JP4334067B2 - E. coli O26 separation medium - Google Patents

Description

【００２３】
表１より、試験した大腸菌Ｏ２６はすべて本培地上で暗紫色の集落を形成し、Ｏ２６以外の血清型の大腸菌および大腸菌以外の腸内細菌とはそれぞれ発色が異なり、鑑別がついた。
デソキシコーレイト寒天培地（ＤＥＳＯ）では、乳糖非分解性のセラチア、プロテウス以外の菌は全て赤色集落を形成し、大腸菌Ｏ２６の鑑別はできなかった。
ラムノース分解能を利用したラムノース加マッコンキー寒天培地（Ｒ−ＭＡＣ）では、大腸菌Ｏ２６とその他の大腸菌の鑑別はできたが、大腸菌以外の腸内細菌のうちラムノース非分解菌（Serratia marcescens、Proteus mirabilis、P.vulgaris）は透明の集落を形成し、大腸菌Ｏ２６との鑑別はできなかった。
【００２４】
実施例３ 食材からの大腸菌Ｏ２６の検出
大腸菌Ｏ２６の汚染を想定した牛挽肉４検体を使用した。牛挽肉２５ｇをｍＥＣブロス２２５ｍＬと混和し、３５℃１８時間前培養し、その培養液を本培地、デソキシコーレイト寒天培地、およびラムノース加マッコンキー寒天培地に１白金耳量塗抹し、３７℃で１６時間培養後、出現した集落を観察し、各培地で大腸菌Ｏ２６と思われる集落を釣菌した。具体的には本培地では暗紫色の集落を、デソキシコーレイト寒天培地では乳糖分解菌を大腸菌として釣菌した。ラムノース加マッコンキー培地ではラムノース非分解菌として透明の集落を釣菌した。
各培地から釣菌した１〜１０個の集落をそのまま用いて、生化学性状検査および抗血清検査を行い、大腸菌Ｏ２６の同定を行った。
判定は釣菌した集落のほとんどが大腸菌Ｏ２６であった場合をＡ、大腸菌Ｏ２６として同定するのにいくつかの集落の試験が必要で、釣菌した集落のうち一部が大腸菌Ｏ２６と確認されるように検出が困難であった場合をＢ、大腸菌Ｏ２６を検出できなかった場合をＣとして判定を行った。結果を表２に示す。
【００２５】
【表２】

【００２６】
得られた集落を生化学性状検査および大腸菌Ｏ２６抗血清で確認したところ、本培地で大腸菌Ｏ２６と思われた集落から釣菌した菌株はすべて大腸菌Ｏ２６であった。
ラムノース加マッコンキー寒天培地（Ｒ−ＭＡＣ）でもすべての検体から本菌を検出することができたが、そのうち３検体は判定Ｂを示し、釣菌した菌の大部分が大腸菌Ｏ２６以外の菌であった。
デソキシコーレイト寒天培地（ＤＥＳＯ）では１検体のみから大腸菌Ｏ２６を検出でき（判定Ｂ）、この場合も釣菌した菌の大部分が大腸菌Ｏ２６以外の菌であった。デソキシコーレイト培地上で大腸菌Ｏ２６を疑って釣菌したにもかかわらず大腸菌Ｏ２６ではなかった菌（判定Ｃ）は、その後の確認試験の結果より、他の血清型の大腸菌であった。
従って、本発明の培地の使用により、１回の選択分離培養で食材由来の大腸菌Ｏ２６が検出可能となった。
【００２７】
【発明の効果】
従来の大腸菌Ｏ２６の検出は熟練された技術と３〜５日間の長時間が必要であった。これは大腸菌Ｏ２６用の選択分離培地はなく、一般の腸内細菌あるいは大腸菌の検出を目的とした培地を使わざるを得なく、このような培地では大腸菌Ｏ２６のみではなく、Ｏ２６以外の大腸菌を含む多くの腸内細菌が発育するためであった。そのため従来の選択分離培養では、疑わしい集落を出来るだけ多く釣菌し、確認培地や同定キットあるいは抗血清試験により大腸菌Ｏ２６のスクリーニングあるいは同定を行い、大腸菌Ｏ２６を疑う集落を鑑別するには多くの経験と熟練が必要であった。
しかし、本培地を用いることでそうした経験などを必要とせず非常に高い確率で大腸菌Ｏ２６の検出が可能になり、また、大腸菌Ｏ２６以外の菌の試験に要していた試薬類、操作の手間と時間も削減できる。
【００２８】
さらに本培地では一回の選択分離培養により、暗緑色から暗紫色集落の有無で大腸菌Ｏ２６の存在が簡単に判断できるので、汚染された食材や食品を早期に排除でき、また品質管理期間の短縮により賞味期間も延長可能で食品流通の経済性に大きく貢献できる。
また、糞便検査や嘔吐物の検査に用いれば、大腸菌Ｏ２６による食中毒患者の早期診断が可能になることから、早期に有効な治療の一助ともなる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a medium for separating enterohemorrhagic Escherichia coli serotype O26 (E. coli O26), which is a food poisoning bacterium.
[0002]
In the present invention, the following abbreviations may be used. The enzyme color-developing substrate represented by -Glc includes a glucuronic acid form and an appropriate salt.
[Abbreviation Table]
X-Glc: 5-Bromo-4-Chloro-3-Indolyl-β-D-Glucopyranoside
X-Gal: 5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside
Bluo-Glc: 5-Bromo-3-Indolyl-β-D-Glucopyranoside
Bluo-Gal: 5-Bromo-3-Indolyl-β-D-Galactopyranoside
MU-Glc: 4-Methyl-Umbelliferyl-β-D-Glucopyranoside
MU-Gal: 4-Methyl-Umbelliferyl-β-D-Galactopyranoside
ONP-Glc: o-Nitrophenyl-β-D-Glucopyranoside
ONP-Gal: o-Nitrophenyl-β-D-Galactopyranoside
Magenta-Glc: 5-Bromo-6-Chloro-3-Indolyl-β-D-Glucopyranoside
Magenta-Gal: 5-Bromo-6-Chloro-3-Indolyl-β-D-Galactopyranoside
Salmon-Glc: 6-Chloro-3-Indolyl-β-D-Glucopyranoside
Salmon-Gal: 6-Chloro-3-Indolyl-β-D-Galactopyranoside
[0003]
[Prior art]
E. coli O26 belongs to the family Enterobacteriaceae and is a Gram-negative, facultative anaerobic Aspergillus oryzae, and is distinguished from normal E. coli by the antigen structure on the surface of the cell. Food poisoning caused by Escherichia coli O26 is an infectious food poisoning caused by ingesting meat, milk, eggs and processed foods contaminated with this bacterium and growing in the intestinal tract. Escherichia coli O26 food poisoning is said to tend to increase. ing.
[0004]
The detection of E. coli O26 is generally performed as follows. First, a specimen is smeared on an intestinal bacteria selective medium such as MacConkey agar medium (MAC), desoxycholate agar medium (DESO) or the like. Incubate at 37 ° C. for 18-24 hours and observe colonies on the plate medium. Furthermore, the colony which seems to be colon_bacillus | E._coli on a flat plate culture medium is fished, and it inoculates into colon_bacillus | E._coli identification kit or a biochemical property test | inspection discrimination medium, culture | cultivates at 37 degreeC for 18-24 hours, and identifies. After being identified as Escherichia coli, it is confirmed that the serotype is O26 by an immunological test using antiserum using these colonies or the expanded bacteria. When the number of bacteria is small, enrichment culture using a modified EC (mEC) medium or EC medium may be performed before the separation culture.
[0005]
As described above, the conventional E. coli O26 detection method has a problem that it requires a skillful technique and a long time of 3 to 5 days. The various selective separation media described above are not intended for selective separation of only E. coli, and many enteric bacteria grow on most of the media.
Therefore, skill and experience are required to distinguish a strain suspected to be Escherichia coli from such a selective separation medium, and it is necessary to further identify a serotype after confirmation of E. coli. Usually, the E. coli O26, which causes food poisoning, is often mixed with normal E. coli in the materials to be inspected. Therefore, it is necessary to check many colonies and confirm the cause. It is extremely. Therefore, if the presence or absence of E. coli O26 can be determined from the findings in the selective separation medium that can identify only E. coli O26, contaminated foods and foods can be eliminated early, and early diagnosis of food poisoning patients becomes possible. It is an important problem for food companies and general consumers to detect E. coli O26 in a short time and eliminate contaminated foods and processed raw materials. Finding will help early treatment of patients.
[0006]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a specific medium composition that can detect Escherichia coli O26 in various specimens including foods in a short time, easily and accurately without requiring any special skill or technique. There is.
[0007]
[Means for Solving the Problems]
Conventionally, a method using an enzyme possessed by a bacterium for the identification of the bacterium is known. It is a method using an enzyme chromogenic substrate that is decomposed by the enzyme and generates a pigment.
For example, documents [1] and [2] published in 1975 and 1976 include ONP-Gal and ONP-Glc, which are decomposed by bacteria-derived enzymes to generate orthonitrophenol which is a yellow pigment, fluorescent There is a description of an enzyme chromogenic substrate medium in which MU-Gal or MU-Glc that generates methylumbelliferone, which is a dye, is added as an enzyme chromogenic substrate, and is used for identification of microorganisms such as Escherichia coli.
In addition, U.S. Pat. No. 3,870,601, JP-A-64-2596, JP-A-4-51900 and the like have enteric bacteria added with enzyme chromogenic substrates such as Magenta-Gal, X-Glc, Bluo-Gal, X-Gal. An identification medium is described.
However, an example of a medium in which an enzyme chromogenic substrate is used for separation / differentiation of E. coli O26 has not yet been reported.
[1] J. Clin. Pathol. (1975), 28 (8), 686-7
[2] ACTA PATHOL. MICROBIOL. SCAND. SECTION B, (1976 Oct) 84B (5) 245-51.
[0008]
In this situation, the inventor noted that E. coli O26, like other E. coli, has glucosidase and galactosidase, but unlike other E. coli, this property and pH indicator. As a result of diligent efforts, the present invention was completed by combining the enzyme coloring substrate. In other words, in E. coli O26, the rhamnose in the medium is not decomposed, so the pH indicator shows a neutral color, and the enzyme coloring substrate is decomposed and colored by the enzyme possessed, but the apparent color is the pH indicator. The neutral color and the color of the substrate are added.
In normal Escherichia coli, rhamnose in the medium is decomposed to produce an acid, so the pH indicator shows an acidic color, and the enzyme chromogenic substrate is decomposed and colored by the enzyme it has, but the apparent color is the pH indicator The acid color and the color of the substrate are added.
In the present invention, Escherichia coli O26 is separated and differentiated by the difference in coloration added by the two.
As the basal medium used in the present invention, SS agar medium, Macconkey agar medium, DHL agar medium, desoxycholate agar medium, etc., which distinguish enteric bacteria by sugar resolution can be used.
[0009]
(1) The present invention is an Escherichia coli O26 separation medium containing rhamnose, a pH indicator, and an enzyme chromogenic substrate.
(2) As a pH indicator, indicators having different neutral colors and acidic colors can be used in the present invention. From the group consisting of phenol red, neutral red, bromophenol purple, bromothymol blue, bromophenol red, and chlorophenol red The pH indicator chosen is suitable for the present invention.
(3) Among them, phenol red is most suitable as a pH indicator.
(4) The enzyme coloring substrate is selected from a glucopyranoside derivative or a galactopyranoside derivative, the glucopyranoside derivative is decomposed by glucuronidase, and the galactopyranoside derivative is decomposed by galactosidase to cause color development. In the present invention, fluorescent substrates such as MU-Gal and MU-Glc are also handled as enzyme coloring substrates.
(5) Indolyl derivatives are also useful in the present invention as enzyme coloring substrates. An indolyl derivative is decomposed and oxidized by an enzyme, and a corresponding indolyl dye, for example, X-Gal · X-Glc, generates blue bromochloroindigo.
(6) The enzyme chromogenic substrate is selected from the group consisting of X-Glc, X-Gal, Bluo-Glc, Bluo-Gal, MU-Glc, MU-Gal, ONP-Glc, ONP-Gal, Salmon-Glc, Salmon-Gal. One type or a plurality of types may be selected.
(7) Furthermore, the selectivity of the present invention increases when one or more anionic surfactants having 10 to 30 carbon atoms are added to the medium of the present invention.
(8) The anionic surfactant is selected from bile salts and alkyl sulfates, and more specifically, bile powder, sodium cholate, sodium lauryl sulfate and the like are preferred in the present invention.
(9) The addition of tellurite as a selective agent increases the selectivity of the present invention.
(10) Addition of cefixime as a selective agent increases the selectivity of the present invention.
(11) More specifically, in the present invention, per 1,000 mL of the medium, 5-15 g of peptone, 2-5 g of yeast extract, 0.1-10 g of sodium chloride, 0.05-0.5 g of sodium lauryl sulfate, X- Glc 0.05-0.5 g and / or X-Gal 0.05-0.5 g, rhamnose 1-20 g, potassium tellurite 1-5 mg, cefixime 0.005-0.1 mg, phenol red 0.01-0. It is a culture medium for E. coli O26 separation containing 1 g and 10-20 g of agar.
X-Glc / X-Gal may be used alone or both may be used.
[0010]
[Action]
The action of the medium of the present invention will be described according to the medium composition described in (11) above.
The present invention utilizes the rhamnose non-resolution, galactopyranoside resolution, and glucopyranoside resolution of E. coli O26, and uses Escherichia coli O26 as a pH indicator to distinguish E. coli O26 from the presence of dark green to dark purple colonies. .
[0011]
Many E. coli other than serotype O26 degrade rhamnose added to the medium to produce acid. The pH of the normally used medium is around neutral (7), and the medium color is red to red due to phenol red added as a pH indicator. Since Escherichia coli that degrades rhamnose degrades rhamnose in the medium and lowers the pH of the medium, the colonies of these fungi and the periphery of the colony turn yellow.
However, since rhamnose non-degrading bacteria such as Escherichia coli O26 do not produce acid, the pH of the medium does not decrease. Rather, the pH is increased by the degradation product of peptone in the medium due to the growth of the bacteria, so the settlement is more strongly red. As a result, the color of the medium around the village remains red.
The amount of rhamnose to be added is suitably 1 to 20 g per liter of medium, and the amount of phenol red is suitably 0.01 to 0.1 g.
[0012]
In general, E. coli containing serotype O26 possesses galactosidase and glucuronidase, decomposes the enzyme chromogenic substrate, and generates a pigment. X-Glc used in the present invention is specifically decomposed by E. coli containing serotype O26 and exhibits a blue color. Similarly, X-Gal is also specifically decomposed by bacteria of the coliform group including serotype O26 and exhibits a blue color. In this case, as described above, Escherichia coli O26 does not decompose rhamnose, so the color of the culture medium around the colony and the colony becomes red, and the blue color due to the degradation of X-Gal or X-Glc is superimposed on this color. From green to dark purple. On the other hand, the colony of rhamnose-degrading Escherichia coli other than E. coli O26 and the surrounding color of the colony are yellow, and the yellow color is overlapped with blue due to the degradation of X-Gal or X-Glc, resulting in a pale green to green colony. In addition, bacteria other than E. coli that cannot degrade X-Gal or X-Glc will be yellow or red colonies depending on the presence or absence of the rhamnose resolution of the bacteria, and can be easily distinguished from E. coli O26. That is, on the culture medium of the present invention, color development of each colony is different between E. coli O26, other E. coli, rhamnose degrading bacteria, and rhamnose non-degrading bacteria, so that each bacteria can be easily separated and differentiated. X-Gal and X-Glc may be used alone or in combination. The amount of X-Gal and X-Glc to be added is suitably 0.05 to 0.5 g per liter of medium.
[0013]
In the present invention, the growth of Gram-positive bacteria such as Bacillus is suppressed by adding an anionic surfactant to the medium. Anionic surfactants useful for this medium include bile salts such as desoxycholic acid and alkyl sulfates such as sodium lauryl sulfate.
[0014]
In this medium, the growth of E. coli other than serotype O26 is suppressed by the addition of the selective agent potassium tellurite. The amount of potassium tellurite added to this medium is suitably 1 to 5 mg per liter of medium.
[0015]
In this medium, the growth of Proteus, which is an enteric bacterium, is suppressed by adding the selective agent cefixime. An appropriate amount of cefixime to be added to the medium is 0.005 to 0.1 mg per liter of the medium. Below this, the growth of Proteus spp. Cannot be suppressed, and above this, E. coli O26 is also suppressed.
[0016]
Hereinafter, the present invention will be described in more detail based on examples. It should be noted that the following examples are merely illustrative and do not limit the present invention.
【Example】
[0017]
Example 1 Preparation of Main Medium and Conventional Medium The medium of the present invention (main medium), desoxycholate agar medium (DESO), and rhamnose-added MacConkey agar medium (R-MAC) having the following composition were prepared. Purified water was added to each composition to make a total volume of 1000 mL. After heating and dissolving, the mixture was cooled to about 50 ° C. and dispensed into a petri dish at a rate of 20 mL to prepare a flat plate.
[0018]
[Composition 1]
Composition of this medium (per 1,000 mL of medium)
───────────────────
Peptone 12g
Yeast extract 3g
Sodium chloride 5g
Sodium lauryl sulfate 0.1g
X-Gal 0.1g
X-Glc 0.1g
Rhamnose 10g
Potassium tellurite 2.5mg
Cefixime 0.01mg
Phenol red 0.05g
Agar 15g
───────────────────
pH 7.2 ± 0.2
[0019]
[Composition 2]
DESO medium composition (per 1,000 mL of medium)
───────────────────
Peptone 10g
Lactose 10g
Sodium desoxycholate 1g
Sodium chloride 5g
2g dipotassium phosphate
2g ammonium iron citrate
Neutral crimson 0.033g
Agar 15g
───────────────────
pH 7.2 ± 0.2
[0020]
[Composition 3]
R-MAC medium composition (per 1,000 mL medium)
───────────────────
Peptone 20g
Rhamnose 10g
Bile salt no. 2 1.5g
Sodium chloride 5g
Neutral crimson 0.03g
Crystal Violet 0.001g
Agar 15g
───────────────────
pH 7.0 ± 0.2
[0021]
Example 2 Separation of Escherichia coli O26 using this medium and conventional medium (1) Test bacteria Nine E. coli strains of serotype O26 stored in-house, 5 other E. coli strains of serotype, and 8 intestinal bacteria other than E. coli and 10 strains Using. The names of the bacteria used and the color of the colonies in each medium are shown in “Table 1”.
(2) Inoculation and determination The test bacteria cultured overnight in the heart infusion broth were smeared onto each medium prepared in Example 1 in an amount of one platinum loop, and cultured at 37 ° C. for 16 hours, and then the emerged colonies were observed. The results are shown in Table 1.
[0022]
(3) Results [Table 1]

[0023]
From Table 1, all tested E. coli O26 formed dark purple colonies on this medium, and the serotypes of E. coli other than O26 and intestinal bacteria other than E. coli were different in color and differentiated.
In the desoxycholate agar medium (DESO), all bacteria other than lactose non-degradable Serratia and Proteus formed red colonies, and Escherichia coli O26 could not be differentiated.
The rhamnose-added MacConkey agar medium (R-MAC) using rhamnose resolution was able to distinguish between E. coli O26 and other E. coli, but among the intestinal bacteria other than E. coli, Serumia marcescens , Proteus mirabilis , P vulgaris ) formed a transparent colony and could not be differentiated from E. coli O26.
[0024]
Example 3 Detection of Escherichia coli O26 from Foods Four ground beef specimens assuming contamination of E. coli O26 were used. 25g of minced beef is mixed with 225mL of mEC broth and pre-cultured at 35 ° C for 18 hours. The culture solution is smeared on this medium, desoxycholate agar medium, and rhamnose-added McConkey agar medium at 37 ° C. After culturing for 16 hours, the emerged colonies were observed, and the colonies considered to be Escherichia coli O26 were caught in each medium. Specifically, dark purple colonies were picked as Escherichia coli on the present medium, and lactose-degrading bacteria as Escherichia coli on the desoxycholate agar medium. In the rhamnose-added MacConkey medium, a clear colony was caught as a rhamnose non-degrading bacterium.
Using 1 to 10 colonies fished from each medium as they were, biochemical property test and antiserum test were performed to identify E. coli O26.
Judgment is based on the fact that most of the fishing colonies are E. coli O26, and some colony tests are required to identify as E. coli O26, and some of the fishing colonies are identified as E. coli O26. Thus, it was determined as B when detection was difficult, and as C when E. coli O26 could not be detected. The results are shown in Table 2.
[0025]
[Table 2]

[0026]
The obtained colonies were confirmed by biochemical characterization and E. coli O26 antiserum. As a result, E. coli O26 was all the strains caught from the colonies considered to be E. coli O26 in this medium.
The rhamnose-added MacConkey agar medium (R-MAC) was able to detect this bacteria from all specimens, but 3 specimens showed judgment B, and most of the bacteria that were caught were bacteria other than E. coli O26. It was.
In the desoxycholate agar medium (DESO), E. coli O26 could be detected from only one sample (decision B), and in this case as well, most of the bacteria that were caught were bacteria other than E. coli O26. Bacteria that were not Escherichia coli O26 even though Escherichia coli O26 was suspected on Desoxycholate medium were judged to be Escherichia coli of other serotypes as a result of the subsequent confirmation test.
Therefore, by using the medium of the present invention, E. coli O26 derived from foodstuffs can be detected by one selective separation culture.
[0027]
【The invention's effect】
Conventional detection of E. coli O26 required skilled techniques and a long time of 3-5 days. There is no selective separation medium for E. coli O26, and a medium for the detection of general intestinal bacteria or E. coli must be used. Such a medium contains not only E. coli O26 but also E. coli other than O26. This was because many intestinal bacteria developed. Therefore, in the conventional selective isolation culture, as many suspicious colonies as possible are cultivated, and E. coli O26 is screened or identified using a confirmation medium, identification kit, or antiserum test, and a lot of experience is found to distinguish colonies suspected to be Escherichia coli O26. And skill was necessary.
However, the use of this medium makes it possible to detect E. coli O26 with a very high probability without requiring such experience, etc. In addition, the reagents and labor required for testing bacteria other than E. coli O26 are eliminated. Time can be saved.
[0028]
Furthermore, in this medium, the presence of E. coli O26 can be easily determined by the presence or absence of dark green to dark purple colonies by one selective separation culture, so that contaminated food and food can be eliminated early, and the quality control period can be shortened. The shelf life can be extended, which can greatly contribute to the economics of food distribution.
In addition, if it is used for stool tests and vomit tests, early diagnosis of food poisoning patients by E. coli O26 becomes possible, which helps early effective treatment.

Claims (3)

A medium for separating Escherichia coli O26, which comprises rhamnose, phenol red, enzyme chromogenic substrate, tellurite and cefixime , wherein the enzyme chromogenic substrate is X-Glc and / or X-Gal . The culture medium for separating Escherichia coli O26 according to claim 1, further comprising bile salt and / or sodium lauryl sulfate .   Per 1,000 mL of medium, 5-15 g of peptone, 2-5 g of yeast extract, 0.1-10 g of sodium chloride, 0.05-0.5 g of sodium lauryl sulfate, 0.05-0.5 g of X-Glc and / or X- E. coli O26 separation containing Gal 0.05-0.5 g, rhamnose 1-20 g, potassium tellurite 1-5 mg, cefixime 0.005-0.1 mg, phenol red 0.01-0.1 g, agar 10-20 g Medium.