JP5878303B2 - Antibiotic-containing fraction, its antibiotic and method for producing the antibiotic - Google Patents

Description

  The present invention relates to a fraction containing an antibiotic fractionated from a culture produced by a novel microorganism belonging to the genus Rhizobacter, a method for producing the antibiotic, and an antibiotic obtained therefrom, and further obtained from the above fraction Furthermore, the present invention relates to antibiotics that exhibit antibacterial activity but no therapeutic effect, methods of using antibiotics that exhibit antibacterial activity and also have a therapeutic effect, and methods for producing them.

  Antibiotics are indispensable for controlling microorganisms and treating infectious diseases. However, when the method of using antibiotics is inappropriate, resistant bacteria are likely to appear, and as a result, multidrug-resistant bacteria having resistance to many drugs have emerged, which is a major clinical problem. Therefore, in order to find new effective antibiotics, investigation by chemical synthesis and search for microorganisms that produce new antibiotics are continually performed.

  Many types of microorganisms have already been reported as microorganisms that produce new antibiotics, and microorganisms belonging to the genus Lysobacter of the Xanthomonas family used in the present invention are also reported in Patent Documents 1 to 3 as such microorganisms. Yes. Among these, Patent Documents 1 and 3 describe bacteria that produce antibiotics that exhibit antibacterial activity against the multidrug-resistant bacteria and methods for producing antibiotics using the bacteria.

  Among multidrug-resistant bacteria, antibacterial activity against methicillin-resistant Staphylococcus aureus (hereinafter abbreviated as “MRSA”) and vancomycin-resistant enterococci (hereinafter abbreviated as “VRE”), which are clinically problematic. It is strongly desired to provide a new antibiotic having an excellent therapeutic effect. However, most of the antibiotics that have been reported to date against multidrug-resistant bacteria are antibiotics that exhibit antibacterial activity only against MRSA, and there are few reports of antibiotics that also exhibit antibacterial activity against VRE. Examples of antibiotics effective for both MRSA and VRE include antibacterial activity in vitro against the above-mentioned multidrug-resistant bacteria among the antibiotics reported in Patent Document 3 (hereinafter, unless otherwise specified) Antibacterial activity is described in the sense of “in vitro antibacterial activity.”) Antibiotics are described.

  On the other hand, the present inventor has constructed a silkworm Staphylococcus aureus infection model that uses silkworms (larvae of Bombyx mori) as experimental animals, which is useful for searching for antibiotics (Patent Document 4), and has been proceeding with its investigation.

Japanese Examined Patent Publication No. 6-99444 Japanese Patent No. 3339235 Japanese Patent No. 4054576 JP 2007-327964 A

  As mentioned above, there are many antibiotics that target multidrug-resistant bacteria, but there are few antibiotics that clearly show efficacy against VRE, and antibiotics for VRE that are in practical use are oxazolidinone antibiotics. To some extent, a linezolid is known. However, linezolid is a synthetic antibiotic created by complete chemical synthesis, and considering the safety issues to living organisms and the possibility of emergence of resistant bacteria to existing drugs, antibiotics for multi-drug resistant bacteria effective for VRE There was a need to increase the options.

  On the other hand, if antibiotics that are effective against VRE can be obtained from substances produced by microorganisms, there is a high possibility that the hurdles required for the development and production of pharmaceuticals can be lowered as compared to fully chemically synthesized products. Because at least the production can be carried out by a culture method, it is considered possible to carry out safe production at a lower cost than the production by chemical synthesis methods that require chemicals such as special catalysts or high temperature and pressure. It is.

  Among the antibiotics described in Patent Document 3 described above, antibiotics exhibiting antibacterial activity for both MRSA and VRE are described as antibiotics effective for VRE as substances produced by such microorganisms. However, do they exhibit an in vivo therapeutic effect on an infectious disease (hereinafter, unless otherwise specified, “therapeutic effect” means the therapeutic effect of the infectious disease confirmed in an in vivo evaluation system)? It has not been described until now. This is because the effectiveness of antibiotic treatment in the past is reflected in the magnitude of the therapeutic effect of the antibiotic, as evaluated by the minimum inhibitory concentration (hereinafter abbreviated as “MIC”). It was because it was evaluated. The lack of a method for simply evaluating the “therapeutic effect” in the past is also considered to be the reason why the evaluation by the MIC had to be performed. However, it was confirmed from the examination results of the present inventors that will be described later that “the magnitude of antibacterial activity” evaluated by MIC does not necessarily reflect the “magnitude of therapeutic effect” of those antibiotics. .

  In other words, most of the antibiotics that have been reported so far are selected based on the evaluation of antibacterial activity by MIC, and only the magnitude of “antibacterial activity” is disclosed. It could not be said that it was disclosed.

  Therefore, in providing fractions containing antibiotics produced by microorganisms (hereinafter referred to as “antibiotic-containing fractions”) and antibiotics obtained from the fractions, not only the antibacterial activity exhibited by them, It is important to select the product after evaluating its therapeutic effect. Therefore, it is important to provide an unprecedented highly useful antibiotic-containing fraction and an antibiotic obtained from the fraction. This is a problem to be solved by the invention.

  And by solving the above-mentioned problems, it is possible to select an antibiotic-containing fraction that exhibits antibacterial activity and also exhibits a therapeutic effect and an antibiotic obtained therefrom, and an antibiotic-containing fraction that exhibits antibacterial activity but does not exhibit a therapeutic effect I realized that it would be possible to select antibiotics. Furthermore, it is natural that the former is highly useful as a therapeutic agent for infectious diseases, but when the latter antibiotic-containing fraction, which has not been noticed in the past, is also used as a microbial control agent, these are therapeutic agents. Therefore, it is not necessary to substantially consider the emergence of multi-drug resistant bacteria, and thus it has been conceived that it has the property of being suitably used as a microorganism control agent.

  That is, an object of the present invention is to provide an antibiotic-containing fraction produced by a microorganism having an unprecedented effectiveness, an antibiotic obtained therefrom, and a method for producing the same, and further, the antibiotic-containing fraction, It is to provide antibiotic-containing fractions and antibiotics that are evaluated and selected not only for their antibacterial activity but also their therapeutic effects. Furthermore, it is providing the antibiotic containing fraction and antibiotics which show a therapeutic effect with respect to multidrug resistant bacteria. Moreover, it is providing the microorganism control agent containing the said antibiotic-containing fraction or antibiotics.

  In view of the above circumstances, the present inventor obtained a novel microorganism producing an antibiotic, an antibiotic-containing fraction obtained from the microorganism, and an antibiotic obtained from the microorganism from the culture supernatant of the microorganism. The fractions containing antibiotics and methods for evaluating antibiotics obtained by separating and purifying the fractions were examined. As a result, when selecting methods for separating and purifying antibiotics in microorganisms and culture supernatants, the antibacterial activity of each sample was evaluated by MIC against Staphylococcus aureus, and at the same time, the therapeutic effect was also evaluated. I came up with a way to do it. And as an evaluation method of the magnitude of therapeutic effect of each sample, the silkworm Staphylococcus aureus infection model using the silkworm as an experimental animal of Patent Document 4 described above is applied, and the target microorganism strain and the above highly useful A study was conducted to obtain antibiotic-containing fractions.

  As a result, 3487 strains of antibacterial activity against Staphylococcus aureus were found in the culture supernatant of 14346 strains of soil bacteria isolated from soil collected in various places in Japan. When the therapeutic effect in the Staphylococcus aureus infection model was examined, the number of microorganisms that showed the therapeutic effect decreased to 45 strains. From this, it was confirmed that the antibacterial activity evaluated by MIC does not guarantee the “therapeutic effect” of the evaluated antibiotic against the infection caused by the MIC target bacteria.

  And, when the antibacterial spectrum of the antibiotic-containing fraction obtained from the culture supernatant of 45 strains was examined, 45 strains contained microorganisms showing antibacterial activity against both MRSA and VRE multidrug resistant bacteria. In addition, the antibiotic-containing fraction fractionated from the culture of the microorganism contains an antibiotic that exhibits a therapeutic effect equivalent to or higher than that of vancomycin in a mouse S. aureus infection model. I found out. And when the antibiotic which showed the said high therapeutic effect was examined in detail including the chemical structure, it confirmed that the antibiotic was a novel antibiotic.

  In addition, when we examined the antibacterial spectrum of other antibiotic-containing fractions and the antibiotics in that fraction, the antibacterial spectrum of other antibiotic-containing fractions was the same as the above-mentioned new antibiotics, The chemical structure was also revealed to be a related compound of the novel antibiotic, and it was confirmed that the fraction containing antibiotics other than the novel antibiotic in the final fractionation stage was a highly useful fraction.

In addition, the therapeutic effect (ED ₅₀ : 50% effective dose) of the fraction containing the novel antibiotics is concentrated 300 times that of the purified initial fraction, whereas the antibacterial activity (MIC) is 5 times higher. It was confirmed that it was only concentrated.

  This fact indicates that the fraction containing antibiotics at the initial stage of purification obtained by the fractionation procedure described below contains a high proportion of antibiotics that have relatively high antibacterial activity but show little therapeutic effect. Show. Therefore, it was considered that these antibiotics contained in the fraction containing antibiotics at the early stage of purification are difficult to use as therapeutic agents for infectious diseases.

  However, from a different point of view, the fact that antibacterial activity is high but does not show a therapeutic effect means that such antibiotics are practically not used as therapeutic agents for infectious diseases. It can be evaluated that it is suitable for an agent. Because it is not used as a therapeutic agent, it is not necessary to consider the emergence of multi-drug resistant bacteria that are a concern when used as an antibacterial agent. In addition, since it is excluded from the fractions in the latter half of the purification, it has been confirmed that there is no similarity in chemical structure with the above-mentioned novel antibiotics, and cross-reaction with antibiotics selected as infectious disease treatment agents It is not necessary to consider the emergence of multidrug-resistant bacteria due to.

  In other words, antibiotic-containing fractions with high antibacterial activity but weak therapeutic effects obtained from fractions in the early stages of purification, and antibiotics obtained from these fractions are also very useful for microbial control purposes. Minutes.

  Furthermore, the antibiotic-containing fractions that exhibit antibacterial activity against MRSA and VRE as described above, and microorganisms that produce antibiotics can be obtained from the results of analysis of the properties, analysis of 16S rRNA base sequence, etc. It was also found that it was a novel microorganism belonging to the genus Lysobacter (hereinafter referred to as “RH2180-5”), and the antibiotic-containing fraction of the present invention and the antibiotic obtained therefrom were obtained from the novel microorganism. It became clear that the present invention was completed.

That is, the present invention provides the following.
<1> An antibiotic-containing fraction obtained by culturing a microorganism belonging to the genus Lysobacter of Accession No. NITE P-870 and fractionating the culture.
<2> The antibiotic-containing fraction according to <1>, wherein the antibiotic-containing fraction is a fraction containing an antibiotic exhibiting antibacterial activity.
<3> The antibiotic according to <1> or <2>, wherein the antibiotic-containing fraction exhibits antibacterial activity at least against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Substance-containing fraction.

<4> The antibiotic-containing fraction according to any one of <1> to <3>, wherein the antibiotic-containing fraction contains at least an antibiotic having a therapeutic effect on an infection caused by Staphylococcus aureus. .
<5> The antibiotic according to <4>, wherein the antibiotic-containing fraction is a fraction containing an antibiotic that exhibits at least a therapeutic effect equivalent to or higher than that of vancomycin against an infection caused by Staphylococcus aureus Containing fraction.
<6> The antibiotic-containing fraction according to <1> or <2>, wherein the antibiotic-containing fraction contains an antibiotic that exhibits antibacterial activity but does not substantially exhibit a therapeutic effect.
<7> The antibiotic-containing fraction according to <6>, wherein the antibiotic that exhibits antibacterial activity but does not substantially exhibit a therapeutic effect is used as a microorganism control agent.

<8> A microorganism belonging to the genus Lysobacter of accession number NITE P-870 is cultured, and antibiotics having antibacterial activity and / or antibiotics having a therapeutic effect on infectious diseases are separated and purified from the culture. And a method for producing an antibiotic.
<9> The method for producing an antibiotic according to <8>, wherein the antibiotic exhibits antibacterial activity at least against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
<10> The method for producing an antibiotic according to <8> or <9>, wherein the antibiotic has a therapeutic effect on at least an infection caused by Staphylococcus aureus.
<11> The method for producing an antibiotic according to <8>, wherein the antibiotic is an antibiotic that exhibits antibacterial activity but does not substantially exhibit a therapeutic effect.

［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。］ <12> An antibiotic obtained by culturing a microorganism belonging to the genus Lysobacter having an accession number of NITE P-870 and obtaining the culture.
<13> The antibiotic according to <12>, wherein the antibiotic exhibits antibacterial activity at least against both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
<14> The antibiotic according to <12> or <13>, wherein the antibiotic has a therapeutic effect on at least an infection caused by Staphylococcus aureus.
<15> The antibiotic according to <14>, wherein the antibiotic exhibits at least a therapeutic effect equivalent to or higher than that of vancomycin against an infection caused by Staphylococcus aureus.
<16> A microorganism belonging to the genus Lysobacter of Accession No. NITE P-870 is cultured, and obtained from the culture, at least for both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). An antibiotic having antibacterial activity and at least a therapeutic effect on infection caused by Staphylococcus aureus.
<17> The antibiotic according to any one of <12> to <16> represented by the following formula (1).

[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. . ]

<18> A microorganism control agent comprising the antibiotic-containing fraction according to <1> or <2>.
<19> A microorganism control agent comprising the antibiotic according to <12>.

  According to the present invention, it is possible to provide a fraction containing a useful antibiotic produced by a new microorganism and the antibiotic. Further, some of the antibiotics show antibacterial activity not only on MRSA but also on VRE, and some show high therapeutic effects on Staphylococcus aureus. Therefore, it has the effect that the fraction containing a very useful novel antibiotic and the novel antibiotic can be provided.

  In addition, antibiotic-containing fractions that show antibacterial activity but no therapeutic effect, and antibiotics that do not show the therapeutic effect obtained from them, are not usually used for treatment, so it is necessary to consider the emergence of multidrug-resistant bacteria And has an effect that it can be suitably provided as an antibacterial agent such as a microorganism control agent.

  Furthermore, the production method of the present invention has an effect that a useful antibiotic can be produced by a method of culturing a microorganism that is simpler than the chemical synthesis method. Specifically, because it is a culture method, it does not require organic solvents, special metal catalysts, reagents, and many synthetic steps that require high temperature and pressure, so it must be cheap and environmentally friendly. Can do.

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). In addition, the numbers on each peak column displayed in a separate frame indicate the molecular weight of each peak substance. It is a figure which shows the analysis result of the amino acid composition analysis of RH2180-5Peak5 substance. It is a figure which shows the analysis result of ¹ H-NMR of a RH2180-5Peak5 substance, and a ¹³ C-NMR analysis. The vertical axis represents the signal intensity, and the horizontal axis represents the chemical shift value (ppm). It is a figure which shows the 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.

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of the technical idea.

  The antibiotic-containing fractions of the present invention and the microorganisms that produce the antibiotics obtained from each fraction include antimicrobial activity by MIC and silkworm described in Patent Document 4 among many microorganisms isolated from Okinawa soil. It is a microorganism selected by evaluating the therapeutic effect by a method using a Staphylococcus aureus infection model.

  As such a microorganism, a microorganism named “RH2180-5 strain” belonging to the genus Rysobacter (hereinafter referred to as “RH2180-5”) was newly discovered. This RH2180-5 is a patent microorganism deposit center of the National Institute of Technology and Evaluation (hereinafter abbreviated as “NITE”) 2-5-8, Kazusa Kamashita, Kisarazu City, Chiba Prefecture, National Institute of Technology and Evaluation (hereinafter abbreviated as “NITE”) NPMD), a microorganism deposited on January 25, 2010 and deposited under the deposit number “NITE P-870”.

  “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: RH2180-5 is a Gram-negative gonococci, with no flagellum 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: The physiological and chemical taxonomic properties of 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 “16S rRNA region”.

  When this base sequence was searched for homology with BLAST of NCBI, the base sequence of the 16S rRNA region of this RH2180-5 showed 99% homology with the Lysobacter enzyme gene DSN 2043T strain belonging to the genus Lysobacter. This Lysobacter enzyme genesis is different from RH2180-5 of the present invention because there is no report of antibiotic production.

  The above properties of RH2180-5 as a strain are compared with the classification according to Bergeys' Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology, vol. 3 1989), and other literature. As a result of comprehensive determination in consideration of the results of 16S rRNA analysis, RH2180-5 was determined to be a microorganism belonging to the genus Lysobacter.

  In addition, microorganisms having a 16S rRNA region base sequence that matches the base sequence of the 16S rRNA region of RH2180-5 are not known, and some of the antibiotics produced by RH2180-5 described below are not only MRSA. VRE also has an antibacterial spectrum with few precedents exhibiting antibacterial activity, exhibits higher therapeutic effect compared to vancomycin for infections caused by Staphylococcus aureus, and an antibiotic produced by RH2180-5 is novel As a result of comprehensive evaluation and examination of having a chemical structure and the like, RH2180-5 was determined to be a novel microbial strain belonging to the genus Rhizobacter.

  The above-mentioned RH2180-5 is domestically deposited at the National Institute for Product Evaluation and Technology (NITE) and the Patent Microorganism Depositary Center (NPMD), and has received the deposit number: NITE P-870 and is available. The antibiotic-containing fraction of the present invention and the antibiotics obtained therefrom can be obtained from this culture of RH2180-5.

  The present invention is obtained by culturing a microorganism belonging to the genus Lysobacter having accession number NITE P-870 having the base sequence of the 16S rRNA region shown in SEQ ID NO: 1 in the sequence listing, and fractionating the culture. This is a fraction containing antibiotics.

  In addition, since the property as a bacterial strain is easily mutated as a general property of bacteria, there is a possibility that RH2180-5 may not remain within the above property range. However, even if the microorganism is mutated from RH2180-5 (accession number NITE P-870), as long as it is a microorganism belonging to the genus Lysobacter and has the ability to produce an antibiotic-containing fraction, RH2180-5 (consignment) No. NITE P-870).

  In addition, it goes without saying that the mutation includes both natural mutation and artificial mutation.

  The antibiotic-containing fraction of the present invention refers to a product obtained by culturing the above RH2180-5 and subjecting the culture to some fractionation treatment. Here, the “culture” means any of a culture supernatant, cultured cells, cultivated cells, and the like. “Fractionation” usually includes all treatments performed for the purpose of separation / purification of the target substance, such as extraction, precipitation, membrane separation, transfer dissolution, chromatogram, etc., usually performed on the culture. .

  Regarding the use of the antibiotic-containing fraction of the present invention, antibiotics contained therein exhibit antibacterial activity, and further exhibit therapeutic effects, and can be used for the production of infectious disease therapeutic agents and exhibit antibacterial activity. However, those that do not show a therapeutic effect can be used for the production of a microbial control agent. Antibiotic-containing fractions containing antibiotics that exhibit antibacterial activity but substantially no therapeutic effect are an aspect of the present invention.

  The method for using the microorganism control agent is not particularly limited, and examples thereof include a method of applying, immersing and moistening on the surface of materials, tools, etc. that require antibacterial properties. More specifically, for example, it is preferably used as an antibacterial component of an adhesive or an antibacterial component in a coating used for various types of adhesive sheets for skin, such as adhesive gauze, bandages, etc. Can be used. In the antibiotic-containing fraction of the present invention, an antibiotic that exhibits antibacterial activity but does not substantially exhibit a therapeutic effect is used as a microorganism control agent (used for use as a microorganism control agent and / or a microorganism control agent). It is preferable that it has the performance used as a).

  Usually, antibiotics are difficult to use as a microorganism control agent because it is necessary to pay attention to the emergence of multi-drug resistant bacteria, but antibiotic-containing fractions that do not show therapeutic effects in the present invention and antibiotics obtained therefrom are It can be used as a microbial control agent without such concerns. When used as a microbial control agent, the antibiotic-containing fraction can be used as a fraction or simply by concentrating the fraction, and the antibiotic can be separated and purified from the fraction. Antibiotics can also be used. Those processes may be selected according to each application.

  As described above, regardless of the presence or absence of therapeutic effect, the antibiotic-containing fraction of the present invention can be obtained by culturing RH2180-5 and fractionating the culture. Thus, first, a method for culturing RH2180-5, which is the premise of each fractionation process, is described below.

(Culture method of RH2180-5)
The RH2180-5 culturing method for producing the antibiotic-containing fraction of the present invention may be carried out in accordance with a general culturing method carried out on a microorganism belonging to the genus Lysobacter. 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 aerobic conditions, 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 around 30 ° C. The culture pH is preferably 6-8, particularly preferably around 7. The culture period is a factor that varies depending on the medium composition used, the culture temperature, the degree of stirring and shaking, etc., but in the case of RH2180-5, it is usually about 1 to 20 days, preferably about 4 to 7 days. A sufficient amount of the object can be secured in between.

(Method for purifying antibacterial active ingredients)
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.

  Usually, the extraction solvent and the separation / purification technique used at the time of separation / purification of the target substance are selected using the concentration of antibacterial activity determined by MIC as an index. There is basically no problem with such a method as long as it is simply separation and concentration of antibacterial active ingredients. However, in the separation and purification of antibiotic-containing fractions and antibiotics produced by RH2180-5 of the present invention, not only the MIC result but also the therapeutic effect on the silkworm Staphylococcus aureus infection model shown in Patent Document 4 is used as an index. Have made a choice. As a result, concentration and purification of “a novel antibiotic produced by RH2180-5”, which was considered difficult to concentrate and purify only by looking at the results of MIC, was accomplished. Therefore, this point will be described next.

(Separation and purification of antibiotics using therapeutic effect as an index in the silkworm Staphylococcus aureus infection model)
<Selection of useful strains by MIC)
Also in the present invention, a method for evaluating antibacterial activity is suitably used for the primary screening for narrowing down useful strains from a very large number of tens of thousands of strains. This is because a strain that does not show any antibacterial activity is not considered. Any method may be used as long as it is a method for evaluating antibacterial activity, but a method of picking up strains having antibacterial activity against Staphylococcus aureus in the culture supernatant of various test strains using MIC as an index Is preferred. Thereafter, when the number of strains is narrowed down to several hundred thousands, evaluation of the therapeutic effect of antibiotic-containing fractions and antibiotics derived from them to measure their ED ₅₀ using silkworms S. aureus infection model Can be considered.

  For the strains that have been confirmed to have a therapeutic effect on the silkworm Staphylococcus aureus infection model, various separation and purification operations are further performed, and the therapeutic effect is evaluated with the silkworm Staphylococcus aureus infection model for each operation. In addition, the type of separation and purification operations performed by administering the antibiotic-containing fractions at each purification stage and the concentration of therapeutic effect by the technique are also confirmed in the silkworm S. aureus infection model. Then, the types and methods of separation / purification operations that can effectively concentrate the “therapeutic effect” will be selected.

  It is practically impossible to conduct such “selection of strains and separation / purification method using therapeutic effect as an index” using laboratory animals such as mice due to technical, cost, and ethical issues. Yes, it was achieved for the first time by using the silkworm Staphylococcus aureus infection model (see Example 1).

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.

The results in Table 1 show that ED ₅₀ is concentrated 300 times from 90 in the acetone extraction stage to the RP-HPLC fractional peak 0.3 in the final purification stage. Shows that it is only concentrated 5 to 5 from 25 to 5. In addition, the recovery rate of therapeutic activity shows that when the acetone extract is 100%, the recovery rate of mass% has reached nearly 30% with only two peaks of only 0.1%. Yes.

  In addition, the residue after the acetone extraction is considered to contain a relatively large amount of antibiotics that show antibacterial activity but no therapeutic effect. Can be used as a fraction containing antibiotics.

  RH2180-5 was effectively found by using a separation / purification method with the therapeutic effect using a silkworm Staphylococcus aureus infection model as an index. This is because when the separation and purification method of the target substance is performed using only the conventional antibacterial activity as an index, antibacterial active ingredients that are not effective in terms of therapeutic effect are concentrated, and the therapeutic effect in vivo. This is because it is inferred from the results in Table 1 that there is a high possibility of overlooking high antibiotics. In this case, the usefulness of RH2180-5 cannot be confirmed, and there is a high possibility that RH2180-5 itself has been missed. Therefore, in providing a novel microorganism (RH2180-5), it is a very important point in the present invention that selection was performed using the therapeutic effect using the silkworm S. aureus infection model as an index.

(Evaluation of antibacterial activity by minimum growth inhibitory concentration MIC)
Antibacterial activity of the antibiotic-containing fraction at each fractional stage from the culture produced by RH2180-5, and the antibacterial activity of the antibiotic separated and purified therefrom can be evaluated by MIC. MIC is performed according to generally accepted standard methods. For example, it is carried out by a micro liquid dilution method based on CLSI (formerly NCCLS American Clinical Laboratory Standards Committee).

  Moreover, since the judgment criteria of sensitivity differ for every strain, each category of S: sensitivity, I: intermediate, R: tolerance is performed based on the judgment criteria defined by the CLSI.

(About antibiotic-containing fractions)
Here, the “antibiotic-containing fraction” of the present invention obtained by culturing RH2180-5 (Accession No. NITE P-870) and fractionating the culture refers to the culture of RH2180-5, Fractions containing antibiotics that exhibit antibacterial activity and / or therapeutic effects, fractions from which some substances have been separated / extracted from the fractions, and antibiotics in the fractions that have been separated / extracted partially This means any fraction that has been purified to a pure substance and further contains an antibiotic that has been purified to a pure substance.

  In addition, the acetone extract and butanol extract of Table 1 and particularly the butanol extract have the low concentration of antibacterial activity in the subsequent purification process and, conversely, the high concentration of therapeutic effect, as shown in Table 4 below. Think of it as an antibiotic-containing fraction that shows antibacterial activity but no therapeutic effect or a lot of very weak antibiotics compared to the antibiotics contained in the nine peaks (antibiotic-containing fractions) Can do. Such an antibiotic-containing fraction is usually judged to be less useful, but is highly useful for the following reasons, and can be judged as an antibiotic-containing fraction included in the present invention.

  In other words, when antibiotics with high antibacterial activity are used as therapeutic agents for infectious diseases, they should be used after careful examination of their necessity in consideration of the emergence of multidrug-resistant bacteria. . On the other hand, when an antibiotic is used as an antibacterial agent such as a microbial control agent, it is inevitably abused because of its usage, and it is difficult to limit its use. The antibiotic used is not used for microbial control.

  However, the antibacterial activity of the present invention, but the antibiotic-containing fraction containing a large amount of antibiotics evaluated as having no therapeutic effect or weak in the silkworm Staphylococcus aureus infection model and other microbial infection models, and the antibacterial activity therefrom Antibiotics that are shown but have no therapeutic effect or are weak can be used for in vitro applications such as microbial control agents. This is because these antibiotic-containing fractions are not used as an infectious disease therapeutic agent, and thus it is not necessary to consider the emergence of multidrug-resistant bacteria. In the present invention, the term “substantially exhibits no therapeutic effect” although it exhibits antibacterial activity means that it does not exhibit a therapeutic effect that can be used as a therapeutic agent.

  For the separation and purification of antibiotics that do not exhibit a therapeutic effect, the same evaluation method as that for the antibiotics that exhibit the therapeutic effect may be used. Specifically, MIC exhibits antibacterial activity and is used in an infectious disease model. What is necessary is just to select and implement the method by which the antibiotic which has a weak therapeutic effect or does not show the therapeutic effect is separated and purified. Although there is no restriction | limiting in particular in the infectious disease model in this case, The infectious disease model using silkworms, such as a silkworm Staphylococcus aureus infection model like the antibiotics which show a therapeutic effect, can be used conveniently.

  In addition, the separation and purification of antibiotics that do not exhibit therapeutic effects may be performed only for that purpose, but a method that uses the results of examination of methods for separating and purifying antibiotics that exhibit therapeutic effects is more effective. preferable. In other words, the antibiotic-containing fraction is treated with a method of collecting antibiotics that exhibit therapeutic effects, and the residue is subjected to a method of concentrating, separating, and purifying antibiotics that do not exhibit therapeutic effects. Antibiotics that exhibit antibacterial activity but no therapeutic effect can be isolated and purified without further examination.

  Next, the method for separating and purifying antibiotics from the antibiotic-containing fraction will be described by way of an example of the method for separating and purifying antibiotics showing therapeutic effects.

(Separation and purification method of antibiotics)
There are no particular limitations on the separation / purification method selected using the therapeutic effect in the silkworm Staphylococcus aureus infection model as an index, but solvent extraction, phase transfer, water precipitation, chromatography using ODS columns, etc., and ODS columns are used. For example, fractionation by RP-HPLC. 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 filled with a carrier modified with an octyl group or a butyl group, a polystyrene-based polymer carrier, or the like may be used (see Table 1).

  The separation / purification method described above is merely an example, and any separation / purification method may be used as long as the method can finally obtain a target antibiotic-containing fraction or antibiotic.

(Structural analysis of separated and purified antibiotics)
By the above RP-HPLC, a compound divided into nine peaks (corresponding to the antibiotic-containing fraction) 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 separation / purification method prior to RP-HPLC.

  There is no particular limitation on the analysis method of the chemical structure of the antibiotic contained in these peaks, and any method may be used. For example, RP-HPLC of peak 5 which is the main peak among these nine peaks The structural analysis of the purified sample 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, two antibiotics each for Thr, Glu, Glu, and Arg and one molecule each for Ser, Gly, and Ile were detected (FIG. 4), 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 antibiotic contained in each peak can be examined by the MIC described above. Thereby, for example, as shown in Table 3 of Test Example 1, the RH2180-5Peak5 substance exhibited antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus and enterococci. Furthermore, it was confirmed that it has antibacterial activity against not only methicillin-resistant Staphylococcus aureus (MRSA) but also vancomycin-resistant enterococci (VRE) and is effective against multidrug-resistant bacteria (Table 3). reference).

  Antibiotics contained in peak 6 and peak 9 also showed the same antibacterial spectrum as RH2180-5Peak5 substance against MRSA and VRE (see Table 3).

  Furthermore, when antibacterial activity of antibiotics contained in peak 2, peak 3, peak 4, peak 7, peak 8 except peak 1 against multidrug-resistant bacteria and bacteria not showing drug resistance was examined, The antibiotics contained also showed the same antibacterial activity against multidrug-resistant bacteria and bacteria that did not show drug resistance as in peak 5, peak 6, and peak 9, and confirmed that they were not affected by multidrug resistance (See Table 4 of Test Example 2).

  From the above, the antibiotic having therapeutic effect contained in the antibiotic-containing fraction of the present invention is an antibiotic produced by the microorganism obtained from the culture solution of RH2180-5 (Accession No. NITE P-870). Thus, it was confirmed that the antibiotic was characterized by having antibacterial activity against MRSA and VRE.

(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 4, it is confirmed that the substance which shows a therapeutic effect with respect to a silkworm Staphylococcus 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 5 in Test Example 3)

  In addition, as a result of an acute toxicity test on mice, mice were not killed at a dose of 50 mg / kg. Therefore, the RH2180-5 Peak 5 substance is low toxic.

From the above results, the antibiotic RH2180-5Peak5 substance produced by RH2180-5 in the present invention exhibits an excellent ED ₅₀ value against Staphylococcus aureus as compared with vancomycin, exhibits a high therapeutic effect, and is clinically very It has been confirmed that not only MRSA, which is a problem, but also VRE has an excellent feature of showing effectiveness.

  That is, the antibiotic-containing fraction of the present invention produced by RH2180-5 contains at least an antibiotic having a therapeutic effect on infection caused by Staphylococcus aureus, and the antibiotic is used for the above infection. Compared to vancomycin, the therapeutic effect is equivalent or higher.

  In addition, since the substance in the other peak (corresponding to the antibiotic in the antibiotic-containing fraction) also shows the same antibacterial spectrum (confirmed by peak 6 and peak 9) and UV spectrum as RH2180-5Peak5 substance, RH2180 It is considered to be a compound similar to the -5 Peak5 substance. Therefore, it is presumed that the therapeutic effect shows the same tendency as that of the RH2180-5Peak5 substance.

Similar to the purified RP-HPLC sample from peak 5, other peaks were also measured for molecular weight by accurate mass spectrometry, amino acid analysis after acid hydrolysis, analysis by ¹ H-NMR and ¹³ C-NMR, TOF type MS As a result of structural analysis by analysis, UV spectrum, infrared absorption spectrum (IR), it was a compound represented by the following formula (1) and Table 2.

［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２中、「高分解能質量分析」は、ＨＲ ＴＯＦ ＭＳ ｍ／ｚ（Ｍ＋Ｈ）^＋

In Table 2, “high resolution mass spectrometry” is HR TOF MS m / z (M + H) ⁺

  In the present invention, the antibiotics represented by the above formula (1) are preferable, and the antibiotics represented by the above formula (1) are particularly preferably those shown in Table 2 above.

  A method for culturing the above RH2180-5 (Accession No. NITE P-870) and separating and purifying antibiotics having antibacterial activity and / or antibiotics having therapeutic effects on infectious diseases from the culture is useful antibiotics. It can be used as a method for producing a substance. The RH2180-5 (Accession number NITE P-870) has the base sequence of the 16S rRNA region represented by SEQ ID NO: 1 in the Sequence Listing.

  In addition, the method can be used as a method for producing an antibiotic having antibacterial activity against both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).

  Furthermore, the present invention can be used as a method for producing an antibiotic that exhibits antibacterial activity against MRSA and VRE, and at least has a therapeutic effect on infection caused by S. aureus.

  An antibiotic-containing fraction obtained by culturing a microorganism having the deposit number NITE P-870 and fractionating the culture, and a microorganism control agent containing an antibiotic obtained from the culture are useful. It is. A microorganism control agent containing a fraction before being isolated as a substance is also useful.

  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
<Separation 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 “Silkworm model”) 3487 specimens with the above antibacterial activity were used for the silkworm model. As a result, the therapeutic effect was observed in 45 culture supernatants.

(3) Separation and purification of therapeutic effect substances using the therapeutic effect as an index using a silkworm model Among RH2180-5 isolated from soil collected in Okinawa among the 45 strains, the therapeutic active substances from the culture supernatant Purification was performed. RH2180-5 was determined to be a new microorganism belonging to the genus Lysobacter from the analysis of 16S rRNA sequence described later, the chemical structure of the produced antibiotic, the antibacterial spectrum of the produced antibiotic, and the like.

  RH2180-5 was inoculated and cultured in 1200 mL of YME medium, and a method for purifying antibiotics showing the 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 most of the antibacterial activity exhibited by the acetone extract of the culture supernatant, which is the starting material for purification, is derived from substances other than antibiotics that show the therapeutic effect of the final purified product. It is presumed that antibacterial active substances other than the indicated antibiotics 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 above 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 sample was suspended in 80 mL of water, and after centrifugation, water was removed and the precipitate was collected.

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

  The antibiotic-containing fractions showing the above therapeutic effects are pooled and dried, and 22 mg of them are dissolved in 50% methanol, and 75% -95 using a Senshu PaK SP-100 ODS column (diameter 20 mm × length 250 mm). Elute with% 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. It was confirmed (see FIG. 1). 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.

Test example 1
<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 3. 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. In addition, it exhibited an antibacterial activity equivalent to or higher than not only methicillin-resistant Staphylococcus aureus (MRSA) but also vancomycin-resistant enterococci (VRE). The value of this MIC is not a low value compared to a conventionally reported antibiotic, and it does not show a particularly high antibacterial activity. It was confirmed to have a therapeutic effect over vancomycin.

Test example 2
<Comparison study of antibacterial activity of multi-drug resistant bacteria and non-drug resistant bacteria of each peak substance>
A comparative study of antibacterial activity against multidrug-resistant bacteria and bacteria that do not have drug resistance was performed on 8 substances from peak 2 substances to peak 9 substances excluding peak 1 substance with a small amount of sample.
The same staphylococcus aureus and enterococci as in Test Example 1 were selected as test target bacteria, and the MICs indicating the peak substances for the same MSSA1 and EF1, MRSA3, MRSA4, and VRE were also determined by CLSI (formerly NCCLS US Clinical Laboratory Standard Committee). ) Based on the micro sample dilution method. The results are shown in Table 4.

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 itself indicated by each peak substance is a large value compared to the conventionally reported antibacterial agents, EF1 and EF1 and the multidrug-resistant bacteria (MSSA1, MRSA3, MRSA4, In comparison between VRE), MIC showed almost the same value, and it was confirmed that the antibacterial activity of each peak substance was not influenced by multidrug resistance.

It has been confirmed that the antibacterial activity and therapeutic effect of each peak substance do not necessarily match. For example, the RH2180-5Peak5 substance having an MIC against MSSA of 6.3 μg / mL in the case of Table 4 and 5 μg / mL in the case of Table 3 has a therapeutic effect on the mouse S. aureus infection model described later (ED ₅₀ ). The results of this study showed an ED ₅₀ of about 1/3 that of vancomycin, indicating a high therapeutic effect.

  MICs of 5 μg / mL and 6 μg / mL are levels that are considered to be MICs of resistant bacteria in vancomycin, and each peak substance may have a therapeutic effect with a mechanism of action different from that of the conventional one. . When the therapeutic effect is shown with a different mechanism of action from the conventional one, it can be expected that the frequency of resistant bacteria in the future will be low.

Test example 3
<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 strain by the method described 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 ) was examined (5 mice per group).
Similarly, for vancomycin, the ED ₅₀ 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 was determined by the probit method.

The results obtained are shown in Table 5. 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 of} 1.6 mg / kg of vancomycin studied at the same time. It was confirmed that this is a highly therapeutic antibiotic.

In addition, the RH2180-5 Peak5 substance was subcutaneously administered to mice, and the acute toxicity of the RH2180-5 Peak5 substance to the mice was also examined by observation the next day after administration.
As a result, there was no acute toxicity at a dose of 50 mg / kg which corresponds to 80 times the _{ED 50.} Therefore, it was suggested that the RH2180-5 Peak5 substance has low toxicity.

Test example 4
<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.

Test Example 5
<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.

  From these results, the antibiotic-containing fraction obtained by culturing the microorganism with the deposit number NITE P-870 and fractionating the culture, and the antibiotic obtained from the culture, It has been found that the contained microbial control agent is useful. A microbial control agent containing the RH2180-5Peak5 substance is useful, but a microbial control agent containing a fraction other than that of the RH2180-5Peak5 substance and containing a fraction prior to isolation as a substance is also useful. I found out.

Test Example 6
<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.

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, excluding some bases on the 5 ′ end side and 3 ′ end side, was determined. The base sequence is shown in SEQ ID NO: 1 in the sequence listing.
Based on this nucleotide sequence, homology search with existing strains was performed using NCBI BLAST. As a result of the homology search, RH2180-5 was 99% homologous to 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 is different in the antibacterial spectrum of the physiologically active substance produced from the Lysobacter enzyme zymogenes DSM 2043T strain, and is different in that it produces a new and useful substance such as RH2180-5Peak5 that has not been reported so far. . This difference is a big difference. Therefore, from the above results, RH2180-5 was determined to be a novel strain belonging to the genus Lysobacter.

  INDUSTRIAL APPLICABILITY The present invention provides a novel antibiotic-containing fraction and antibiotics contained therein, and an industry capable of providing the antibiotic-containing fraction and a novel and useful method for producing antibiotics obtained therefrom. Has the above applicability.

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

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

Claims (14)

A compound or antibiotic-containing fraction containing a salt represented by the following formula (1), by culturing a microorganism belonging to the Risobakuta (Lysobacter) genus accession number NITE B P-870, Fraction The culture An antibiotic-containing fraction, characterized in that it is obtained by

[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 antibiotic-containing fraction according to claim 1, wherein the antibiotic-containing fraction is a fraction containing an antibiotic exhibiting antibacterial activity.   The antibiotic-containing fraction according to claim 1 or 2, wherein the antibiotic-containing fraction exhibits antibacterial activity at least against both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Minutes.   The antibiotic-containing fraction according to any one of claims 1 to 3, wherein the antibiotic-containing fraction contains at least an antibiotic having a therapeutic effect on an infection caused by Staphylococcus aureus. .   The antibiotic-containing fraction according to claim 4, wherein the antibiotic-containing fraction is a fraction containing an antibiotic that exhibits at least a therapeutic effect equivalent to or higher than that of vancomycin against infection caused by Staphylococcus aureus. .   The antibiotic-containing fraction according to claim 1 or 2, wherein the antibiotic-containing fraction is a fraction containing an antibiotic that exhibits antibacterial activity but does not substantially exhibit a therapeutic effect.   The antibiotic-containing fraction according to claim 6, wherein the antibiotic that exhibits antibacterial activity but does not substantially have a therapeutic effect is used as a microorganism control agent. Compounds represented by the following formula (1) or a method for producing the antibiotic is a salt thereof, by culturing a microorganism belonging to the Risobakuta (Lysobacter) genus accession number NITE B P-870, from the culture, antibacterial activity A method for producing an antibiotic, comprising separating and purifying an antibiotic having a therapeutic effect and / or an antibiotic having a therapeutic effect on an infectious disease.

[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 method for producing an antibiotic according to claim 8, wherein the antibiotic exhibits antibacterial activity at least against both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).   The method for producing an antibiotic according to claim 8 or 9, wherein the antibiotic has a therapeutic effect on at least an infection caused by Staphylococcus aureus.   The method for producing an antibiotic according to claim 8, wherein the antibiotic is an antibiotic that exhibits antibacterial activity but does not substantially exhibit a therapeutic effect. Even without low, shows the antimicrobial activity both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), characterized in that it is a compound or a salt thereof represented by the following formula (1) Antibiotics.

[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 antibiotic according to claim 12, wherein the antibiotic exhibits a therapeutic effect on at least an infection caused by Staphylococcus aureus. The antibiotic according to claim 13 , wherein the antibiotic exhibits at least a therapeutic effect equivalent to or higher than that of vancomycin against an infection caused by Staphylococcus aureus.

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