JP2014128264A - Medium for detecting enterohemorrhagic e. coli - Google Patents
Medium for detecting enterohemorrhagic e. coli Download PDFInfo
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- SRRYZMQPLOIHRP-UHFFFAOYSA-L dipotassium;tellurate Chemical compound [K+].[K+].[O-][Te]([O-])(=O)=O SRRYZMQPLOIHRP-UHFFFAOYSA-L 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
Description
本発明は、食中毒菌である腸管出血性大腸菌を検出できる培地に関するものである。また、本発明は、前記培地を用いた、被検試料からの腸管出血性大腸菌の検出方法に関するものである。 The present invention relates to a medium capable of detecting enterohemorrhagic Escherichia coli, which is a food poisoning bacterium. The present invention also relates to a method for detecting enterohemorrhagic Escherichia coli from a test sample using the medium.
近年、腸管出血性大腸菌による食中毒が世界各国で報告されており、国内においても腸管出血性大腸菌による食中毒の発生が認められる。この腸管出血性大腸菌は一般の大腸菌と異なり、下痢症や溶血性尿毒症性症候群に関わるベロ毒素を産生する特徴がある。そのため、腸管出血性大腸菌による食中毒は大きな社会問題となってきており、その原因究明と予防対策の確立が急務となっている。 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. This enterohemorrhagic E. coli is different from general E. coli in that it produces verotoxin related to diarrhea and hemolytic uremic syndrome. 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.
本毒素を産生する腸管出血性大腸菌の多くは血清型がＯ１５７であるが、Ｏ１５７の他に本毒素を産生する多数の種類の血清型の腸管出血性大腸菌が知られており、国内ではＯ２６、Ｏ１１１、Ｏ１０３、Ｏ１２１、Ｏ１４５等の血清型が下痢患者から検出されている。 Most enterohemorrhagic Escherichia coli that produces this toxin has a serotype of O157, but in addition to O157, many types of enterohemorrhagic Escherichia coli that produce this toxin are known. Serotypes such as O111, O103, O121, and O145 have been detected from diarrhea patients.
これらの血清型の異なる腸管出血性大腸菌を同一の方法で検出するため、変異型を含めたベロ毒素遺伝子を標的とし、オリゴヌクレオチドを用いて検出する方法が開示されている（特許文献１および特許文献２）。また、ベロ毒素を抗原抗体反応によって検出する方法も開示されている（特許文献３）。しかし、これらの検査方法は通常の微生物検査とは異なり、特別な装置や設備を必要とする。 In order to detect enterohemorrhagic Escherichia coli having different serotypes by the same method, a method for detecting a verotoxin gene including a mutant type using an oligonucleotide is disclosed (Patent Document 1 and Patent) Reference 2). A method for detecting verotoxin by antigen-antibody reaction is also disclosed (Patent Document 3). However, these inspection methods are different from the normal microorganism inspection and require special devices and equipment.
一方、直接培養法により腸管出血性大腸菌を検出する方法として、酸化還元色素および糖を含む培地で培養する検査方法が開示されている（特許文献４）。しかしながら、この培地を用いた培養方法では、検出出来る腸管出血性大腸菌の血清型が限られており、腸管出血性大腸菌とその他の大腸菌との区別が十分に行なえない。 On the other hand, as a method for detecting enterohemorrhagic Escherichia coli by a direct culture method, a test method for culturing in a medium containing a redox dye and a sugar is disclosed (Patent Document 4). However, in the culture method using this medium, the serotypes of enterohemorrhagic Escherichia coli that can be detected are limited, and it is not possible to sufficiently distinguish enterohemorrhagic Escherichia coli from other Escherichia coli.
そこで、腸管出血性大腸菌を明確に検出できる培地、すなわち、他の類似菌等の発育を抑制するか、発育してもコロニーの色調や形状が異なる培地の開発が待たれていた。 Therefore, development of a medium that can clearly detect enterohemorrhagic Escherichia coli, that is, a medium that suppresses the growth of other similar bacteria or has a different colony color and shape even after the growth has been awaited.
食中毒の原因菌が腸管出血性大腸菌であるか否かにより、その治療法や予後が異なる。そのため、ベロ毒素の産生の有無を遺伝子検査等で調べる、腸管出血性大腸菌の検出法が実施されている。しかし、これらの検査を行うためには特別な装置や設備を必要とする。 The treatment method and prognosis differ depending on whether or not the causative agent of food poisoning is enterohemorrhagic Escherichia coli. Therefore, a method for detecting enterohemorrhagic Escherichia coli, in which the presence or absence of verotoxin production is examined by genetic testing or the like, has been implemented. However, special equipment and facilities are required to perform these inspections.
また、腸管出血性大腸菌が有する酵素の発色基質を添加する培養法では、全ての腸管出血性大腸菌を類似細菌と判別するためには複数の培地を用い、それらを組み合わせる必要がある。それゆえ、比較的簡便な培養法を用い、広い範囲の腸管出血性大腸菌を検出することが出来る方法、すなわち培地が必要とされる。 In addition, in the culture method in which the chromogenic substrate for the enzyme of enterohemorrhagic Escherichia coli is added, in order to distinguish all enterohemorrhagic Escherichia coli from similar bacteria, it is necessary to combine a plurality of media. Therefore, a method capable of detecting a wide range of enterohemorrhagic Escherichia coli using a relatively simple culture method, that is, a medium is required.
大腸菌とその他の腸内細菌科細菌が有している酵素は類似しているため、酵素活性を利用する培養法によってこれらを判別することは困難である。すなわち、大腸菌およびその他の腸内細菌科細菌はガラクトシダーゼを有しており、その発色基質であるガラクトシド誘導体を培地中に添加したとき、その発育に伴い、発色基質が分解したときの色調を呈する。 Since the enzymes possessed by Escherichia coli and other Enterobacteriaceae bacteria are similar, it is difficult to discriminate them by a culture method using enzyme activity. That is, Escherichia coli and other Enterobacteriaceae bacteria have galactosidase, and when a galactoside derivative, which is a chromogenic substrate, is added to the medium, it exhibits a color tone when the chromogenic substrate is decomposed along with its growth.
例えば、発色基質であるガラクトシド誘導体として５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドを選択して培地中に添加したとき、その発育に伴い、５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドが分解してコロニーが緑色を呈する。 For example, when 5-bromo-4-chloro-3-indolyl-β-D-galactoside is selected and added to the medium as a galactoside derivative that is a chromogenic substrate, 5-bromo-4-chloro- 3-Indolyl-β-D-galactoside is decomposed and the colony becomes green.
また、白糖およびｐＨ指示薬を培地中に添加したとき、大腸菌およびその他の腸内細菌科細菌が白糖を分解することにより生じる酸により、ｐＨ指示薬の色が酸性側の色に変色する。 Further, when sucrose and a pH indicator are added to the medium, the color of the pH indicator changes to an acidic color due to the acid generated by the decomposition of sucrose by Escherichia coli and other Enterobacteriaceae bacteria.
例えば、ｐＨ指示薬としてニュートラルレッドを選択して培地中に添加したとき、その発育に伴い、白糖が分解して生じる酸によりコロニーが赤色を呈する。 For example, when neutral red is selected as a pH indicator and added to the medium, the colony becomes red due to the acid produced by the decomposition of sucrose as the growth proceeds.
それゆえ、発色基質であるガラクトシド誘導体の５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド並びに白糖およびｐＨ指示薬のニュートラルレッドを添加した培地で大腸菌およびその他の腸内細菌科細菌を培養すると、ともにコロニーが紫色を呈することとなる。 Therefore, Escherichia coli and other Enterobacteriaceae bacteria can be treated in a medium supplemented with galactoside derivative 5-bromo-4-chloro-3-indolyl-β-D-galactoside, which is a chromogenic substrate, and neutral red, a sucrose and a pH indicator. When cultured, both colonies are purple.
しかしながら、白糖の添加量を減ずることにより、その量にしたがって大腸菌のコロニーの呈色が紫色から緑色に変化する反面、その他の腸内細菌科細菌のコロニーの呈色は紫色のままであった。 However, the coloration of the colonies of E. coli changed from purple to green according to the amount of sucrose added, but the coloration of colonies of other Enterobacteriaceae bacteria remained purple.
すなわち、培地中に５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド並びに白糖およびニュートラルレッドを添加するとき、白糖の添加量を少なくすることにより、大腸菌とその他の腸内細菌科細菌を判別することが可能となった。 That is, when 5-bromo-4-chloro-3-indolyl-β-D-galactoside and sucrose and neutral red are added to the medium, the amount of sucrose added is reduced, so that E. coli and other enterobacteriaceae It became possible to distinguish bacteria.
次に、腸管出血性大腸菌とその他の大腸菌を判別する方法であるが、培地中に亜テルル酸カリウムを添加しても腸管出血性大腸菌は発育する反面、その他の大腸菌は発育が抑制された。それゆえ、培地中に亜テルル酸カリウムを添加することにより、腸管出血性大腸菌とその他の大腸菌を判別することが可能となる。 Next, there is a method for discriminating enterohemorrhagic E. coli from other E. coli, but even when potassium tellurite was added to the medium, enterohemorrhagic E. coli grew, but growth of other E. coli was suppressed. Therefore, it is possible to distinguish enterohemorrhagic E. coli from other E. coli by adding potassium tellurite to the medium.
これらの結果より、発色基質であるガラクトシド誘導体の５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド並びに減じた量の白糖およびｐＨ指示薬のニュートラルレッドを添加した培地中に、さらに亜テルル酸カリウムを添加した培地を用いることにより、腸管出血性大腸菌を判別することが可能となる。 From these results, it was found that the galactoside derivative of 5-bromo-4-chloro-3-indolyl-β-D-galactoside and a reduced amount of sucrose and the pH indicator neutral red were further added to the medium. By using a medium supplemented with potassium tellurate, enterohemorrhagic Escherichia coli can be discriminated.
すなわち、本発明は以下の構成からなる。
（１）白糖、ラムノース、ｐＨ指示薬、酵素発色基質および亜テルル酸塩を含有する培地であって、酵素発色基質がガラクトシド誘導体であることを特徴とする、腸管出血性大腸菌の検出培地。
（２）さらに、胆汁酸塩および／またはラウリル硫酸ナトリウム、並びにノボビオシンを含有することを特徴とする、（１）記載の腸管出血性大腸菌の検出培地。
（３）前記ｐＨ指示薬が、フェノールレッド、ニュートラルレッド、ブロモクレゾールパープル、ブロモチモールブルー、ブロモフェノールレッド、クロロフェノールレッドより選ばれるｐＨ指示薬である、（１）または（２）記載の腸管出血性大腸菌の検出培地。
（４）前記ｐＨ指示薬が、ニュートラルレッドである、（１）または（２）記載の腸管出血性大腸菌の検出培地。
（５）前記ガラクトシド誘導体が、５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド、３−インドリル−β−Ｄ−ガラクトシド、４−メチル−ウムベリフェリル−β−Ｄ−ガラクトシド、ｐ−ニトロフェニル−β−Ｄ−ガラクトシド、６−クロロ−３−インドリル−β−Ｄ−ガラクトシド、５−ブロモ−６−クロロ−３−インドリル−β−Ｄ−ガラクトシドより選ばれる酵素発色基質である、（１）〜（４）のいずれかに記載の腸管出血性大腸菌の検出培地。
（６）前記ガラクトシド誘導体が、５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドである、（１）〜（４）のいずれかに記載の腸管出血性大腸菌の検出培地。
（７）前記白糖の濃度が、１−２６ｇ／Ｌである、（１）〜（６）のいずれかに記載の腸管出血性大腸菌の検出培地。
（８）前記白糖の濃度が、１−１８ｇ／Ｌである、（１）〜（６）のいずれかに記載の腸管出血性大腸菌の検出培地。
（９）前記白糖の濃度が、１−６ｇ／Ｌである、（１）〜（６）のいずれかに記載の腸管出血性大腸菌の検出培地。
（１０）培地１，０００ｍＬあたり、ペプトン５−１５ｇ、酵母エキス２−５ｇ、塩化ナトリウム０．１−１０ｇ、胆汁酸塩０．１−２ｇ、白糖１−２６ｇ、５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド０．０５−０．５ｇ、ラムノース０．１−８ｇ、亜テルル酸カリウム０．５−５ｍｇ、ノボビオシン０．０５−１ｍｇ、ニュートラルレッド０．００１−０．０１ｇ、寒天１０−２０ｇを含有する、腸管出血性大腸菌の検出培地。
（１１）培地１，０００ｍＬあたり、ペプトン５−１５ｇ、酵母エキス２−５ｇ、塩化ナトリウム０．１−１０ｇ、胆汁酸塩０．１−２ｇ、白糖１−１８ｇ、５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド０．０５−０．５ｇ、ラムノース０．１−８ｇ、亜テルル酸カリウム０．５−５ｍｇ、ノボビオシン０．０５−１ｍｇ、ニュートラルレッド０．００１−０．０１ｇ、寒天１０−２０ｇを含有する、腸管出血性大腸菌の検出培地。
（１２）培地１，０００ｍＬあたり、ペプトン５−１５ｇ、酵母エキス２−５ｇ、塩化ナトリウム０．１−１０ｇ、胆汁酸塩０．１−２ｇ、白糖１−６ｇ、５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド０．０５−０．５ｇ、ラムノース０．１−８ｇ、亜テルル酸カリウム０．５−５ｍｇ、ノボビオシン０．０５−１ｍｇ、ニュートラルレッド０．００１−０．０１ｇ、寒天１０−２０ｇを含有する、腸管出血性大腸菌の検出培地。
（１３）被験試料から腸管出血性大腸菌を検出する方法において、（１）〜（１２）のいずれかに記載の検出培地を用いて試料を培養する工程を少なくとも含む、腸管出血性大腸菌の検出方法。
That is, the present invention has the following configuration.
(1) A medium for detecting enterohemorrhagic Escherichia coli, comprising a medium containing sucrose, rhamnose, a pH indicator, an enzyme chromogenic substrate and tellurite, wherein the enzyme chromogenic substrate is a galactoside derivative.
(2) The detection medium for enterohemorrhagic Escherichia coli according to (1), further comprising bile salt and / or sodium lauryl sulfate and novobiocin.
(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 Detection medium.
(4) The enterohemorrhagic Escherichia coli detection medium according to (1) or (2), wherein the pH indicator is neutral red.
(5) The galactoside derivative is 5-bromo-4-chloro-3-indolyl-β-D-galactoside, 3-indolyl-β-D-galactoside, 4-methyl-umbelliferyl-β-D-galactoside, An enzyme chromogenic substrate selected from p-nitrophenyl-β-D-galactoside, 6-chloro-3-indolyl-β-D-galactoside, and 5-bromo-6-chloro-3-indolyl-β-D-galactoside A detection medium for enterohemorrhagic Escherichia coli according to any one of (1) to (4).
(6) The enterohemorrhagic Escherichia coli detection medium according to any one of (1) to (4), wherein the galactoside derivative is 5-bromo-4-chloro-3-indolyl-β-D-galactoside.
(7) The enterohemorrhagic Escherichia coli detection medium according to any one of (1) to (6), wherein the sucrose concentration is 1-26 g / L.
(8) The enterohemorrhagic Escherichia coli detection medium according to any one of (1) to (6), wherein the concentration of the sucrose is 1-18 g / L.
(9) The enterohemorrhagic Escherichia coli detection medium according to any one of (1) to (6), wherein the concentration of the sucrose is 1-6 g / L.
(10) 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, 1-26 g of sucrose, 5-bromo-4-chloro- 3-indolyl-β-D-galactoside 0.05-0.5 g, rhamnose 0.1-8 g, potassium tellurite 0.5-5 mg, novobiocin 0.05-1 mg, neutral red 0.001-0.01 g A detection medium for enterohemorrhagic Escherichia coli containing 10-20 g of agar.
(11) 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, 1-18 g of sucrose, 5-bromo-4-chloro- 3-indolyl-β-D-galactoside 0.05-0.5 g, rhamnose 0.1-8 g, potassium tellurite 0.5-5 mg, novobiocin 0.05-1 mg, neutral red 0.001-0.01 g A detection medium for enterohemorrhagic Escherichia coli containing 10-20 g of agar.
(12) 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, 1-6 g of sucrose, 5-bromo-4-chloro- 3-indolyl-β-D-galactoside 0.05-0.5 g, rhamnose 0.1-8 g, potassium tellurite 0.5-5 mg, novobiocin 0.05-1 mg, neutral red 0.001-0.01 g A detection medium for enterohemorrhagic Escherichia coli containing 10-20 g of agar.
(13) In the method for detecting enterohemorrhagic Escherichia coli from a test sample, the method for detecting enterohemorrhagic Escherichia coli comprising at least a step of culturing the sample using the detection medium according to any one of (1) to (12) .
本発明を実施することにより、被検試料中に含まれる、食中毒菌として重要な腸管出血性大腸菌を、一種類の培地を用いて検出することが可能となる。 By carrying out the present invention, enterohemorrhagic Escherichia coli that is important as a food poisoning bacterium contained in a test sample can be detected using one type of medium.
細菌の増殖を支持するための栄養素および塩濃度、ならびに平板培地とするためのゲル化剤を含む基礎培地に、細菌を鑑別するための白糖およびｐＨ指示薬並びに酵素発色基質のガラクトシド誘導体、検出対象外の細菌の増殖を抑制する亜テルル酸塩、ノボビオシン、ならびに胆汁酸塩および／またはクエン酸ナトリウムを加えることにより、腸管出血性大腸菌を検出することが出来る。 Nutrient and salt concentrations to support bacterial growth, and basal medium containing gelling agent to make plate medium, saccharose and pH indicator to differentiate bacteria, galactoside derivative of enzyme chromogenic substrate, not subject to detection Enterohemorrhagic Escherichia coli can be detected by adding tellurite, novobiocin, and bile salts and / or sodium citrate, which inhibit the growth of bacteria.
本発明を、一例としてｐＨ指示薬にニュートラルレッドを、ガラクトシド誘導体に５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドを、発育抑制剤に亜テルル酸カリウムを用い、白糖の濃度を６ｇ／Ｌの条件で説明するが、本例に限定されることはない。 As an example of the present invention, neutral red is used as a pH indicator, 5-bromo-4-chloro-3-indolyl-β-D-galactoside is used as a galactoside derivative, potassium tellurite is used as a growth inhibitor, and the concentration of sucrose is adjusted. Although described under the condition of 6 g / L, it is not limited to this example.
ガラクトシダーゼ酵素を有している大腸菌およびその他の腸内細菌科細菌を、０．１ｇ／Ｌの５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドを添加した培地で培養すると、その発育に伴い、５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドが分解して、コロニーが緑色を呈する。 When E. coli and other Enterobacteriaceae bacteria having a galactosidase enzyme are cultured in a medium supplemented with 0.1 g / L of 5-bromo-4-chloro-3-indolyl-β-D-galactoside, As the cell grows, 5-bromo-4-chloro-3-indolyl-β-D-galactoside is decomposed and the colony becomes green.
また、大腸菌およびその他の腸内細菌科細菌を、６ｇ／Ｌの白糖および５ｍｇ／Ｌのニュートラルレッドを添加した培地で培養すると、その発育に伴い、白糖が分解されて酸が生じることによりｐＨが低下し、コロニーが赤色を呈する。 In addition, when Escherichia coli and other Enterobacteriaceae bacteria are cultured in a medium supplemented with 6 g / L sucrose and 5 mg / L neutral red, the pH is reduced by decomposition of the sucrose and generation of acid during the development. The colony is red.
しかし、これらを組み合わせて添加した培地、すなわち、６ｇ／Ｌの白糖および５ｍｇ／Ｌのニュートラルレッド並びに０．１ｇ／Ｌの５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドを添加した培地で大腸菌およびその他の腸内細菌科細菌を培養すると、大腸菌のコロニーは、白糖およびニュートラルレッドを添加しなかったときと同様に緑色〜青緑色を呈するが、その他の腸内細菌科細菌のコロニーは、緑色と赤色の中間色の紫色を呈した。 However, a medium containing a combination of these was added, ie 6 g / L sucrose and 5 mg / L neutral red and 0.1 g / L 5-bromo-4-chloro-3-indolyl-β-D-galactoside. When Escherichia coli and other Enterobacteriaceae bacteria are cultured on the prepared medium, the colonies of Escherichia coli are green to blue-green as when no sucrose and neutral red are added, but other Enterobacteriaceae bacteria are present. The colony had a purple color that was intermediate between green and red.
このことから、白糖およびニュートラルレッド並びに５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドを添加した培地において、白糖の濃度を低濃度にすることにより、大腸菌とその他の腸内細菌科細菌を判別することが可能であることが解った。 Therefore, in a medium supplemented with sucrose and neutral red and 5-bromo-4-chloro-3-indolyl-β-D-galactoside, the concentration of sucrose is reduced to reduce the concentration of E. coli and other enteric bacteria. It was found that it is possible to discriminate bacteria.
また、培地中に亜テルル酸カリウムを０．００１ｇ／Ｌ添加すると、腸管出血性大腸菌は発育する反面、その他の大腸菌の発育が抑制された。このことから、亜テルル酸カリウムを添加した培地において、腸管出血性大腸菌とその他の大腸菌を判別することが可能であることが解った。 Moreover, when 0.001 g / L of potassium tellurite was added to the medium, enterohemorrhagic Escherichia coli grew, but growth of other Escherichia coli was suppressed. From this, it was found that enterohemorrhagic Escherichia coli and other Escherichia coli can be discriminated in a medium supplemented with potassium tellurite.
すなわち、６ｇ／Ｌの白糖、５ｍｇ／Ｌのニュートラルレッド、０．１ｇ／Ｌの５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド、および０．００１ｇ／Ｌの亜テルル酸カリウムを添加した培地を用いることにより、発育してそのコロニーが緑色〜青緑色を呈色したものについて、腸管出血性大腸菌と判断出来る。 6 g / L sucrose, 5 mg / L neutral red, 0.1 g / L 5-bromo-4-chloro-3-indolyl-β-D-galactoside, and 0.001 g / L potassium tellurite By using a medium supplemented with, the colonies that grew and the colonies colored green to blue-green can be judged as enterohemorrhagic Escherichia coli.
なお、培地に添加するｐＨ指示薬を、ニュートラルレッドからフェノールレッド、ブロモクレゾールパープル、ブロモチモールブルー、ブロモフェノールレッド、またはクロロフェノールレッドに変更しても、細菌の発育に伴う培地の酸性化を色の変化で捉える機序は同じであるから、同等の結果が得られる。 Even if the pH indicator added to the medium is changed from neutral red to phenol red, bromocresol purple, bromothymol blue, bromophenol red, or chlorophenol red, the acidification of the medium accompanying the growth of the bacteria Since the mechanism of capturing changes is the same, equivalent results are obtained.
また、培地に添加する発色基質であるガラクトシド誘導体を、５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドから３−インドリル−β−Ｄ−ガラクトシド、４−メチル−ウムベリフェリル−β−Ｄ−ガラクトシド、ｐ−ニトロフェニル−β−Ｄ−ガラクトシド、６−クロロ−３−インドリル−β−Ｄ−ガラクトシド、または５−ブロモ−６−クロロ−３−インドリル−β−Ｄ−ガラクトシドに変更しても、細菌の発育に伴う酵素活性を色の変化で捉える機序は同じであるから、同等の結果が得られる。 Further, a galactoside derivative which is a chromogenic substrate added to the medium is changed from 5-bromo-4-chloro-3-indolyl-β-D-galactoside to 3-indolyl-β-D-galactoside, 4-methyl-umbelliferyl- β-D-galactoside, p-nitrophenyl-β-D-galactoside, 6-chloro-3-indolyl-β-D-galactoside, or 5-bromo-6-chloro-3-indolyl-β-D-galactoside Even if the change is made, the same mechanism can be obtained because the mechanism of capturing the enzyme activity associated with the growth of bacteria by the change in color is the same.
対象細菌を鑑別するための白糖および薬剤の濃度の確認
１．白糖の濃度の違いによる発育コロニーの呈色
白糖の濃度の違いによる白糖の分解性の違いを調べるために、以下の組成の培地に白糖を添加し、白糖の濃度が１、２、６、１０、１４、１８、２２、２６、および３０ｇ／Ｌとなるように培地を調製した。試験菌として大腸菌およびその他の腸内細菌科細菌を用い、ハートインフュージョンブイヨン培地で１夜培養してその１白金耳を各培地に塗抹接種し、３７℃１８時間培養した後のコロニーの呈色を確認した。
Confirmation of sucrose and drug concentrations to identify target bacteria
1. To investigate the degradation of the difference in sucrose due to the difference in the concentration of coloration sucrose developmental colonies by the concentration of sucrose difference, it added white sugar to the medium having the following composition, the concentration of sucrose 1,2,6,10 , 14, 18, 22, 26, and 30 g / L. Using Escherichia coli and other Enterobacteriaceae bacteria as test bacteria, culturing overnight in a heart infusion broth medium, smearing one platinum ear into each medium, and colorizing colonies after culturing at 37 ° C. for 18 hours It was confirmed.
培地の組成（培地１Ｌあたり）
ペプトン １０ｇ
塩化ナトリウム ５ｇ
５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド ０．１ｇ
ニュートラルレッド ０．０３ｇ
寒天 １５ｇ
ｐＨ ７．２±０．２
Medium composition (per liter of medium)
Peptone 10g
Sodium chloride 5g
5-Bromo-4-chloro-3-indolyl-β-D-galactoside 0.1 g
Neutral red 0.03g
Agar 15g
pH 7.2 ± 0.2
コロニーの呈色について表１に示すが、白糖濃度が２６ｇ／Ｌで大腸菌が全て青紫色を呈し、その他の腸内細菌科細菌は桃色または紫色を呈したため、大腸菌の判別が可能となる。また、白糖濃度を１８ｇ／Ｌまで下げると大腸菌が全て青緑色を呈し、その他の腸内細菌科細菌は桃色または紫色を呈したため、より大腸菌の判別が可能となる。さらに、白糖濃度を６ｇ／Ｌまで下げると大腸菌が青緑色〜緑色を呈し、その他の腸内細菌科細菌は桃色または紫色を呈したため、さらに大腸菌の判別が可能となる。それゆえ、白糖濃度が、好ましくは２６ｇ／Ｌ以下、より好ましくは１８ｇ／Ｌ以下、さらに好ましくは６ｇ／Ｌ以下の場合に、発育したコロニーの呈色の違いにより、大腸菌とその他の腸内細菌科細菌を判別することが可能であった。 The coloration of the colonies is shown in Table 1. As the sucrose concentration is 26 g / L, all the E. coli are blue-purple, and the other Enterobacteriaceae bacteria are pink or purple. Further, when the sucrose concentration is lowered to 18 g / L, all the E. coli are blue-green, and the other Enterobacteriaceae bacteria are pink or purple, so that E. coli can be more discriminated. Furthermore, when the sucrose concentration is lowered to 6 g / L, Escherichia coli exhibits blue-green to green, and other Enterobacteriaceae bacteria are pink or purple, so that it is possible to further distinguish Escherichia coli. Therefore, when the concentration of sucrose is preferably 26 g / L or less, more preferably 18 g / L or less, and even more preferably 6 g / L or less, E. coli and other enteric bacteria may differ depending on the coloration of the grown colonies. It was possible to discriminate family bacteria.
２．亜テルル酸カリウムの濃度の違いによる腸管出血性大腸菌およびその他の大腸菌の発育抑制効果
亜テルル酸カリウムの濃度の違いによる腸管出血性大腸菌およびその他の大腸菌の発育抑制効果を調べるために、実施例１の１．で示した組成の培地に亜テルル酸カリウムを添加し、亜テルル酸カリウムの濃度が０．２、０．４、０．６、０．８、１．０、１．２、および１．４ｍｇ／Ｌとなるように培地を調製した。試験菌として腸管出血性大腸菌およびその他の大腸菌を用い、ハートインフュージョンブイヨン培地で１夜培養してその１白金耳を各培地に塗抹接種し、３７℃１８時間培養した後のコロニーの呈色を確認した。
2. To investigate the growth inhibition effects of the concentration differences enterohemorrhagic Escherichia coli and by other E. coli growth inhibitory effect nitrite enterohemorrhagic Escherichia coli due to the difference in the concentration of potassium tellurite and other E. coli potassium tellurite, Example 1 1. And potassium tellurite is added to the medium having the composition shown in the above, and the concentration of potassium tellurite is 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mg. The medium was prepared so as to be / L. Intestinal hemorrhagic Escherichia coli and other E. coli were used as test bacteria, cultured overnight in a heart infusion broth medium, the platinum ears were smeared on each medium, and the coloration of colonies after culturing at 37 ° C. for 18 hours confirmed.
腸管出血性大腸菌の発育について表２に、その他の大腸菌の発育について表３に示すが、０．２〜１．０ｍｇ／Ｌの濃度の亜テルル酸カリウムを添加した培地で全ての腸管出血性大腸菌の発育が認められたが、０．８ｍｇ／Ｌの濃度の亜テルル酸カリウムを添加した培地で９０％を超えるその他の大腸菌の発育を抑制することが出来た。 Table 2 shows the growth of enterohemorrhagic Escherichia coli, and Table 3 shows the growth of other Escherichia coli. However, all enterohemorrhagic Escherichia coli were grown in a medium supplemented with potassium tellurite at a concentration of 0.2 to 1.0 mg / L. The growth of other Escherichia coli exceeding 90% could be suppressed with a medium supplemented with potassium tellurite at a concentration of 0.8 mg / L.
腸管出血性大腸菌検出培地を用いた細菌の培養
実施例１の結果を基に、表４に示す組成の培地を用いて、種々の細菌を培養し、腸管出血性大腸菌の検出の可否を調べた。なお、試験菌は、ハートインフュージョンブイヨン培地で１夜培養してその１白金耳を各培地に塗抹接種し、３７℃１８時間培養した後のコロニーの発育およびその呈色を確認した。
Culture of bacteria using enterohemorrhagic Escherichia coli detection medium Based on the results of Example 1, various bacteria were cultured using the medium having the composition shown in Table 4 to examine whether or not enterohemorrhagic Escherichia coli could be detected. . The test bacteria were cultured overnight in a heart infusion broth medium, and one platinum loop was smeared on each medium, and the growth and coloration of colonies after culturing at 37 ° C. for 18 hours were confirmed.
腸管出血性大腸菌およびその他の細菌のコロニーの発育およびその呈色を、各々表５および表６に示すが、腸管出血性大腸菌の他の大腸菌２例（ＥＫＮ４６４およびＥＫＮ６８８）を除き、コロニーの発育およびその呈色により、腸管出血性大腸菌およびその他の細菌を判別することが可能であった。 The growth and coloration of enterohemorrhagic Escherichia coli and other bacterial colonies are shown in Tables 5 and 6, respectively, except for two other cases of enterohemorrhagic E. coli (EKN464 and EKN688). It was possible to distinguish enterohemorrhagic Escherichia coli and other bacteria by the coloration.
培地の処方中のｐＨ指示薬および発色基質の検討
実施例２の培地の処方中のｐＨ指示薬のニュートラルレッドをフェノールレッド、ブロモクレゾールパープル、ブロモチモールブルー、ブロモフェノールレッドまたはクロロフェノールレッドに、および発色基質の５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド（Ｘ−ｇａｌ）を３−インドリル−β−Ｄ−ガラクトシド（Ｙ−ｇａｌ）、４−メチル−ウムベリフェリル−β−Ｄ−ガラクトシド（ＭＵＧ）、ｐ−ニトロフェニール−β−Ｄ−ガラクトシド（ＰＮＰＧ）、６−クロロ−３−インドリル−β−Ｄ−ガラクトシド（Ｓａｌ−ｇａｌ）または５−ブロモ−６−クロロ−３−インドリル−Ｄ−ガラクトシド（ｍｅｇｅｎｔａ−ｇａｌ）に置き換えて、種々のｐＨ指示薬および発色基質を培地の処方として用いたときの腸管出血性大腸菌の検出能を検討した。
Examination of pH indicator and chromogenic substrate in the formulation of the medium Neutral red of the pH indicator in the formulation of the medium of Example 2 to phenol red, bromocresol purple, bromothymol blue, bromophenol red or chlorophenol red, and chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) of 3-indolyl-β-D-galactoside (Y-gal), 4-methyl-umbelliferyl-β-D -Galactoside (MUG), p-nitrophenyl-β-D-galactoside (PNPG), 6-chloro-3-indolyl-β-D-galactoside (Sal-gal) or 5-bromo-6-chloro-3-indolyl -Substituting D-galactoside (menta-gal) with various pH indicators and And the ability to detect enterohemorrhagic Escherichia coli when a chromogenic substrate was used as a medium formulation.
表４に示す組成の中、ｐＨ指示薬および発色基質を置き換えて作製した培地を用いて、種々の細菌を培養し、腸管出血性大腸菌の検出の可否を調べた。この時のｐＨ指示薬および発色基質の濃度は、実施例２のニュートラルレッドおよび５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシドと同量の、各々０．００５ｇ／Ｌおよび０．１ｇ／Ｌとした。なお、試験菌は、ハートインフュージョンブイヨン培地で１夜培養してその１白金耳を各培地に塗抹接種し、３７℃１８時間培養した後のコロニーの発育およびその呈色を確認した。 Among the compositions shown in Table 4, various bacteria were cultured using a medium prepared by replacing a pH indicator and a chromogenic substrate, and whether or not enterohemorrhagic Escherichia coli was detected was examined. The concentrations of the pH indicator and the chromogenic substrate at this time were 0.005 g / L and 0.005 g, respectively, in the same amount as that of Neutral Red and 5-Bromo-4-chloro-3-indolyl-β-D-galactoside of Example 2. 1 g / L. The test bacteria were cultured overnight in a heart infusion broth medium, and one platinum loop was smeared on each medium, and the growth and coloration of colonies after culturing at 37 ° C. for 18 hours were confirmed.
腸管出血性大腸菌およびその他の細菌のコロニーの発育およびその呈色を、ｐＨ指示薬として用いたニュートラルレッド（表７−１）、フェノールレッド（表７−２）、ブロモクレゾールパープル（表７−３）、ブロモチモールブルー（表７−４）、ブロモフェノールレッド（表７−５）およびクロロフェノールレッド（表７−６）に分けて示すが、発色基質として５−ブロモ−６−クロロ−３−インドリル−Ｄ−ガラクトシドを用いたとき、並びにｐＨ指示薬としてフェノールレッドまたはクロロフェノールレッドおよび発色基質としてｐ−ニトロフェニール−β−Ｄ−ガラクトシドを用いたときのＥｎｔｅｒｏｂａｃｔｅｒ ｃｌｏａｃａｅ等を除き、コロニーの発育およびその呈色により、腸管出血性大腸菌およびその他の細菌を判別することが可能であった。 Neutral red (Table 7-1), phenol red (Table 7-2), bromocresol purple (Table 7-3) used as pH indicators for the growth of colony of enterohemorrhagic E. coli and other bacteria and their coloration , Bromothymol blue (Table 7-4), bromophenol red (Table 7-5) and chlorophenol red (Table 7-6), but 5-bromo-6-chloro-3-indolyl as a chromogenic substrate Colony growth and its appearance, except when -D-galactoside was used and when phenol red or chlorophenol red was used as a pH indicator and p-nitrophenyl-β-D-galactoside was used as a chromogenic substrate Identifies enterohemorrhagic E. coli and other bacteria by color It was possible to
すなわち、本願発明の培地を用いることにより、腸管出血性大腸菌を、その他の細菌と判別して検出することが可能となった。 That is, by using the culture medium of the present invention, enterohemorrhagic Escherichia coli can be distinguished from other bacteria and detected.
従来、腸管出血性大腸菌の検出には、血清型毎に特異的な培地が使用されてきた。すなわち、腸管出血性大腸菌を検出するためには複数の培地を用いる必要があった。 Conventionally, a medium specific to each serotype has been used for detection of enterohemorrhagic E. coli. That is, in order to detect enterohemorrhagic Escherichia coli, it was necessary to use a plurality of media.
しかし、本願発明の培地では、１枚の平板培地を用いることにより腸内出血性大腸菌の存在を容易に判断できる。それゆえ、食品工業分野においては、原材料や製品中の腸管出血性大腸菌の有無を検証するための工程において、接種培地数を減らすことによる培養スペースや作業を簡便化でき、汚染品の排除や製品の出荷を速やかに行うことが可能となり、食品流通の経済性に大きく貢献できる。 However, in the medium of the present invention, the presence of enterohemorrhagic Escherichia coli can be easily determined by using a single plate medium. Therefore, in the food industry, in the process of verifying the presence of enterohemorrhagic Escherichia coli in raw materials and products, it is possible to simplify the culture space and work by reducing the number of inoculated media, and to eliminate contaminated products and products. Can be shipped promptly and can greatly contribute to the economics of food distribution.
また、医療分野においては、糞便や嘔吐物を被検試料として用いることにより、腸管出血性大腸菌による食中毒患者の早期診断が可能となり、早い段階より有効な治療法の選択に寄与できる。 In the medical field, the use of feces or vomit as a test sample enables early diagnosis of patients with food poisoning due to enterohemorrhagic Escherichia coli, which can contribute to the selection of an effective treatment method from an early stage.
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