How to search for antibacterial agents

Abstract

To provide a search method of searching excellent antibacterial agent and antibiotic agent.SOLUTION: There is provided a search method of an antibacterial agent comprising following all steps: a step 1 for spreading a supernatant of a suspension liquid of soil on an oligotrophic solid medium and culturing the supernatant for forming a colony of bacteria A; a step 2 for adding an agar which is a liquid state at a survival temperature of bacteria A on a medium of bacteria B being subjected to sterilization, then adding bacteria B to the acquired liquid agar medium for preparing a layer liquid, then flowing the layer liquid on the solid medium on which the colony of the bacteria A is formed for forming a layer solid medium; a step 3 for detecting an inhibition circle in a plane of grown bacteria B, then selecting, collecting and purifying the bacteria A from the colony of the bacteria A which exist in a substantially center of the inhibition circle; a step 7 for culturing the bacteria A in a liquid medium for preparing a sample, then selecting a sample whose MIC value is 1 / 10 or lower with respect to the bacteria B; and a step 8 for using a silk worm infection model, for selecting a sample having an antibacterial treatment effect to the bacteria B.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for screening antibacterial agents, and more particularly to a method for efficiently screening antibacterial agents derived from soil bacteria. [Background technology]

[0002] Antibiotics, or antibacterial agents, are extremely important in the medical field. However, there is a risk of new infectious diseases emerging, and resistant bacteria that are resistant to existing antibacterial agents have already appeared. Furthermore, there is a high possibility that new resistant bacteria will emerge in the future, which are becoming major social issues. However, conventional methods for discovering antibacterial agents are not cost-effective and are becoming less attractive due to strict pharmaceutical regulations. For these reasons, the discovery and exploration of new antibacterial agents by pharmaceutical companies and other organizations has been slowing down in recent years.

[0003] The conventional discovery method involves isolating bacteria that produce antibacterial agents, culturing the bacteria in pure culture in liquid medium, detecting the antibacterial activity of the substance produced, purifying the substance and determining its structure, and then conducting experiments using animals (such as mice) to confirm its pharmacokinetics, therapeutic effects, efficacy, side effects, etc. In other words, conventional methods are based on the technical idea of ​​finding a bacterium that produces a substance with antibacterial properties, finding a single substance with antibacterial activity produced by that bacterium, and purifying that substance. However, since it is not possible to evaluate multiple bacteria in parallel or multiple produced substances in parallel, this is thought to place limitations on the discovery and search for new antibacterial agents.

[0004] Furthermore, with conventional methods, when a single substance with antibacterial activity is discovered over a long period of time and then purified and evaluated using a large number of mice, it is often rejected at the later stages of investigation and evaluation due to reasons such as poor pharmacokinetics, poor therapeutic effect despite high antibacterial activity, or significant side effects.

[0005] Meanwhile, the present inventors have established a silkworm infection model (Patent Documents 1 and 2, Non-Patent Documents 1 to 3), and have used it to discover antibacterial agents and antibiotics with new backbones (Patent Documents 3 to 7, Non-Patent Documents 4 to 6). Specifically, it has been confirmed that the ED50 value, which is an index of the therapeutic effect of antibacterial agents and antibiotics, is closely consistent between silkworms and mammals (Non-Patent Document 2). This is because the pharmacokinetics of antibiotics in silkworms is common to that in mammals (Non-Patent Document 3).

[0006] In fact, we have already discovered new antibacterial agents and antibiotics with new targets using a silkworm infection model (Non-Patent Document 4). Furthermore, we have discovered a new antibacterial agent and antibiotic called lysocin E, which is effective against MRSA, from a bacterium of the genus Lysobacter isolated from soil in Okinawa (Patent Documents 3-6, Non-Patent Document 5). We have also discovered a new antifungal drug called ASP2397 (Non-Patent Document 6). In this way, the silkworm infection model has been shown to be effective in discovering new antibacterial agents and antibiotics that also have therapeutic effects.

[0007] In recent years, there has been a lack of new antibacterial agents with novel structures, and there is a strong desire to invent new methods for discovering superior antibacterial agents and antibiotics to replace the existing methods that have the major problems mentioned above. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-327964 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-133975 [Patent Document 3] Japanese Patent Application Laid-Open No. 2012-006917 [Patent Document 4] Japanese Patent Application Laid-Open No. 2012-005480 [Patent Document 5] Japanese Patent Application Laid-Open No. 2012-005481 [Patent Document 6] International Publication No. 2011 / 148959 [Patent Document 7] Japanese Patent Publication No. 2016-210776

Non-licensed literature

[0009]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

Non-patent document 6

[0010] The present invention has been made in view of the above-mentioned background art, and an object of the present invention is to provide a method for searching for excellent antibacterial agents and antibiotics. [Means for solving the problem]

[0011] As a result of extensive research into solving the above problems, the inventors of the present invention discovered that when using a silkworm infection model to select a sample containing a "compound that has an antibacterial therapeutic effect against Bacterium B, such as a pathogenic bacterium," if a "sample containing an antibacterial substance" is obtained by a specific method, it is possible to screen multiple types of bacteria and multiple types of antibacterial agents / antibiotics in parallel, and to search for antibacterial agents efficiently and without omissions, thereby arriving at the present invention.

[0012] That is, the present invention provides a method for screening antibacterial agents, which is characterized by comprising all of the following steps 1, 2, 3, 7, and 8. Step 1: A soil fungus colony formation step in which the supernatant of the soil suspension is spread on a sterilized, nutrient-poor solid medium and cultured to form colonies of the fungus A contained in the soil. Step 2: A step of forming a layered solid medium by adding the bacterium B to a liquid agar medium obtained by adding agar that is in a liquid state at "at least one temperature at which the bacterium B does not die when added to the medium" to prepare a layered liquid, and pouring the layered liquid onto the solid medium on which the colonies of bacterium A formed in step 1 above and cooling it to form a layered solid medium. Step 3: A purification step of finding an inhibition zone formed around the colony on the surface of the target bacteria B that has grown over the entire surface of the above-mentioned multilayer solid medium, and selecting and extracting bacteria A from the colony of bacteria A that exists approximately in the center of the inhibition zone, and purifying only bacteria A. Step 7: Bacterium A is cultured in a liquid medium, an organic solvent is added, and the mixture is stirred and mixed. The soluble matter is dried and solidified, and water is added to prepare a sample for measuring the MIC value. A "sample derived from bacteria A" with an MIC value of 1 / 10 or less against bacteria B, which is the target of the antibacterial treatment, is selected (Sample Selection Step A). Step 8: A sample selection step B in which a sample having an antibacterial therapeutic effect against the target bacterium B is selected from the samples selected in step 7 using a silkworm infection model.

[0013] The present invention also provides the above-mentioned method for screening antibacterial agents, which further comprises the following step 4 after the above-mentioned step 3: Step 4: A step of purely culturing the colonies of bacteria A purified in step 3 on a solid medium.

[0014] The present invention also provides the above-mentioned method for screening antibacterial agents, which further comprises the following steps 5 and 6 after step 4: Step 5: A step of preparing a layer solution by adding the bacteria B to a liquid agar medium obtained by adding agar that remains liquid at "at least one temperature at which the bacteria B does not die when added to the medium" to prepare a layer solution for the bacteria B, which is the target of the antibacterial treatment, and pouring the layer solution onto the solid medium in which the bacteria A has been purely cultured in step 4 above and cooling it to form a layer solid medium. Step 6: A step of confirming that an inhibition zone is formed around the pure cultured bacteria A on the surface of the target bacteria B, which has grown over the entire surface of the multilayer solid medium formed on the pure cultured bacteria A.

[0015] The present invention also provides the method for screening an antibacterial agent, wherein the following step a and / or step b is carried out in and / or after step 7: Step a: A step of dissolving the compound in a poorly water-soluble or non-water-soluble organic solvent Step b: Column fractionation step

[0016] The present invention also provides the above-mentioned method for screening antibacterial agents, which further comprises the following step 9 after step 8: Step 9: An antibacterial agent obtaining step of isolating and obtaining an antibacterial agent from the sample selected in step 8. [Effects of the Invention]

[0017] The present invention solves the above-mentioned problems and issues, and enables the search for antibacterial agents and antibiotics that take into account pharmacokinetics in humans from the early search stage. In other words, by using a silkworm infection model and selecting antibacterial agents based on the mortality of silkworms, it is possible to eliminate from the outset as "antibacterial candidate agents" those that will be rejected in later stages of clinical trials (e.g., those with no therapeutic effect), thereby saving costs and time and making the search more efficient.

[0018] Furthermore, by using the silkworm infection model, it is not necessary to narrow down candidate antibacterial substances to a single substance from the initial search stage. Therefore, purification to a single substance and structural determination can be carried out at a later stage once good substances, including those with therapeutic effects, have been narrowed down, thereby saving costs and time. Furthermore, since silkworms are used, which are ethically less problematic than vertebrates (mammals) such as mice, it is possible to evaluate a large number of samples.

[0019] The silkworm infection model allows for the screening and evaluation (searching) of samples containing multiple types of antibacterial compounds or samples containing impurities. To fully utilize this advantage, the present invention has developed and adopted a method for obtaining samples that are particularly suitable for evaluation in the silkworm infection model. In other words, the method of utilizing the inhibition zone in the present invention is extremely well matched to the silkworm infection model for the above reasons, and furthermore, since it captures a large number of antibacterial substances in a crude state without any omissions, the "method having steps 1, 2, 3, 7, and 8" in the present invention is extremely effective.

[0020] The present invention achieves remarkable effects by synergistically combining a nutrient-poor solid medium (soil medium), layered solid medium formation (soft agar screening), and a silkworm infection model (silkworm in vivo evaluation).

[0021] Furthermore, by combining steps 4, 5, and 6, the above-mentioned matching properties and features can be further enhanced, and multiple candidate substances can be efficiently isolated before evaluation using the silkworm infection model. Furthermore, even if bacteria A that do not form inhibition zones are mixed in up to step 3, they can be removed, reducing noise.

[0022] Furthermore, by combining step a and / or step b, for example, if a "substance that suppresses innate immunity" is contaminated in the "substance produced by bacterium A," the substance can be removed. That is, it is possible to eliminate cases in which, despite having antibacterial activity and therapeutic activity against bacterium B, the innate immune activity of the silkworm is reduced due to the contamination with a substance that suppresses the innate immunity of the silkworm, resulting in the subsequent death of the silkworm due to bacterium B. In other words, it is possible to eliminate cases in which the silkworms are killed by other factors when confirming the therapeutic effect of silkworms. Step a and step b can be performed whether the fungus B is a bacterium or a fungus, but the above-mentioned case often occurs when the fungus B is a fungus. Therefore, the addition of step a and / or step b is particularly effective when the fungus B is a fungus.

[0023] In the present invention, the colony of bacteria A, which is a soil bacterium, is used as the central substance (object) of the inhibition zone that is formed when the growth of bacteria B, such as a pathogenic bacterium, is inhibited, so at this stage, neither the bacteria nor the antibiotic is isolated. This allows multiple bacteria A and their products (antibiotics) to be screened simultaneously in parallel, and since neither the bacteria A nor the antibiotics are identified in the early stages of the search, there is no waste, and costs and time are saved.

[0024] The conventional method for discovering antibacterial agents and antibiotics involves isolating the producing bacteria, culturing them in large quantities in liquid medium, detecting the antibacterial activity of the product, and then using mice, etc., to confirm the pharmacokinetics, therapeutic effects, efficacy, side effects, etc. However, this method does not allow for the parallel evaluation of multiple bacteria or multiple produced substances, and further requires purification and isolation of single substances from the initial discovery stage, which has limited the discovery and discovery of new antibacterial agents due to cost and time considerations. In current screening, when soil bacteria are randomly selected and cultured to prepare an extract, it is not easy to obtain a sample with an MIC value of 1 / 10 or less (described below). The present invention was made based on the discovery of the technical concept of collecting antibiotic-producing bacteria that form inhibition zones from soil, etc., in the early steps of steps 1, 2, and 3.

[0025] The method of the present invention, which includes steps 1, 2, 3, 7, and 8, and further includes a method in which these steps are combined with steps 4, 5, and 6 and / or step 9, overcomes all of the drawbacks of the conventional methods described above, and enables the discovery of excellent antibacterial agents with extremely high efficiency.

[0026] According to the method of the present invention, which includes steps 1, 2, 3, (4), (5), (6), 7, (a), (b), 8, and (9) (where the parentheses indicate preferred embodiments), a large number of types of antibacterial agents or antibiotics can be used in small amounts in the initial search (and mixtures may also be used). The first priority is to identify Bacterium A that produces an excellent antibacterial substance and to confirm the very existence of said antibacterial substance. The present invention focuses on this point, allowing for extensive and thorough search and selection of antibacterial agents and antibiotics.

[0027] Considering actual use on humans, it is desirable that the antibacterial agent be water-soluble. In step 7, by adjusting the type of organic solvent used to dissolve and extract the antibacterial agent and its mixing ratio with water, it is possible to extract water-soluble substances (antibacterial agents), and efficient screening is also carried out here. Furthermore, the formation of an inhibition zone means that the antibacterial agent or antibiotic that caused the inhibition zone to form is water-soluble. Therefore, screening is also carried out efficiently here (in step 3).

[0028] Specifically, by carrying out step 1, it is possible to efficiently evaluate a large number of bacteria over a wide range, and it is also possible to find (select) difficult-to-culture bacteria and minor bacteria that have previously been overlooked. Furthermore, in steps 2 and 3, an inhibition zone is not formed (utilized) by a "single identified antibacterial agent," so efficient searching is possible without identifying either bacterium A or the antibacterial agent, i.e., purification and identification can be postponed.

[0029] In addition, in step 7, screening based on the smallness of the MIC value is important, but as long as the MIC value, which is the dilution ratio, is taken into consideration, the crude product can be advanced to the "evaluation stage using the silkworm infection model," which is extremely efficient. Furthermore, the silkworm infection model in step 8 synergistically exerts the above-mentioned excellent effects due to the fact that it allows testing even with crude samples and uses silkworms, which have few ethical issues.

[0030] As mentioned above, the silkworm infection model makes it possible to screen for antibacterial agents with therapeutic effects at an early stage of research, reducing the number of substances that are discarded due to poor pharmacokinetics, etc., at later stages of investigation such as clinical trials. Therefore, the silkworm infection model itself is an excellent method for researching antibacterial agents and antibiotics.

[0031] In the present invention, the nature of the samples used in the "test using the silkworm infection model" and the method for obtaining and selecting the samples have been further investigated and improved. As described above, the sample obtaining and selection method of the present invention (steps 1 to 7) is extremely well-suited for the silkworm infection model, making the most of the features of the silkworm infection model. Therefore, the silkworm infection model has been refined by the present invention.

[0032] As a method for selecting samples to be used in "screening (searching) for 'antibacterial agents with therapeutic effects' using a silkworm infection model," a comparison is shown in Figure 1 between using steps 1, 2, 3, (4, 5, 6), 7, and (9) of the present invention and not using the above steps of the present invention, depending on the silkworm infection model. In Figures 1(a), (b), and (c), the populations used for the search are different, namely, "compound library," "natural products," and "soil," respectively. However, as shown in Figure 1(c), the probability of discovering "antibiotics with therapeutic effects" using the present invention is far higher than that in Figures 1(a) and (b), and it can be said that the present invention is the first to make this search method practical.

[0033] Specifically, as described in the Examples, acetone extracts of the culture medium were prepared from 791 strains (up to step 6) that formed an inhibition zone against Staphylococcus aureus, and 20 samples (up to step 8) that had a therapeutic effect on silkworms were obtained (Figure 1(c)). This is a discovery probability that is 1,000 times higher than when searching from a compound library (Figure 1(a)) and 10 times higher than when searching from natural products (Figure 1(b)).

[0034] Furthermore, as described in the Examples, acetone extracts of culture medium were prepared from 329 strains (up to step 6) that formed inhibition zones against E. coli W3110, and 18 samples (up to step 8) with anti-Pseudomonas aeruginosa therapeutic effects in silkworms were obtained (Figure 1(c)). Until now, no substances with therapeutic effects against Gram-negative bacteria have been discovered (Figure 1(a)). Therefore, the present invention is effective as a method for searching (screening) for substances with anti-Gram-negative bacterial therapeutic effects.

[0035] In the present invention, screening is performed taking into account not only the MIC value, which is an index of antibacterial activity, but also the therapeutic effect using a silkworm infection model. Therefore, it is possible to pick up all of the bacteria A (its products) (antibacterial drug candidates) that have previously been missed from screening because they have a slightly high MIC value (small denominator), i.e., slightly low antibacterial activity, despite having a therapeutic effect. Furthermore, since the present invention allows the search for novel antibacterial agents, the searched for antibacterial agents are highly likely to exhibit antibacterial activity against bacteria B that have acquired resistance to existing antibacterial agents (resistant bacteria B). Therefore, the present invention makes it possible to search for antibacterial agents for resistant bacteria. [Brief explanation of the drawings]

[0036] [Figure 1] This is a table showing the difference in the discovery efficiency of antibacterial substances depending on the selected (obtained) form of the "sample to be administered to silkworms" when using a silkworm infection model. (a) Discovery efficiency using the conventional method without steps 1 to 8 when starting from a compound library. (b) Discovery efficiency using the conventional method without steps 1 to 8 when starting from natural products. (c) Discovery efficiency using the method of the present invention with steps 1 to 8 when starting from soil. [Figure 2] 1 is a process flow diagram of an example of a preferred embodiment of the present invention. [Figure 3] 1 is a photograph showing an example of a colony of soil bacterium A formed on a nutrient-poor solid medium in step 1. [Figure 4] This is a photograph showing an example of an inhibition zone formed around a colony of soil bacteria A against Staphylococcus aureus (bacteria B) in step 3. [Figure 5] 10 is a photograph showing an example of an inhibition zone formed around a colony of soil bacteria A against Escherichia coli (bacterium B) in step 3. [Figure 6] This is a photograph showing an example of an inhibition zone formed around a colony of soil bacteria A against baker's yeast (bacterium B) in step 3. [Figure 7] 10 is a photograph showing an example of an inhibition zone formed around a colony of soil bacteria A against Staphylococcus aureus (bacteria B) in step 6. [Figure 8] 10 is a photograph showing an example of an inhibition zone formed around a colony of soil bacteria A against Escherichia coli (bacterium B) in step 6. [Figure 9] This is a photograph showing an example of an inhibition zone formed around a colony of soil bacteria A against baker's yeast (bacterium B) in step 6. [Figure 10]FIG. 3 is a process flow diagram of one example of a preferred embodiment of the present invention, which is a flow diagram in which butanol transfer (step a) and column fractionation (step b) are further performed in addition to the flow diagram of FIG. 2. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention will be described below, but the present invention is not limited to the following specific embodiments and can be modified as desired within the scope of the technical concept.

[0038] The method for screening antibacterial agents of the present invention comprises at least the following steps: Steps 4 to 6 and Step 9 are not essential but are preferably included. Step 1: A soil fungus colony formation step in which the supernatant of the soil suspension is spread on a sterilized, nutrient-poor solid medium and cultured to form colonies of the fungus A contained in the soil. Step 2: A step of forming a layered solid medium by adding the bacterium B to a liquid agar medium obtained by adding agar that is in a liquid state at "at least one temperature at which the bacterium B does not die when added to the medium" to prepare a layered liquid, and pouring the layered liquid onto the solid medium on which the colonies of bacterium A formed in step 1 above and cooling it to form a layered solid medium. Step 3: A purification step of finding an inhibition zone formed around the colony on the surface of the target bacteria B that has grown over the entire surface of the above-mentioned multilayer solid medium, and selecting and extracting bacteria A from the colony of bacteria A that exists approximately in the center of the inhibition zone, and purifying only bacteria A. Step 4: A step of purely culturing the colonies of bacteria A purified in step 3 on a solid medium. Step 5: A step of preparing a layer solution by adding the bacteria B to a liquid agar medium obtained by adding agar that remains liquid at "at least one temperature at which the bacteria B does not die when added to the medium" to a medium for the bacteria B to be treated with the antibacterial agent, and then pouring the layer solution onto the solid medium in which the bacteria A has been purely cultured in step 4 above and cooling it to form a layer solid medium. Step 6: A step of confirming that an inhibition zone is formed around the pure cultured bacteria A on the surface of the target bacteria B, which has grown over the entire surface of the multilayer solid medium formed on the pure cultured bacteria A. Step 7: Bacterium A is cultured in a liquid medium, an organic solvent is added, and the mixture is stirred and mixed. The soluble matter is dried and solidified, and water is added to prepare a sample for measuring the MIC value. A "sample derived from bacteria A" with an MIC value of 1 / 10 or less against bacteria B, which is the target of the antibacterial treatment, is selected (Sample Selection Step A). Step 8: A sample selection step B in which a sample having an antibacterial therapeutic effect against the target bacterium B is selected from the samples selected in step 6 using a silkworm infection model. Step 9: An antibacterial agent obtaining step of isolating and obtaining an antibacterial agent from the sample selected in step 7 above.

[0039] <Process 1> Step 1 is a soil bacterial colony formation step in which the supernatant of a suspension of soil S is spread on a sterilized, nutrient-poor solid medium and cultured to form colonies of bacteria A contained in the soil S. It should be noted that when "bacteria A" is written, the type of bacteria may be one type or two or more types at the same time. The letter "A" is used merely to distinguish "bacteria A," which is a soil bacterium, from "bacteria B," which is the target of antibacterial treatment. When "bacteria A" is written, the bacteria is not limited to one type, and may be multiple types. In fact, it is preferable that multiple types are present simultaneously up to step 3 or before step 6. The "suspension of soil S" is preferably a suspension prepared by adding sterilized water to soil S.

[0040] The solid medium is not particularly limited, but an agar medium is preferred. The term "poor nutrition" as used above refers to a lack of nutrients that prevents bacteria with high growth rates from forming large colonies and prevents bacteria with low growth rates from forming colonies (does not inhibit the colony formation of other bacteria), and refers to the absence of active addition of so-called "nutrients" when forming the solid medium.

[0041] When using conventional nutrient media such as YME agar, the growth rate varies significantly depending on the strain, resulting in the formation of large colonies that inhibit the colony formation of other strains. Furthermore, simply lowering the concentration of nutrients can limit the types of colonies that appear. By using a nutrient-poor solid medium, it became possible to simultaneously obtain a large number of small colonies (preferably of various types), resulting in an ideal shape for the object (colony) present approximately in the center of the inhibition zone in step 3.

[0042] The size of the colonies of Bacterium A formed in step 1 preferably has an average diameter of 0.5 mm to 10 mm, more preferably 1 mm to 5 mm, and particularly preferably 2 mm to 3 mm.

[0043] If the colonies of Bacterium A are too large, it may be possible to evaluate only a small number of colonies or Bacterium A in one container (such as a petri dish), or it may be impossible to obtain an inhibition zone with a suitable diameter or shape in step 3. On the other hand, if the colony of Bacterium A is too small, Bacterium A that should have been selected may not be able to produce a sufficient amount of antibacterial agent, and may not be able to form an inhibition zone in step 3.

[0044] A particularly preferred nutrient-poor solid medium is one obtained by adding water to soil R, stirring thoroughly, sterilizing using an autoclave or the like, and then adding solid medium materials such as agar to the supernatant (hereinafter sometimes referred to as "soil extract") and allowing it to solidify. In the method for screening antibacterial agents of the present invention, the "poorly nutrient solid medium" preferably contains a soil extract.

[0045] The soil R for preparing the solid medium may be the same as or different from the soil S containing the bacteria A that produce the antibacterial agent to be searched for. Although there is no particular limitation, the supernatant of a mixed suspension of different soils S, S', S"... may be spread on one petri dish, and in that case, it is preferable that the soil R is the same as any one of the soils S, S', S"... When the supernatant of a suspension of one type of soil S is spread in one petri dish, there are no particular limitations, but it is particularly preferable that the soil R is soil S. In other words, it is particularly preferable to use the same soil.

[0046] In the present invention, "soil" refers to a naturally occurring solid substance as a whole, and includes a mixture of inorganic matter such as minerals, rocks, and sand; organic matter such as biological products, excrement, corpses, and corroded substances; natural liquids such as petroleum, petroleum precursors, petroleum spring water, mineral water, hot spring water, river water, lake water, and seawater; and living organisms (living organisms). It includes not only soil that exists on the ground, but also soil that exists at the bottom of bodies of water such as rivers, lakes, and seas.

[0047] Soil contains inorganic and organic substances specific to that soil, and if the inorganic and organic substances contained in soil S, which contained bacteria A that produces the antibacterial agent being sought, are the same as those contained in soil R, which is the raw material for the soil extract of the fixed medium, then this will be a suitable culture environment for bacteria A. In particular, if the mineral components, trace element components, metal (ion) components, acid-alkali components, etc. are the same, this is thought to be a more suitable culture environment for bacteria A. This makes it possible to easily use even a small amount of bacteria A contained in the soil S or bacteria A that are difficult to form colonies as colonies (i.e., antibiotic-producing substances) without leakage.

[0048] The sterilization treatment in step 1 can be carried out by a known method (conventional method) such as autoclaving. Alternatively, the supernatant of a suspension prepared by adding water to soil S can be spread and cultured to form soil bacterial colonies by a known method.

[0049] That is, the supernatant is preferably diluted with sterilized physiological saline, sterilized distilled water, sterilized deionized water, or the like and spread on the medium. Sterilization is not necessarily required for the purpose of obtaining antibiotic-producing bacteria, but if the medium is not sterilized, the route by which the obtained antibiotic-producing bacteria were obtained becomes unclear. The dilution ratio depends on the amount of water added when preparing the suspension of soil S (concentration of the suspension), but when calculated by adding the same mass of water as soil S to prepare the suspension, the dilution ratio is preferably 10 to 10,000 times, more preferably 50 to 5,000 times, and particularly preferably 100 to 2,000 times.

[0050] The culture conditions for forming colonies of Bacterium A are not particularly limited, but a temperature of 4° C. to 40° C. is preferred, and a temperature of 27° C. to 32° C. is particularly preferred. The culture time is not particularly limited, but a temperature of 24 hours to 120 hours is preferred, and a temperature of 48 hours to 96 hours is particularly preferred.

[0051] <Process 2> Step 2 is a step of forming a layer solid medium, in which a layer liquid is prepared by adding the bacteria B that are the target of the antibacterial treatment to a "liquid agar medium obtained by adding agar that is in a liquid state at 'at least one temperature at which the bacteria B that are the target of the antibacterial treatment do not die when added to the medium'" and pouring the layer liquid onto the solid medium on which the colonies of bacteria A have formed in step 1 above and cooling it to form a layer solid medium.

[0052] Bacterium B, which is the target of antibacterial treatment, is preferably pathogenic, and unlike bacteria A in steps 1 to 3, is usually of a single type. Bacterium B in the "method for searching for antibacterial agents" of the present invention is preferably a pathogenic microorganism, and particularly preferably a pathogenic bacterium or fungus. Furthermore, as for Bacterium B, not only bacteria that have not acquired resistance to existing antibacterial agents, but also bacteria that have acquired resistance or low to moderate resistance are particularly preferred in the present invention.

[0053] Bacterium B in step 2 and bacterium B in step 8 described below may be different as long as their antibacterial mechanisms are considered to be similar. This is because they are considered to share effective antibacterial agents, and the screening results are considered to be similar or identical.

[0054] In order to pour the "stratification liquid containing bacteria B" for forming a solid stratification medium onto the solid medium on which bacteria A colonies have formed in step 1 above, the stratification liquid must be liquid at a temperature at which bacteria B present in the stratification liquid do not die. Therefore, the agar used here must be an agar that is liquid when added to the medium for bacteria B at at least one temperature at which bacteria B do not die.

[0055] The temperature of the stratification liquid when actually pouring it is above the "melting point of the agar or the melting point of the stratification liquid" and below the "maximum temperature at which bacteria B does not die." Here, the "melting point" refers to the lowest temperature within the temperature range at which the bicarbonate solution has sufficient fluidity to be poured into a container such as a petri dish. Furthermore, when simply written as "agar," it also means agarose (purified agar, etc.), and also includes agarose mixed with other substances to form what is known as "agar."

[0056] When the bacterium B is a bacterium that can survive at 56°C, such as Staphylococcus aureus, the agar used in step 2 and / or step 5 is preferably agar with a melting point of 30°C or higher and 60°C or lower, more preferably agar with a melting point of 30°C or higher and 56°C or lower, and particularly preferably agar with a melting point of 30°C or higher and 53°C or lower. When bacterium B is a bacterium other than Staphylococcus aureus, such as Escherichia coli or Saccharomyces cerevisiae, which can survive at 45°C but cannot survive at 55°C, agar with a melting point of 28°C or higher and 50°C or lower is preferred, and agar with a melting point of 30°C or higher and 45°C or lower is particularly preferred. If the agar is within the above melting point range, the layer solution containing Bacterium B satisfies both the above immortal conditions for Bacterium B and the liquid conditions for the layer solution.

[0057] For example, if bacterium B is Staphylococcus aureus, S. aureus can survive at 58°C, so agar with a melting point of 56°C can be used. However, if bacterium B is Escherichia coli or baker's yeast, E. coli and baker's yeast survive at 45°C but die at 58°C. Therefore, if, for example, "agar with a melting point of 56°C is used and the layer solution is heated to 58°C" and poured into a petri dish, the bacterium will die, and an inhibition zone cannot be formed using the process described below.

[0058] As long as the above conditions are met, there are no particular limitations on the agar or agarose used, but what is called low-melting point agar, soft agar, etc. are preferably used. Hereinafter, steps 2 and 5, which use agar or the like, may be abbreviated as "soft agar overlay."

[0059] The agar medium obtained by adding agar to the medium for bacteria B is in a liquid state (at the temperature at that time), and it is preferable to sterilize it using an autoclave or the like before adding bacteria B to prepare a stratification solution, in order to eliminate bacteria other than the ``bacterium B that is the target of antibacterial treatment.''

[0060] The layer solution obtained as described above is poured onto the solid medium on which the colonies of Bacterium A have formed in step 1, and cooled to form a solid layer medium. After pouring, the mixture is cooled to form a solid layer medium, but the physical properties, concentration, and composition of the layer liquid (or solid layer medium) are adjusted so that it remains solid or non-fluid at the subsequent incubation temperature (the temperature at which bacteria B grows).

[0061] The thickness of the solid overlay medium obtained by the overlay liquid alone is preferably 0.5 mm to 10 mm, more preferably 1 mm to 5 mm, and particularly preferably 2 mm to 3 mm. If the thickness of the overlay solid medium is too thin, the amount of bacteria B may be insufficient, making it difficult for bacteria B to grow over the entire surface of a container such as a petri dish, and in such cases, an inhibition zone may not be observed. On the other hand, if the thickness of the overlay solid medium is too thick, the antibiotic secreted from the producing bacteria may be diluted by diffusion, making it impossible to observe an inhibition zone.

[0062] <Process 3> Step 3 is a purification step of bacteria A, in which an inhibition zone formed around the colony is found on the surface of the target bacteria B that has grown all over the surface of the multilayer solid medium, and bacteria A is selected and isolated from the colony of bacteria A that exists approximately in the center of the inhibition zone, and bacteria A is purified to obtain only bacteria A. Note that when "bacterium A" is written, the bacteria A may be one type or multiple types.

[0063] Prior to step 3, incubation is carried out to grow bacteria B. The temperature varies depending on the type of bacteria B, but is preferably 4°C or higher and 40°C or lower, and particularly preferably 30°C or higher and 37°C or lower. If the temperature is too low, bacteria B may not grow over the entire surface, and an inhibition zone may not form. On the other hand, if the temperature is too high, the multilayer solid medium using agar with a low melting point may become fluid, and bacteria B may not grow, making it impossible to observe an inhibition zone.

[0064] The incubation time varies depending on the type of bacterium B, but is preferably 6 hours to 48 hours, and particularly preferably 12 hours to 24 hours. If the time is too short, the bacterial growth may be insufficient and the inhibition zone may not be observable, whereas if the time is too long, the formed inhibition zone may become unobservable due to bacterial growth.

[0065] Once the inhibition zone is found, bacteria A is selected and collected from the "colony of bacteria A" that exists approximately in the center of the inhibition zone. The collected bacteria A may contain a small amount of overlay solid medium or antibiotic non-producing bacteria. These will be removed in the purification process described below.

[0066] There are no particular limitations on the solid medium used for purifying Bacterium A, and general-purpose medium such as YME agar medium can be used. There are also no particular limitations on the growth conditions, and the purification can be carried out by a conventional method.

[0067] <Step 4> Step 4 is a step of purely culturing the colonies of Bacterium A purified in Step 3 above on a solid medium. Step 4 is not essential, but is preferable because there may be cases where bacteria without antibacterial properties (bacteria that cannot form an inhibition zone) are mixed in. Note that "Step 4" or "Steps 4 to 6" can be repeated.

[0068] The solid medium used in step 4 is not particularly limited, and a general-purpose medium such as YME agar medium can be used. The growth conditions are also not particularly limited, and can be carried out by a conventional method.

[0069] <Process 5> Step 5 is a step of preparing a layer solution by adding the bacteria B, which is the target of the antibacterial treatment, to a liquid agar medium obtained by adding agar that is in a liquid state at 'at least one temperature at which the bacteria B, which is the target of the antibacterial treatment, does not die when added to the medium', and pouring the layer solution onto the solid medium in which bacteria A has been purely cultured in step 4 above and cooling it to form a layer solid medium.

[0070] As mentioned above, the "bacteria A" selected and collected in step 3 often contains a mixture of multiple species of bacteria, and some of these multiple types of "bacteria A" do not form inhibition zones. It is considered that the colonies of bacteria A formed in step 1 contain a mixture of bacteria A that cannot form inhibition zones and bacteria A that do form inhibition zones, and that inhibition zones may be formed. Step 5 is performed to eliminate "bacteria A that cannot form an inhibition zone" that was mixed in with "bacteria A that formed an inhibition zone" at the stage of step 3. Note that "steps 5 and 6" can be repeated.

[0071] In step 5, the type of agar, preparation conditions for the overlay solution, type of bacteria B, concentration of bacteria B, temperature conditions, thickness of the solid overlay medium, incubation conditions for the solid overlay medium, etc. do not need to be exactly the same as in step 2, but the preferred ranges, etc. are the same as those in step 2 described above.

[0072] <Process 6> Step 6 is a step of confirming that an inhibition zone is formed around the pure cultured bacteria A on the surface of the bacteria B, which is the target of the antibacterial treatment and has grown over an entire surface on the layer solid medium formed on the pure cultured bacteria A.

[0073] Prior to step 6, incubation is performed to grow the bacterium B. The incubation conditions may be the same as or different from the incubation conditions performed prior to step 3, but the range of conditions is the same as the range of conditions for the incubation performed prior to step 3 described above.

[0074] In the present invention, the bacteria referred to as "bacterium A" is not limited to one type and may be a mixture of multiple types, but in step 6, "bacterium A that does not form an inhibition zone against bacterium B" are eliminated, and only bacteria A that form an inhibition zone against bacterium B are isolated, purified, and selected. In other words, "bacterium A that does not produce an antibacterial agent" that was selected and collected in step 3 is eliminated.

[0075] <Process 7> Step 7 is a sample selection step A in which bacterium A is cultured in a liquid medium, an organic solvent is added and mixed by stirring, the soluble matter is dried to solid, water is added to prepare a sample for measuring the MIC value, and a "sample derived from bacterium A" is selected that has an MIC value of 1 / 10 or less against the target bacterium B. Step 7 is a step in which antibacterial activity is evaluated and samples are selected using antibacterial activity as an index. The bacteria A used in step 7 is the bacteria A that formed an inhibition zone (purified) in step 3.

[0076] The liquid medium used in step 7 is not particularly limited, and any known medium can be used. In addition, it is preferable that the liquid medium is "nutritious" rather than "poorly nutritious" as in step 1. A preferred liquid medium is, for example, YME liquid medium.

[0077] The organic solvent is not particularly limited as long as it dissolves the antibacterial agent or antibiotic that is the target component present in the liquid medium. However, it is preferable that the organic solvent is compatible with water (contained in the liquid medium) at the ratio used, or is compatible to some extent with water. Specific examples include ketones such as acetone and methyl ethyl ketone (MEK); alcohols such as methanol, ethanol and propanol; esters such as ethyl acetate; chlorine-based solvents such as chloroform; hydrocarbons such as hexane; etc. Particularly preferred are acetone, methanol, ethanol, etc.

[0078] The "MIC value" here is defined as follows: The colony of Bacterium A obtained in the step preceding step 7 is cultured in 20 mL of YME liquid medium at 30°C for 4 days, to which an equal volume of acetone is added and centrifuged to obtain a supernatant. The supernatant is evaporated to obtain a solid component, which is then dissolved in 1 mL of pure water, and the resulting solution is used as the "sample for measuring the MIC value." Next, the sample liquid is diluted with pure water in stages, starting from a dilution rate of 10 times, to prepare multiple test liquids with different dilution rates. The antibacterial properties of the test liquid are measured by measuring the decrease in turbidity and the decrease in viable cell count against the target bacterium B using a suitable method. The reciprocal of the maximum dilution rate exhibiting antibacterial activity obtained by the above method, i.e., the smallest value among the "reciprocals of dilution rates exhibiting antibacterial activity," is defined as the "MIC value."

[0079] In step 7, samples with an "MIC value" defined above of 1 / 10 or less are selected as "samples derived from bacteria A." In other words, only samples that show antibacterial activity even when the "sample for measuring the MIC value" is diluted 10 times or more are selected.

[0080] It goes without saying that the above-mentioned sample solution preparation method and antibacterial activity measurement method are preparation and measurement methods used to define the "MIC value" of the present invention, and that the sample preparation method and measurement method used to actually practice the present invention are not limited to the above-mentioned "method used to define the MIC value." In other words, regardless of the preparation method or measurement method actually used, any obtained value that falls within the range of the "MIC value defined above" is within the technical scope of the present invention.

[0081] The selection criterion is to select "samples derived from Bacterium A" with an MIC value of 1 / 10 or less. This is based on the principle that when 50 μL of sample is injected into silkworms (5th instar silkworms weighing 2 g) with a blood volume of 500 μL, it is diluted 10-fold. It is not necessary to select all samples with an MIC value of 1 / 10 or less, but it is most preferable to select all of them. The selection criteria may be tightened from this stage. In this case, the threshold MIC value used as the selection criteria is preferably set to a value of 1 / 1000 to 1 / 10 or less, more preferably a value of 1 / 500 to 1 / 10 or less, and particularly preferably a value of 1 / 100 to 1 / 10 or less.

[0082] If the above value is too small, there are cases where actually excellent antibacterial agents are screened out in step 7, or where the screening is too strict and the number of samples taken up until evaluation in the silkworm infection model in step 8 is too small. On the other hand, if the above value is too large, there are cases where agents that do not have practical antibacterial activity are selected, or where the screening is too lenient and the samples taken up in step 8 contain a lot of noise.

[0083] The sample solution to be injected into silkworms is preferably approximately 50 μL, taking into account the body fluids and body weight of the silkworms. The blood (body fluid) of 5th instar, 1-day-old silkworms used in infection treatment experiments is approximately 500 μL per silkworm. Therefore, the sample solution is diluted 1 / 10 in the silkworm blood (body fluid). Therefore, the antibacterial activity of the sample used in infection treatment experiments must be 1 / 10 or less in terms of MIC value. Samples with higher MIC values ​​cannot be expected to be therapeutically effective. In cases where antibacterial activity is enhanced by substances derived from body fluids, therapeutic effects may be obtained even with MIC values ​​greater than 1 / 10, but such cases are unusual.

[0084] <Common to process a and process b> Step a is a step of dissolving the compound in a slightly water-soluble or water-insoluble organic solvent. Step b is a column fractionation step. Step a and / or step b are performed "in step 7, i.e., during step 7" and / or "after step 7." Here, "performed after step 7" includes not only performing the step after step 7, but also performing the step after step 8 described below. Hereinafter, "step a and / or step b" may be abbreviated as "step ab" regardless of the order of step a and step b (they may be reversed).

[0085] Step ab may be performed using a sample prepared in step 7 by culturing bacterium A in a liquid medium, adding an organic solvent such as acetone, stirring and mixing, drying the soluble matter, and adding water (hereinafter, this procedure may be simply abbreviated as "acetone extraction," regardless of whether it is acetone or not); or it may be performed after measuring the MIC value of a "sample prepared by adding an organic solvent, stirring and mixing, drying the soluble matter, and adding water to dissolve it."

[0086] The MIC value may be measured every time step a or step b is performed, or may be measured before or after step ab. In step 7, it is preferable to measure the MIC value after step ab is performed. Furthermore, the therapeutic effect of step 8 may be measured every time step a or step b is performed, or may be measured before or after step ab is performed. If the MIC value measurement in step 7 and the therapeutic effect measurement in step 8 are performed frequently during step 7 and step ab, the accuracy of antibacterial agent search will increase, and by identifying samples that are not expected to have a therapeutic effect at an early stage, the number of samples to be subjected to the subsequent steps will be reduced, leading to a reduction in time.

[0087] Although not limited thereto, preferred examples include the following: Note that, hereinafter, when only one of step a and step b is described, they can be interchanged, and when both are described, the order can be reversed. Acetone extraction ⇒ Step a ⇒ Step b ⇒ MIC value measurement ⇒ Step 8 Acetone extraction ⇒ Step a ⇒ MIC value measurement ⇒ Step b ⇒ MIC value measurement ⇒ Step 8 Acetone extraction ⇒ Step a ⇒ MIC value measurement ⇒ Step 8 Acetone extraction ⇒ MIC value measurement ⇒ Step a ⇒ Step b ⇒ MIC value measurement ⇒ Step 8 Acetone extraction ⇒ MIC value measurement ⇒ Step a ⇒ MIC value measurement ⇒ Step b ⇒ MIC value measurement ⇒ Step 8 Acetone extraction ⇒ MIC value measurement ⇒ Step 8 ⇒ Step a ⇒ MIC value measurement ⇒ Step 8 Acetone extraction ⇒ MIC value measurement ⇒ Step 8 ⇒ Step a ⇒ MIC value measurement ⇒ Step 8 ⇒ Step b ⇒ MIC value measurement ⇒ Step 8 Acetone extraction ⇒ MIC value measurement ⇒ Step 8 ⇒ Step a ⇒ Step b ⇒ MIC value measurement ⇒ Step 8

[0088] Although not limited thereto, a particularly preferred procedure is acetone extraction ⇒ MIC value measurement ⇒ step a ⇒ MIC value measurement ⇒ step b ⇒ MIC value measurement ⇒ step 8.

[0089] In the present invention, step a and step b are not essential, but by performing them, samples with therapeutic effects can be consistently picked up without omission in step 8. In other words, "sensitivity" is improved.

[0090] The "sample obtained in step 3 or step 6" and the "sample prepared in step 7 by adding an organic solvent, stirring and mixing, drying the soluble matter, and adding water to measure the MIC value" may contain "substances that suppress natural immunity" or "toxic substances" to silkworms. In such cases, it is preferable to perform step a and / or step b to remove the substances.

[0091] In the case of such a sample, even if the therapeutic effect is subsequently confirmed using silkworms in step 8, the silkworms may die not because of Bacterium B but because of the above-mentioned substance. That is, even if the injected Bacterium B actually has therapeutic activity against Bacterium B, it is conceivable that the silkworms may die from the injected Bacterium B due to, for example, the contamination with a substance that suppresses the innate immunity of the silkworms. Such cases can be eliminated by performing step a and / or step b to remove "substances that suppress innate immunity" or "toxic substances." In other words, it is possible to eliminate cases in which the silkworms are killed by other factors when confirming the therapeutic effect using silkworms.

[0092] Steps a and b can be performed whether the microorganism B is a bacterium or a fungus (see Example 1), but are particularly preferably performed when the microorganism B is a fungus (see Example 2).

[0093] <Process a> Step a is a step of dissolving the compound in a slightly water-soluble or water-insoluble organic solvent. An organic solvent (such as acetone) is added, stirred, and mixed, and the soluble portion is dried to dryness. To the resulting mixture, water is added to prepare a sample. To this, a "poorly water-soluble or water-insoluble organic solvent" is added, and by stirring, for example, substances that are more soluble in the "poorly water-soluble or water-insoluble organic solvent" than in "water" are dissolved in the "poorly water-soluble or water-insoluble organic solvent." For example, the liquids are separated using a separatory funnel or centrifuge, and the "poorly water-soluble or water-insoluble organic solvent" layer is separated and collected.

[0094] Here, the "slightly water-soluble or water-insoluble organic solvent" is not particularly limited, but examples thereof include alcohols such as n-butanol, sec-butanol, tert-butanol, pentanol, hexanol, and cyclohexanol; ketones such as methyl ethyl ketone (MEK) and tert-butyl methyl ketone; esters such as ethyl acetate and butyl acetate; chlorinated solvents such as chloroform and dichloromethane; aliphatic hydrocarbons such as hexane and heptane; and aromatic hydrocarbons such as toluene and xylene. More preferred are n-butanol, ethyl acetate, chloroform, toluene, etc., and particularly preferred is n-butanol, etc.

[0095] Since antibacterial agents are generally highly oil-soluble and (natural) immunosuppressants are generally highly water-soluble, it is conceivable that step a removes the (natural) immunosuppressants dissolved in the aqueous phase, and only the antibacterial agents dissolved in the "poorly water-soluble or water-insoluble organic solvent" are obtained.

[0096] <Process b> Step b is a column fractionation step. The column to be used is not particularly limited, but examples include ODS C8 and C18. The elution solvent is not particularly limited, but an alcohol-based solvent is preferred.

[0097] <Process 8> Step 8 is a sample selection step B in which a sample having an antibacterial therapeutic effect against the target bacterium B is selected from the samples selected in step 7 above using a silkworm infection model.

[0098] The silkworm infection model is a method in which a sample containing a candidate antibacterial substance and the bacteria that are the target of the antibacterial treatment are administered to silkworms, and antibacterial agents (containing samples) are screened from the candidate antibacterial agents based on criteria such as the death, discoloration, and weakness of the silkworms. The silkworm infection model is clearly defined and known to be effective, as described in, for example, Patent Documents 1 and 2 and Non-Patent Documents 1 to 3. Furthermore, it has been confirmed that the LD50, ED50, etc. of the silkworm infection model are similar to those of mammals such as mice.

[0099] The samples selected (screened) using the silkworm infection model contain antibacterial agents that have antibacterial therapeutic effects against the target bacterium B. In step 8, the selection (screening) is carried out taking into account not only the antibacterial effect but also the therapeutic effect.

[0100] It is also preferable to carry out the above-mentioned step a and / or step b after step 8. Furthermore, by inserting step 8 after (each time) carrying out step a and / or step b, it is possible to check each time whether or not there is a therapeutic effect, whether or not the silkworms have been killed by inhibiting natural immunity, etc. The expression "carrying out step a and / or step b after step 7" includes "carrying out step a and / or step b after step 8."

[0101] <Process 9> Step 9 is an "antibacterial agent obtaining step" in which an antibacterial agent is isolated and obtained from the sample selected in step 8 above. The antibacterial agent isolated and obtained in step 9 has been screened for antibacterial activity up to step 8, and is therefore an antibacterial agent that has antibacterial therapeutic effects against bacteria B. The sample selected in step 8 contains substances derived from bacterium B in addition to the antibacterial agent, and such "unnecessary substances derived from bacterium B" are removed in step 8. Examples of such unnecessary substances include various substances that were present in the cells of the producing bacterium, components derived from the culture medium, and substances that were dissolved in water / organic solvents in step 7.

[0102] The method for separating the antibacterial agent is not particularly limited, and examples thereof include two-phase partitioning with an organic solvent, organic solvent precipitation, salting out, ion exchange chromatography, activated carbon chromatography, hydrophobic chromatography, thin layer chromatography, HPLC, UPLC, recrystallization, etc. Separation can also be confirmed by known methods.

[0103] In the present invention, screening is extremely efficient because there is no need to isolate the antibacterial agent / antibiotic until step 9. Furthermore, in step 9, there is no need to secure an amount of the antibacterial agent / antibiotic sufficient to administer to, for example, mice, etc., so screening is extremely efficient. That is, compared with conventional methods, the present invention is extremely efficient in screening for "the very presence of an antibacterial agent," which is the most important point. [Example]

[0104] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples as long as they do not depart from the gist of the invention. In "Example 1," only an example in which all steps 1 to 9 were performed is described, and only representative bacterial species were used, but the present invention is not limited to this Example 1 as long as it does not deviate from the gist of the present invention. In "Example 2," only the example in which the steps shown in Table 9 were performed was described, but the present invention is not limited to Example 2 as long as it does not depart from the gist of the invention. Furthermore, when "%" refers to the amount of a substance, it means "% by mass" unless otherwise specified.

[0105] Example 1 <Process 1> <Preparation of solid medium containing soil extract and cultivation of Bacterium A (formation of soil bacterial colonies)> To 2 L of soil R, 2.5 L of water (RO water) was added, and the mixture was autoclaved (121°C, 20 minutes), then centrifuged (8000 rpm, 5 minutes) to obtain the supernatant, which was used as the "soil extract." Agar (final concentration 1.5% by mass) was added thereto, and after autoclaving, the mixture was poured into a 10 cm diameter plastic dish and allowed to solidify at room temperature, thus preparing a nutrient-poor solid medium.

[0106] An equal volume of water (RO water) was added to soil S, stirred, and left for 5 minutes. The supernatant was diluted 1000 times with saline, and 100 μL of this was spread with a cone rod onto the above-mentioned solid medium (agar medium containing soil extract from soil R) and cultured at 30°C for 4 days to form soil colonies of bacteria A.

[0107] Soil R and soil S were considered to be essentially identical because they were collected from the same location and used separately.

[0108] The culture conditions were adjusted and set (as described above) so that the colony size would be an average diameter of 2 mm or less, resulting in an average diameter of 0.5 mm to 3 mm. The number of colonies in a 10 cm diameter petri dish was 50 to 500.

[0109] <Process 2> <Formation of a layered solid medium (layering soft agar containing Bacterium A)> For bacteria B, LB75 medium (containing 75 g / L of NaCl) was used for Staphylococcus aureus, LB10 medium (containing 10 g / L of NaCl) for Escherichia coli, and YPD medium for baker's yeast. 8 g / L of agar (low-melting point agarose for Escherichia coli and baker's yeast) was added to each medium, and the mixture was autoclaved (121°C, 20 minutes) to obtain a liquid overlay liquid (soft agar) at the pouring temperature described below. The melting point of the agar used for Staphylococcus aureus was 55°C, and the melting point of the agar used for Escherichia coli and baker's yeast was 35°C.

[0110] The above three types of bacteria B were cooled to 58°C, 45°C, and 45°C in that order, respectively, and full growth of the bacteria was added at 1 / 100, 1 / 40, and 1 / 80 of the volume, respectively, and stirred to obtain a layer solution containing each type of bacteria B. 10 mL of each of the layer solutions was poured into a 10 cm diameter Petri dish containing colonies of soil bacteria, and left to stand at room temperature for 30 minutes or more to obtain a solid layer medium. Then, the mixture was incubated at 30°C for 1 day.

[0111] <Process 3> <Observation of inhibition zones and purification of bacteria A that formed inhibition zones> After the incubation, the formation of an inhibition zone was observed. Bacterium A was collected from the colony of Bacterium A, which was present in the center of the inhibition zone and was thought to be the antibacterial agent / antibiotic producer. However, Bacterium A does not necessarily represent a single type of bacterium.

[0112] Since the collected bacteria A may have contained small amounts of solid overlay medium and antibiotic non-producing bacteria, these were removed in the following purification process. YME agar medium was used as the solid medium for purifying Bacterium A, and purification was carried out according to standard methods.

[0113] <Step 4> <Pure culture of Bacterium A obtained in step 3> Bacterium A in the colony obtained in step 3 was pure cultured on YME agar medium.

[0114] <Process 5> <Formation of a solid medium by layering soft agar containing bacterium B on purely cultured bacterium A> In step 2, a solid medium was formed by layering soft agar containing bacterium B on top of the purely cultured bacterium A in the same manner as in step 2, except that in step 2, "a colony of soil bacteria was formed" was replaced with "bacterium A was cultured in pure culture."

[0115] <Process 6> <Observation of the inhibition zone> In the pure culture of bacteria A, there were cases where bacteria B that grew over an entire surface did not form an inhibition zone, so these were eliminated and only bacteria A that formed an inhibition zone was selected.

[0116] <Process 7> <Selection of samples with low MIC values (extraction with an organic solvent and measurement of the MIC value of the obtained sample solution)> The candidate strains for producing antibacterial agents and antibiotics purified in Step 3 were shake-cultured at 30 °C for 4 days in 20 mL of YME liquid medium. To the obtained culture solution, 20 mL of acetone (equal volume to the YME liquid medium) was added, stirred, and centrifuged (8000 rmp, 5 minutes) to obtain a supernatant. The supernatant was dried (liquid removed, dried) using a high-speed evaporator (manufactured by Labconco), and 1 mL of water (1 mL of Milli-Q water (registered trademark)) was added thereto and dissolved by vortexing. The pH was adjusted with 10N NaOH to be between 5 and 8, and a sample (solution) for MIC value measurement was obtained.

[0117] Regarding Staphylococcus aureus, which is a Gram-positive bacterium, the same strain was used in Step 7. However, for Gram-negative bacteria, Escherichia coli was evaluated. Regarding Saccharomyces cerevisiae, which is a fungus, in Step 7, it was changed to Candida albicans, a pathogenic fungus (ATCC 10231).

[0118] The above sample (solution) was diluted in an 8-step dilution series from 1 / 2 to 1 / 256 with physiological saline, and 50 μL thereof was mixed with 50 μL of Staphylococcus aureus and Escherichia coli diluted 1 / 1000 with Mueller-Hinton medium (MH medium), incubated at 37 °C for 24 hours, and the presence or absence of turbidity was visually determined to obtain the MIC value.

[0119] Regarding Candida albicans, a suspension of bacteria with an absorbance of 0.5 at 530 nm was diluted 1 / 1000 with RPMI medium, mixed with 100 μL of the sample, incubated at 37 °C for 48 hours, and the presence or absence of turbidity was determined.

[0120] Samples (liquids) that showed MIC values ​​of 1 / 10 or less were selected for the next step, a therapeutic test using silkworms. In other words, sample solutions that showed antibacterial activity even when diluted 10 times or more were selected. The reason for selecting samples with a certain level of antibacterial activity is that the body fluid of the fifth-instar larvae (weighing 2 grams) used is 500 μL, and the sample volume used in the injection test is 50 μL. Therefore, the sample is diluted 10 times immediately after injection, and therapeutic effect can only be expected if the MIC value is 1 / 10 or less. This situation does not apply to antibiotics whose antibacterial activity is enhanced by substances in the body fluid, but such cases are exceptional.

[0121] <Process 8> <Treatment test using a silkworm infection model (selection of samples with antibacterial therapeutic effects against Bacillus subtilis B)> For samples showing MIC values ​​of 1 / 10 or less, the original solution and various dilutions were prepared and subjected to a therapeutic test (screening (exploration) using a silkworm infection model). For the reasons mentioned above, the MIC values ​​of the diluted samples were made to be no greater than 1 / 10.

[0122] In steps 2 to 6, Staphylococcus aureus (MSSA1 strain) was used as the Gram-positive bacterium, Escherichia coli (W3110 strain) was used as the Gram-negative bacterium, and baker's yeast (Saccharomyces cerevisiae, BY4741 strain) was used as the fungus. However, in the treatment test in step 8 (screening (exploration) using a silkworm infection model), Staphylococcus aureus (MSSA1 strain), Pseudomonas aeruginosa (PAO1 strain), and Candida albicans (ATCC10231) were used, respectively.

[0123] Fifth instar silkworms were fed 1g of artificial food per silkworm for one day. The average weight of the silkworms used was 2.0g.

[0124] Staphylococcus aureus (MSSA1 strain), Pseudomonas aeruginosa (PAO1 strain), and Candida albicans (ATCC10231) were cultured in liquid medium until they reached full growth, and then diluted with saline to 1 / 10, 1 / 1000, and 1 / 1, respectively, in the above order. 50 μL of each diluted solution was injected into the blood of silkworms. Immediately after the injection, 50 μL of the sample solution diluted with water (sterilized Milli-Q water) ("Milli-Q" is a registered trademark) was injected into the blood of each silkworm. The total number of silkworms per group was four.

[0125] For each of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida, the survival and death of silkworms was determined 24 to 48 hours after injection, and the ED50 was calculated from the survival curve. The negative control for the therapeutic effect was 0.9% by mass NaCl, and the positive controls were 100 μg / mL vancomycin, 2 mg / mL gentamicin, and 1 mg / mL amphotericin B for Staphylococcus aureus, Pseudomonas aeruginosa, and Candida, respectively, in that order.

[0126] Samples with antibacterial therapeutic effects against each of the Bacillus species (Staphylococcus aureus (Table 1), Pseudomonas aeruginosa (Table 4), and Candida species) were selected.

[0127] When bacterium B was Staphylococcus aureus, the MIC values ​​against multiple strains of MRSA (methicillin-resistant Staphylococcus aureus) were also determined for samples that showed a therapeutic effect in the silkworm infection model (Table 2). MRSA is known to be resistant to antibiotics currently in clinical use. The candidate compounds to which MRSA is resistant can be considered to be similar to antibiotics already in clinical use. Therefore, samples containing resistant MRSA strains were determined at this stage not to be novel antibiotics.

[0128] When the bacterium B used up to step 7 was Staphylococcus aureus, the MIC value against Streptococcus pneumoniae was also determined for samples that showed a therapeutic effect in the silkworm infection model (Table 3).

[0129] <Process 9> <Isolation and acquisition of antibacterial agents> LC-MS analysis of therapeutically active fractions The acetone-extracted fraction of t4-11, which showed therapeutic effects, was dissolved in butanol and separated by HPLC on a C18-ODS column. The fraction that showed therapeutic effects in the silkworm infection model (eluting at 18-19 min) was further analyzed by LC-MS (Waters, Xevo G2-XS Q-TOF). The exact mass of each peak was determined and compared with known antibiotics using the Dictionary of Natural Products.

[0130] [Results of Example 1] An outline of the operational flow of Example 1 is shown in Figure 2. Figure 2 shows the "overall flow of steps 1 to 8" including steps 4 to 6 (referred to as "2nd screening" in Figure 2), and if all steps 1 to 8 are performed, a total of four screenings will be performed.

[0131] <Process 1> A nutrient-poor soil agar medium was prepared by adding water to soil R, autoclaving it, and then adding agar to the supernatant.Using this medium, it became possible to obtain a large number of small colonies at once (Figure 3). As mentioned above, because a nutrient-poor solid medium containing soil extract was used, the size of the colonies of bacteria A formed was small enough to serve as the central object (antibiotic source) for the formation of the inhibition zone.In step 3, the inhibition zones could be observed favorably in terms of size, number, etc., and bacteria A could be collected from the center of the inhibition zone (Figures 4 to 6). On the other hand, when conventional nutrient media such as YME agar were used, the growth rates varied significantly among the strains, resulting in the formation of large colonies, which inhibited the colony formation of other strains (not shown).Furthermore, simply lowering the nutrient concentration limited the types of colonies that appeared.

[0132] <Process 2, Process 3> In order to determine the therapeutic effect of the test sample solution injected into the blood of silkworms in the silkworm infection model, it is necessary to prepare a sample solution with sufficiently high antibacterial activity. This is because a silkworm weighing 2g has 500μL of blood, and if the usual injection volume of 50μL is used, the blood will be diluted 10-fold immediately after injection. Therefore, if antibacterial activity is the cause of treatment, the MIC value must be 1 / 10 or less, because otherwise the antibacterial activity will be lost due to the 10-fold dilution with blood immediately after injection.

[0133] In actual screening, it is not easy to obtain a large number of samples with MIC values ​​of 1 / 10 or less. When soil bacteria are randomly selected and cultured, and an acetone extract is prepared, it is difficult to obtain samples with MIC values ​​of 1 / 10 or less. This led to the present invention, in which antibiotic-producing bacteria that form inhibition zones are collected from soil.

[0134] When soft agar containing Staphylococcus aureus (MSSA1 strain), Escherichia coli (W3110 strain), and baker's yeast (Saccharomyces cerevisiae (BY4741 strain)) was layered on top of a colony of soil bacterium A on soil R agar solid medium and incubated, inhibition zones were observed for all bacteria (Figures 4 to 6). From the center of the inhibition circle, "bacteria A that form the inhibition circle" could be suitably collected (bacteria A may be a mixture).

[0135] <Steps 4, 5, 6> The bacteria A that formed the inhibition zone observed (obtained) in step 6 of Example 1 were 791 strains of Staphylococcus aureus, 329 strains of Escherichia coli, and 433 strains of baker's yeast.

[0136] <Process 7> For the gram-positive bacterium Staphylococcus aureus, 229 strains (samples), or 29% of the 791 strains that formed inhibition zones, showed MIC values ​​of 1 / 10 or less. For Escherichia coli, a gram-negative bacterium, 27 strains (samples), or 8.2% of the 329 strains that formed inhibition circles, showed MIC values ​​of 1 / 8 or less. Among fungi, 147 strains (samples), or 34% of the 433 strains that formed inhibition circles with baker's yeast, showed MIC values ​​of 1 / 10 or less against the pathogenic fungus Candida albicans (ATCC10231).

[0137] <Process 8> <<Staphylococcus aureus>> Twenty samples were obtained that showed therapeutic effects in the silkworm infection model (Table 1). Furthermore, the MIC values ​​of these bacteria against eight types of MRSA were calculated and compared with those of Staphylococcus aureus (MSSA1). As a result, it was found that none of the 11 samples contained resistant MRSA strains (Table 1, Table 2).

[0138] [Table 1]

[0139] [Table 2]

[0140] These results suggest that these 11 samples are either novel antibiotics, already in clinical use as anti-MRSA infection treatments, or antibiotics under development. These 11 samples are at least independently obtained novel antibacterial agents (novel antibiotics) candidates in the present invention. From the above, it was found that the present invention is an excellent method for screening antibiotics, including resistant bacteria.

[0141] Of the 20 samples that showed therapeutic effects in the silkworm infection model (MSSA1 infection model), four samples (sample Nos. 6-39, 6-40, 10-27, and 14-7) showed high antibacterial activity against Streptococcus pneumoniae (Table 1, Table 3). From the above, it was found that the present invention is an excellent method for discovering antibacterial agents and antibiotics for all Gram-positive bacteria.

[0142] [Table 3]

[0143] Of the 791 strains that formed inhibition zones against Staphylococcus aureus, 226 samples with MIC values ​​of 1 / 10 or less were obtained, and 20 samples that had a therapeutic effect in silkworms were obtained (Table 1, Table 6, Figure 1(c)). This result shows that the discovery efficiency is 1000 times higher than when searching from a compound library using conventional methods, and 10 times higher than when searching from natural products using conventional methods (Figure 1(a)(b)(c)). The main reason for this is that the adoption of the inhibition zone formation method in this invention has made it easier to obtain many samples with high antibacterial activity.

[0144] <<Pseudomonas aeruginosa>> Table 4 shows the antibacterial activity, bacterial species, and butanol solubility of the therapeutically active substances selected in step 8 of the samples that showed a therapeutic effect against Pseudomonas aeruginosa PAO1. In the silkworm infection model (step 8), 18 samples were obtained that showed therapeutic effects against Pseudomonas aeruginosa (Table 4). All 18 samples shown in Table 4 showed significant therapeutic effects when administered to silkworms one day before inoculation with Pseudomonas aeruginosa (not shown in Table 4). This result suggests that the therapeutic activity of these samples is due to the immune stimulating effect of silkworms.

[0145] [Table 4]

[0146] Table 5 shows the yields of the acetone-extracted fraction, butanol-soluble fraction, and activity for the 10 samples that showed a therapeutic effect against Pseudomonas aeruginosa in the silkworm infection model (step 8).

[0147] [Table 5]

[0148] Of the 329 strains that formed an inhibition zone against E. coli W3110, 27 samples with MIC values ​​of 1 / 8 or less were obtained, and 18 samples with anti-Pseudomonas aeruginosa therapeutic effects in silkworms were obtained (Table 4, Figure 1(c)). Until now, conventional methods have not been able to discover substances that are effective against Gram-negative bacteria (Figure 1(a)). The inhibition zone formation method of the present invention has been shown to be effective as a method for discovering substances that are effective against Gram-negative bacteria.

[0149] <<Fungi>> For the fungus Candida, the number of samples with MIC values ​​of 1 / 10 or less was 147, but has since increased to 232. From these, samples that show therapeutic effects can be screened using the silkworm infection model. The evaluation of fungi is described below in Example 2.

[0150] <Process 9> <<Staphylococcus aureus>> The therapeutic effect of the butanol-soluble fraction was tested on 10 of the 11 samples in which the MRSA strains were determined not to be resistant. As a result, it was found that the therapeutically effective substance was soluble in butanol for six samples (sample No. 14-7, t4-11, t5-2, t5-3, t5-18, and t5-32) (Table 1, item "Butanol-soluble").

[0151] Substances that dissolve in butanol can be purified in organic solvents such as HPLC, and are therefore considered easy to purify.Furthermore, these six samples were analyzed by HPLC.

[0152] Based on the HPLC elution time and UV absorption pattern, sample No. t5-32 was found to contain lysocin E, a known therapeutic drug (Patent Documents 3-7, Non-Patent Documents 4-6). The discovery of a known, excellent antibiotic demonstrates that the present invention is effective in the discovery of antibacterial agents and antibiotics.

[0153] Samples t5-2, t5-3, and t5-18 all had identical absorption patterns in HPLC analysis and were produced by the same species of bacteria (Streptomyces capoamus), suggesting that they are derived from the same bacteria. These may all be novel antibacterial and antibiotic agents.

[0154] For sample No. t4-11, the acetone extract fraction was dissolved in butanol and fractionated at 1-minute elution intervals using HPLC on a C18 column, and the therapeutic activity of each fraction was examined in a silkworm infection model. As a result, therapeutic activity was found in the fraction eluting between 18 and 19 minutes. Furthermore, this fraction was analyzed by LC-MS, and the exact mass of each peak was determined. The masses were found to match those of WAP-8294 A1 and WAP-8294 A2, which have already been reported as antibiotics with therapeutic effects. The discovery of known, excellent antibiotics demonstrates that this invention is effective in the discovery of antibacterial agents and antibiotics.

[0155] <<Pseudomonas aeruginosa>> The therapeutic effect of the butanol-transferred fraction was tested on the 18 samples that showed a therapeutic effect, and therapeutic effects were suggested in 12 samples (sample Nos. w2-19, w2-25, w2-26, w3-9, w6-13, w7-16, w7-29, w8-11, w16-2, w16-10, w17-7, and w17-10) (Table 1, item "Butanol-transferred").

[0156] Example 2 <Example in which step a and step b were performed with fungi> As shown in the results of Example 1, when bacterium B was the bacterium Staphylococcus aureus, the results shown in Table 6 below were obtained. Note that, as for the bacteria that formed an inhibition zone, steps 4, 5, and 6 were carried out as described above, and the bacteria that were confirmed to have formed an inhibition zone were counted (after step 6).

[0157] [Table 6]

[0158] In contrast, the same evaluation and exploration as in Example 1 was carried out except that bacteria B was replaced with a fungus belonging to the genus Candida (Candida albicans, ATCC10231), i.e., the evaluation and exploration was carried out without carrying out steps a and b. The results shown in Table 7 below were obtained. Note that the "bacteria that formed an inhibition zone" were counted by carrying out steps 4, 5, and 6 and confirming that an inhibition zone had been formed (i.e., after step 6).

[0159] [Table 7]

[0160] From the results in Table 7, in the screening of therapeutic drugs for Candida (bacterium B) (result: 0 samples), even if a substance with antibacterial therapeutic effect (with an MIC value of 1 / 10 or less) was present in the sample up to step 7, it was thought that the sample may also contain immunosuppressant substances or toxic substances, etc., which would cause the silkworms to die, and therefore the true antibacterial therapeutic effect in the silkworm infection model may not be apparent.

[0161] In fact, when Candida fungus was used, the results shown in Table 8 below were obtained. As described in step 8 of Example 1, the cells were cultured in a liquid medium until full growth was achieved, diluted with saline, and 50 μL of the diluted solution was injected into the blood of silkworms. Immediately after the injection, 50 μL of the sample solution diluted with sterilized Milli-Q water was injected into the blood of silkworms.

[0162] That is, when the fungus B in Example 1 was Candida, the results shown in Table 8 were obtained. In Table 8, "+" indicates that the silkworms were injected in step 8, and "-" indicates that the silkworms were not injected. "○" indicates that all (e.g., 3) survived, and "×" indicates that more than half died.

[0163] [Table 8]

[0164] Without the injection of Candida, the mice survived on both the first and second days (No. A, C). On the other hand, when Candida was injected, the animals died on the second day regardless of whether they received a sample injection (No. B, D), but on the first day they survived without a sample injection (No. B) but died with a sample injection (No. D). This suggests that even if a substance with antibacterial therapeutic effects was present in the sample, the silkworms may have died due to the action of natural immune suppressants or toxic substances contained in the sample. In other words, it is thought that the natural immune suppressants in the sample may have suppressed the silkworms' natural immunity, causing the silkworms to die from Candida albicans.

[0165] If this is the case, adding a process for separating antifungal active substances from toxic substances should increase the efficiency of discovering substances that have antibacterial therapeutic effects, especially against fungi.

[0166] Specifically, in Example 1, the acetone-extracted fraction (acetone-soluble matter) was used to measure the antifungal activity (MIC value) in step 7 and to test the antibacterial therapeutic effect in step 8, whereas in Example 2, the "acetone-extracted fraction (acetone-soluble matter)" was further subjected to two additional purification steps: step a, "butanol transfer," and step b, "fractionation using an ODS cartridge column" (see Table 9).

[0167] In the following Table 9, the operations (tests) were performed in the order from top to bottom. Operations (contents) not listed in Table 9 were performed in substantially the same manner as in Example 1. As the bacterium B, a Candida bacterium (Candida albicans, ATCC10231) was used.

[0168] [Table 9]

[0169] <<Selection of Bacterium A against Bacterium B (Candida)>> As mentioned above, when Bacterium B is a fungus, Candida, there were 232 samples after step 7, but 0 samples after step 8. Therefore, from the 232 samples remaining after step 7, 10 bacteria that met the following two conditions were selected. These 10 bacteria are designated "#1" to "#10" respectively (see Table 10).

[0170] <<<Condition 1>>> In step 7, the acetone-extracted sample had high anti-Candida activity (low MIC value (large denominator of MIC value)). <<<Condition 2>>> In step 8, in a therapeutic efficacy test using silkworms, while all members of the control group injected with only Candida survive, more than half of the members of the group injected with both the sample and Candida die.

[0171] <<Step 7: Preparation of acetone extract fraction>> Glycerol stocks of the bacteria selected under the above two conditions were spread onto YME agar medium and cultured at 30°C for 2 days. The bacteria grown on the YME agar medium were inoculated into four 100 mL eggplant flasks containing 20 mL of YME liquid medium, and cultured with shaking at 30°C and 200 rpm for four days. After the liquid culture, the same volume of acetone was added, and the mixture was centrifuged at 8000 rpm for 5 minutes. The centrifuged supernatant was dried and dissolved in the above-mentioned Milli-Q water in an amount 1 / 20 of the liquid medium. The pH was then adjusted to 7.

[0172] <<Step a: Preparation of butanol-soluble fraction>> The same volume of butanol as the acetone extract was added, and the mixture was centrifuged at 8000 rpm for 5 minutes to obtain the supernatant. This process took less than 10 minutes per sample. The resulting centrifuged supernatant was evaporated to dryness for 12 hours using a centrifugal evaporator, and then dissolved in the same amount of physiological saline as before the addition of butanol.

[0173] <<Step 7: MIC Measurement>> Candida was cultured in Sabouraud liquid medium for 2 days. The culture medium was centrifuged to collect the bacteria, and then the bacteria were added to physiological saline until the bacterial concentration reached OD 530 It was adjusted to be 0.5. The obtained OD 530 The bacterial solution at 0.5 μg / ml was diluted 1 / 1000 with RPMI1640-MOPS and 100 μL of each was dispensed into a round-bottom 96-well plate. 100 μL of the sample was added to the first row of the round-bottom 96-well plate, and serial dilutions were performed. After that, the bacteria were cultured at 37°C for 2 days, and the minimum concentration at which bacterial growth was inhibited was taken as the MIC value.

[0174] <<Step b: Fractionation using an ODS cartridge column>> An ODS Sep-Pak cartridge column was used. The column was washed with acetone, methanol, and water, and the sample was applied to the column. It was then eluted with water, 10% MeOH, 20% MeOH, 30% MeOH, 40% MeOH, 50% MeOH, 60% MeOH, 70% MeOH, 80% MeOH, 90% MeOH, 100% MeOH, and acetone. This process took less than 10 minutes per sample. The collected sample was dried for 12 hours in a centrifugal evaporator, and then dissolved in physiological saline to a concentration four times that of the butanol-dissolved sample.

[0175] <<Step 8: Measurement of therapeutic effect using silkworms>> Candida was cultured in Sabouraud liquid medium for 2 days. The culture medium was centrifuged and the bacterial cells were collected. They were suspended in 10 times the volume of physiological saline, and 50 μL of the suspension was injected intravenously into fifth-instar silkworm larvae that had been fed for one day. Immediately afterwards, 50 μL of the sample diluted with physiological saline was injected intravenously into the blood of each larva. The survival of the silkworms was assessed 1 and 2 days after the injection.

[0176] In therapeutic efficacy evaluation (test) using silkworms, the MIC value of the sample must be 1 / 10 or less in order to expect a therapeutic effect. This is because when 50 μL of the sample is injected into the blood of a silkworm (weighing 2 g), it is immediately diluted to 1 / 10. In this study, we looked at the antibacterial therapeutic effect of samples with an MIC of 1 / 10 or less in silkworms.

[0177] [Results of Example 2] <Step 7: Antibacterial activity test> Table 10 shows the MIC values ​​(dilution ratio) against Candida of the acetone extract fraction, butanol-soluble fraction, and ODS column fraction prepared from 10 bacteria. Although not shown in Table 10, the MIC values ​​of the samples eluted with 10% MeOH, 20% MeOH, 30% MeOH, 40% MeOH, 50% MeOH, and acetone were all large, at ">1 / 2" or "1 / 2" (low antibacterial activity).

[0178] [Table 10]

[0179] As can be seen from Table 10, of the 10 samples #1 to #10, 6 samples (No. 1, 6 to 10) had MIC values ​​of the butanol-soluble fraction of 1 / 10 or less. These six samples were fractionated using an ODS cartridge column and subjected to antibacterial activity tests. As a result, the high-concentration MeOH elution fractions of all six samples showed antibacterial activity with an MIC value of <1 / 10 (see Table 10).

[0180] <Step 8: Evaluation of therapeutic effect (test)> In the above process, the ODS fractions with "MIC values ​​< 1 / 10" were subjected to a therapeutic effect test in silkworms. The results are shown in Table 11.

[0181] [Table 11]

[0182] As can be seen from Table 11, all of the controls (No. 102 and No. 137 in Table 11) injected with 1 / 10 diluted Candida survived (3 / 3) on the first day, but more than half died on the second day. In other words, the results are similar to those of No. B in Table 8 above.

[0183] One day after injection of the acetone-extracted fractions (1 / 1 (undiluted) and 1 / 10 diluted), more than half of the silkworms died. That is, as described above, when a sample that had not undergone either step a or step b was injected and Candida was injected, more than half of the silkworms died on the first day, as shown in No. D of Table 8. In contrast, in the samples injected with "the sample subjected to step a: butanol dissolution and / or step b: column fractionation" and then injected with Candida, more than half were still alive even after one day (survival rate of "2 / 3" or "3 / 3"), as can be seen from Table 11. This suggests that natural immune suppressants or toxic substances were removed by performing step a or step b.

[0184] Furthermore, among the samples injected with Candida, not only were more than half still alive after one day, but more than half were still alive on the second day. The sample-injected groups in which more than half of the silkworms were alive on the second day after injection were the following three groups: "Sample #1" 80% MeOH (1 / 1) (No. 105 in Table 11), "Sample #1" 90% MeOH (1 / 1) (No. 106 in Table 11), and 0.5 mg / mL AmpB (control). In addition, in No. D in Table 8, where neither step a nor step b was performed, more than half of the animals had died on the first day, so naturally more than half had also died on the second day (see Table 8). In Table 11, "AmpB" represents "amphotericin B," an antifungal drug widely used against fungal infections, which was used as a positive control.

[0185] When bacteria B was Candida, if steps a and b were not performed, the number of samples in which a therapeutic effect was confirmed in step 8 was 0 out of 232 (see Table 7). However, by performing steps a and b, the number of samples in which a therapeutic effect was confirmed in step 8 was 1 out of 10 selected samples (#1) (see Table 12).

[0186] [Table 12]

[0187] This "sample #1" was determined to have no therapeutic effect when steps a and b were not performed, but a therapeutic effect was found when steps a and b were performed. In other words, performing steps a and b increased the sensitivity of the "method for screening antibacterial agents" of the present invention.

[0188] Even when step a and / or step b were added, all steps from step 1 to this point could be carried out in a short time. Moreover, simultaneous screening of many strains of Bacterium A and samples from Bacterium A was possible.

[0189] Furthermore, as shown in Table 7, 232 samples had antibacterial activity (MIC value of 1 / 10 or less) after step 7. As mentioned above, 10 samples were selected from these, and one sample (#1) was found to have a therapeutic effect (a rate of about 1 / 10) (see Table 12). This suggests that of the 222 untested samples in Example 2, there may be about 22 samples (about 1 / 10) that have a therapeutic effect.

[0190] <Step 9: Isolate and obtain antibacterial agents> Sample #1, which was found to have a therapeutic effect in step 8, can be purified using HPLC to determine its novelty. The major components in the HPLC fraction of Sample #1 can be confirmed to be known antifungal agents.

[0191] The "substance obtained in step a (e.g., dissolving in butanol) and / or the substance obtained in step b (e.g., adsorbed and eluted on an ODS column)" can be easily further purified in step 9, and the novelty of the substance as an antibacterial agent can be easily determined. That is, such a substance can be more easily purified by HPLC using a C18 column or the like, and its novelty can be determined based on the molecular weight determined by LC / MS. [Industrial Applicability]

[0192] By using the screening method of the present invention, it is possible to search for and select many "candidate compounds that have therapeutic activity and are also effective against resistant bacteria." Furthermore, since it is known that the therapeutic effects of antibiotics are consistent between silkworms and mice, it is believed that antibiotics that have shown therapeutic effects in silkworms in the present invention will also have therapeutic effects in mice (and therefore in humans). Furthermore, some of the compounds obtained using the screening method of the present invention are "expected to have a therapeutic effect against resistant bacteria," which suggests that they may be novel antibiotics that have not yet been used clinically.

[0193] If the crude fraction of the antibiotics discovered in this invention that are effective against resistant bacteria and have therapeutic effects is purified and their structures are determined, such novel antibiotics are expected to be clinically effective as therapeutic agents for infections caused by resistant bacteria.

[0194] Therefore, the "method for searching for antibacterial agents" of the present invention can be widely used in fields where antibacterial agents, antibiotics, etc. are searched for, developed, and manufactured.

Claims

1. A method for discovering an antibacterial agent, comprising all of the following steps 1, 2, 3, 7 and 8. Step 1: A soil fungus colony formation step in which the supernatant of a soil suspension is spread and cultured on a sterilized "soil extract, which is the supernatant of the sterilized soil, which is mixed with water, and sterilized" and a nutrient-poor solid medium containing agar, thereby forming colonies of fungus A contained in the soil. Step 2: A step of forming a layered solid medium by adding the bacteria B to a liquid agar medium obtained by adding agar that is in a liquid state at "at least one temperature at which the bacteria B does not die when added to the medium" to prepare a layered liquid, and pouring the layered liquid onto the solid medium on which the colonies of bacteria A were formed in step 1 and cooling it to form a layered solid medium. Step 3: A purification step of finding an inhibition zone formed around the colony on the surface of the target bacteria B that has grown over the entire surface of the multilayer solid medium, and selecting and extracting bacteria A from the colony of bacteria A present approximately in the center of the inhibition zone, and purifying only bacteria A. Step 7: A sample selection step A in which bacteria A is cultured in a liquid medium, an organic solvent is added and mixed by stirring, the soluble matter is dried, and water is added to prepare a plurality of samples with different concentrations of bacteria A, and the antibacterial activity against the target bacteria B for each sample is measured, and a "sample derived from bacteria A" that shows antibacterial activity even when diluted 10 times or more is selected. Step 8: A sample selection step B of selecting a sample having an antibacterial therapeutic effect against the target bacterium B from among the samples selected in the above step 7 using a silkworm infection model.

2. 2. The method for screening antibacterial agents according to claim 1, wherein the size of the colonies of said bacterium A formed in said step 1 has an average diameter of 0.5 mm or more and 10 mm or less.

3. The method for screening antibacterial agents according to claim 1 or 2, further comprising the following step 4 after step 3: Step 4: A step of purely culturing the colonies of bacteria A purified in step 3 on a solid medium.

4. The method for discovering an antibacterial agent according to claim 3, further comprising the following steps 5 and 6 after step 4: Step 5: A step of preparing a layered liquid by adding the bacteria B to a liquid agar medium obtained by adding agar that is in a liquid state at "at least one temperature at which the bacteria B does not die when added to the medium" to a medium for the bacteria B to be antibacterial, and then pouring the layered liquid onto the solid medium in which the bacteria A was purely cultured in step 4 above and cooling it to form a layered solid medium. Step 6: A step of confirming that an inhibition zone is formed around the pure cultured bacteria A on the surface of the target bacteria B, which has been grown over an entire surface on the solid layer medium formed on the pure cultured bacteria A.

5. The method for screening an antibacterial agent according to any one of claims 1 to 4, further comprising carrying out the following step a and / or step b in and / or after step 7: Step a: A step of dissolving in a poorly water-soluble or water-insoluble organic solvent Step b: Column fractionation step

6. A method for searching for an antibacterial agent described in any one of claims 1 to 4, comprising carrying out the following step a and / or step b before carrying out step 7. Step a: A step of dissolving in a poorly water-soluble or water-insoluble organic solvent Step b: Column fractionation step

7. The method for screening an antibacterial agent according to any one of claims 1 to 6, further comprising the following step 9 after step 8: Step 9: An antibacterial agent obtaining step of isolating and obtaining the antibacterial agent from the sample selected in step 8.

8. 8. The method for searching for an antibacterial agent according to any one of claims 1 to 7, wherein the agar in step 2 or step 5 is agar having a melting point of 30°C or higher and 60°C or lower when the bacterium B is Staphylococcus aureus, and is agar having a melting point of 30°C or higher and 45°C or lower when the bacterium B is Escherichia coli or Saccharomyces cerevisiae.

9. 9. The method for screening antibacterial agents according to claim 1, wherein the thickness of the overlay solid medium in step 2 or step 5 is 0.5 mm or more and 10 mm or less.

10. The method for screening antibacterial agents according to any one of claims 1 to 9, wherein the bacterium B is a pathogenic bacterium or fungus.

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