JP2000135077A - Selective culture medium of helicobacter pylori and selective culture of helicobacter pylori using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a selective culture medium for Helicobacter pylori that can selectively culture Helicobacter pylori as, a pathogenic bacterium causing digestive ulcer, which is derived from animals, particularly from materials of rodent animals by admixing bacitracin to a basal medium. SOLUTION: A basal medium, for example, GC medium, Brucella medium or BHI(Brain Heart Infusion) agar medium is autoclave-fertilized and dissolved. Then, the basal medium is cooled down to 45 deg.C, then equine defibrinated blood having the final concentration of 7%, Dent supplement, bacitracin having the final concentration of 2.5-10 U/L of the basal medium are added, mixed, then dispensed to sterilized petri dishes or the like, and naturally cooled down to solid whereby the objective Helicobacter pylori selective culture medium is obtained. This culture medium can selectively culture Helicobacter pylori, as a pathogenic bacterium causing digestive ulcer or the like and is useful for development of an anti-Helicobacter pylori drug and an antiulcer drug.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a helicobacter
The present invention relates to a selective medium for H. pylori and a selective culture method. The present invention is directed to selectively culturing Helicobacter pylori in a culture sample containing Helicobacter pylori obtained from a gastrointestinal tract such as the stomach and duodenum of an animal such as a human or a rodent for the purpose of diagnosis or research. Useful to

[0002]

2. Description of the Related Art It has been known that gastric ulcer patients have a higher gastric ammonia concentration than healthy persons and have urease activity in the gastric mucosa. In 1983, Marshall and Warren of Australia succeeded in isolating new gram-negative bacilli from the gastric mucosa of gastric ulcer patients (Warre
n, JR and Marshall, BJ, Lancet, 1, 1273-1275, 198
3) It was revealed that urease activity in gastric ulcer patients was derived from this bacterium. This bacterium was initially discovered in Campylobacter pyloridis (Campylobacter p.
ylolidis), which was later named Helicobacter pylori.
Oridis), and Helicobacter pylori is now the official scientific name.

In recent years, research on Helicobacter pylori has progressed, and it has been revealed that this bacterium is not only a gastric ulcer but also a pathogenic factor in human upper gastrointestinal diseases such as chronic gastritis, duodenal ulcer, gastric cancer and lymphoma. .

The NIH held in the United States in February 1994
In Consensus Conference, "Peptic ulcer disease and Helicobacter pylori infection"
A unified view on the issue was published (NIH Consensus, Helicoba
cter pylori in peptic ulcer diseases, JAMA, 272, 6
5-69, 1994), a recommendation was made that `` peptic ulcers with confirmed Helicobacter pylori infection, regardless of their onset or recurrence, require eradication therapy with antisecretory drugs and antimicrobial agents. '' Was done.

In the same year, the World Health Organization (WHO)
Concludes that Helicobacter pylori is a risk factor for the development of human gastric cancer, and
e carcinogen (WHO Interna
national Agency for Research on Cancer, IARC monograp
hs on evaluation of carcinogenic risks to humans.S
chistosomes, liver flukes and Helicobacter pylor
i., IARC, 61, 177-241, 1994).

[0006] Under such circumstances, it is desired to develop a drug for safely and surely removing Helicobacter pylori, but it has not yet been realized.

[0007] At present, combinations of various drugs, mainly antibiotics, have been attempted to eradicate Helicobacter pylori. Specific combinations of drugs include conventional bismuth preparations, triple therapy using metronidazole and existing antibiotics, and dual therapy using proton pump inhibitors and amoxicillin, which have become mainstream in recent years. Or a combination therapy of a gastric mucosa protective agent such as sucralfate and an antibiotic. However, a sufficient eradication rate has not been obtained in any of these therapies. In addition, the administration of antibiotics not only raises concerns about side effects such as diarrhea and metabolism, but also begins to point out the danger of emergence of resistant bacteria if eradication fails.

[0008] In order to develop a new drug for safely and surely eliminating Helicobacter pylori, a drug in vivo is required.
A general-purpose Helicobacter pylori-infected animal model for screening the eradication effect of E. coli is essential.

[0009] Many of the animal models of Helicobacter pylori infection reported so far use sterile animals such as sterile pigs, sterile beagle dogs, and sterile mice. When Helicobacter pylori is isolated and cultured from these Helicobacter pylori alone-infected animals, no special selective culturing method is required because no bacteria are present. However, helicobacter pylori alone infected animals have low utility as routine drug screening systems because they require special equipment and complicated aseptic operations for maintaining animals, and are inferior in experimental efficiency and economic efficiency.

On the other hand, recently, the present inventors have proposed that Helicobacter pylori be replaced by SPF (specific pathogen-fr).
ee) and conventional common laboratory animals, specifically rodents such as gerbils, mice and rats. These infected animals have excellent experimental efficiency and economic efficiency, and are expected to be useful as drug screening systems in the future. However, in normal germ-free animals, there are a large number of species-specific resident bacteria and foreign bacteria in the digestive tract including the stomach. A culture method is required.

As a method for selective cultivation of Helicobacter pylori, a method using a selective medium containing various antibiotics and antibacterial agents as a component for suppressing various bacteria has been devised. Representative Helicobacter pylori selection media include M-BHM containing vancomycin, amphotericin B, nalidixic acid and triphenyltetrazolium chloride (TTC).
Examples include a medium, a Skirow medium containing trimethoprim, vancomycin and polymyxin B, and a Dent medium containing vancomycin, amphotericin B, trimethoprim and cefsulodin.

[0012]

SUMMARY OF THE INVENTION These selective media are:
Although it has already been put into practical use in human clinical and research fields, it is not suitable for the purpose of isolating and culturing the same bacterium from animal-derived materials. The contained antimicrobial component suppresses the growth of various bacteria present in the human stomach, but resident bacteria that have colonized the animal's stomach, especially lactic acid, which is present in large amounts in the stomach of rodents This is because the growth of bacilli cannot be suppressed.

In fact, when the rodent gastric mucosa is homogenized and cultured in the above selective medium, clear lactobacillus colonization is observed. In such a state, even if Helicobacter pylori is present therein, it is difficult to visually discriminate the two. Methods for distinguishing Helicobacter pylori from the coloration of colonies formed on the culture medium have also been introduced in conference presentations, etc., but depending on the degree of development, not all Helicobacter pylori colonies are necessarily colored, Since the colonization of Helicobacter pylori is inhibited by the presence of various bacteria, the results of determination or quantification by this method are hardly accurate.

That is, it has been impossible or difficult to selectively culture Helicobacter pylori from materials derived from animals, particularly rodents, using the conventional techniques.

Accordingly, an object of the present invention is to provide a selective culture medium for Helicobacter pylori which can selectively culture Helicobacter pylori from a material derived from an animal, particularly a rodent, and a method for selective culture of Helicobacter pylori using the same. To provide.

[0016]

Means for Solving the Problems As a result of intensive studies on the above problems, the present inventors have found that Helicobacter pylori can be selectively cultured by using a medium containing bacitracin, and completed the present invention. did.

That is, the present invention provides a Helicobacter pylori selection medium containing bacitracin in a basal medium. The present invention also provides a method for selectively culturing Helicobacter pylori, which comprises culturing a culture sample containing Helicobacter pylori in the selective medium of the present invention.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The method for selective cultivation of Helicobacter pylori of the present invention is an antibiotic which can selectively inhibit the growth of Gram-positive bacteria such as lactobacilli without inhibiting the growth of Helicobacter pylori. Bacitracin (U.S. Pat. No. 2,498,165 and U.S. Pat. No. 2,828,2)
No. 46). The content of bacitracin is not particularly limited, but preferably 1 L of basal medium.
It may be in the range of 2.5 to 10 U.

As the basal medium constituting the selective medium of the present invention, any basal medium conventionally used for culturing Helicobacter pylori can be used. For example, GC medium (manufactured by Difco), Brucella medium (Dif
Co., Ltd.), BHI (Brain Heart Inf)
usion) medium (manufactured by Difco) or the like can be preferably used. Preferably, the basal medium contains 5-10% animal defibrinated blood or serum, more preferably 7% horse defibrinated blood. These basal media may be liquid media, or may contain 12 to 15 g of agar per liter of basal media as a component for solidifying the media.

The selective medium of the present invention contains Dent supplem as an auxiliary component for suppressing the growth of various bacteria.
ent (manufactured by Oxoid) or Skyrow su
It is preferable to add a bacteriostatic agent such as a supplement (manufactured by Oxoid).

The selective medium of the present invention may contain, in addition to the above-mentioned components, other components such as other antibiotics as long as the effects of the present invention are not impaired.

The culture sample containing Helicobacter pylori referred to in the present invention means any sample to be cultured, including Helicobacter pylori, and is preferably a human sample such as gastric ulcer, chronic gastritis, duodenal ulcer, gastric cancer or lymphoma. Diagnosis of Helicobacter pylori infection in patients with upper gastrointestinal tract disease, or biomaterials of animals including humans collected for the purpose of developing and researching medicines for eradication of Helicobacter pylori or prevention of Helicobacter pylori infection, More preferably, it is collected from a gastrointestinal tract such as the stomach or duodenum of an animal including a human, a biological material such as mucous membrane, mucus, and contents, and more preferably, is collected from a gastrointestinal tract of a rodent infected with Helicobacter pylori. Biological materials such as mucous membranes, mucus, and contents.

The method for culturing Helicobacter pylori using the selective medium of the present invention can be performed in the same manner as the conventional method for culturing Helicobacter pylori. For example, under aerobic conditions of 5% O ₂ , 10% CO ₂ and 85% N ₂ ,
Culture can be performed preferably at 37 ° C.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

BHI (Brain Heart Inf)
usion) agar medium (manufactured by Difco) is heated and dissolved by autoclave sterilization, and then cooled to 45 ° C., followed by a 7% final concentration of horse-defibrated blood (manufactured by Nikken Biomedical Research Institute Co., Ltd.), Dent supplement (Oxoid) Was added, and a final concentration of 2.5, 5, or 10 U of bacitracin (Sigma) was added. After mixing, 20 ml each was dispensed into a sterile petri dish and solidified by natural cooling. As a control medium, a medium having the same composition but not containing bacitracin was prepared.

A male ddY mouse bred in a conventional environment was euthanized by cervical dislocation, and the stomach was aseptically removed. The stomach was incised, the contents were washed and removed with physiological saline, and the gastric mucosa was peeled and collected using a scalpel.
Then, 1 ml of BHI broth (D
ifco), and using a sterilized glass mortar,
A gastric mucosal suspension was prepared.

The Helicobacter pylori-containing sample was added to the above gastric mucosal suspension in a Mac Farland standard solution of 10 ⁵ /
ml of Helicobacter pylori (TK1402 strain)
Was prepared by mixing. As a control sample, the above gastric mucosal suspension not mixed with Helicobacter pylori was used.

A medium containing Helicobacter pylori or a control sample 1 was added to a medium containing bacitracin or a control medium.
0 μl using a platinum loop, 5% O ₂ , 10% CO 2
_2. Cultured at 37 ° C. for 7 days under aerobic atmosphere of 85% N ₂ . After completion of the culture, the total number of colonies formed in each medium was counted, and then the number of urease-positive colonies was counted as the number of Helicobacter pylori. The number of urease-positive colonies
Put the surface of the culture medium in urea / phenol red solution (urea 50g /
l, NaCl 5 g / l, NaH ₂ PO ₄ 0.8 g /
l, citric acid 0.4 g / l, phenol red 0.
(1 g / l, pH 5.8 ± 0.2) was stamped on a filter paper soaked, and the number of spots that developed pink was counted. Table 1 shows the results.

[0029]

[Table 1]

From the above results, it has been clarified that by using a medium containing bacitracin, only Helicobacter pylori can be selectively cultured.

[0031]

According to the culturing method of the present invention for culturing in a medium containing bacitracin, it was revealed that Helicobacter pylori, which is a causative bacterium such as peptic ulcer, can be selectively cultured. Therefore, by using the selective medium and the selective culture method of the present invention, a drug Helicobacter using an animal such as SPF or a conventional rodent is used.
Screening for eradication of H. pylori becomes possible,
It can make a significant contribution to the development of anti-Helicobacter pylori and anti-ulcer drugs.

 ────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Naohiro Yamada 1111 Tehiro, Kamakura-shi, Kanagawa F-term in Toray Industries, Inc. Basic Research Laboratory 4B065 AA01X AC10 BB37 BC12 CA46

Claims (6)

[Claims] 1. A selective medium for Helicobacter pylori containing bacitracin in a basal medium. 2. The medium according to claim 1, wherein the medium contains 2.5 to 10 U of bacitracin per liter of the medium. 3. The selective medium for Helicobacter pylori according to claim 1, wherein the basal medium is a GC medium, a Brucella medium, or a BHI medium. 4. A method for selectively culturing Helicobacter pylori, comprising culturing a culture sample containing Helicobacter pylori in the medium according to any one of claims 1 to 3. 5. The method for selectively culturing Helicobacter pylori according to claim 4, wherein the culture sample containing Helicobacter pylori is a material derived from the digestive tract of an animal including human. 6. The method for selective culture of Helicobacter pylori according to claim 4, wherein the culture sample containing Helicobacter pylori is a material derived from the digestive tract of a rodent infected with Helicobacter pylori.