JP4196318B2 - Selective separation medium for E. coli and separation method - Google Patents

Description

【００４４】
〔実施例３〕
代表腸内細菌発育阻害能結果
実施例１で調製した培地を用い、代表的な腸内細菌の発育阻害能を比較した。試験方法は、実施例２と同様に行った。結果を表２に示す。表２は、各培地における試験菌株懸濁液の各希釈倍率における発育菌数、色及びコロニーのサイズを示している。
表２より、培地Ａ、培地ＢともにＣＴ添加培地と同様に各種腸内細菌の発育を抑制することが分った。
【００４５】
【表２】

【００４６】
〔実施例４〕
緑膿菌発育阻害能結果
実施例１で調製した培地を用い、緑膿菌の発育阻害能を比較した。試験方法は、実施例２と同様に行った。表３は、各培地における緑膿菌の試験菌株懸濁液の各希釈倍率における発育菌数、色及びコロニーのサイズを示している。発育菌数の計測ができない場合は、＋＋（一部コロニー融合のため計測不可）および＋＋＋（ほとんどコロニー融合のため計測不可）で表記した。発育コロニーが微小のため計測できないものは、「±」で示した。
表３より、培地Ａはいずれの希釈倍率においても緑膿菌の発育が抑制されることがわかった。また、培地Ｂでは、希釈倍率１０^−４で緑膿菌の発育を完全に抑制できることがわかった。したがって、ＣＴ培地と比較して、目的とする大腸菌Ｏ１５７を選択的に分離することができた。
【００４７】
【表３】

【００４８】
〔実施例５〕
腸管出血性大腸菌Ｏ１５７の分離例
成人便１ｇに対して１０^３−１０^４ＣＦＵになるように腸管出血性大腸菌Ｏ１５７を接種し、滅菌生理食塩水を用いて１０倍希釈して擬似的に陽性便を作成する。１０μＬを採取して本発明培地に直接塗抹接種し、３５℃で１８時間培養した。本発明培地では腸管出血性大腸菌Ｏ１５７と推定されるコロニーが純培養状に生育した。これらのコロニーを釣菌し、大腸菌Ｏ１５７検出キット「ＵＮＩ」（商品名：関東化学（株））を用いて免疫学的にＯ１５７抗原を調べたところ、全て陽性であることが確認された。
【００４９】
【発明の効果】
以上のように本発明は、大腸菌、特に腸管出血性大腸菌Ｏ１５７を特異的、高感度かつ簡便に分離することができ、他の偽陽性菌の試験に要していた試薬類の費用、操作の手間および時間を削減できる。また、保菌者検索や汚染された食材や食品を的確に検出でき、食品の安全性および検査費用削減に大きく貢献できる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a selective separation medium for Escherichia coli, particularly enterohemorrhagic Escherichia coli O157, which is a causative bacterium of the third class of infectious diseases.
[0002]
[Prior art]
In the treatment of infectious diseases caused by Escherichia coli, it is important to identify the causative bacteria of the infectious diseases and to quickly carry out treatment suitable for the causative substances. Among enterohemorrhagic E. coli, serotype O157, in particular, is responsible for many food poisonings in the United States, Canada and Europe. In Japan as well, various food ingredients (particularly beef) are involved in these food poisonings since the large-scale food poisoning in 1996.
This Escherichia coli causes hemorrhagic colitis characterized by bloody diarrhea and can lead to very severe renal complications (hemolytic uremic disease), which is a major public health challenge Yes. In particular, enterohemorrhagic Escherichia coli O157 is carried out with emphasis on carrier search (fecal inspection) and food quality inspection mainly by food workers.
[0003]
When infection of enterohemorrhagic Escherichia coli O157 is suspected or when searching for carriers, it is necessary to separate intestinal hemorrhagic Escherichia coli O157 using fecal material or the like as a test material for a definitive diagnosis. In this case, the enterohemorrhagic Escherichia coli O157 selective separation medium is required. The selective separation medium is a general term for media devised so that only the target microorganism can grow as much as possible. When using a test material that may contain a large amount of various intestinal bacteria such as fecal material, Selection performance is particularly important. In general, a selective medium is used as a selective separation medium in which a substance that acts as a selective substance and acts on the microorganisms that coexist and does not affect the growth of the target microorganism is added.
[0004]
For example, food samples and fecal samples contain many microorganisms other than enterohemorrhagic Escherichia coli O157, and in order to separate and culture enterohemorrhagic Escherichia coli O157 from these, a selective separation medium must be used. . Prior art separation media or selective separation media are as follows:
(1) Separation medium {circle around (1)} SMAC (Sorbitol MacConkey) agar medium It is used for testing all microorganisms that are gram-negative and non-fermentable or slow-fermentable with sorbitol. Antibiotics may be added due to low selectivity, but false positives due to non-sorbitol or slow-fermenting bacteria cannot be excluded. By adding a fluorogenic compound or a chromogenic compound as a differentiation reagent, microorganisms (essentially typical E. coli) producing β-glucuronidase can be removed.
Most of the enterohemorrhagic E. coli O157 isolated from the patient does not ferment sorbitol after 24 hours. Moreover, it does not have β-glucuronidase activity. Utilizing these properties, screening of enterohemorrhagic Escherichia coli O157 in clinical or food specimens uses sorbitol / Macconkey agar (SMAC) as the primary separation medium.
The sorbitol non-fermentable or slow fermentable suspicious colonies (colorless to white) are visually observed, and the suspicious colonies are confirmed as enterohemorrhagic Escherichia coli O157 by biochemical and serological tests.
[0006]
(2) MUG-added SMAC agar medium A medium for clarifying sorbitol-negative and β-glucuronidase-negative colonies. MUG (4-methylumbelliferyl-β-glucuronide) is added for confirmation of β-glucuronidase activity.
[0007]
(3) BCIG-added SMAC agar medium It is a medium for clarifying sorbitol negative and β-glucuronidase negative colonies. BCIG (5-bromo-4-chloro-3-indoxyl-D-β-glucuronide) is added for confirmation of β-glucuronidase activity.
[0008]
(4) Fluorocult E. coli O157: H7 agar medium (trade name: Kanto Chemical Co., Inc.) In a medium containing sorbitol, MUG and sodium thiosulfate to reveal sorbitol negative, MUG negative and H ₂ S negative colonies is there.
[0009]
(2) Selective separation medium CT-SMAC agar medium A SMAC-derived medium containing two types of inhibitors (cefixime and potassium tellurite). These inhibitors prevent the growth of many microorganisms that do not ferment sorbitol, such as Proteus, Morganella morganii, Providencia, Hafnia albei, etc., so that the medium is more selective To.
[0010]
However, the separation medium of (1) contains a large number of false-positive bacteria that have a small amount of enterohemorrhagic Escherichia coli O157 in carrier feces and food samples as compared with other coexisting microorganisms and show similar properties. Therefore, there is a problem that sensitivity and specificity are poor.
[0011]
In the selective separation medium (2), sorbitol non-fermenting bacteria and slow-fermenting bacteria other than enterohemorrhagic Escherichia coli O157, particularly Pseudomonas aeruginosa, are separated at a high rate. Therefore, it is difficult to determine the presence or absence of enterohemorrhagic Escherichia coli O157 from the colony properties because it is necessary to use a skillful technique to distinguish colonies suspected to enterenterohemorrhagic Escherichia coli O157 from such a selective separation medium. Therefore, there was a problem that the result of the subsequent identification test had to be waited.
[0012]
Furthermore, in the culture medium of JP 2000-342249, the growth of Pseudomonas aeruginosa cannot be suppressed, and when the contamination with Pseudomonas aeruginosa is high, the target enterohemorrhagic Escherichia coli O157 cannot be separated. Moreover, since growth inhibition of enterohemorrhagic Escherichia coli O157 was observed, false negatives tended to be high.
[0013]
On the other hand, conventional detection of enterohemorrhagic Escherichia coli O157 is generally performed by the following method.
(1) In the case of a food sample The sample is pre-cultured at 42 ° C. for 18 to 24 hours in a preculture medium novobiocin-added mEC broth. A portion of this preculture is smeared on the enterohemorrhagic Escherichia coli O157 selective separation medium or separation medium, cultured at 37 ° C. for 18-24 hours, and colonies on the plate medium are observed. Further, colonies that appear to be enterohemorrhagic Escherichia coli O157 on a flat plate medium are picked and inoculated into an enterohemorrhagic Escherichia coli O157 identification kit or a differential culture medium for biochemical property inspection, and cultured and identified at 37 ° C. for 18 to 24 hours. In addition, immunological tests using antiserum and PCR methods have been performed using these colonies and enrichment solutions.
[0014]
(2) In the case of stool specimen A part of the stool specimen is directly smeared on the enterohemorrhagic Escherichia coli O157 selective separation medium or separation medium, cultured at 37 ° C for 18-24 hours, and colonies on the plate medium are observed. Further, colonies that appear to be enterohemorrhagic Escherichia coli O157 on a flat plate medium are picked and inoculated into an enterohemorrhagic Escherichia coli O157 identification kit or a differential culture medium for biochemical property inspection, and cultured and identified at 37 ° C. for 18 to 24 hours. In addition, immunological tests using antiserum and PCR methods are also performed using these colonies.
In such a method, since an operation of separation culture using a separation medium or a selective separation medium is required for the inspection process, it is difficult to identify colonies similar to enterohemorrhagic Escherichia coli O157 as described above. In particular, there was a problem that Pseudomonas aeruginosa was isolated as a similar colony and had to wait for the result of the subsequent identification test.
[0015]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to solve the above problems and provide a medium and a separation method that can detect Escherichia coli in various samples including stool, particularly enterohemorrhagic Escherichia coli O157 with high sensitivity, in a specific and simple manner. There is.
[0016]
[Means for Solving the Invention]
The present inventors have found that the above-mentioned problems can be solved by including a selective agent in the basic medium while advancing intensive studies to solve the above-mentioned problems, and have completed the present invention.
[0021]
Furthermore, the present invention relates to a kit for the medium.
[0022]
Furthermore, the present invention provides a method for cultivating a sample in a medium containing a selective agent that does not inhibit the growth of the target Escherichia coli and inhibits the growth of Pseudomonas bacteria, A method for selective separation of Escherichia coli, characterized in that the sample is stool, Escherichia coli is enterohemorrhagic Escherichia coli O157, and the selective agents are cefixime, tellurite and cefsulosin The present invention relates to a method for selective separation of E. coli.
[0023]
Therefore, according to the present invention, it is possible to avoid spillage due to the mixture of false positive bacteria (high sensitivity and specificity), and inoculate Escherichia coli, particularly enterohemorrhagic Escherichia coli O157 identification kit and differential culture medium for biochemical property inspection. Therefore, it can greatly contribute to the reduction of the cost of identification, and further, it can reduce the labor and time of operation, which can be said to be significant in practical use.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
The target Escherichia coli in the present invention can be applied to various Escherichia coli such as enterohemorrhagic Escherichia coli, enterotoxigenic Escherichia coli, intestinal invasive Escherichia coli, enteropathogenic Escherichia coli, intestinal agglutinating Escherichia coli, and the cause of food poisoning Enterohemorrhagic Escherichia coli O157, which is considered to be a bacterium, is preferably used.
[0025]
Pseudomonas bacteria whose growth is suppressed by the selective agent of the present invention are Psedomonas. aeruginosa (Pseudomonas aeruginosa), P. aeruginosa. fluorescens, P.M. Although there are various bacterial species such as cepacia, growth of Pseudomonas aeruginosa is suitably suppressed when the enterohemorrhagic Escherichia coli O157 is selectively separated.
[0026]
The medium according to the present invention may be any medium as long as the target Escherichia coli grows with high sensitivity. Specifically, CT-SMAC agar medium, SMAC medium, MUG-added SMAC agar medium, BCIG-added SMAC agar medium, fluorocult E. coli O157: H7 agar medium (trade name: Kanto Chemical Co., Ltd.), A medium with modification, for example, a medium modified with sugar or the like is used. Among these, a CT-SMAC agar medium that can suppress the growth of other coexisting microorganisms is preferable. These media can be used in any state of agar, powder or liquid as long as they do not affect the culture.
[0027]
The method for preparing the selective separation medium used in the present invention can be prepared by adding purified water to the basal medium to make a constant amount, cooling after autoclaving, and adding a constant amount of additives. Specifically, for example, 51.5 g of SMAC agar medium is made up to 1000 mL with purified water and sterilized at 121 ° C. for 15 minutes. After sterilization, it can be prepared by cooling to about 50 ° C. and adding to a concentration of cefixime 0.05 mg / L, potassium tellurite 2.5 mg / L, and cefsulodin 5 mg / L.
[0028]
The selective agent used in the present invention refers to a substance containing an antibiotic that inhibits the growth of cefixime, tellurite and Pseudomonas and does not inhibit the growth of E. coli, and has an adverse effect on the isolation and culture of the target E. coli. Anything else may be included as long as it is not.
[0029]
As the tellurite used as the selective agent, alkali metal or alkaline earth metal salts such as potassium salt, calcium salt, sodium salt, magnesium salt and the like are used. A potassium salt is preferably used from the viewpoint of ease of use. The concentration may be any concentration that does not affect the isolation and culture of the target E. coli, preferably 0.5 to 5 mg / L, more preferably 1 to 3 mg / L, and even more preferably 2 to 2.5 mg. / L.
[0030]
The concentration of cefixime used in the selection agent may be any concentration that does not affect the separation of the target Escherichia coli, preferably 0.01 to 0.19 mg / L, more preferably 0.03 to 0.10 mg / L. L.
[0031]
In the case of a basic medium that already contains cefixime and tellurite, such as CT-SMAC agar medium, the selective agent according to the present invention can be added if it does not affect the isolation and culture of the target E. coli. You may add only antibiotics. On the other hand, in a basic medium that does not contain cefixime and tellurite, such as SMAC agar medium, only the target Escherichia coli can be selectively separated by adding the selective agent according to the present invention.
[0032]
Antibiotics used in the present invention may be any antibiotics that do not inhibit the growth of the target microorganism, and inhibit the growth of Pseudomonas bacteria, such as cefsulosin, ceftriaxone, cefpyramide, cefotaxime, ceftazidime, cefpimizole, Cephems such as ceftizoxime and cefmenoxime, tetracyclines such as novobiocin and vancomycin, and penicillins such as piperacillin and carbenicillin are used. If the target Escherichia coli is enterohemorrhagic Escherichia coli O157, cefsulodin or novobiocin that suppresses the growth of Pseudomonas aeruginosa almost completely is preferable, and cefsulosin is more preferably used. These antibiotics can also be used in the form of salts. Moreover, the antibiotics to be used may be used alone or in combination.
[0033]
The concentration of the antibiotic used as the selection agent may be any concentration that suppresses the growth of Pseudomonas bacteria, and in the case of cefrosin, it is preferably 0.5 to 100 mg / L, more preferably 0.78 to 50 mg / L, More preferably, it is 1.56-25 mg / L. In the case of novobiocin, it is preferably 5 to 50 mg / L, more preferably 15 to 30 mg / L, and still more preferably 20 to 25 mg / L.
[0034]
In the method for separating microorganisms according to the present invention, a biologically-derived sample including stool, a food-derived sample, an enrichment culture solution of these samples and the like are smeared onto the selective separation medium prepared as described above using a platinum loop or the like, 35 The target microorganism can be isolated by culturing at a temperature of from 37 to 37 ° C. for 18 to 24 hours.
[0035]
In addition, it is preferable in terms of convenience that the reagents and equipment for carrying out the present invention are in the form of a portable kit. In this kit, in addition to reagents for selectively separating E. coli, for example, selective agents, basal media, etc., a stomac necessary for enrichment culture such as stool collection containers and stool collection rods necessary for stool specimen intake, food inspection A bag, an enrichment medium, or a platinum ear necessary for separation culture stored in a portable container is more preferable in terms of simplification of work and efficiency. In addition, a selective agent such as cefsulodin, cefixime, and tellurite containing only three agents in a portable container is also preferable in terms of simplification and efficiency.
[0036]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this.
[Example 1]
Preparation of medium Purified water was added to SMAC agar medium (51.5 g) to make a total volume of 1000 mL, followed by high-pressure sterilization (121 ° C., 15 minutes). After cooling to about 50 ° C., a predetermined amount of the present medium A-added reagent, the present medium B-added reagent, and the target CT having the following composition were added. The reagent was mixed well, and 18 mL each was dispensed into a petri dish to prepare a flat plate.
[0037]
Basic composition of this medium (per 1000 mL of medium)
Peptone (20 g), sorbitol (10 g), bile salt (1.5 g), sodium chloride (5 g), neutral red (0.03 g), crystal violet (0.001 g), agar (15 g), pH = 7. 1 ± 0.1
[0038]
Reagent added to medium A (per 1000 mL of medium)
Potassium tellurite (2.5 mg), cefixime (0.05 mg), cefsulodin (5 mg)
[0039]
Reagent added to medium B (per 1000 mL of medium)
Potassium tellurite (2.5 mg), cefixime (0.05 mg), novobiocin (20 mg)
[0040]
Target CT additive reagent (per 1000 mL of medium)
Potassium tellurite (2.5 mg), cefixime (0.05 mg)
[0041]
[Example 2]
Enterohemorrhagic Escherichia coli O157 growth support ability result The dilution ratio of the test strain suspension was changed, and the enterohemorrhagic Escherichia coli O157 growth support ability in each medium was compared. The test method was performed as follows.
Various test strains are precultured (35 ° C., overnight) on tryptone soya agar medium (trade name: Kanto Chemical Co., Inc.), and the resulting colonies are suspended in sterile physiological saline. Each test strain suspension is ^serially diluted to 10 ⁻⁷ , and each dilution series is added dropwise (20 μL) to the evaluation medium and cultured (35 ° C., 18 to 24 hours).
[0042]
The results are shown in Table 1. Table 1 shows the number of growing bacteria, color, and colony size at each dilution rate of the test strain suspension in each medium. When the number of viable bacteria could not be measured, it was expressed in ++ (not measurable due to partial colony fusion) and ++++ (mostly not measurable due to colony fusion). From Table 1, it was found that intestinal hemorrhagic Escherichia coli O157 develops in medium A and medium B as compared with medium CT or higher. The colony size was also equivalent.
[0043]
[Table 1]

[0044]
Example 3
Results of inhibition of growth of representative intestinal bacteria Using the culture medium prepared in Example 1, the growth inhibition of representative enteric bacteria was compared. The test method was the same as in Example 2. The results are shown in Table 2. Table 2 shows the number of growing bacteria, color, and colony size at each dilution rate of the test strain suspension in each medium.
From Table 2, it was found that both medium A and medium B inhibit the growth of various intestinal bacteria in the same manner as the medium supplemented with CT.
[0045]
[Table 2]

[0046]
Example 4
Results of inhibition of growth of Pseudomonas aeruginosa Using the medium prepared in Example 1, the growth inhibition of Pseudomonas aeruginosa was compared. The test method was the same as in Example 2. Table 3 shows the number of viable bacteria, color and colony size at each dilution rate of the test strain suspension of Pseudomonas aeruginosa in each medium. When the number of viable bacteria could not be measured, it was expressed in ++ (not measurable due to partial colony fusion) and ++++ (mostly not measurable due to colony fusion). Those that cannot be measured due to small growth colonies are indicated by “±”.
From Table 3, it was found that growth of Pseudomonas aeruginosa was suppressed at medium A at any dilution rate. Moreover, in the culture medium B, it turned out that the growth of Pseudomonas aeruginosa can be completely suppressed by dilution factor ^10-4 . Therefore, the target Escherichia coli O157 could be selectively separated as compared with the CT medium.
[0047]
[Table 3]

[0048]
Example 5
Example of Isolation of Enterohemorrhagic Escherichia coli O157 Intestinal hemorrhagic Escherichia coli O157 is inoculated to 10 ³ -10 ⁴ CFU per 1 g of adult stool, diluted 10-fold with sterile physiological saline, and pseudo positive stool Create 10 μL was collected, smeared directly on the medium of the present invention, and cultured at 35 ° C. for 18 hours. In the culture medium of the present invention, a colony presumed to be enterohemorrhagic Escherichia coli O157 grew in a pure culture state. These colonies were picked and immunologically examined for O157 antigen using an E. coli O157 detection kit “UNI” (trade name: Kanto Chemical Co., Inc.), all were confirmed to be positive.
[0049]
【The invention's effect】
As described above, the present invention can isolate Escherichia coli, particularly enterohemorrhagic Escherichia coli O157 specifically, highly sensitively and easily, and the cost and operation of reagents required for testing other false-positive bacteria. Save time and effort. In addition, searching for carriers and accurately detecting contaminated foods and foods can greatly contribute to food safety and cost reduction.

Claims (1)

The sample is cultured in a medium containing a selective agent that does not inhibit the growth of the target Escherichia coli and that inhibits the growth of Pseudomonas bacteria, and the growth of the Pseudomonas bacteria is suppressed to selectively isolate E. coli. A method for selective separation of E. coli, characterized in that the sample is feces , E. coli is enterohemorrhagic E. coli O157, and the selective agent is cefixime, tellurite and cefsulosin.