JP5823733B2 - Antibiotic-producing microorganism and antibiotic produced by the same - Google Patents

Description

  The present invention relates to a microorganism producing an antibiotic, a method for producing a novel antibiotic by culturing the microorganism, and the novel antibiotic.

  In recent years, it has resistance to many antibiotics such as methicillin-resistant Staphylococcus aureus (hereinafter sometimes abbreviated as “MRSA”) and vancomycin-resistant enterococci (hereinafter abbreviated as “VRE”). There are many cases where multi-drug resistant bacteria are isolated from clinical sites, which is a serious problem. In particular, the emergence of VRE that is resistant to vancomycin effective against MRSA is taken more seriously. This is because enterococci themselves are resident in the intestinal tract, so it is easy to generate potential carriers, and resistance genes are transmitted to other bacteria by being in the patient's body for a long time. This is because the risk of generating vancomycin-resistant Staphylococcus aureus (VRSA) and the like is expected to increase.

  In addition to the problem of the emergence of resistant bacteria, there is a large demand for antibiotics that are superior in safety compared to vancomycin, etc., and have a substantially high therapeutic effect. Therefore, they are effective for MRSA and VRE and are effective for existing drugs. Studies are underway to obtain new antibiotics with fewer side effects and higher safety.

  For the purpose of the previous, along with the search by the synthesis such as the creation of antibiotics with a new chemical structure and the improvement of existing antibiotics, new with the ability to produce new and useful antibiotics that have not been reported so far The search for microorganisms is also widely conducted. This is because antibiotics produced by microorganisms are natural products produced in vivo, and therefore, it is considered that many antibiotics are safer to the living body than chemically synthesized products.

  For example, Patent Literature 1 and Patent Literature 2 include a novel microorganism belonging to the genus Lysobacter, Patent Literature 3 to the genus Flavobacterium, and Patent Literature 4 to the genus Streptomyces and its microorganisms. New antibiotics that are produced and manufactured are described.

  On the other hand, the present inventor has constructed a silkworm infection model (Patent Document 5) using a silkworm as an experimental animal, which is useful for searching for antibiotics, and has been proceeding with its investigation.

Japanese Patent No. 3339235 Japanese Patent No. 4054576 JP 2003-113192 A JP 2007-131552 A JP 2007-327964 A

  The antibiotics described in the previous patent documents are targeted against multi-drug resistant bacteria, and are mainly considered effective against MRSA. Among them, the antibiotics described in Patent Document 1 and Patent Document 3 are characterized by low toxicity and faster action time than vancomycin, and the antibiotics described in Patent Document 4 are different in chemistry from existing antibacterial drugs. Since it has a structure, it can be expected that the appearance of resistant bacteria is small. In addition, the antibiotics described in Patent Document 2 include antibiotics that exhibit antibacterial properties not only in MRSA but also in VRE. However, this antibiotic has been evaluated mainly for its antibacterial properties in vitro. Hereinafter, unless otherwise specified, “antibacterial activity” indicates that evaluated by an in vitro evaluation system, and “therapeutic effect” indicates that evaluated by an in vivo evaluation system using experimental animals.

  Linezolid, an oxazolidinone antibiotic, is well known as an antibiotic that has shown therapeutic effects against MRSA and VRE and has been created by complete chemical synthesis. is there.

  On the other hand, it is expected that antibiotics obtained from substances produced by microorganisms are likely to be superior to synthetic products in terms of safety to living organisms, and such antibiotics can reduce the development steps to become pharmaceuticals. It is done. For example, in the production method, raw materials that are gentler to the living body than in the case of chemical synthesis can be used, and the process and equipment can be performed by simpler and safer processes and equipment because the production conditions are close to normal temperature and pressure. It has the merit that. Therefore, there is a high possibility that the drug development steps can be reduced compared to antibiotics created by chemical synthesis.

  However, for microorganisms that produce antibiotics that not only show effectiveness against MRSA but also show antibacterial properties against VRE, although there are examples like Patent Document 2, the therapeutic effects in actual treatment and Considering the emergence of resistant bacteria, etc., it was necessary to further investigate. In other words, not only MRSA but also VRE has antibacterial activity, and a new microorganism having the characteristic of actually producing an antibiotic having a therapeutic effect is found and effective for both MRSA and VRE using the microorganism. It has been desired to provide a new antibiotic that exhibits sex and a method for producing the same.

  In addition, in order to obtain a wide range of new antibiotics regardless of the presence or absence of antibacterial activity against MRSA, VRE, etc., provision of new microorganisms has been desired.

  The present invention has been made in view of the above, and an object thereof is to provide a novel microorganism capable of producing an antibiotic, and to provide a novel microorganism used in a method for producing an antibiotic. There is to do. In particular, it is an object of the present invention to provide a novel microorganism capable of producing a novel antibiotic effective against a multi-drug resistant bacterium that is effective for at least both MRSA and VRE.

  In view of the above circumstances, the present inventor conducted a search for microorganisms with the object of providing a novel microorganism capable of producing antibiotics. In particular, the search for microorganisms was started with the further object of providing new microorganisms that are antibiotics that are also effective against multidrug-resistant bacteria. As a search method thereof, a silkworm S. aureus infection model using a silkworm larva as an experimental animal (see Patent Document 5) is used to produce various microorganisms whose antibacterial activity has been confirmed. The search for microorganisms that can solve the above-mentioned problems was made by conducting screening using the therapeutic effect as an index.

  As a result, among the 14346 strains of soil bacteria, there were 3487 strains that showed antibacterial activity in the culture solution, and the "therapeutic effect in silkworm Staphylococcus aureus infection model" indicated by the culture solution of these bacteria was A therapeutic effect was observed for 45 of them. Thereafter, it was confirmed that 45 microorganisms having antibacterial activity and having a therapeutic effect in the above infection model contained microorganisms belonging to the genus Rhizobacter.

  Then, when the antibacterial spectrum of the antibiotic purified from the culture medium of the microorganism belonging to the genus Rhizobacter was examined in detail, this antibiotic showed antibacterial activity on both MRSA and VRE, and the previous silkworm yellow grape It was confirmed that it has a high therapeutic effect not only against the cocciacterial infection model but also against the mouse Staphylococcus aureus infection model compared to the existing drug vancomycin. Furthermore, since it was found that this antibiotic is structurally novel, the “microorganism belonging to the genus Lysobacter that produces antibiotics” of the present invention is a highly useful novel that can solve the problem. It was confirmed that it was a microorganism, and the present invention was completed.

That is, the present invention exists as follows.
(1) A microorganism having the accession number NITE P-870 belonging to the genus Lysobacter or a microorganism mutated naturally or artificially thereof, and having the ability to produce an antibiotic having antibacterial activity.
(2) The microorganism according to (1), wherein the antibiotic has antibacterial activity against at least methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
(3) The microorganism according to (1) or (2), which has the base sequence of the 16S rRNA region represented by SEQ ID NO: 1 in the sequence listing.

［式（１）中、Ｒ^１は水酸基を１つ有する炭素数が７、８又は９のアシル基を示し、Ｒ^２はメチル基又は水素原子を示し、Ｒ^３はエチル基又はメチル基を示す。］ (4) A method for producing an antibiotic, comprising obtaining the antibiotic produced by the microorganism from the culture solution of the microorganism according to any one of (1) to (3).
(5) Antibacterial activity against at least methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) produced by the microorganism from the culture solution of the microorganism according to any one of (1) to (3) An antibiotic production method comprising obtaining an antibiotic having
(6) An antibiotic produced by the microorganism, which is obtained from the microorganism culture solution according to any one of (1) to (3), and is at least methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci ( Antibiotics characterized by having antibacterial activity against VRE).
(7) The antibiotic of (6), which is a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[In formula (1), R ¹ represents an acyl group having one hydroxyl group, 7, 8 or 9 carbon atoms, R ² represents a methyl group or a hydrogen atom, and R ³ represents an ethyl group or a methyl group. . ]

  According to the present invention, a novel microorganism capable of producing an antibiotic having antibacterial activity can be provided. In particular, some antibiotics produced by the microorganisms of the present invention have antibacterial activity in MRSA and VRE and have been confirmed to have a high therapeutic effect. Therefore, they are effective against multidrug-resistant bacteria. This has the effect of providing a novel microorganism capable of producing a highly novel antibiotic.

  In addition, the method for producing antibiotics of the present invention can produce particularly novel antibiotics, and can further produce antibiotics that have not only antibacterial activity but also high therapeutic effects. In addition, since the method for producing an antibiotic of the present invention is a method of culturing microorganisms that is simpler than chemical synthesis methods, organic solvents, metal catalysts, reagents, many chemical synthesis steps, high temperature, high pressure Etc. are not required, so that the cost is low and it is environmentally friendly.

  In addition, the antibiotic of the present invention is a novel antibiotic and has an antibacterial activity at least against MRSA and VRE and has been confirmed to have a high therapeutic effect, and thus can be effective in the treatment of diseases caused by multidrug-resistant bacteria. .

It is a figure which shows the fractionation result by the ODS column of the antibiotic containing fraction which shows the therapeutic effect obtained from the culture of RH2180-5. The vertical axis represents absorption intensity, and the horizontal axis represents elution time (minutes). Further, the numbers in the column of each peak displayed in a separate frame indicate the molecular weight of each peak substance. It is a figure which shows the amino acid composition analysis result of RH2180-5Peak5 substance. It is a figure which shows the 1H and 13C-NMR analysis result of RH2180-5Peak5 substance. The vertical axis represents the signal intensity, and the horizontal axis represents the chemical shift value (ppm). It is a figure which shows the MS-MS analysis result of TOF (Time of Flight: Time-of-flight type) MS of RH2180-5Peak5 substance. It is a figure which shows the chemical structure of RH2180-5Peak5 substance derived from each analysis result. It is a figure which shows the chemical structure which also clarified the three-dimensional structure of the amino acid of RH2180-5Peak5 substance derived from each analysis result. It is a figure which shows the bactericidal activity of RH2180-5Peak5 substance.

  The microorganism of the present invention is a microorganism having the accession number NITE P-870 belonging to the genus Lysobacter or a microorganism that is naturally or artificially mutated and has the ability to produce antibiotics having antibacterial activity. is there. The novel microorganism belonging to the genus Rhizobacter was identified from the properties of the strain described later and the sequence of the 16S rRNA region.

  The microorganism of the present invention is a microorganism having an ability to produce an antibiotic having antibacterial activity, and specifically, the accession number “NITE P− isolated by the present inventors from soil collected in Okinawa” 870 ", Kisarazu City Kazusa Kamashiga 2-5-8, National Institute of Technology and Evaluation (hereinafter referred to as" NITE ") Patent Microorganism Depositary Center (NPMD) (Deposit date: January 25, 2010) and named as “RH2180-5” (hereinafter referred to as “RH2180-5”).

  “RH2180-5” then submitted the original deposit application form to the patent microorganisms deposit center (NPMD) of Kazusa Kama feet 2-5-8, Kisarazu City, Chiba Prefecture, and National Institute of Technology and Evaluation (NITE). Apply for transfer from domestic deposit (original deposit date: January 25, 2010) to deposit under the Budapest Treaty (transfer date (international deposit date: May 20, 2011)) As a result of receiving an application for transfer to a deposit under the Budapest Treaty (international deposit), the deposit number “NITE BP-870” has been received.

  The properties of RH2180-5 (Accession No. NITE P-870) as a strain are as follows.

  Form: This RH2180-5 is a Gram-negative bacilli and has no flagella but gliding properties. Formation of fruiting bodies is not permitted. In addition, it does not show acidity.

Growth status in medium:
(1) A pale yellow colony is formed on a broth agar plate. Diffusible dyes are not observed.
(2) In the broth gelatin puncture culture, it grows while liquefying gelatin over the inside.

Physiological properties: Physiological and chemical taxonomic properties of this RH2180-5 are as follows: (1) Growth pH (optimum growth pH): 5-9 (6-8)
(2) Growth temperature (optimum growth temperature): 10 to 40 ° C. (25 to 30 ° C.)
(3) Attitude toward oxygen: aerobic (4) MR red test (-)
(5) VP test (Voges-Proscauer test): +
(6) Pigment production (Pigment): +
(7) Oxidase test: +
(8) Catalase test: +
(9) Urease test:-
(10) Phosphatase test: +
(11) Casein hydrolysis: +
(12) Cellulose hydrolysis:-
(13) Gelatin hydrolysis: +
(14) Starch hydrolysis:-
(15) Deoxyribonuclease test: +
(16) Nitrate reduction:-
(17) Denitrification:-
(18) Hydrogen sulfide production (H ₂ S production): −
(19) Indole production:-
(20) Citrate utilization: +
(21) OF-test: oxidation
(22) Ability to produce acid and gas from the following saccharides etc. L-arabinose:-
D-xylose:-
D-glucose: +
D-mannose: +
D-fructose: +
D-galactose:-
D-maltose: +
D-sucrose: +
D-lactose: +
D-trehalose: +
D-sorbitol:-
Glycerol:-
Starch:-

Molecular biological analysis result: The analysis result of RH2180-5 of 16S rRNA used as an index of molecular biological systematic classification is as follows.
(12) Analysis result of 16S rRNA sequence From colony of RH2180-5, the base sequence of 16S rRNA region was amplified by colony PCR and analyzed by a sequencer. As a result, some of 5 'end side and 3' end side were analyzed. A base sequence corresponding to almost the entire length of 16S rRNA shown in SEQ ID NO: 1 in the sequence listing was found. Since the base sequence of SEQ ID NO: 1 in this sequence listing is not the full length of 16S rRNA, it was designated as a 16S rRNA “region”. When the homology search of this base sequence was performed by BLAST of NCBI, the base sequence of the 16S rRNA region of RH2180-5 of the present invention showed a homology rate of 99% with the Lysobacter enzyme gene DSN2043T strain belonging to the genus Lysobacter. Therefore, RH2180-5 of the present invention is a microorganism belonging to the genus Lysobacter.

  The above-mentioned Lysobacter enzyme is not different from RH2180-5 of the present invention since no antibiotic production has been reported.

  The above properties of the strain were compared with the classification according to Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology, vol. 3 1989) and the contents of other documents, and the results of 16S rRNA analysis. As a result of comprehensive determination in view of the above, this strain was determined to be a microorganism belonging to the genus Lysobacter.

  Effective for multidrug-resistant bacteria including VRE, as indicated by the absence of microorganisms having a 16S rRNA region base sequence that matches the base sequence of the 16S rRNA region of RH2180-5 of the present invention, and antibiotics produced by this strain This strain was judged to be a novel microbial strain from the tendency of the novel antibacterial spectrum and the property of producing a novel antibiotic with high utility that has not been reported so far.

  Based on the above results, the present strain found this time was judged to be a new and useful strain of the genus Lysobacter that has not been reported so far, and was named the genus Lysobacter, “RH2180-5”. RH2180-5 has been deposited domestically with the Patent Microorganism Deposit Center (NPMD) of the National Institute for Product Evaluation and Technology (NITE) under the accession number: NITE P-870 (date of deposit: January 25, 2010). Yes. In addition, as a general property of bacteria, the property as a strain is easily mutated, and there is a possibility that it does not stay within the range shown above. However, even a microorganism mutated from RH2180-5 (Accession No. NITE P-870) is included in the microorganism of the present invention as long as it has the ability to produce antibiotics.

  That is, the present invention is a microorganism having the accession number NITE P-870 belonging to the genus Lysobacter or a microorganism that is naturally or artificially mutated and has the ability to produce an antibiotic having antibacterial activity. is there. Moreover, it is said microorganisms which have a base sequence of 16S rRNA area | region shown by sequence number 1 of a sequence table.

  More preferably, the present invention is the above microorganism, wherein the produced antibiotic has antibacterial activity against at least methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). That is, a microorganism of NITE P-870 having an ability to produce an antibiotic having antibacterial activity at least for MRSA and VRE, and a microorganism mutated from NITE P-870 are further included in the microorganism of the present invention. It is. Needless to say, the mutation includes natural mutation and artificial mutation.

(Method for culturing microorganisms of the present invention)
The culture method of RH2180-5 of the present invention may be performed according to a general culture method performed for a microorganism belonging to the genus Rhizobacter. Specifically, RH2180-5 is inoculated into a nutrient source-containing medium such as a YME medium, an SGM medium, or a CDY medium, and cultured under aerobic conditions. Examples of the carbon source in the medium include organic carbon compounds such as D-glucose, D-fructose, sucrose, starch, dextrin, glycerin, molasses, starch syrup, and fats and oils, and nitrogen sources include meat extract, Organic / inorganic nitrogen compounds such as casein, peptone, yeast extract, dry yeast, germ, soy flour, urea, amino acid, ammonium salt can be used. Further, as the salts, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, phosphate, iron salt, copper salt, zinc salt, cobalt salt and the like are appropriately added as necessary. Furthermore, it is preferable to add a growth promoting substance such as biotin, vitamin B1, cystine, methyl oleate, lard oil or the like in terms of increasing the production amount of the target product. Moreover, you may add antifoamers, such as a silicone oil and surfactant.

  As described above, the culture is preferably performed under an aerobic condition as described above, and aeration and agitation culture is desirable for the liquid culture method. If it is a small scale, a shaking culture method using a flask may be used. The culture temperature can be 20 ° C. to 40 ° C., but is preferably kept between 25 ° C. and 35 ° C., more preferably 27 ° C. to 32 ° C. The culture pH is preferably 6-8, particularly preferably around 7. The culture period is a factor that varies depending on the composition of the medium used, the culture temperature, and the like. In the case of RH2180-5, a sufficient amount is usually obtained in a short period of about 1 to 20 days, preferably about 4 to 7 days. Things can be secured.

(Purification method of antibacterial active ingredient)
The recovery of the antibacterial active ingredient from the culture of RH2180-5 may be performed by a method of recovering and purifying the physiologically active substance from a normal microorganism culture. Here, the culture includes culture supernatant, cultured cells, and disrupted cultured cells. For example, after adding a suitable organic solvent such as acetone as an extraction treatment to the culture and suspending it, the extraction supernatant usually separated from the cells by centrifugation or filtration membrane separation is usually used for isolation. What is necessary is just to add a purification process. Further, if necessary, the remaining bacterial cell residue may be subjected to a grinding treatment and then extracted again.

  Solvents and separation methods usually used for isolation and purification are selected using the antibacterial activity determined by the minimum growth inhibitory concentration (hereinafter abbreviated as “MIC”) as an index. There is basically no problem with such a method as long as it is simply separation and concentration of antibacterial activity. However, in the isolation and purification of antibiotics produced by RH2180-5 of the present invention, not only the results of MIC but also the selection based on the therapeutic effect on the silkworm Staphylococcus aureus infection model shown in Patent Document 5 is performed. Yes. As a result, concentration and purification of a novel antibiotic produced by “RH2180-5 of the present invention”, which was considered difficult to concentrate and isolate only by the MIC result, was achieved. Therefore, this point will be described next.

(Isolation and purification of antibiotics using therapeutic effect in silkworm Staphylococcus aureus infection model)
The primary screening for narrowing down useful strains from many strains can be performed by any method as long as it is a method for evaluating antibacterial activity. For example, S. aureus is contained in the culture supernatant of various strains using MIC as an index. It is possible to pick up specimens with antibacterial activity. Thereafter, the specimen obtained in each purification step is subjected to a silkworm Staphylococcus aureus infection model, whereby the therapeutic effect of the specimen can be examined.

  Isolation and purification were performed on specimens with therapeutic effects on silkworm Staphylococcus aureus infection models by further performing isolation and purification procedures, and administering specimens at each purification stage to silkworm Staphylococcus aureus infection models. Confirm the concentration of treatment effect by operation type and method. Such a “selection method of specimen and purification operation method using therapeutic effect as an index” was accomplished for the first time by using a silkworm Staphylococcus aureus infection model.

The results in Table 1, summarizes the antibacterial activity MIC of the analyte in each purification step to be purified from the culture solution of RH2180-5, the therapeutic effect _{ED 50} of silkworm infection model. In Table 1, the total activity “unit” is defined as the amount of activity necessary for survival of a silkworm infected with S. aureus weighing 1 g with a probability of 50%.

As shown in Table 1, it was extremely interesting that it was found that the concentration of antibacterial activity and the concentration of therapeutic effect did not always match due to the isolation and purification operations performed. The results in Table 1 show that ED ₅₀ is 300 times concentrated, whereas MIC is only 5 times concentrated in the isolation and purification method performed in this case.

  That is, RH2180-5 of the present invention can be considered to have been found effectively by using an isolation and purification method using the therapeutic effect using a silkworm Staphylococcus aureus infection model as an index. This is because when the target substance is isolated and purified using only the conventional antibacterial activity as an index, antibacterial active ingredients that are not effective in terms of therapeutic effect are concentrated, and thus the therapeutic effect is in vivo. This is because it is considered from the results in Table 1 that there is a high possibility of missing a high antibiotic. In that case, the usefulness of RH2180-5 of the present invention cannot be confirmed, and it is highly likely that the existence of RH2180-5 has been missed. Therefore, in providing the new microorganism (RH2180-5) of the present invention to the world, it was very important that selection was performed using the therapeutic effect using the silkworm Staphylococcus aureus infection model as an index and a new antibiotic was obtained. It is considered important.

  Hereinafter, an antibiotic produced by the microorganism according to the present invention is obtained from the culture solution of the microorganism, and the microorganism is produced from the microorganism culture solution of the present invention. The specific contents of the method for producing antibiotics, characterized in that antibiotics having antibacterial activity at least against MRSA and VRE are described.

(Method for isolation and purification of antibiotics)
There is no particular limitation on the isolation and purification method selected using the therapeutic effect in the silkworm Staphylococcus aureus infection model as an index. Solvent extraction, phase transfer, water precipitation, chromatography using an ODS column, etc. Examples include fractionation by RP-HPLC used. The solvent for solvent extraction and the solvent for transfer are not particularly limited, but water-soluble solvents such as acetone; hydrophilic solvents such as butanol; mixed solvents thereof; mixed solvents of water and hydrophilic solvents; water and water-soluble A mixed solvent with an organic solvent; Instead of the ODS column, a column packed with a carrier modified with an octyl group or a butyl group, a polystyrene polymer carrier, or the like may be used. See Table 1.

  The isolation and purification method described above is merely an example, and any isolation and purification method may be used as long as the target antibiotic is finally obtained.

(Structural analysis of isolated and purified antibiotics)
By the above RP-HPLC, a compound divided into 9 peaks could be separated from the culture of RH2180-5 (FIG. 1). These compounds are similar to each other because of their similar UV absorption patterns, and are single components depending on the isolation and purification method prior to RP-HPLC.

The structural analysis of the purified RP-HPLC preparation of peak 5, which is the main peak among these nine peaks, was performed by the following analysis means. That is, molecular weight measurement by accurate mass spectrometry, amino acid analysis after acid hydrolysis treatment (FIG. 2), ¹ H-NMR analysis and ¹³ C-NMR analysis (FIG. 3), TOF (Time of Flight) MS analysis (FIG. 4) and the like, as a result, two molecules each of Thr, Glu, Glu, and Arg and one molecule each of Ser, Gly, and Ile were detected (FIG. 2), and a novel antibiotic having the novel skeleton structure shown in FIG. (Hereinafter referred to as “RH2180-5Peak5 substance”).

(Antimicrobial spectrum)
The antibacterial spectrum exhibited by the antibiotics in each of the above peaks can be examined by MIC obtained by a micro liquid dilution method or the like. Thereby, for example, as shown in Table 2, it was confirmed that the RH2180-5Peak5 substance exhibited antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus and enterococci. Furthermore, to confirm not only methicillin-resistant Staphylococcus aureus (MRSA) but also vancomycin-resistant enterococci (VRE) have the same antibacterial activity as normal bacteria and are effective against multidrug-resistant bacteria. I was able to. See Table 2 and Table 6 below.

  As described above, the antibiotic of the present invention is an antibiotic produced by the microorganism, which is obtained from the microorganism culture solution, and has at least antibacterial activity against MRSA and VRE. It is.

(Therapeutic effect in mouse Staphylococcus aureus infection model)
Antibiotics produced by RH2180-5 have been confirmed to have therapeutic effects in silkworm Staphylococcus aureus infection models in each purification step. In the said patent document 5, it is confirmed that the substance which shows a therapeutic effect with respect to a silkworm S. aureus infection model also shows a therapeutic effect also with respect to a mouse infection model. Therefore, was subjected to the same check against RH2180-5Peak5 material, RH2180-5Peak5 material also shows a therapeutic effect in a mouse model of infection, the _{ED 50} values of 0.6 mg / kg next vancomycin 1.6 mg / It was confirmed that the therapeutic effect was clearly lower than kg. See Table 3.

  Moreover, since the mouse | mouth was not killed as a result of the acute toxicity test with respect to a mouse | mouth at the dosage of 50 mg / kg, it can be anticipated that RH2180-5 Peak5 substance is low toxicity.

From the above results, the antibiotic RH2180-5Peak5 produced by the RH2180-5 of the present invention exhibits an excellent ED ₅₀ value against MRSA compared to vancomycin and exhibits a high therapeutic effect, and is also very problematic in clinical practice. It has been confirmed that it has an excellent feature of showing effectiveness not only in MRSA but also in VRE. In addition, substances in other fractions showing therapeutic effects are also considered to be similar compounds because they exhibit the same UV spectrum as the RH2180-5Peak5 substance, and therefore the antibacterial spectrum is expected to show the same tendency. The above results indicate that RH2180-5 found by the inventors is a highly useful novel microorganism.

  As described above, the compound produced by RH2180-5 is a novel compound having a novel cyclic peptide structure as a basic skeleton, and the compound exhibits antibacterial activity not only on MRSA but also on VRE. As a result of comprehensive judgment including the fact that the therapeutic effect was confirmed in mice against infection and such antibacterial spectrum was not known, RH2180-5 was judged to be a novel microbial strain.

Similar to the RP-HPLC purified sample from peak 5, which is the main peak of the nine peaks, other peaks were also measured for molecular weight by accurate mass spectrometry, amino acid analysis after acid hydrolysis, ¹ H-NMR And ¹³ C-NMR analysis, TOF-type MS analysis, UV spectrum, and infrared absorption spectrum (IR). As a result, compounds giving nine peaks were compounds represented by the following formula (1) and Table 4. It was.

［式（１）中、Ｒ^１は水酸基を１つ有する炭素数が７、８又は９のアシル基を示し、Ｒ^２はメチル基又は水素原子を示し、Ｒ^３はエチル基又はメチル基を示す。］

[In formula (1), R ¹ represents an acyl group having one hydroxyl group, 7, 8 or 9 carbon atoms, R ² represents a methyl group or a hydrogen atom, and R ³ represents an ethyl group or a methyl group. . ]

表４中、「高分解能質量分析」は、ＨＲ ＴＯＦ ＭＳ ｍ／ｚ（Ｍ＋Ｈ）^＋

In Table 4, “high resolution mass spectrometry” is HR TOF MS m / z (M + H) ⁺

  The present invention relates to an antibiotic produced by a microorganism of the microorganism having the accession number NITE P-870 belonging to the genus Lysobacter or a naturally or artificially mutated microorganism, which is at least methicillin. It is also an antibiotic which is a compound represented by the above formula (1) having antibacterial activity against resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE) or a pharmaceutically acceptable salt thereof.

  As shown in Study Example 2, although the MIC value itself indicated by each peak substance is a large value compared to the conventionally reported antibacterial agents, in comparison between normal bacteria and their multidrug resistant bacteria The MIC values were almost equivalent, and it was confirmed that the antibacterial activity of each peak substance was not affected by multidrug resistance.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example and an examination example, this invention is not limited to a following example.

Example 1
<Isolation and purification of RH2180-5 Peak5 substance>
(1) Search for microorganisms having antibacterial activity by MIC Soil collected from various places is suspended in physiological saline, the supernatant is applied to GA medium and HV medium, and the bacteria grown after incubation at 30 ° C. are separated, The cells were cultured in a YME medium, an SGM medium, or a CDY medium at 30 ° C. for 5 days. An equal amount of acetone was added, the suspension was centrifuged, and the supernatant was evaporated. After the obtained residue was diluted with physiological saline, the antibacterial activity against Staphylococcus aureus was evaluated by MIC using a micro liquid dilution method. As a result, antimicrobial activity was observed in the culture supernatant of 3487 strains out of 14346 strains.

(2) Examination of microbial strains that produce therapeutically active substances using a silkworm Staphylococcus aureus infection model (hereinafter abbreviated as “the silkworm model”) The sample of 3487 strains with the above-mentioned antibacterial activity was used as the silkworm model. When the therapeutic effect was examined, a therapeutic effect was observed in 45 culture supernatants.

(3) Purification of therapeutically active substance using therapeutic effect using silkworm model as an index Of 45 strains above, RH2180-5 isolated from soil collected in Okinawa was purified from the culture supernatant. Carried out. RH2180-5 was determined to be a novel microorganism belonging to the genus Lysobacter based on the analysis of 16S rRNA sequence described later, the tendency of acidic substances, and the like.

  RH2180-5 was inoculated and cultured in 1200 mL of YME medium, and a method for purifying antibiotics showing therapeutic effect from a 50% acetone extract of the culture was examined using the therapeutic effect in a silkworm model as an index. As a result, antibiotics showing therapeutic effects were purified by butanol transfer, water precipitation, chromatography using an ODS column, and RP-HPLC (reverse phase HPLC) using an ODS column.

As shown in Table 1, the specific activity of the therapeutic effect (ED ₅₀ value) increased 300 times that of the acetone extract by the above purification process. On the other hand, the increase in antibacterial activity (MIC) stayed only 5 times. This is because the antibacterial activity of the acetone extract of the culture supernatant, which is the starting material for purification, also involves substances other than antibiotics that show the therapeutic effect of the final purified product. Presumed to have been removed.

  In addition, since 9 compounds having similar UV absorption patterns were detected in the RP-HPLC, it was considered that RH2180-5 produced at least 9 related compounds.

Example 2
<Culture of RH2180-5 and production of RH2180-5 Peak5 substance>
Bacteria were scraped from the RH2180-5 slant culture with a platinum loop, inoculated into a 500 mL Erlenmeyer flask containing 100 mL of YME medium, and cultured at 30 ° C. with shaking for 3 days to obtain a seed culture solution. Next, 1.0 mL of this seed culture solution was inoculated into 12 500 mL Erlenmeyer flasks containing 100 mL of the previous liquid medium, and cultured with shaking at 30 ° C. for 5 days.

  An equal amount of acetone was mixed with the culture broth thus obtained, and the mixture was sufficiently stirred and then centrifuged, and the supernatant was evaporated to remove acetone. The sample was then subjected to butanol transfer. In butanol inversion, the acetone extract was suspended in 80 mL of water, and after adding an equal amount of butanol and shaking well, the butanol layer was separated by standing, separating funnel, and dried under reduced pressure. Subjected to precipitation. For water precipitation, the previous sample was suspended in 80 mL of water, the water was removed after centrifugation, and the precipitate was collected.

  Dissolve a 75 mg sample of butanol extract residue in 60% methanol and use Waters, Sep-paK® C18, 25 mL, 20 mL every 10% with 60% -100% methanol containing 0.1% TFA. It was eluted with the solution. As a result, an antibiotic-containing fraction showing a therapeutic effect was eluted in the 70% -80% methanol fraction.

  Antibiotic-containing fractions showing the above therapeutic effect were pooled and dried, and 22 mg of the fraction was dissolved in 50% methanol, and 75% − using a Senshu PaK SP-100 ODS column (diameter 20 mm × length 250 mm). Elute with 95% methanol + 0.1% TFA. Antibiotic-containing fractions showing therapeutic effects by PR-HPLC under these conditions were fractionated into substances each showing nine peaks consisting of a single substance.

  In the above RP-HPLC, the antibiotic-containing fraction showing a therapeutic effect is composed of a group of compounds having nine similar UV absorption patterns (UV apparatus Waters 2996 photodiode array) having the peak 5 as a main component. confirmed. See FIG. By the above operation, 5.3 mg of RH2180-5Peak5 substance was obtained.

<Structural analysis of RH2180-5 Peak5 substance>
As a result of subjecting the RH2180-5 Peak5 substance to accurate mass spectrometry using a Bruker Daltonics BioTOF-Q mass spectrometer, the molecular weight was 1616.9 [ESI-TOF-MS, (M + H) ⁺ was m / z. = 1617.8755]. In addition, after hydrolyzing at 105 ° C. overnight under 6N hydrochloric acid, amino acid analysis was performed using a Hitachi amino acid analyzer, and each of the two molecules Thr, Glu, and Arg, and one molecule each of Ser, Gly, and Ile. Was detected (FIG. 2). Furthermore, as a result of ¹ H, ¹³ C-NMR analysis (FIG. 3) and TOF-MS analysis (FIG. 4) by JEOL ECA-500 NMR, the RH2180-5Peak5 substance is a compound having the novel skeleton shown in FIG. Became clear.

Furthermore, when the RH2180-5Peak5 substance subjected to acid hydrolysis was determined for D and L isomers using a chiral column, Ile, Ser, Leu, 2 Thr were L isomer, N-MePhe, 2 Arg , Trp was found to be D-form.
For Gln and Glu, both were Glu as a result of acid hydrolysis, and D and L isomers were detected at 1: 1. Therefore, bis (1,1-trifluoroacetoxy) was detected against peptides containing glutamine and glutamic acid. By converting iodobenzene to glutamine into diaminobutyric acid, and after hydrolysis, the absolute configuration of the unreacted glutamic acid was determined using a chiral column. As a result, Gln was in D form and Glu was in L form. Became clear. (The method for determining D and L forms of peptides by this decomposition method is a novel method first performed in the present application.)
For the hydroxyl group of the fatty acid chain, the absolute configuration R was determined by the modified Mosher method.
As a result, it was found that the RH2180-5Peak5 substance is a novel cyclic peptide compound in which each amino acid has the three-dimensional structure shown in FIG.

<Antimicrobial spectrum of RH2180-5 Peak5 substance>
The antibacterial spectrum of various substances of RH2180-5Peak5 substance was examined by MIC using a micro liquid dilution method. The results are shown in Table 2. From the results of MIC, it was clarified that the substance exhibits antibacterial activity against Gram-positive bacteria since it exhibits antibacterial activity against Staphylococcus aureus and enterococci. Moreover, since it showed antibacterial activity against not only methicillin-resistant Staphylococcus aureus (MRSA) but also vancomycin-resistant enterococci (VRE), it was considered effective against multidrug-resistant bacteria. The value of MIC shown in this result is not lower than that of the previously reported antibiotics and does not show a particularly high antibacterial activity. When compared with vancomycin, it was confirmed to have a therapeutic effect higher than that of vancomycin.

<Investigation of therapeutic effect and toxicity of RH2180-5 Peak5 substance in mouse S. aureus infection model (mouse model)>
S. aureus Smith strain was suspended in 7% mucin + 0.2 mM iron (III) ammonium citrate (Ferric ammonium citrate), and 6.2 × 10 ⁶ mice (20 × LD50) were grouped into 5 mice (ICR female). 4 weeks old). Each drug was injected subcutaneously 2 hours after administration of the previous strain by the method shown below.

RH2180-5Peak5 substance dissolved in PBS was subcutaneously injected into the mouse model to 25 mg / kg, 12.5 mg / kg, and 6.3 mg / kg, and the number of surviving the next day was measured after administration. The therapeutic effect (ED ₅₀ value) was examined (5 per group). Similarly, for vancomycin, the ED ₅₀ value for the mouse model used this time was examined. In the case of RH2180-5Peak5 substance, the survival of all mice was confirmed at a dose of 25 mg / kg under the condition of no survival by PBS administration. The ED ₅₀ value was determined by the probit method.

The obtained results are shown in Table 3. The RH2180-5 Peak 5 substance has a therapeutic effect on the mouse model, and its ED ₅₀ value is 0.6 mg / kg, which is clearly lower than the ED ₅₀ value of vancomycin examined at the same time, 1.6 mg / kg. It was confirmed that this is a highly therapeutic antibiotic.

In addition, RH2180-5 Peak 5 substance was subcutaneously administered to mice, and the acute toxicity of RH 2180-5 Peak 5 substance to mice was also examined by observation the next day after administration. The acute toxicity was not observed at a dose of 50 mg / kg corresponding to 80 times the ED ₅₀ value, suggesting that the RH2180-5 Peak 5 substance is low toxic.

Study example 1
<16S rRNA analysis>
The base of 16S rRNA of RH2180-5 was amplified by a colony PCR method, and the amplified RNA fragment was analyzed by a sequencer. As a result, a base sequence corresponding to almost the entire length of the 16S rRNA region shown in SEQ ID NO: 1 in the sequence listing, excluding some bases on the 5 ′ end side and 3 ′ end side, was determined. Based on this base sequence, homology search with existing strains was performed using NCBI BLAST. As a result, RH2180-5 showed 99% homology with the Lysobacter enzyme genes DSM 2043T strain, and thus was considered to be a microorganism belonging to the genus Lysobacter.

<About novelty of RH2180-5>
RH2180-5 has many similarities in chemical properties to Lysobacter enzyme zymogenes DSM 2043T strain, but the antibacterial spectrum of the bioactive substance produced is different, producing a new and useful RH2180-5Peak5 substance that has not been reported so far The difference is that it has been confirmed. Therefore, from the above results, RH2180-5 was determined to be a novel strain belonging to the genus Lysobacter.

Study example 2
<Comparison study of antibacterial activity of multi-drug-resistant bacteria and non-drug-resistant bacteria for each peak substance>
Comparison of antibacterial activity against multidrug-resistant bacteria and bacteria without drug resistance was conducted on 8 novel cyclic peptide compounds of the present invention with 2 to 9 peaks, excluding 1 substance with a small amount of sample. .

  S. aureus and enterococci are selected as the test bacteria, and the microorganisms are also based on CLSI (formerly NCCLS US Clinical Laboratory Standards Committee) based on the MIC values of each peak substance for MSSA1 and EF1, MRSA3, MRSA4 and VRE. It was measured by a micro sample dilution method. The results are shown in Table 5.

ＭＩＣ（μｇ／ｍＬ）

MIC (μg / mL)

MSSA1: Staphylococcus aureus MRSA3: OX, FL, KM, TC, EM-resistant Staphylococcus aureus MRSA4: OX, FL, KM, CP, CPLX-resistant Staphylococcus aureus EF1: Enterococci VRE: Vancomycin-resistant enterococci

OX: Oxacillin, FL: Fromomoyev, KM: Kanamycin,
TC: Tetracycline, CP: Chloramphenicol, EM: Erythromycin CPLX: Ciprofloxacin

  As a result, although the MIC value itself shown by each peak substance is larger than that of the conventionally reported antibacterial agents, the EF1 between normal bacteria and their multidrug-resistant bacteria (MSSA1 and MRSA3, MRSA4) In comparison between VRE and VRE), the MIC values were almost equivalent, and it was confirmed that the antibacterial activity of each peak substance was not affected by multidrug resistance.

It has been confirmed that the antibacterial activity and the therapeutic effect do not necessarily match. For example, the RH2180-5Peak5 substance having an MIC value for MSSA of 6.3 μg / mL in the case of Table 5 and 5 μg / mL in the case of Table 2 has a therapeutic effect (ED ₅₀ value) on a mouse S. aureus infection model. The examination results show an ED ₅₀ value of about 1/3 of vancomycin, and it has been confirmed that a high therapeutic effect is exhibited.

  Although the MIC values of 5 μg / mL and 6 μg / mL are the levels that are indicated as MIC values of resistant bacteria in vancomycin, one of the reasons why the compound represented by the formula (1) exhibits a sufficient therapeutic effect. In addition, it is possible that this compound exhibits antibacterial activity with a mechanism of action different from that of existing drugs, which may be reflected in the therapeutic effect. When antibacterial activity is shown with a mechanism of action different from that of existing drugs, at least the current resistant bacterial isolation frequency is predicted to be low, which is considered to be an advantage of the compound represented by the formula (1). .

Study example 3
<Investigation of antibacterial spectrum of RH2180-5 Peak5 substance>
The antibacterial spectrum of various microorganisms including MRSA and VRE of the above RH2180-5 Peak5 substance was examined. When 10% serum was added and when serum was not added, the respective MIC values were measured by a micro sample dilution method based on CLSI (former NCCLS American Clinical Laboratory Standards Committee). Table 6 shows the measurement results.

  From the results shown in Table 6, it was confirmed that the RH2180-5Peak5 substance was effective against Gram-positive bacteria, and the activity increased with the addition of serum.

Study example 4
<Examination of bactericidal activity of RH2180-5 Peak 5 substance>
CA-Mueller Hinton Broth medium was inoculated with 1.8 × 10 ⁸ Staphylococcus aureus, and the above RH2180-5 Peak5 substance (25 μg / mL), vancomycin (VM, 5 μg / mL), gentamicin (GM, 2.5 μg / mL) ), And the number of viable cells after 15 minutes, 30 minutes, 60 minutes and 120 minutes was determined as CFU (colony forming unit / mL). The results are shown in Table 7.

  From the results of Table 7, it was confirmed that the addition of RH2180-5Peak5 substance immediately reduced the number of Staphylococcus aureus as compared with the case where vancomycin and gentamicin were added.

Study example 5
<Examination of lytic activity of RH2180-5 Peak 5 substance>
A Staphylococcus aureus solution was diluted in CA-Mueller Hinton Broth medium, RH2180-5Peak5 substance, vancomycin and daptomycin were added at a concentration 5 times that used in Test Example 1 and cultured at 37 ° C. Using an absorptiometer (manufactured by Shimadzu Corporation), the absorbance at 600 nm after addition (OD ₆₀₀ ) was measured over time. The results are shown in FIG.

From the results shown in FIG. 7, it was confirmed that the RH2180-5Peak5 substance exhibited a lytic activity because the decrease in absorbance (OD ₆₀₀ ) was larger than that of vancomycin and daptomycin.

  The novel microbial strain RH2180-5 of the present invention has industrial applicability such as a method for producing a new antibiotic and the provision of the antibiotic.

Domestic deposit: NITE P-870
Deposit under the Budapest Treaty: NITE BP-870

  SEQ ID NO: 1 in the sequence listing is a base sequence corresponding to almost the entire length of 16S rRNA of an unknown strain belonging to the genus Lysobacter.

Claims (5)

Risobakuta (Lysobacter) Accession No. NITE B P-870 microorganism belonging to the genus or antibiotic is a compound represented by the following formula (1) with its natural or artificially mutated a microorganisms with antibacterial activity Microorganisms capable of producing

[In formula (1), R ¹ represents an acyl group having one hydroxyl group, 7, 8 or 9 carbon atoms, R ² represents a methyl group or a hydrogen atom, and R ³ represents an ethyl group or a methyl group. . ]   The microorganism according to claim 1, wherein the antibiotic has antibacterial activity against at least methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).   The microorganism according to claim 1 or 2, which has the base sequence of the 16S rRNA region represented by SEQ ID NO: 1 in the sequence listing. A method for producing an antibiotic which is a compound represented by the above formula (1) or a salt thereof, wherein the microorganism is produced from the culture solution of the microorganism according to any one of claims 1 to 3. A method for producing an antibiotic, comprising obtaining an antibiotic. The method for producing an antibiotic according to claim 4, wherein the antibiotic has antibacterial activity against at least methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).

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