JP5354564B2 - Medium for simultaneous detection of E. coli O26 and O157 and detection method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a new culture medium capable of simultaneously identifying Escherichia coli O26, O157, and ordinary Escherichia coli through solving the problem that conventional culture media and techniques, Escherichia coli O26, O157, and ordinary Escherichia coli to be examined cannot be identified simultaneously, and the examinations have been needed to conduct using individual culture media respectively. <P>SOLUTION: The new culture medium includes nutrients, a discriminatory agent, a selective agent, and a solid ingredient; wherein the discriminatory agent includes rhamnose, such an enzymatic substrate that Escherichia coli O157 is non-color-developable, and a pH indicator. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a medium capable of simultaneously separating enterohemorrhagic Escherichia coli O26 and O157, which are food poisoning bacteria, and a detection method thereof.

  Enterohemorrhagic Escherichia coli is a pathogenic bacterium that causes severe gastroenteritis and may have a lethal course if it is accompanied by hemolytic uremic syndrome. In 2006, 2,154 isolates were isolated nationwide. Information was reported to the Institute for Infectious Diseases. Among them, enterohemorrhagic Escherichia coli O26 and O157 (hereinafter sometimes simply referred to as “O26” and “O157”) account for 23.8% and 68.1%, respectively. The Ministry of Health, Labor and Welfare emphasized that the number of cases of infection caused by O26 has been conspicuous in recent years. In November 2006, the Ministry of Health, Labor and Welfare notified the inspection method for enterohemorrhagic Escherichia coli not only O157 but also O26. In this notice, as a rule, two types of agar medium for O26 (CT-RMAC, medium that can distinguish E. coli) and two types for O157 (CT-RMAC, enzyme substrate medium for O157) should be used. However, if there is a medium that can distinguish both types of bacteria, O26 and O157 should be detected efficiently using three types of medium (O26 selection medium, O157 selection medium, medium that can select both types). Is possible.

  In addition, enterohaemorrhagic Escherichia coli targets in stool tests for gastroenteritis patients and cooking workers are currently being narrowed down to O157, but a medium that can distinguish O26 and O157 has been developed. Therefore, O26 can be targeted without increasing labor and cost, and the detection efficiency of enterohemorrhagic Escherichia coli infection can be increased.

At present, several types of media for detecting enterohemorrhagic E. coli O26 and general E. coli, or O157 and general E. coli have been commercialized, and many patent applications and papers are recognized (patent documents and non-patent documents).
Therefore, in order to selectively separate both O26 and O157 E. coli from samples such as food and feces, at least two types of agar medium are required.
For O26, CT-RMAC agar medium (Denka Seika etc.) and CT-RX O26 agar medium (Eiken Chemical), for O157 CT-SMAC agar medium (Nissui Pharmaceutical etc.), BCMO157 agar medium ( Eiken Chemical Co., Ltd.), chromoagar O157 medium (chromoager), CT-O157: H7ID agar medium (Nihon Biomeryu), etc. have been commercialized.

The medium for O26 utilizes the property that O26 is non-degrading rhamnose (a kind of sugar) unlike general E. coli, and many of the mediums for O157 are non-degradable or sorbitol (a kind of sugar) of O157 or β -Utilizing the negative nature of glucuronidase activity, one of O26 and O157 can be detected, but the other cannot be distinguished from general E. coli.
JP 2000-210076 A JP 2000-342249 A Special Table 2000-508176 JP 2001-8679 A JP 2002-281959 A Journal of Infectious Diseases, Vol. 73, No. 5, pp. 407-412 Journal of Infectious Diseases, Vol.75, No.4, 291-299

  In view of the current state of the art, it is to develop a medium capable of simultaneously distinguishing O26 and O157 and general E. coli and a detection method thereof.

That is, the Escherichia coli O26 and O157 simultaneous detection medium according to claim 1 is a medium comprising nutrients, a discrimination agent, a selection agent, and a solid agent. As a discrimination agent, rhamnose, Escherichia coli O157 is a non-chromogenic enzyme substrate and neutral and acidic colors. Is characterized in that it contains different pH indicators and contains as a selective agent what inhibits the growth of E. coli other than O26 and O157 and bacterial microorganisms other than E. coli .

Furthermore, the Escherichia coli O26 and O157 simultaneous detection medium according to claim 2 is a Gram-positive medium that suppresses the growth of Escherichia coli other than O26 and O157 and bacterial microorganisms other than Escherichia coli as a selective agent in addition to the requirement of claim 1 above. It is characterized by containing one or more selected from among fungus inhibitors, tellurite and antibiotics.

Further, the Escherichia coli O26 and O157 simultaneous detection medium according to claim 3 is characterized in that, in addition to the requirement according to claim 1 or 2, the pH indicator having a neutral color and an acidic color is phenol red or neutral red. Is.

  The E. coli O26 and O157 simultaneous detection medium according to claim 4 is a chromogenic compound in which E. coli O157 is a non-chromogenic enzyme substrate is a β-glucuronidase substrate in addition to the requirements of claim 1, 2 or 3. It is characterized by being.

Further, the Escherichia coli O26 and O157 simultaneous detection medium according to claim 5 is characterized in that, in addition to the requirement of claim 1, 2, 3 or 4, E. coli O157 is non-chromogenic enzyme substrate is 5-bromo-4-chloro-3-indolyl It is characterized by being β-D-glucuronide.

The method for simultaneous detection of Escherichia coli O26 and O157 according to claim 6 is such that rhamnose and Escherichia coli O157 are non-chromogenic enzyme substrates and neutral and acidic colors as a distinguishing agent in a medium comprising nutrients, a distinguishing agent, a selective agent and a solid agent. It is characterized by using a medium containing different pH indicators and containing as a selective agent what suppresses the growth of E. coli other than O26 and O157 and bacterial microorganisms other than E. coli .

  According to the present invention, since O26 and O157 can be identified simultaneously, O26 can be further identified without increasing labor and cost, so that the detection efficiency of enterohemorrhagic Escherichia coli infection can be increased.

  The present inventor is able to distinguish between O26 and O157 and general E. coli when using rhamnose, E. coli O157 non-chromogenic enzyme substrate and pH indicator as a discrimination agent in a medium composed of nutrients, discrimination agents, selection agents and solid agents. As a result of intensive research and examination, a separation medium capable of simultaneously detecting E. coli O26 and O157 and a detection method thereof were completed.

  As the nutrient used in the medium of the present invention, any component necessary for the growth of microorganisms can be used. Casein peptone, animal meat peptone, soybean peptone, gelatin peptone, casein / animal meat peptone, etc., meat extract, heart Extracts such as leachate, yeast extract, malt extract, and potato extract, and inorganic salts such as sodium chloride and dipotassium hydrogen phosphate can be used.

In the medium of the present invention, rhamnose and Escherichia coli O157 are non-chromogenic enzyme substrates and pH indicators are used as a discrimination agent.
The non-chromogenic enzyme substrate of E. coli O157 is a chromogenic compound that is a substrate of β-glucuronidase, for example, X-Gluc: 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, MUG: 4-methylumbelliferyl-β-D-glucuronide, Magenta-Gluc: 5-bromo-6-chloro-3-indolyl-β-D-glucuronide, Salmon-Gluc: 6-chloro-3-indolyl-β -D-glucuronide and the like are used. These are preferably used in an amount of about 0.05 to 0.5 g per liter of medium.

  As the pH indicator, indicators having different neutral and acidic colors such as phenol red, neutral red, bromophenol purple, bromothymol blue, bromophenol red, and chlorophenol red are suitable. Although these differ depending on the indicator used, it is preferable to use about 0.01 to 0.1 g per liter of the medium.

  As the selective agent, one that suppresses the growth of E. coli other than O26 and O157 and bacterial microorganisms other than E. coli is used. For example, surfactants such as bile salts, sodium desoxycholate and sodium laurate, organic acid salts such as sodium citrate, pigments such as eosin Y, methylene blue, brilliant green, crystal violet, malachite green, selenite, tetrathion Inorganic salts such as acid salts and sodium thiosulfate, and antibacterial agents such as tellurite and cefixime can be used as necessary. These differ depending on the selection agent used, but it is better to use about 0.0001 to 2 g per liter of the medium.

As a solid agent, agar is most conveniently used.
EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited at all.

  Deoxycholate / rhamnose / X-Gluc agar (DRX agar), cefixime / potassium tellurite-added DRX agar (CT-DRX agar) and bile acids / rhamnose / X-Gluc agar (BRX agar medium) was prepared. Purified water was added to the composition of each medium to make a total volume of 1000 ml, heated and dissolved, cooled to 50-60 ° C., and dispensed into a petri dish at 20 ml. Cefixime and potassium tellurite were added after cooling to 50-60 ° C.

  Intestinal hemorrhagic Escherichia coli O26 isolate (S4433), enterohemorrhagic Escherichia coli O157 isolate (S4099) and general E. coli O103 isolate (S5163) were collected on DRX agar, CT-DRX agar and BRX agar. After colonizing and culturing at 37 ° C. for 20 hours, colonies that grew on each medium were observed.

  As shown in Table 4, in the colonies on DRX agar medium, O26 is not rhamnose-degraded, so it decomposes to alkaline due to degradation products such as peptone, and the phenol red turns red. Furthermore, the β-glucuronidase activity causes the chromogenic enzyme substrate (X -Gluc) turned blue-green and mixed to turn blue. O157 was weakly rhamnose-degraded under the conditions of this medium, phenol red remained almost the medium color, and β-glucuronidase activity was negative, so a transparent white colony was formed. O103 tends to be acidic by decomposing rhamnose, turns pale yellow due to yellowing of phenol red and deoxycholic acid precipitation, and further turns blue-green due to β-glucuronidase activity, resulting in a turbid green color. Became.

In CT-DRX agar medium with enhanced inhibitory power against bacteria other than O26 and O157, O26 and O157 formed colonies similar to DRX agar medium, but O103 could not grow.
On the BRX agar medium, O26 is a green colony with X-Gluc color (blue green) and alkaline pale yellow (neutral red alkali color). It became a slightly reddish white village (transparent). O103 was mixed with reddish color due to rhamnose decomposition (neutral red acidic color) and X-Gluc color (blue-green), resulting in a purple colony.
When three types of E. coli were mixed and smeared, the DRX agar medium and the BRX agar medium had three color colonies, but the CT-DRX agar medium did not show O103 growth, so the two color colonies I could only confirm.

  The growth status and colony color of E. coli on DRX agar medium and CT-DRX agar medium prepared in Example 1 were designated as CT-RMAC agar medium and CT-SMAC agar designated by No. 1102004 on November 2, 2006. The culture medium, CT-RXO26 agar medium and chromoagar O157TAM agar medium were compared. The bacterial solution of each of the 6 strains of O26, O157 and other E. coli was diluted 10-fold with PBS (-) to 10-8, and 50 μl was smeared on each selective medium and heart infusion (HI) agar medium. After culturing at 20 ° C. for 20 hours, confirmation of settlement color and measurement of the number of settlements were performed. The numbers in parentheses indicate the number of bacteria in each medium when the number of bacteria measured on the HI agar medium is 100.

  As shown in Table 5, in the colonies on DRX agar medium and CT-DRX agar medium, all 6 strains of O26 turned blue and all 6 strains of O157 turned white. In addition, all O26 strains grow on CT-RMAC agar and CT-RXO26 agar, which are selective media for O26, and all O157 strains grow on CT-SMAC agar and chromoagar O157TAM agar for O157. All of the media became the set colors. E. coli other than O26 and O157 showed green colonies in 6 strains on the DRX agar medium, but 4 out of 6 strains on the CT-DRX agar medium were supplemented with other cefixime and potassium tellurite (CT-SMAC agar). Growth was not observed as in the case of the medium, CT-RXO26 agar medium). The number of O26 and O157 strains grown on DRX and CT-DRX agar media tended to be slightly lower than that on HI agar media due to the effects of inhibitors, but individual strains compared to other selective media However, although there was some increase or decrease, there was no significant difference in the number of growing bacteria.

  Six strains of major enterobacteriaceae other than E. coli were streaked on DRX agar medium and CT-DRX agar medium, cultured at 37 ° C. for 20 hours, and the growth colonies were observed.

    As shown in Table 6, none of these six species formed a blue colony similar to O26 on DRX agar medium and CT-DRX agar medium. Two species of the genus Proteus were white (transparent) colonies similar to O157 on DRX agar medium, but became non-developed or microcolonized on CT-DRX agar medium and could be distinguished from O157 colonies. .

Claims (6)

In a medium consisting of a nutrient, a distinguishing agent, a selective agent and a solid agent, rhamnose, E. coli O157 as a distinguishing agent contains a non-chromogenic enzyme substrate and a pH indicator with different neutral and acidic colors , and as a selective agent with O26 A medium for simultaneous detection of Escherichia coli O26 and O157, which contains E. coli other than O157 and those that inhibit the growth of bacterial microorganisms other than E. coli . Those that suppress the growth of Escherichia coli other than O26 and O157 and bacterial microorganisms other than Escherichia coli as selective agents contain one or more selected from Gram-positive bacteria inhibitors, tellurite and antibiotics The medium for simultaneous detection of E. coli O26 and O157 according to claim 1. The medium for simultaneous detection of Escherichia coli O26 and O157 according to claim 1 or 2, wherein the pH indicator having a different neutral color and acidic color is phenol red or neutral red.   The E. coli O26 and O157 simultaneous detection medium according to claim 1, 2, or 3, wherein E. coli O157 is a chromogenic compound in which the non-chromogenic enzyme substrate is a substrate of β-glucuronidase. The medium for simultaneous detection of Escherichia coli O26 and O157 according to claim 1, 2, 3 or 4, wherein the non-chromogenic enzyme substrate of E. coli O157 is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide. Rhamnose, E. coli O157 as a distinguishing agent in a medium composed of nutrients, a distinguishing agent, a selective agent and a solid agent contains a non-chromogenic enzyme substrate and a pH indicator having a different neutral and acidic color , and O26 and O157 as selective agents. A method for simultaneous detection of Escherichia coli O26 and O157, which comprises using a medium containing a substance that inhibits the growth of Escherichia coli other than Escherichia coli and bacterial microorganisms other than Escherichia coli .