JP2608301B2 - Toxogenic Escherichia coli prophylactic / therapeutic agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a prophylactic / therapeutic agent for diarrhea caused by enterotoxigenic Escherichia coli.

(Conventional technology and problems to be solved) Toxigenic Escherichia coli is known as a causative agent of traveler's diarrhea which bothers travelers to the tropics and subtropics, and is also called infant diarrhea or so-called "per food" The involvement of diarrhea that occurs on a daily basis has been speculated, and in recent years it has been particularly important.

The main cause of diarrhea caused by toxinogenic Escherichia coli is enterotoxins produced by this bacterium, but the structure is very similar to cholera toxin, and it is insensitive to heat when heated at 60 ° C for 10 minutes. Enterotoxin (LT) increases intracellular cyclic AMP levels by activating adenylate cyclase, causing severe diarrhea similar to cholera diarrhea.

Toxogenic Escherichia coli is mainly manifested by cholera-like diarrhea and abdominal pain in humans, and diarrheal stool is often stomach-like or watery stool of rice, and relatively few cases complain of fever. On the other hand, animals are known to cause diarrhea in domestic animals such as pigs, cows, and sheep. For example, this diarrhea is 1
It occurs regularly in suckling pigs aged 3 weeks and sporadically occurs in piglets immediately after weaning. Symptoms are mainly characterized by diarrhea, the hair grows bad and the growth almost stops. When diarrhea becomes chronic, she died of melancholy or suffered a secondary infection and died of sepsis. Further, even if the recovery proceeds, the subsequent growth is delayed, and it often becomes a so-called porcine pig and loses its economic value in many cases.

In animals, antibiotics are commonly used to prevent and treat this diarrhea. However, the use of antibiotics has led to the emergence of resistant bacteria and has not always been an effective treatment.

The present inventors have isolated bacteria from soil and conducted intensive studies on the effects of the isolated strains on diarrhea caused by toxinogenic Escherichia coli. The present inventors have found that the present invention is effective against primary colibacillosis and completed the present invention.

(Means for Solving the Problems) Therefore, the above-described object is to prevent virulent or dead cells of Clostridium sp. Strain NIP1201 or to prevent toxinogenic Escherichia coli containing a dead cell or a culture thereof as an active ingredient. Achieved by therapeutic agents.

(Action) Clostridium sp. Strain N used in the present invention
IP1201 proliferates as a vegetative body, forms durable spores as the growth environment deteriorates, and sleeps. It germinates again in a favorable environment and becomes a vegetative body and grows in a life cycle.

After administration, the viable cells (spores, vegetative cells) proliferate in the digestive tract and produce active ingredients inside and outside the cells. The active ingredient is also contained in a culture solution or dead cells obtained by culturing the present bacterium.

Therefore, Clostridium is used in the present invention.
live cells (spores and vegetative bodies) of sp. strain NIP1201,
Dead cells and the culture solution.

The prophylactic / therapeutic agent for toxigenic Escherichia coli of the present invention is prepared by diluting a cell of Clostridium sp. Strain NIP1201 or a culture solution thereof as it is, or diluting it with a solid or liquid diluent. For example, dosage forms such as powders, granules, fine granules, tablets, sugar-coated tablets, capsules, water-soluble or soluble film coating agents, dry syrups and the like, and suspensions and syrups as solutions may be used. Diluents include excipients, binders, disintegrants, etc. used in general pharmaceuticals, but in addition to these, colorants, flavors, stabilizers, preservatives, emulsifiers, pH adjusters, etc. May be.

In addition, for animals, it is used without dilution, or used as a powder, powder, granules, tablets, liquids, capsules, etc. with a diluent, or directly or once into drinking water, feed, artificial milk, breast milk, etc. Those dispersed in a diluent may be added. Any diluent may be used if it is physiologically harmless, but more preferable is a feed or nutrient.
In addition, any of these preparations for animals and the like may contain antidiarrheal agents, astringents, mucosal protective agents, antibacterial agents, antibiotics, enzyme agents, other viable bacterial preparations, and the like.

Administration of the prophylactic / therapeutic agent for toxinogenic Escherichia coli of the present invention is performed orally. Further, as shown in Example 3, the dose is very low in toxicity, and thus can be appropriately determined depending on the administration subject, body weight, age (day of age), purpose of administration, and the like. Usually, the dose is 1 kg of body weight as the number of viable bacteria.
The range is from 10 ⁶ to 10 ¹⁰ per.

The properties and the like of Clostridium sp. Strain NIP1201 are shown below, and Examples show that the present invention has an excellent effect as a prophylactic / therapeutic agent for toxinogenic Escherichia coli and has high safety.

・ Properties of Clostridium sp. Strain NIP1201 This bacterium is a Gram-positive obligate anaerobic bacillus and forms spores.

Bacterial morphology: The vegetative somatic cells are rods of 0.9 μm × 1.2 × 4.0 μm (GAM medium) μm linear and blunt at both ends. Sometimes it can be double or multi-chain. The spores are oval from 1.0 to 1.2 × 2.0 to 2.2 μm and are subexistent. In addition, some vegetative body cells have swelled at one end.

Colony morphology: Irregular and marginal rhizoid, prominent in the center (GAM plate medium).

 This is the so-called “Medusa head” colony. The color is light tan.

Fermentation products: Produce acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, isocaproic acid, etc.

Motility: Motility is observed in GAM medium.

Aerobic growth: No aerobic growth on GAM plate medium.

Gelatin Liquefaction: Positive.

Casein digestion: positive.

Sulfate reduction: negative.

Glycolytic: It degrades glucose, fructose and maltose and slightly degrades cellobiose, sorbitol, sucrose and glycerol, but galactose, mannose, raffinose, rhamnose, ribose, starch, xylose, Does not degrade lactose.

From the above results, Clostridium sp. Strain N
IP1201 is for Clostridium sporogenes ( Clostridiu
m sporogenes ).

Deposition of microorganism The present inventors have proposed a Clostridium sp.N used in the present invention.
IP1201 ( Clostridium sp. Strain NIP1201) was deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.

 Accession No .: No. 9570 (FERM p-9570) Date of receipt: September 7, 1987 Toxicity test No acute toxicity was observed in mice.

(Examples) Hereinafter, the present invention will be described in more detail based on examples.

Example 1 After culturing Clostridium sp. Strain NIP1201 in a GAM medium at 37 ° C. for 72 hours, the culture obtained by centrifugation was freeze-dried, and this was used as a NIP1201 culture FD product (yield 5
6 mg / ml) for the following experiments.

Escherichia coli ( Escherichia coli IFM3043 or less, hereinafter referred to as E. coli LT), which is a heat-labile enterotoxin-producing bacterium, was used as the toxinogenic Escherichia coli . In CAYE medium,
NIP1201 culture solution FD product each final concentration 1,2,5,10mg / ml
E. coli LT was shake-cultured at 37 ° C. for 24 hours, and then the cell concentration and the production of heat-labile enterotoxin were measured. In addition, instead of NIP1201 culture solution FD product, GAM
A group to which the medium was lyophilized and added to a final concentration of 10 mg / ml was used as a control.

Measurement of cell concentration is optical in 620nm density (OD ₆₂₀₎
Was measured.

The measurement of the amount of the heat-labile enterotoxin was measured using the reverse passive latex agglutination method, and the sample was serially diluted two-fold, and the result was indicated by the highest dilution showing positive.

 Table 1 shows the results.

The experimental results are shown in Table 1.
Addition of 1,2,5,10 mg / ml does not affect the growth of E. coli LT, but the production of heat-labile enterotoxin from E. coli LT is 1 / 4, 1/16, 1/6
It was suppressed in a dose-dependent manner at 4, 1/128. Thus, the FD product of the NIP1201 culture solution was found to have the effect of suppressing the production of heat-labile enterotoxin, which is the main cause of diarrhea in toxogenic Escherichia coli. In this test, the culture solution of NIP1201, that is,
Although the product from the cells was used, it goes without saying that the cells that produce and contain this product are also effective.

Example 2 E. coli LT was shake-cultured for 6 hours in a CAYE medium using E. coli LT, and the supernatant was removed by centrifugation. After washing once,
A suspension in a fresh GAM medium at 2 × 10 ⁸ cells / ml was used as a control group. On the other hand, NIP1201 times nutrient solution FD in GAM medium
An E. coli LT suspension in which the product was easily dissolved (56 mg / ml) was used as a test group and used in the following rabbit intestinal loop experiment.

Japanese white male rabbits (body weight 1.5 kg to 2.0 kg) were fasted for 2 days (water intake was not restricted), and then general anesthesia (20 mg / kg) was performed with pentobarbital. The abdomen was opened in accordance with the usual method (Test method for cholera and Escherichia coli, written by Miwaya et al., Published by Nane).
Seven were created with an interval of m. During this loop, 2 ml of each of the two groups of test solutions prepared as described above was alternately administered and infected between the loops. After the laparotomy, the rabbit was left indoors at 24 ° C., 20 hours later, sacrificed under anesthesia, and the laparotomy was performed to remove the loop. Next, the weight (g) and length (cm) of the contents of each loop were measured, and the liquid storage activity (W / L ratio) was calculated according to the following equation.

In addition, the significant difference between each control group and the test group was tested by the student t-test.

 Table 2 shows the results.

As shown in Table 2, the W / L ratio of the control group to which E. coli LT was applied was 1.306 ± 0.154, whereas the W / L ratio of the test group treated with the NIP1201 culture solution FD product. Was 0.210 ± 0.053, and the suppression rate was 83.9%. That is, E. Collie LT
It was shown that the intestinal water retention induced by infection with N.p.a. was significantly suppressed by the NIP1201 broth FD (bacterial product). The findings obtained in the above in vitro and in vivo systems indicate that the product (culture solution) from NIP1201
The cells that produce and contain this are themselves E. coli,
This indicates that the drug is an excellent drug for preventing and treating diarrhea caused by LT-producing bacteria.

Example 3 Clostridium sp. Strain NIP1201 was cultured at 37 ° C. for 72 hours by the GasPak method using a GAM plate medium, and the resulting colonies were collected and adjusted to a predetermined concentration with tap water. The above test solution was administered to a group of 10 ICR mice (sex and male, weight 3
0 g to 50 g) orally or intraperitoneally 10 ¹⁰ / kg, 10
¹¹ / kg were each administered. General condition was observed for 7 days after administration of the test solution, and necropsy was performed 8 days later.

As a result, in the control group and the group to which the test solution was administered, there was no noticeable change in the general state and the autopsy findings in the oral administration. However, in the intraperitoneal administration group, in each of the test liquid administration groups of each concentration, hairiness became worse and spontaneous movement decreased, but recovered after 48 hours. Based on the above, it was concluded that the toxicity of this drug was extremely low, and the bacteria themselves were not pathogenic.

(Effects of the Invention) The present invention relates to Clostridium sp. Strain NIP12
01 ( Clostridium sp. Strain NIP1201) is a prophylactic / therapeutic agent for toxinogenic Escherichia coli containing live cells, dead cells, or a culture of the cells as an active ingredient.
It has an extremely excellent effect as a preventive and therapeutic agent for toxigenic Escherichia coli.

Claims (1)

(57) [Claims] [Claim 1] Clostridium sp. Strain NIP12
01 ( Clostridium sp. Strain NIP1201), a prophylactic and therapeutic agent for toxinogenic Escherichia coli containing live cells, dead cells, or a culture thereof as an active ingredient.