 12 strains of O26, the cells were cultured on the above medium. At that time, the temperature of the constant temperature bath for culture was 37 ° C., and the culture time was 24 hours. Table 3 shows the number of colonies and the color of the colonies on the medium after the culture.

[0052]

[Comparative Examples 1-4] Under exactly the same conditions as in Examples 1-12, O
Culture was performed using 4 strains of O157 instead of 26. Table 3 shows the number of colonies and the color of the colonies on the medium after the culture.

[0053]

Comparative Example 5 O26 under the same conditions as in Examples 1 to 12
Instead, culture was performed using rhamnose-degrading Escherichia coli other than enterohemorrhagic Escherichia coli. Table 3 shows the number of colonies and the color of the colonies on the medium after the culture.

[0054]

[Table 3]

[0055]

Examples 13 to 24 The same O used in Examples 1 to 12 was used.
26 strains, and under the same conditions as in Examples 1 to 12, except that a selective separation medium having the composition shown in Table 4 below was used.
Each culture was performed. Table 5 shows the number of colonies and the color of the colonies on the medium after the culture.

[0056]

[Table 4]

[0057]

Comparative Examples 6 to 9 The same strains as used in Comparative Examples 1 to 4 were cultured under the same conditions as in Comparative Examples 1 to 4 except that a medium having the composition shown in Table 4 was used. Table 5 shows the number of colonies and the color of the colonies on the medium after the culture.

[0058]

Comparative Example 10 Culture was performed under the same conditions as in Comparative Example 5 except that the same strain used in Comparative Example 5 was used and a medium having the composition shown in Table 4 was used. Table 5 shows the number of colonies and the color of the colonies on the medium after the culture.

[0059]

[Table 5]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification code FI C12R 1:19) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 1/20 C12Q 1/04 WPI (DIALOG) ) BIOSIS (DIALOG)

Claims (11)

(57) [Claims] 1. A selective isolation medium for enterohemorrhagic Escherichia coli O26, wherein rhamnose is added as a sugar component to a basal medium for detecting Escherichia coli. 2. The selective isolation medium for enterohemorrhagic Escherichia coli O26 according to claim 1, wherein the rhamnose is contained in an amount of 0.1 g to 50 g per 1000 ml of the culture medium. 3. The selective isolation medium for enterohemorrhagic Escherichia coli O26 according to claim 1 or 2, further comprising tellurite. 4. The selective isolation medium for enterohemorrhagic Escherichia coli O26 according to claim 3, wherein the tellurite is potassium tellurite. 5. The method according to claim 1, wherein the potassium tellurite is contained in a medium 100.
The enterohemorrhagic Escherichia coli O2 according to claim 4, wherein the E. coli O2 is added in an amount of 0.1 mg to 25 mg per 0 ml.
6 selective separation medium. 6. The selective isolation medium for enterohemorrhagic Escherichia coli O26 according to claim 3, further comprising an antibiotic which inhibits the growth of intestinal bacterial flora. 7. The enterohemorrhagic Escherichia coli O26 according to claim 6, wherein the antibiotic is cefixime.
Selective separation medium. 8. The selective isolation medium for enterohemorrhagic Escherichia coli O26 according to claim 7, wherein the antibiotic is added in an amount of 0.01 mg to 1.0 mg per 1000 ml of the medium. 9. The selective isolation medium for enterohemorrhagic Escherichia coli O26 according to any one of claims 1 to 8, wherein the basal medium is an intestinal bacterial isolation medium using bile salts. 10. An enterohemorrhagic Escherichia coli O26 from intestinal bacteria.
A method for identifying enterohemorrhagic Escherichia coli O26, comprising at least a step of culturing a sample using the selective separation medium according to any one of claims 1 to 9. 11. An enterohemorrhagic Escherichia coli O26 from intestinal bacteria.
A method for the isolation and identification of Enterohemorrhagic Escherichia coli O26, which comprises at least a step of culturing a sample using the selective separation medium according to any one of claims 1 to 9. .