JPH01153645A - Preventive and treating agent for toxinogenic escherichia coli infection - Google Patents

Abstract

PURPOSE:To obtain the title preventive and treating agent, containing live or dead microbial cells of Clostridium sp. strain NIP1201 or a cultivation filtrate thereof as an active ingredient, having high safety and capable of exhibiting excellent effects. CONSTITUTION:A preventive and treating agent containing live or dead microbial cells of Clostridium sp. strain NIP1201 (FERM P-9570) which is a Gram-positive, strict anaerobic and sporulating bacillus or a cultivation filtrate thereof as an active ingredient. The above-mentioned live microbial cells are capable of propagating in the digestive tracts after administration and intracellularly or extracellulary producing the active ingredient. The active ingredient is also contained in the cultivation filtrate obtained by cultivating the microorganism or dead microbial cells. The afore-mentioned preventive and treating agent is orally administered and the dose thereof is within the range of 10<6>-10<10> cells expressed in terms of viable cell number per kg body weight.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a prophylactic and therapeutic agent for diarrhea caused by toxigenic Escherichia coli.

(Conventional technology and problems to be solved) Toxigenic Escherichia coli is known as the causative agent of traveler's diarrhea, which plagues travelers to tropical and subtropical regions, and is also responsible for infantile diarrhea and so-called "food-induced diarrhea." It is also suspected to be involved in diarrhea that occurs on a daily basis, and has received particular attention in recent years.

The main cause of diarrhea caused by toxigenic Escherichia coli is enterotoxins produced in Japan, among which the structure is extremely similar to cholera toxin, and it is
Heat-labile enterotoxin (L-cho) that is inactivated by heating for 0 minutes
increases intracellular cyclic A) IP levels by activating adenylate cyclase, causing severe diarrhea similar to cholera diarrhea.

Toxigenic colibacillosis occurs in humans, and in 5A patients, the main symptoms are cholera-like diarrhea and abdominal pain, and the diarrhea is often rice-soak-like or watery, and there are relatively few cases of fever.

On the other hand, in animals, it is known to cause diarrhea in livestock such as pigs, cows, and sheep. For example, this diarrhea is 1 to 33! It occurs regularly in suckling pigs of I age, and also occurs sporadically in piglets immediately after weaning. Symptoms include diarrhea, hair growth becomes poor, and growth almost stops. If diarrhea becomes chronic, the patient may become weakened and die, or may suffer from secondary infection and die from sepsis.

Furthermore, even if the pigs start to recover, their subsequent growth is delayed and they often become so-called pigs, losing their economic value.

Antibiotics are commonly used for preventive treatment of this diarrheal disease in animals. However, the use of antibiotics has seen the emergence of resistant bacteria and is no longer necessarily an effective treatment method.

The present inventors isolated bacteria from soil and conducted intensive research on the effects of the isolated strains on diarrhea caused by toxigenic E. coli.
We found that Clostridium Sp, strain NIP1201) is effective against toxin-spreading coliosis,
The invention has been completed.

(Means to solve the problem) Therefore, the above objective is to treat Clostridium sp.

Strain N I P 1201 (Clostrid
This can be achieved by using a therapeutic agent for preventing and treating toxigenic coliosis, which contains as an active ingredient live or dead cells or a culture filtrate thereof.

(Function) The Clostridium sp strain NIP1201 used in the present invention proliferates as a vegetative body, forms durable spore bodies as the growth environment deteriorates, and becomes dormant. It has a life cycle in which it germinates again in a favorable environment and multiplies with nutrition.

After administration, the viable bacterial cells (spores, vegetative bodies) proliferate in the digestive tract and produce active ingredients inside and outside the bacterial cells. Also,
Active ingredients are also contained in cultured tear fluid or dead bacterial cells obtained by culture in Japan.

Therefore, what is used in the present invention is Clostridium s
p, live bacterial cells, spores and vegetative bodies of strain NIP1201), dead bacterial cells and the culture filtrate.

The prophylactic and therapeutic agent for toxigenic coliosis of the present invention is prepared by using Clostridium sp, strain NIP1201 cells or their culture filtrate as is or by diluting them with a solid or liquid diluent. For example, powders, granules, fine granules,
It may also be administered in the form of tablets, sugar-coated tablets, capsules, water-soluble or positively soluble film-coated agents, dry syrups, etc., or aqueous solutions, suspensions, syrups, and the like. Diluents include excipients, binders, and
In addition to disintegrants, coloring agents, flavoring agents, stabilizers, preservatives, emulsifiers, 1) H regulators, etc. may be added.

For animals, it can be used undiluted or made into powders, powders, granules, tablets, liquids, capsules, etc. with a diluent, or used in drinking water, feed, artificial milk, breast milk, etc. It may be added directly or once dispersed in a diluent. The diluent may be one that is physiologically harmless, but it is better if it is a feed or nutritious diluent. Further, any of these preparations for animals may also contain antidiarrheal agents, astringents, mucosal protectants, antibacterial agents, antibiotics, enzyme preparations, other viable bacterial preparations, and the like.

The preventive and therapeutic agent for toxigenic coliosis of the present invention is administered orally. In addition, as shown in Example 3, its toxicity is extremely low, so the dose may vary depending on the subject, body weight,
It can be determined as appropriate depending on age (age in days), purpose of administration, etc. Usually, the dose is 1 body weight as the number of viable bacteria.
It ranges from 106 to 1010 pieces per kg.

Below, Clostridium sp, Strain NIPL20
Examples show that the present invention has excellent effects as a prophylactic and therapeutic agent for toxigenic coliosis and has high safety.

The native species is a Durham-positive obligate anaerobic bacillus that forms spores. □ Bacterial morphology: trophozoite cells are 0.9-1.2X (GA
Y medium) 4, 0 to 7, Ou, m straight bacillus with obtuse circles at both ends. Sometimes there are two or more series. The spores are 1.0-1.2X2. O ~ 2.2 μm, oval, sub-endoscopic. In addition, some trophozoite cells have swollen ends.

Colony morphology: Irregular, periclinal, medium (GA)
H plate medium) The center is raised. so-called”) fed
Llsa head”ro is unique.

The color is pale yellowish brown.

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

Motility: Motility observed in G and AM medium.

Aerobic growth: No aerobic growth occurs on GAM plate medium.

gelatin Liquefaction: Positive.

case'in' Digestion: Positive.

Sulfate reduction: Negative.

Glycolytic properties: Decomposes nigercose, fructose, and maltose, and slightly decomposes cellobiose, sorbitol, sucrose, and glycerol, but does not decompose galactose, mannose, raffinose, rhamnose, ribose, starch, xylose, and lactose.

From the above results, Clostridium sp, Strain
NIP1201 is Clostridium sporogenes (C
lostridium sporogenes).

The inventor of the present invention entrusted the two scattered examples with the Clostridium sp used in the present invention,
N, IP1201 (Clost Salmon Shi), s
train NIP1201) was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Accession number: FER No. 9570 (FER)it)
-9570> Date received; September 7, 1986 Toxicity test No acute toxicity was observed in mice.

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

After culturing Yuculostridium Sp, strain NIPI201 in GAMAM medium at 7°C for 172 hours, the culture filtrate obtained by centrifugation was freeze-dried, and this was used as NIP120.
1 culture sap FD product (yield 56 ml/ml) was used in the following experiment.

Toxigenic E. coli is Escherichia coli (Escherichia c.), which is a heat-labile enterotoxin-producing bacterium.

FM3043 (hereinafter referred to as E, Collie LT)
It was used. Add NIP1201 culture fluid FD to CAYE medium at final concentrations of 1, 2, and 5.10 μ/ml, respectively.
After culturing E. coli LT at 37°C for 24 hours with shaking, the bacterial cell concentration and the amount of heat-labile enterotoxin produced were measured. In addition, NIP1201 culture fluid FD
Instead of GAMAM medium after drying, final concentration 10
The control group was the group in which the solution was added at a concentration of 1/ml.

The bacterial cell concentration was measured using the optical density at 620 nm (0
0620).

The amount of heat-labile enterotoxin produced was measured using a reverse passive latex agglutination method, and the sample was serially diluted 2 times, and the highest dilution factor indicating a positive result was expressed.

The results are shown in Table 1.

Table 1 Experimental results are shown in Table 1. When NIP1201 culture fluid FD was added at 1, 2, 5, and 10 mg/ml, it did not affect the growth of E. coli LT, but E.
, the production amount of heat-labile enterotoxin from Collie LT was 1/4.1/16.1/64.1/1 compared to the control.
28 and was suppressed in a dose-dependent manner. Therefore, N I P'
The 1201 culture filtrate FD product was found to be effective in suppressing the production of heat-labile enterotoxin, which is the main cause of diarrhea in toxigenic colibacillosis. In addition, in this test, NIPl, 20
Although the cultured tear fluid of No. 1, that is, the product from bacterial cells, was used, it goes without saying that the bacterial cells themselves that produce and contain this product are also effective.

Example 2 Toxigenic E. coli was produced by CAYE using E. coli LT.
After culturing E. coli LT in a medium for 6 hours with shaking, the supernatant was removed by centrifugation, and the bacterial cells were diluted with physiological saline.
After washing twice, 2×108 cells/cell were added to fresh G-AM medium.
A control group was prepared by suspending the suspension to a volume of ml. on the other hand,
Add filtrate of NIP1201x FD product to GAM medium (
56 mg/ml).

A suspension of Colli LT was used as a test group and used in the following rabbit intestinal loop experiment.

2 Japanese white male rabbits (weight 1.5kg to 2.0kg)
After fasting for one day (seed water was not restricted), general anesthesia (20 μ/kg) was performed using bendoparvicool. The abdomen was opened according to the conventional method (Testing method for Vibrio cholerae and Escherichia coli, written by Miwatani et al., Nane Publishing), and approximately 10 Cn per head was placed in the small intestine.
Create seven loops, each spaced approximately 1 mark apart.

Into this loop, 2 ml of each of the two groups of test solutions prepared as described above was administered alternately between each loop to cause infection. After laparotomy, the rabbits were left indoors at 24°C, and 20 hours later they were sacrificed under anesthesia.The rabbits were then laparotomyd and the loop removed.

Next, the weight X (tar) and length (mark) of the contents of each loop were measured, and the liquid storage activity (WZL ratio) was calculated according to the following formula.

Liquid storage activity (W/L ratio) Weight of loop (6) Length of loop (am) Significant differences between each control group and test group were tested by Student's t-test.

The results are shown in Table 2.

Table 2 As shown in Table 2, the WZL ratio in the control group treated with E. coli LT was 1.306±0.154, whereas in the test treated with NIP1201 culture fluid FD product The W/L ratio of the group was 0.210±0.053, and the inhibition rate was 83.9%. That is, intestinal water retention induced by infection of E. coli LT is N1. P
1201 culture filtrate FD product (bacterial product) can significantly suppress this product (cultured tear fluid).Furthermore, the bacterial cells that produce and contain this product are themselves useful for preventing and treating diarrhea caused by toxigenic Escherichia coli, especially LT-producing bacteria. This shows that it is an excellent drug.

Dog treatment Clostridium sp, strain NIP1201
using the Ga5Pak method using a GAM plate medium at 37°C.
After culturing for 2 hours, the resulting colonies were collected and adjusted to a predetermined concentration with tap water. The above test solution was administered orally or intraperitoneally to a group of 10 ICR mice (male and female, body weight 3'Og to 50g), with 1010 mice per day and 1011 mice per group.
kg were administered respectively. The general condition was observed for 7 days after administration of the test solution, and autopsy was performed 8 days later.

As a result, no notable changes were observed in the general condition or autopsy findings during oral administration in either the control group or the test solution administration group. However, in the intraperitoneal administration group, hair became unruly and a decrease in spontaneous movement was observed in all test solution administration groups at each concentration, but recovery occurred after 48 hours. From the above, it was concluded that the toxicity of the main rule is extremely low, and that the bacterial species are not pathogenic.

(Effects of the Invention) The present invention provides clostridium sp, Stray 7 NI
P1201 lostridium s, 5trai
NIP1201) is a preventive and therapeutic agent for toxigenic coliosis that contains live bacterial cells, dead bacterial cells, or their culture filtrate as an active ingredient, and is highly safe and extremely effective as a preventive and therapeutic agent for toxigenic coliosis. It has the effect of

Claims (1)

[Claims] Clostridium sp. Strain NIP1201 (C
lostridium sp. strain NIP1
201) A prophylactic and therapeutic agent for toxigenic coliosis containing live bacterial cells, dead bacterial cells, or a culture filtrate thereof as an active ingredient.