JP2997774B1 - Antidote for toxins derived from animal and plant pathogens - Google Patents

Abstract

The present invention relates to a microorganism belonging to the genus Detrasinobacter, Sphingobacterium, Bacillus, or Acinetobacter and having an action of detoxifying a toxin produced by an animal or plant pathogen, and / or a culture thereof. An antidote for toxins produced by animal pathogens or plant pathogens, characterized by being contained as an active ingredient. The present invention provides an antidote capable of effectively detoxifying toxins produced by animal or plant pathogens.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antidote for toxins produced by animal or plant pathogens containing a microorganism and / or a culture thereof as an active ingredient, and an effect of detoxifying toxins produced by animal or plant pathogens. And a microorganism having

[0002]

BACKGROUND OF THE INVENTION In medicine and plant pathology, how to prevent and treat bacterial infections is a major proposition. In general, bacterial infections, regardless of animal or plant,
Treatments that suppress the growth of pathogenic bacteria themselves are being performed by administration of antibiotics. It is a big problem. Also, in the agricultural field, drug-resistant bacteria such as streptomycin-resistant apple blight and polyoxin-resistant pear black spot have emerged, and the emergence of these resistant bacteria has become a major problem.

[0003] Bacterial infections are often caused mainly by toxins produced by bacteria. For example, intestinal bleeding caused by ciguatoxin produced by pathogenic Escherichia coli O-157 has recently been attracting attention. Many human lives have been lost due to this bacterial infection. Under such a background, development of an antidote capable of effectively detoxifying toxins produced by a pathogenic bacterium and making it difficult for drug-resistant bacteria to appear has been desired.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an antidote capable of effectively detoxifying a toxin produced by a pathogenic bacterium with less occurrence of drug-resistant bacteria.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, several types of bacteria isolated from field mushrooms were found to be phytopathogenic bacteria Pseudomonas sp.
They have found that they have an effect of effectively detoxifying the toxin produced by Tracy, and have completed the present invention. That is, the present invention includes the following inventions.

(1) A microorganism belonging to the genus Detrasinobacter, Sphingobacterium, Bacillus, or Acinetobacter and having an action of detoxifying toxins produced by animal or plant pathogens, and / or a culture thereof is effective. An antidote for a toxin produced by an animal pathogen or a plant pathogen, which is contained as a component.

(2) The microorganisms are Detrasinobacter tsukamotoae, Detrasinobacter siratae, Detrasinobacter muratae, Sphingobacterium
The antidote according to the above (1), which is a microorganism belonging to Multiborum or Bacillus pamilus and having an action of detoxifying a toxin produced by an animal or plant pathogen.

[0008] (3) The microorganism is Detrasinobacter tsukamotoae OM-F11, Detrasinobacter siratae OM-D5, Detrasinobacter muratae CM-14, Sphingobacterium multiborum OM-A8. The antidote according to the above (1), which is a strain, Sphingobacterium sp. OM-E81 strain, Bacillus pamilus OM-F6 strain, or Acinetobacter sp. OM-H10 strain.

(4) The antidote according to any one of (1) to (3), wherein the pathogenic bacterium is Pseudomonas tracy. (5) Detrasinobacter tsukamotoae having an action of detoxifying toxins produced by animal or plant pathogens. (6) Detracinbacter silatae having an action of detoxifying toxins produced by animal or plant pathogens. (7) Detracinbacter muratae which has an action of detoxifying toxins produced by animal or plant pathogens.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The antidote of the toxin produced by the animal or plant pathogen of the present invention is a genus Detrasinbacter (Detolaasinbacter),
Sphingobacterium, Bacillus, or Acinetobacter (Ac
inetobacter), a microorganism having an action of detoxifying toxins produced by animal or plant pathogens, and / or
Alternatively, a culture thereof is contained as an active ingredient.

The microorganism used in the present invention is a microorganism belonging to the genus Detrasinobacter, Sphingobacterium, Bacillus, or Acinetobacter, and has a function of detoxifying toxins produced by animal or plant pathogens. Not limited. Here, the "detoxifying effect" of the microorganism used in the present invention includes any effect as long as it is an effect of converting a toxin into a substance having low toxicity or a substance having no toxicity. Examples of such an action include an action of decomposing a toxin and converting the substance into a low-toxic or non-toxic substance, and an action of chemically modifying a toxin to change the substance into a low-toxic or non-toxic substance. No.

Preferred examples of the microorganisms used in the present invention include the following microorganisms. That is, as microorganisms belonging to the genus Detrasinbacter and having an action of detoxifying toxins produced by animal pathogens or plant pathogens, Detrasinobacter tsukamotoae (Detolaas)
Inbacter tsukamotoae), Detrasinbacter shiratae (Detolaasinbacter shiratae), or Detrasinobacter muratae (Detolaasinbacter muratae), microorganisms having an action of detoxifying toxins produced by animal pathogens or plant pathogens are preferable, The Tsukamotoae OM-F11 strain, the Detracinobacter siratae OM-D5 strain, or the Detrasinobacter muratae CM-14 strain is more preferred.

[0013] It also belongs to the genus Sphingobacterium,
Microorganisms that have the effect of detoxifying toxins produced by animal or plant pathogens include Sphingobacterium and
Microorganisms belonging to multiborum (Sphingobacterium multivorum) and having an action of detoxifying toxins produced by animal pathogens or plant pathogens are preferred, and Sphingobacterium multiborum OM-A8 strain or Sphingobacterium sp. OM-E81. Strains are more preferred.

[0014] Bacillus pumilus (Bacillus pumilus) is a microorganism belonging to the genus Bacillus and having an action of detoxifying toxins produced by animal or plant pathogens.
And a microorganism having an action of detoxifying toxins produced by animal pathogens or plant pathogens is preferable, and Bacillus pamilus OM-F6 strain is more preferable. As a microorganism belonging to Acinetobacter and having an action of detoxifying a toxin produced by an animal pathogen or a plant pathogen, Acinetobacter sp. OM-H10 strain is preferable.

[0015] In addition, Detra-Symbacter Tsukamoto Ae
OM-F11 strain as FERM P-16772, Detrasinobacter siratae OM-D5 strain as FERM P-16769, Detrasinobacter muratae CM-14 strain as FERM P-16767, Sphingobacterium multiborum OM-A8 strain is FERM
As P-16768, Sphingobacterium sp. OM-E81
The strain is FERM P-16770, the Bacillus pamilus OM-F6 strain is FERM P-16771, and Acinetobacter sp. OM
-H10 strain has been deposited as FERM P-16773 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Deposit date: 1998
April 20).

The culture of the microorganism used in the present invention is
In addition to those directly obtained as a result of culturing the microorganism, those obtained by processing the same are included. It is not necessary to use a special method for culturing the microorganism, and a method similar to a known aerobic bacterium can be used. For example, as a medium,
Any of a synthetic medium and a natural medium may be used as long as the medium appropriately contains an assimilable carbon source, a nitrogen source, an inorganic substance, and a necessary growth promoting substance. In particular, a potato semi-synthetic medium (potato tuber) 300g of decoctions _{1L, Ca (NO 3) 2} · 4H 2 O:
_{0.5g, Na 2 HPO 4 · 12H} 2 O: 2g, peptone: 5 g, sucrose: 15 g, agar: 18 g) are preferable. During culturing, it is desirable to maintain the temperature at 15 to 32 ° C, preferably 20 to 26 ° C. When the culture is performed for about 1 to 2 days under the above conditions, a sufficient amount of colonies are formed on the surface of the medium.

The method for treating the microorganism directly obtained as a result of culturing the microorganism can be performed by any known method as long as the microorganism does not lose its ability to detoxify toxins produced by animal or plant pathogens. Good. Examples of such a processing method include extraction and filtration. The antidote of the present invention can be used to detoxify toxins produced by animal or plant pathogens.
The antidote of the present invention can also be used to treat diseases caused by toxins produced by animal or plant pathogens.

The toxin to be detoxified by the antidote of the present invention is not particularly limited. Antidote of the present invention, for example,
Animal pathogens such as Bordetella spp.
It can detoxify toxins produced by phytopathogenic fungi such as spot disease fungus and pear black spot fungus), but especially effective against toxins produced by phytopathogenic bacterium Pseudomonas traci. obtain.

As the antidote of the present invention, the above-mentioned microorganisms and / or cultures thereof may be used directly as they are, but generally, they are mixed with a pharmaceutically acceptable solid carrier or liquid carrier,
It is prepared and used in the form of liquids, wettable powders, powders, granules, emulsions, oils, capsules and the like. The concentration of the microorganism and / or a culture thereof, which is an active ingredient of the antidote of the present invention,
Although it can be appropriately determined depending on the amount and type of the toxin to be detoxified, if it is a liquid, it is usually determined as the concentration of the microorganism.
It is appropriate to use 10 ⁶ to 10 ⁸ cells / ml.

[0020]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example 1] Separation of bacteria Using mushrooms (oyster mushrooms, etc.) collected from the field, a mushroom tissue grinding diluent was prepared according to a conventional method. On the other hand, potato semi-synthetic medium (potato tuber 300 g decoction 1L, Ca
0.5 g of (NO ₃ ) ₂ .4H ₂ O, 2 g of Na ₂ HPO ₄ .12H ₂ O, 5 g of peptone, 15 g of sucrose, 18 g of agar) were poured into a Petri dish having a diameter of 9 cm to prepare a plate medium.

[0021] The above mushroom tissue grinding and diluting solution was spread on the plate medium and cultured at 25 ° C for 3 days to form colonies. Various bacteria were isolated as single colonies from colonies formed on the plate medium according to a conventional method. As a result, 2200 strains of bacteria were isolated.

From the obtained 2,200 isolated bacteria, bacterial strains that decompose toxins produced by the phytopathogenic bacterium Pseudomonas traci were screened by potato section inoculation. As a result, seven strains of bacteria capable of efficiently decomposing toxins produced by Pseudomonas traci were obtained. These seven strains were identified as OM-F11, OM-D5, CM-14, OM-A8, OM-
The strains were named E81 strain, OM-F6 strain and OM-H10 strain, and the genus and species of each strain were identified.

Example 2 Identification of Bacteria (1) The 16S rRNA gene of each strain isolated in Example 1 was analyzed by the method of Ausubel et al. (Ausubel et al., Short Protocols
In Molecular Biology 3rd edition p2-11 (1995)). For the 16S rRNA gene of each strain, ABI Pr
ism BigDye Terminator Cycle Sequencing Ready React
After performing sequencing PCR using the ion Kit (manufactured by Perkin Elmer Japan) and the primer set shown in Table 1 below, ABI Prism377 DNASequencer (Perkin Elmer
(Japan)).

[0024]

[Table 1]

(2) The nucleotide sequence of the determined 16S rRNA gene of each strain was analyzed using the phylogenetic analysis program ClustalW ver. 1.7 (ftp:
//ftp.ebi.ac.uk/pub/software/mac/clustalw/clustal
w.17.sea.hpx) and processed by the method of Thompson, JD et al. (Thompson, JD et al., Nucleic Acids Research, 22 (2
2): A phylogenetic tree for each strain was prepared according to 4673-4) (FIGS. 1 to 4). This clarified the taxonomic status of each strain.

That is, of the above seven strains, two strains (OM
-A8 strain and OM-E81 strain) were found to belong to the genus Sphingobacterium (FIG. 2), and one strain (OM-F6 strain) was found to belong to the genus Bacillus (FIG. 3). OM-H10
Strain) was found to belong to the genus Acinetobacter (FIG. 4). On the other hand, the remaining three strains (OM-F11, OM-D5 and CM-
14 strains) were found to belong to the same genus, but were not classified as an existing genus, and were found to constitute a new genus (FIG. 1).

The phylogenetic tree shows the evolution process from left to right. The "branch" in the middle of the phylogenetic tree indicates that it has differentiated into a new microorganism by evolution, and the number (Bootstrap) at the base of each branch indicates the probability of the differentiation occurring as a percentage. Things. The downstream of a branch that has a high probability is called a “cluster”,
Clusters are formed by "genus" units.

(3) As a result of the phylogenetic analysis, three strains (OM-F11 strain, OM-D5 strain and CM-14 strain) found to belong to the new genus
The enzyme activity and assimilation of the strain were examined by a conventional method. The results are shown in Table 2 below.

[0029]

[Table 2]

[0030]

[0031]

The following Table 3 shows the above three strains (OM-F11 strain,
2 shows motility, catalase activity, menaquinone and GC contents of OM-D5 strain and CM-14 strain) and related bacteria. The mycological properties of bacteria that are closely related to the above three strains are described in JGHolt et al., Bergey's Manual of Determina.
tive Bacteriology, 9th edition, 787pp (1994); PHASnea
th et al., Bergey's Manual of Systematic Bacteriolo
gy, vol. 2, 1261-1434 (1986).

[0033]

[Table 3]

Based on Table 3, the above three strains (OM-F11 strain,
The motility, catalase activity, menaquinone (cell wall composition) and GC content of OM-D5 strain and CM-14 strain) were compared with those of closely related bacteria, and the results of phylogenetic analysis showed that the three strains constitute a new genus. Strongly confirmed that the three strains constitute a new genus. Therefore, the new genus was named "Detolaasinbacter". Whether or not the above three strains that were found to belong to the new genus "Detrasinobacter" were independent species was examined by a DNA-DNA hybridization method using all chromosomes of the three strains. The results are shown in Table 4 below.

[0035]

[Table 4]

From the results shown in Table 4, it was found that the three strains were independent new species. Therefore, the OM-F11 strain was transferred to Detolasinbacter tsukamotoae (Detolaasinbacter tsu
kamotoae) and OM-D5 strain were named Detolasinbacter shiratae, and CM-14 strain was named Detrasinbacter muratae.

It should be noted that Detrasinbacter Tsukamoto Ae
OM-F11 strain, Detracinobacter siratae OM-D5 strain and Detrasinbacter muratae CM-14 strain are each FE
RM P-16772, FERM P-16769 and FERM P-16767 have been deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Deposit date: April 20, 1998).

(4) One strain (OM-A8) of the two strains (OM-A8 and OM-E81) found to belong to the genus Sphingobacterium as a result of the phylogenetic analysis,
Bacteriological properties were examined using an API20NE Gram-negative bacteria identification kit (manufactured by Biomerieux Biotech Japan) to identify species.

The results are shown in Table 5 below. Table 5 shows the mycological properties of Sphingobacterium multiborum and Sphingobacterium spiritivorum, which are known species of the genus Sphingobacterium (Yabuu
chi et al. , International
Journalof Systematic Bact
eriology, 33 580-598 (198
3); Holmes et al., International Journal of Syst
ematic Bacteriology, 31 21-34 (1981); Holmes et a
l., International Journal of Systematic Bacteriolo
gy, 38 348-353 (1988); Holmes et al., The Prokaryot
es, 2nd edition, 3620-3630 (1992)).

[0040]

[Table 5]

From the results shown in Table 5, it was found that the OM-A8 strain was identified by Sphingobacterium multiborum with a probability of 99.9% (Excellent Identification to Sp.
hingobacterium multivorum,% ID = 99.9%, T = 1.00). The Sphingobacterium multiborum OM-A8 strain has been deposited as FERM P-16768 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (deposit date: April 20, 1998).

As a result of the phylogenetic analysis, the species of the other strain (OM-E81 strain) of the two strains found to belong to the genus Sphingobacterium has not been identified. The sphingobacterium sp.OM-E81 strain is
It has been deposited as FERM P-16770 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Deposit date: April 20, 1998).

(5) As a result of the phylogenetic analysis, one strain (OM-F6), which was found to belong to the genus Bacillus,
50CHB Bacillus bacterium identification kit (Japan BioMérieux
Mycological properties were examined using Biotech) to identify species. The results are shown in Table 6 below. Table 6 shows the mycological properties of Bacillus pamilus and Bacillus subtilis, known species of the genus Bacillus (Holmes et al.,
The Prokaryotes, 2nd edition, 1664-1696 (1992); Nielsen
et al., FEMS Microbiology Letters, 117 61-66 (199
4); Pretorius et al., Journal of Applied Bacteriol
ogy, 60 351-360 (1986)).

[0044]

[Table 6]

From the results shown in Table 6, it was found that the strain was identified by Bacillus pamilus with a probability of 99.9% (Very Good Identification to Bacillus pumillus,% I
d = 99,9%, T = 0.54). The Bacillus pamilus OM-F6 strain has been deposited as FERM P-16771 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (deposit date: April 20, 1998).

(6) As a result of the phylogenetic analysis, no species has been identified for one strain (OM-H10 strain) that was found to belong to the genus Acinetobacter. The Acinetobacter sp. OM-H10 strain has been deposited as FERM P-16773 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (deposit date: April 20, 1998).

Example 3 Detoxification Effect of Toxin Produced by Phytopathogenic Bacterium Pseudomonas trachii Strain, Detrasinbacter siratae OM-D5 strain, Detrasinbacter muratae CM-
14 strains, Sphingobacterium multiborum OM-A8
Strain, Sphingobacterium sp. OM-E81 strain, Bacillus pamilus OM-F6 strain, Acinetobacter sp. OM-H10 strain)
Was examined for its detoxifying action as follows.

The decoctions 1L potato semi synthetic liquid medium (potato tubers _{300g, Ca (NO 3) 2} · 4H 2 O0.5g, Na 2 HPO 4 · 12H 2 O
30 ml of 2 g, 5 g of peptone, and 15 g of sucrose) were placed in a 300 ml Erlenmeyer flask, each strain was inoculated therein, and cultured with shaking (25 ° C., 130 rpm) for 2 days. Each culture solution and a previously prepared toxin solution of Pseudomonas traci (toxin concentration 40μ
g / ml), and the degree of toxin degradation was measured over time by HPLC. As a result, each of the strains completely decomposed the toxin in the liquid after 6 to 9 hours (Table 7).

[0049]

[Table 7]

The numbers in Table 7 indicate the amount of toxin remaining in the toxin mixed culture (unit: μg / ml). The toxin produced by Pseudomonas traci used for the toxin detoxification assay is a peptide consisting of about 20 amino acids. Peptides having such toxin activity are secreted extracellularly by many bacteria and often affect host tissues. It is considered that the bacterium capable of detoxifying the toxin has an action of efficiently decomposing peptides derived from animal or plant pathogenic bacteria having such toxicity.

[0051]

According to the present invention, there is provided an antidote which can effectively detoxify toxins produced by animal pathogens or plant pathogens. The antidote of the present invention can effectively prevent or treat infectious diseases caused by toxins produced by animal or plant pathogens.

[Brief description of the drawings]

FIG. 1 is a diagram showing a phylogenetic tree of microorganisms.

FIG. 2 is a diagram showing a phylogenetic tree of microorganisms.

FIG. 3 is a diagram showing a phylogenetic tree of microorganisms.

FIG. 4 is a diagram showing a phylogenetic tree of microorganisms.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:01)

Claims (6)

(57) [Claims] 1. A microorganism belonging to the genus Detrasinobacter, Sphingobacterium, Bacillus, or Acinetobacter and having an action of detoxifying a toxin produced by Pseudomonas tracy or a culture thereof as an active ingredient. Characterized as an antidote for toxins produced by Pseudomonas tracy. 2. The microorganism according to claim 1, wherein the microorganism belongs to Detracinbacterium tsukamotoae, Detracinebacter siratae, Detracinebacterium Muratae, Sphingobacterium multiborum, or Bacillus pamilus, and produces a toxin produced by Pseudomonas trachye. The antidote according to claim 1, which is a microorganism having a detoxifying action. 3. The microorganism according to claim 1, wherein the microorganism is Detrasinobacter tsukamotoae OM-F11 strain (FERM P-16772), Detrasinobacter sylatae OM-D5 strain (FERM P-16769), Detrasinobacter muratae CM-14 strain. (FERM P-16767), Sphingobacterium multiborum OM-A8 strain (FERM P-16)
768), sphingobacterium sp. OM-E81 strain (FERM P-
16770), Bacillus pamilus OM-F6 strain (FERM P-1677)
1) or Acinetobacter sp. OM-H10 strain (FERM P-167
73) The antidote according to claim 1, characterized in that: 4. A detracinbacterium tsukamotoae having an action of detoxifying a toxin produced by Pseudomonas tracy. 5. A detracinbacter silatae having an action of detoxifying a toxin produced by Pseudomonas tracy. 6. A detracinbacter muratae having an action of detoxifying a toxin produced by Pseudomonas tracy.