JP7610193B2 - How to discover antibacterial agents - Google Patents

Description

The present invention relates to a method for discovering antibacterial agents, and more specifically, to a method for efficiently discovering antibacterial agents derived from soil bacteria.

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. In addition, it is highly likely that new resistant bacteria will appear 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 discovery of new antibacterial agents by pharmaceutical companies and other organizations has been slowing down in recent years.

Conventional screening methods involve 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, determining its structure, and then conducting experiments using animals (e.g., mice) to confirm its pharmacokinetics, therapeutic effects, pharmacological 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. Since it is not possible to evaluate multiple bacteria in parallel or multiple produced substances in parallel, this is thought to impose limitations on the discovery and exploration of new antibacterial agents.

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, etc., it is often discarded 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.

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 scaffolds (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 well 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).

In fact, they have already discovered a new antibacterial agent/antibiotic with a new target using a silkworm infection model (Non-Patent Document 4). In addition, they have discovered a new antibacterial agent/antibiotic called lysocin E that is effective against MRSA from bacteria of the genus Lysobacter derived from Okinawa soil (Patent Documents 3 to 6, Non-Patent Document 5). They have also discovered a new antifungal drug called ASP2397 (Non-Patent Document 6).
In this way, the silkworm infection model has been proven to be effective in discovering new antibacterial and antibiotic agents that also have therapeutic effects.

In recent years, with the emergence of few antibacterial agents with novel structures, there is a strong need to invent a method for searching for new, superior antibacterial agents and antibiotics to replace the existing search methods that have the major problems mentioned above.

JP 2007-327964 A JP 2010-133975 A JP 2012-006917 A JP 2012-005480 A JP 2012-005481 A International Publication No. 2011/148959 JP 2016-210776 A

Kaito C, Akimitsu N, Watanabe H & Sekimizu K (2002) Microb Pathog 32: 183-190. Hamamoto H, Kurokawa K, Kaito C, et al. (2004) Antimicrob Agents Chemother 48: 774-779. Hamamoto H, Tonoike A, Narushima K, Horie R & Sekimizu K (2009) Comp Biochem Physiol C Toxicol Pharmacol 149: 334-339. Paudel A, Hamamoto H, Panthee S, Kaneko K, Matsunaga S, Kanai M, Suzuki Y, Sekimizu K. (2017) Front. Microbiol., 8: 712. Hamamoto H, Urai M, Ishii K, Yasukawa J, Paudel A, Murai M, Kaji T, Kuranaga T, Hamase K, Katsu T, Jie Su, Adachi T, Uchida R, Tomoda H, Yamada M, Souma M, Kurihara H , Inoue M, Sekimizu K. (2015) Nature Chemical Biology, 11: 127-133. Nakamura I, Kanasaki R, Yoshikawa K, Furukawa S, Fujie A, Hamamoto H, Sekimizu K. (2017) J Antibiot (Tokyo). 70: 41-44.

The present invention was made in consideration of the above background technology, and its objective is to provide a method for discovering superior antibacterial agents and antibiotics.

As a result of extensive research to solve 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 bacteria B such as pathogenic bacteria," 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 without missing any.

That is, the present invention provides a method for screening antibacterial agents, which is characterized by including all of the following steps 1, 2, 3, 7 and 8.
Step 1: A soil bacteria colony formation step in which the supernatant of a soil suspension is spread on a sterilized, nutrient-poor solid medium and cultured to form colonies of bacteria A contained in the soil. Step 2: A liquid agar medium is prepared by adding bacteria B to a medium for bacteria B that is the target of antibacterial treatment and 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 layer liquid. The layer liquid is poured onto the solid medium on which the colonies of bacteria A have been formed in step 1 and cooled to form a layer solid medium. Step 3: A purification step of bacterium A, in which an inhibition circle formed around the colony is found on the surface of the bacterium B, which is the target of antibacterial treatment, grown all over the surface of the above-mentioned solid layer medium, and bacterium A is selected and extracted from the colony of bacterium A present at approximately the center of the inhibition circle, and purified to only bacterium A. Step 7: 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, and 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 bacterium B, which is the target of antibacterial treatment.
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.

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.

The present invention also provides the above-mentioned method for screening antibacterial agents, which further comprises the following steps 5 and 6 after the above step 4:
Step 5: A step of preparing a layer solution by adding the bacteria B to the medium for the bacteria B to be treated with antibacterial agents and 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 liquid agar medium for the bacteria B to be treated with antibacterial agents, and pouring the layer solution onto the solid medium in which the bacteria A was purely cultured in step 4 and cooling it to form a layer solid medium. Step 6: A step of confirming that an inhibition circle is formed around the pure cultured bacteria A on the surface of the layer solid medium formed on the pure cultured bacteria A, which is the target of antibacterial agents, and which has grown all over the surface of the layer solid medium.

The present invention also provides the method for screening an antibacterial agent, comprising carrying out the following step a and/or step b in and/or after step 7:
Step a: Dissolving in a poorly water-soluble or water-insoluble organic solvent Step b: Column fractionation

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

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

Furthermore, by using the silkworm infection model, there is no need to narrow down candidate antibacterial substances to a single substance from the early search stage, so purification to a single substance and determination of its structure can be performed at a later stage after narrowing down the candidates to those with good therapeutic effects, thereby saving costs and time.
In addition, since silkworms are used, which are less ethically problematic than vertebrates (mammals) such as mice, it is possible to evaluate a large number of samples.

In the silkworm infection model, it is possible to screen and evaluate (search) samples containing multiple types of antibacterial compounds or samples containing impurities. In order to fully utilize this advantage, the present invention has developed and adopted a method for obtaining samples that is particularly suitable for evaluation in the silkworm infection model.
In other words, the method of the present invention that utilizes inhibition zones is extremely well matched to the silkworm infection model for the above-mentioned reasons, and furthermore, since it captures a large number of antibacterial substances in a crude state without omission, the "method having steps 1, 2, 3, 7, and 8" in the present invention is extremely effective.

The present invention achieves remarkable results 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).

Furthermore, by combining steps 4, 5, and 6, the above-mentioned matching properties and features can be further achieved, and multiple candidate substances can be efficiently separated before evaluation using the silkworm infection model. Even if bacteria A that do not form an inhibition zone are mixed in by step 3, they can be removed, reducing noise.

Furthermore, by combining step a and/or step b, for example, if a "substance that suppresses natural immunity" is contaminated in the "product of 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 natural immune activity of the silkworm is reduced due to the contamination with a substance that suppresses the natural immunity of the silkworm, which then results in the death of the silkworm due to bacterium B. In other words, it is possible to eliminate cases in which the silkworm is killed by another factor when confirming the therapeutic effect of silkworms.
Although steps a and b can be performed whether the fungus B is a bacterium or a fungus, the above-mentioned case often occurs when the fungus B is a fungus. Therefore, the addition of steps a and/or b is particularly effective when the fungus B is a fungus.

In the present invention, the colony of bacteria A, which is a soil bacterium, is itself used as the central substance (object) of the inhibition zone that is formed when the growth of bacteria B, such as a pathogen, is inhibited, so at this stage, neither the bacteria nor the antibiotics are isolated.
As a result, multiple bacteria A and their products (antibiotics) can be screened simultaneously in parallel, and since neither the bacteria A nor the antibiotics are identified in the early stages of the search, waste is eliminated, and costs and time can be saved.

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

The method of the present invention, which has steps 1, 2, 3, 7, and 8, and further which combines these steps with steps 4, 5, and 6 and/or step 9, eliminates all of the drawbacks of the conventional methods described above, and makes it possible to discover an excellent antibacterial agent that is extremely efficient.

According to the method of the present invention having steps 1, 2, 3, (4), (5), (6), 7, (a), (b), 8, and (9) (where the steps in parentheses indicate preferred embodiments), a large number of types of antibacterial agents/antibiotics are required in small amounts at the initial stage of the search (and mixtures are also possible). First, the top priority is given to confirming the bacterium A that produces an excellent antibacterial substance and the very existence of said antibacterial substance. The present invention focuses on this point and can search for and select them over a wide range without omission.

Considering actual use on humans, it is desirable for the antibacterial agent to 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 can be performed here as well.
In addition, the formation of an inhibition circle means that the antibacterial agent or antibiotic that caused the inhibition circle formation is water-soluble. Therefore, screening is also performed efficiently there (in step 3).

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

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," making the process extremely efficient.
In addition, the silkworm infection model in step 8 synergistically exerts the above-mentioned excellent effects due to the fact that it is possible to test crude samples and that silkworms, which have few ethical issues, are used.

As mentioned above, the silkworm infection model makes it possible to screen (search) for "antibacterial agents that even have therapeutic effects" at an early stage of research, which reduces the number of substances that are discarded due to poor pharmacokinetics, etc., at the later stages of clinical trials and other investigations. Therefore, the silkworm infection model itself is an excellent method for searching for antibacterial agents and antibiotics.

In the present invention, the form of the sample to be used in the "test using the silkworm infection model" and the method of obtaining and selecting the sample have been further studied and improved. As described above, the sample obtaining and selection method in the present invention (steps 1 to 7) is extremely well matched for the silkworm infection model, making the most of the features of the silkworm infection model. Thus, the silkworm infection model has been improved by the present invention.

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 a method using steps 1, 2, 3, (4, 5, 6), 7, and (9) of the present invention and a method not using the above steps of the present invention, although this depends on the silkworm infection model.
In Figures 1(a), (b), and (c), the populations used in the search are different, namely, "compound library,""naturalproducts," and "soil," respectively. However, as shown in Figure 1(c), the probability of discovering "antibiotics with therapeutic effects" according to 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 realistic.

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

As described in the Examples, acetone extracts of culture medium were prepared from 329 strains (up to step 6) that formed inhibition circles against E. coli W3110, and 18 samples (up to step 8) with anti-Pseudomonas aeruginosa therapeutic effects in silkworms were obtained (Figure 1(c)). Up 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.

In the present invention, a silkworm infection model is used to screen candidates that take into account not only the MIC value, which is an index of antibacterial activity, but also the therapeutic effect, making it possible to pick up all of the bacteria A (its products) (antibacterial candidate agents) that have been missed from screening in the past 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, according to the present invention, antibacterial agents for resistant bacteria can be searched for.

1 is a table showing the difference in the discovery efficiency of antibacterial substances depending on the change in the selected (obtained) form of the "sample to be administered to silkworms" when using a silkworm infection model. (a) Discovery efficiency in the conventional method not including steps 1 to 8 when starting from a compound library (b) Discovery efficiency in the conventional method not including steps 1 to 8 when starting from natural products (c) Discovery efficiency in the method of the present invention including steps 1 to 8 when starting from soil 1 is a process flow of one example of a preferred embodiment of the present invention. This is a photograph showing an example of a colony of soil bacteria A formed on a nutrient-poor solid medium in step 1. 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. This 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. 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. 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 6. This 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. 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. 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.

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

The method for screening antibacterial agents of the present invention comprises at least the following steps: Note that steps 4 to 6 and step 9 are not essential but are preferably included.
Step 1: A soil bacteria colony formation step in which the supernatant of a soil suspension is spread on a sterilized, nutrient-poor solid medium and cultured to form colonies of bacteria A contained in the soil. Step 2: A layered solid medium formation step in which bacteria B that is the target of antibacterial treatment is added 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 is the target of antibacterial treatment does not die when added to the medium" to prepare a layered liquid, and the layered liquid is poured onto the solid medium in which the colonies of bacteria A were formed in step 1 and cooled to form a layered solid medium. Step 3: A purification step of bacteria A in which an inhibition circle formed around the colony of bacteria B that is the target of antibacterial treatment and has grown all over the surface of the layered solid medium is found, and bacteria A is selected and harvested from the colony of bacteria A that exists approximately in the center of the inhibition circle, and only bacteria A is purified. Step 4: A colony of bacteria A purified in step 3 is purified. Step 5: A step of adding the bacteria B to be treated with antibacterial agents to prepare a layer liquid, and pouring the layer liquid onto the solid medium in which the bacteria A was purely cultured in step 4 and cooling it to form a layer solid medium. Step 6: A step of confirming that an inhibition circle is formed around the pure cultured bacteria A on the surface of the layer solid medium formed on the pure cultured bacteria A, which is the target of antibacterial treatment, of the bacteria B that has grown all over the surface of the layer solid medium formed on the pure cultured bacteria A. Step 7: A step of culturing the bacteria A in a liquid medium, adding an organic solvent and stirring to mix, drying the soluble matter, and adding water to prepare a sample for measuring the MIC value, and selecting a "sample derived from bacteria A" that has an MIC value of 1/10 or less against the bacteria B that is the target of antibacterial treatment.
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 step 6 using a silkworm infection model.
Step 9: An antibacterial agent obtaining step of isolating and obtaining the antibacterial agent from the sample selected in step 7.

<Step 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.
In addition, 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 only to distinguish "bacteria A", which is a soil bacterium, from "bacteria B", which is the target of antibacterial treatment, and when "bacteria A" is written, the bacteria is not limited to one type, and may be multiple types. Rather, it is preferable that multiple types are present at the same time up to step 3 or before step 6.
The above-mentioned "suspension of soil S" is preferably a suspension prepared by adding sterilized water to soil S.

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

When using conventional nutrient media such as YME agar medium, the growth rate of different strains varies significantly, resulting in the formation of large colonies that inhibit the formation of colonies by other strains. Furthermore, simply lowering the concentration of nutrients may limit the types of colonies that appear.
By using a nutrient-poor solid medium, it is possible to obtain a large number of small colonies (preferably many types) at once, resulting in a shape suitable for the object (colony) present approximately in the center of the inhibition zone in step 3.

The size of the colonies of bacteria A formed in step 1 is preferably an average diameter of 0.5 mm to 10 mm, more preferably an average diameter of 1 mm to 5 mm, and particularly preferably an average diameter of 2 mm to 3 mm.

If the colonies of bacteria A are too large, it may be possible to evaluate only a small number of colonies or bacteria 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 bacteria A is too small, bacteria 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.

A particularly preferred example of a nutrient-poor solid medium is one obtained by adding water to soil R, thoroughly stirring and mixing the mixture, sterilizing the mixture 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 above-mentioned "poorly nutrient solid medium" preferably contains a soil extract.

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 in 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, although there is no particular limitation, it is particularly preferable that the soil R is soil S. In other words, it is particularly preferable to use the same soil.

In the present invention, "soil" refers to a naturally occurring solid substance as a whole, and refers to a mixture of inorganic matter such as minerals, rocks, and sand; organic matter such as biological products, excrement, corpses, and humus; natural liquids such as petroleum, petroleum precursors, petroleum spring water, mineral water, hot spring water, river water, lake water, and sea water; and living organisms (living organisms). In addition to soil that exists on the ground, it also includes soil that exists at the bottom of bodies of water such as rivers, lakes, and seas.

Soil contains inorganic and organic matter specific to that soil, and if the inorganic and organic matter contained in soil S, which contained bacteria A producing the antibacterial agent to be searched for, and soil R, which is the raw material for the soil extract of the fixed medium, are the same, it is suitable as a culture environment for bacteria A. In particular, if the mineral components, trace element components, metal (ion) components, acid-alkali components, etc. are the same, it is considered to be more suitable as a culture environment for bacteria A.
This makes it possible to easily use even trace amounts 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.

The sterilization treatment in step 1 can be carried out by a known method (conventional method) such as autoclaving.
Alternatively, water may be added to the soil S to prepare a suspension, and the resulting supernatant may be spread and cultured to form soil bacterial colonies, using a known method.

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

The culture conditions for forming colonies of Bacterium A are not particularly limited, but a temperature between 4°C and 40°C is preferred, and a temperature between 27°C and 32°C is particularly preferred. There are also no particular limitations on the culture time, but a temperature between 24 hours and 120 hours is preferred, and a temperature between 48 hours and 96 hours is particularly preferred.

<Step 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 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 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 were formed in step 1 above and cooling it to form a layer solid medium.

The bacteria B that is the target of antibacterial treatment is preferably pathogenic, and unlike the 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 more 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/medium resistance to existing antibacterial agents are particularly preferred in the present invention.

Bacterium B in step 2 and bacterium B in step 8 described below may be different if 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.

In order to pour the "stratification liquid containing bacteria B" to form a stratification solid 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, it is essential that the agar used here is an agar that is liquid when added to the medium for bacteria B at at least one temperature at which bacteria B does not die.

The temperature of the stratification liquid when actually poured is equal to or higher than the "melting point of the agar or the melting point of the stratification liquid" and equal to or lower than the "maximum temperature at which bacteria B does not die."
Here, the "melting point" refers to the minimum temperature within the temperature range at which the bilayer liquid 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 the state in which agarose is mixed with other substances to form what is known as "agar".

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

For example, if bacterium B is Staphylococcus aureus, Staphylococcus 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 do not die at 45°C but do die at 58°C. Therefore, for example, if "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 bacteria will die and an inhibition zone cannot be formed using the process described below.

As long as the above conditions are satisfied, the agar or agarose to be used is not particularly limited, but what is called low melting point agarose, soft agar, etc. are preferably used.
Hereinafter, steps 2 and 5, which use agar or the like, may be abbreviated as "soft agar overlay."

The agar medium obtained by adding agar to the medium for bacteria B is in a liquid state (at the temperature at which it is obtained), and is then sterilized in an autoclave or the like before adding bacteria B to prepare a stratification solution. This is preferable from the viewpoint of eliminating bacteria other than "bacteria B, which are the target of antibacterial treatment."

The layer solution obtained as described above is poured onto the solid medium on which the colonies of Bacterium A have been formed in step 1, and cooled to form a layer solid 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 the mixture remains solid or non-fluid at the subsequent incubation temperature (the growth temperature of bacteria B).

The thickness of the solid layer medium obtained by the layer 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 all over the 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 solid overlay medium is too thick, the antibiotic secreted from the producing bacteria may be diluted by diffusion, making it impossible to observe the inhibition zone.

<Step 3>
Step 3 is a purification step of bacteria A, in which an inhibition circle formed with the colony as the center on the surface of bacteria B, which is the target of antibacterial treatment and has grown all over the surface of the multilayer solid medium, is found, and bacteria A is selected and extracted from the colony of bacteria A present approximately in the center of the inhibition circle, and bacteria A is purified only from bacteria A. Note that when "bacterium A" is written, the bacteria A may be one type or multiple types.

Prior to step 3, incubation is performed to grow the bacteria B. The temperature varies depending on the type of the 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, the bacteria B cannot grow over the entire surface, and an inhibition zone may not be formed, etc. 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 the bacteria B may not grow, making it impossible to observe an inhibition zone, etc.

The incubation time varies depending on the type of bacteria B, but is preferably from 6 hours to 48 hours, and particularly preferably from 12 hours to 24 hours.
If the time is too short, the bacteria may not grow sufficiently to make the inhibition zone observable, whereas if the time is too long, the inhibition zone may become observable due to the bacteria growing.

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

The solid medium used for purifying Bacterium A is not particularly limited, and a 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 conventional methods.

<Step 4>
Step 4 is a step of purely culturing the colonies of bacteria A purified in step 3 on a solid medium.
Step 4 is not essential, but is preferable since 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" may be repeated.

The solid medium used in step 4 is not particularly limited, and a general-purpose medium such as YME agar medium can be used. There are also no particular limitations on the growth conditions, and growth can be carried out by conventional methods.

<Step 5>
Step 5 is a step of preparing a layer solution 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' for the bacteria B that is the target of the antibacterial treatment," and then pouring the layer solution onto the solid medium in which the bacteria A was purely cultured in step 4 above and cooling it to form a layer solid medium.

As described above, the "bacteria A" selected and collected in step 3 often contains a mixture of multiple species, 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 thus inhibition zones may be formed.
Step 5 is carried out in order to eliminate "bacteria A that cannot form an inhibition zone" that was mixed with "bacteria A that formed an inhibition zone" at the stage of step 3. Note that "steps 5 and 6" can be repeated.

In step 5, the type of agar, preparation conditions for the stratification liquid, type of bacteria B, concentration of bacteria B, temperature conditions, thickness of the solid stratification medium, incubation conditions for the solid stratification medium, etc. do not have to be exactly the same as in step 2, but the preferred ranges, etc. are the same as those in step 2.

<Step 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 target bacteria B, which has grown over an entire surface on the layer solid medium formed on the pure cultured bacteria A.

Prior to step 6, incubation is performed to grow bacteria B. The incubation conditions may be the same as or different from those of the incubation performed prior to step 3, but the range of conditions is the same as that of the incubation performed prior to step 3 described above.

In the present invention, the bacteria referred to as "bacteria A" is not limited to one type, and may be a mixture of multiple types. However, in step 6, "bacteria A that do not form an inhibition zone against bacteria B" are eliminated, and only bacteria A that form an inhibition zone against bacteria B are isolated, purified, and selected. In other words, "bacteria A that do not produce antibacterial agents" that were selected and collected in step 3 are eliminated.

<Step 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" having an MIC value of 1/10 or less against bacterium B, which is the target of antibacterial treatment, is selected. Step 7 is a step in which antibacterial activity is evaluated and a sample is 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.

The liquid medium used in step 7 is not particularly limited, and any known medium may 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.

The organic solvent is not particularly limited as long as it dissolves the antibacterial agent/antibiotic, which is the target component present in the liquid medium, but it is preferable that the organic solvent is compatible with water (contained in the liquid medium) at the ratio used, or is compatible to a certain extent with water.
Specific examples of the solvent 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, and hydrocarbons such as hexane. Particularly preferred are acetone, methanol, ethanol, etc.

The "MIC value" here is defined as follows:
The colony of bacteria A obtained in the step preceding step 7 is cultured in 20 mL of YME liquid medium at 30° C. for 4 days, and the same volume of acetone is added thereto and centrifuged to obtain a supernatant. The supernatant is evaporated to obtain a solid component, which is dissolved in 1 mL of pure water, and the obtained solution is used as a "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, and the antibacterial properties of the test liquid are measured by measuring the decrease in turbidity or the decrease in viable cell count against the target bacteria B using a suitable method.
The reciprocal of the maximum dilution ratio exhibiting antibacterial activity obtained by the above method, i.e., the minimum value among the "reciprocals of dilution ratios exhibiting antibacterial activity", is defined as the "MIC value".

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

The above-mentioned sample liquid preparation method and the above-mentioned antibacterial measurement method are the preparation method and measurement method used to define the "MIC value" of the present invention, and it goes without saying that the sample preparation method and measurement method used when actually implementing 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 and measurement method actually implemented, as long as the result falls within the range of the "MIC value defined above," it is included in the technical scope of the present invention.

It is essential to select "samples derived from Bacteria A" whose MIC value, which is the selection criterion, is 1/10 or less. This is based on the principle that when 50 μL of the sample is injected into silkworms (5th instar silkworms weighing 2 g used) with a blood volume of 500 μL, the sample is diluted 10 times. It is not necessary to select all samples whose MIC value is 1/10 or less, but it is most preferable to select all of them.
The selection criteria may be made stricter from this stage. In this case, the threshold MIC value used as the selection criteria is preferably "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".

If the above value is too small, antibacterial agents that are actually excellent may be eliminated in step 7, or the screening may be too strict, resulting in too few samples before evaluation in the silkworm infection model in step 8. On the other hand, if the above value is too large, agents that do not have practical antibacterial properties may be selected, or the screening may be too lenient, resulting in too much noise in the samples used in step 8.

Taking into consideration the body fluids and body weight of the silkworms, it is preferable to inject about 50 μL of sample liquid into the silkworms. The blood (body fluids) of 5th instar silkworms on the first day used in the infection treatment experiment is about 500 μL per silkworm. Therefore, the sample liquid is diluted to 1/10 in the blood (body fluids) of the silkworms. Therefore, the antibacterial activity of the sample used in the infection treatment experiment needs to be 1/10 or less in terms of MIC value. With samples with a higher MIC value, no therapeutic effect can be expected. In cases where antibacterial activity is promoted by substances derived from body fluids, a therapeutic effect may be obtained even if the MIC value is greater than 1/10, but such cases are unusual.

<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 step of column fractionation.
Step a and/or step b are performed "in step 7, i.e., during step 7" and/or "after step 7". Here, "after step 7" includes performing the steps following step 7, as well as performing the steps 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).

Step ab may be performed with a sample prepared in step 7 by culturing bacteria A in a liquid medium, adding an organic solvent such as acetone, stirring and mixing, drying the soluble matter, and adding water (hereinafter, this operation may be abbreviated simply as "acetone extraction" without being limited to acetone); it may also be performed after measuring the MIC value with a "sample prepared by adding an organic solvent, stirring and mixing, drying the soluble matter, and adding water to dissolve it."

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

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 replaced with each other, 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

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

In the present invention, steps a and b are not essential, but by performing them, samples with therapeutic effects can be consistently picked up without missing any in step 8. In other words, "sensitivity" is increased.

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 the case of such samples, it is preferable to carry out step a and/or step b to remove the substances.

In the case of such a sample, even if silkworms are subsequently used in step 8 to confirm the therapeutic effect, the silkworms may die not from Bacterium B but from the above-mentioned substance. In other words, even if the bacteria actually has therapeutic activity against Bacterium B, it is possible that the silkworms may die from the injected bacteria B due to, for example, the presence of a substance that suppresses the natural immunity of the silkworms. By carrying out step a and/or step b to remove the "substance that suppresses natural immunity" or the "toxic substance," such cases can be eliminated. 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.

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

<Step 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, the soluble portion is dried and solidified, and water is added to the resulting mixture to prepare a sample. To this mixture, a "poorly water-soluble or water-insoluble organic solvent" is added, and, for example, stirred, to dissolve substances that are more soluble in the "poorly water-soluble or water-insoluble organic solvent" than in "water" in the "poorly water-soluble or water-insoluble organic solvent." For example, the liquids are separated using a separatory funnel or centrifugation, and the "poorly water-soluble or water-insoluble organic solvent" layer is separated and collected.

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; chlorine-based 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.

Because 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.

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

<Step 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.

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 weakening of the silkworms.
The silkworm infection model is described in, for example, Patent Documents 1 and 2 and Non-Patent Documents 1 to 3, and is clearly defined and known to be effective. In addition, it has been confirmed that the LD50, ED50, and the like of the silkworm infection model are similar to those of mammals such as mice.

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 performed taking into account not only the antibacterial effect but also the therapeutic effect.

It is also preferable to carry out the above-mentioned step a and/or step b after step 8. In addition, 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 natural immunity has been inhibited and the silkworms have been killed, 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."

<Step 9>
Step 9 is an “antibacterial agent obtaining step” in which the 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 by step 8, and is therefore an antibacterial agent that has an antibacterial therapeutic effect 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 the 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 the water/organic solvent in step 7.

The method for separating the antibacterial agent is not particularly limited, and may be, for example, 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.

In the present invention, since there is no need to isolate the antibacterial agent/antibiotic until step 9, screening is extremely efficient. Also, in step 9, there is no need to secure an amount of the antibacterial agent/antibiotic to be administered to, for example, a mouse, 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.

The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples as long as the gist of the present invention is not exceeded.
In "Example 1", only an example in which all steps 1 to 9 were performed is described, and only representative bacterial species are 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 this Example 2 as long as it does not depart from the gist of the present invention.
Additionally, when "%" refers to the amount of a substance, it means "% by mass" unless otherwise specified.

Example 1
<Step 1>
<Preparation of solid medium containing soil extract and cultivation of bacteria A (formation of soil bacteria colonies)>
2.5 L of water (RO water) was added to 2 L of soil R, and the mixture was autoclaved (121° C., 20 min), then centrifuged (8000 rpm, 5 min) to obtain a 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 plastic petri dish having a diameter of 10 cm and allowed to solidify at room temperature to prepare a nutrient-poor solid medium.

An equal amount of water (RO water) was added to soil S, stirred and left for 5 minutes, the supernatant was diluted 1000-fold with physiological saline, and 100 μL of this was spread with a cone-larger on the above solid medium (agar medium containing soil extract of soil R) and cultured at 30°C for 4 days to form soil colonies of bacteria A.

Soil R and soil S were taken from the same location and divided for use, so they were considered to be essentially identical.

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

<Step 2>
<Formation of layered solid medium (layering soft agar containing bacteria 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) was used for Escherichia coli, and YPD medium was used 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 stratification solution (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.

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 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 stratification solutions was poured into a petri dish having a diameter of 10 cm on which colonies of soil bacteria had formed, and allowed to stand at room temperature for 30 minutes or more to obtain a solid stratification medium.
Then, the mixture was incubated at 30° C. for one day.

<Step 3>
Observation of inhibition zones and purification of bacteria A that formed inhibition zones
After the above incubation, the formation of an inhibition zone was observed. Bacterium A was collected from a colony of bacteria A that was present in the center of the inhibition zone and was considered to be a producer of antibacterial agents and antibiotics. Here, bacteria A is not necessarily one type of bacteria.

Since there was a possibility that a small amount of solid layer medium or antibiotic non-producing bacteria was present in the collected bacteria A, 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 a conventional method.

<Step 4>
<Pure culture of bacteria A obtained in step 3>
Bacterium A in the colony obtained in step 3 was purely cultured on YME agar medium.

<Step 5>
<Formation of a solid medium by layering soft agar containing bacteria B on purely cultured bacteria A>
In step 2, a solid medium was formed by layering soft agar containing bacterium B on the pure cultured bacterium A in the same manner as in step 2, except that in step 2, "colonies of soil bacteria were formed" was changed to "bacterium A was cultured in pure culture."

<Step 6>
<Observation of inhibition zone>
In the pure culture of bacteria A, there were cases where bacteria B that had grown over an entire surface did not form an inhibition zone. These bacteria were eliminated, and only bacteria A that formed an inhibition zone was selected.

<Step 7>
<Selection of a sample with a small MIC value (extraction with organic solvent and measurement of the MIC value of the obtained sample solution)>
The candidate strains producing the antibacterial agent/antibiotic purified in step 3 were cultured with shaking in 20 mL of YME liquid medium at 30° C. for 4 days.
20 mL of acetone (the same amount as the YME liquid medium) was added to the resulting culture solution, stirred, and centrifuged (8000 rpm, 5 minutes) to obtain a supernatant. The supernatant was dried (dehydrated and 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 to between 5 and 8 with 10 N NaOH to prepare a sample (liquid) for measuring the MIC value.

The same strain of gram-positive bacteria, Staphylococcus aureus, was used in step 7, but gram-negative bacteria, Escherichia coli, were used for evaluation. In addition, the fungus, baker's yeast, was changed to the pathogenic fungus, Candida albicans (ATCC10231), in step 7.

The above sample (liquid) was diluted in 8 stages (1/2 to 1/256) using a 2-fold dilution series with physiological saline, and 50 μL of each 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 determine the MIC value.

For Candida, a bacterial suspension with an absorbance of 0.5 at 530 nm was diluted 1/1000 with RPMI medium, mixed with 100 μL of the sample, and incubated at 37°C for 48 hours to determine the presence or absence of turbidity.

Samples (liquids) that showed MIC values of 1/10 or less were selected for the next step, which was a therapeutic test using silkworms. In other words, sample liquids 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 5th instar larvae (weighing 2 grams) used was 500 μL, and the sample volume used in the injection test was 50 μL, so the sample was diluted 10 times immediately after injection, and unless the MIC value was 1/10 or less, therapeutic effects could not be expected. This situation does not apply to antibiotics whose antibacterial activity is promoted by substances in the body fluid, but such cases are exceptional.

<Step 8>
<Treatment test in silkworm infection model (selection of samples with antibacterial therapeutic effect against Bacillus subtilis)>
For samples showing MIC values of 1/10 or less, the original solution and various dilutions were prepared and subjected to a treatment test (screening (exploration) using a silkworm infection model). For the reasons mentioned above, the MIC value of the diluted sample was made not to be greater than 1/10.

In steps 2 to 6, the gram-positive bacteria used were Staphylococcus aureus (MSSA1 strain), the gram-negative bacteria used were Escherichia coli (W3110 strain), and the fungus used was Baker's yeast (Saccharomyces cerevisiae, BY4741 strain), but 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.

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

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 in the above order at 1/10, 1/1000, and 1/1, and 50 μL of each was injected into the blood of silkworms.
Immediately after the injection, 50 μL of the above sample solution diluted with water (sterile Milli-Q water) ("Milli-Q" is a registered trademark) was injected into the blood of the silkworms. The total number of silkworms per group was 4.

For each of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida, the survival or death of silkworms was judged 24 to 48 hours after injection, and 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.

Samples were selected that had antibacterial therapeutic effects against each of the Bacteria B (Staphylococcus aureus (Table 1), Pseudomonas aeruginosa (Table 4), and Candida albicans).

When bacterium B was Staphylococcus aureus, for samples that showed a therapeutic effect in the silkworm infection model, the MIC values against multiple strains of MRSA (methicillin-resistant Staphylococcus aureus) were also determined (Table 2).
MRSA is known to be resistant to antibiotics 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 strains of MRSA were determined at this stage to not be novel antibiotics.

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).

<Step 9>
<Isolation and acquisition of antibacterial agents>
LC-MS Analysis of Therapeutically Active Fractions
For t4-11, in which a therapeutic effect was observed in the acetone-extracted fraction, the fraction was dissolved in butanol and fractionated by HPLC using a C18-ODS column.
The fraction (eluted at 18-19 min) that showed therapeutic effects in the silkworm infection model was further analyzed by LC-MS (Waters, Xevo G2-XS Q-TOF). The accurate mass of each peak in the LC was determined and compared with known antibiotics using the Dictionary of Natural Products.

[Results of Example 1]
An outline of the operation 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 performing all steps 1 to 8 means performing a total of four screenings.

<Step 1>
A nutrient-poor soil agar medium was prepared by adding water to soil R, autoclaving it, and then adding agar to the supernatant. By using this medium, it is possible to obtain a large number of small colonies at once (Figure 3).
As described 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 circle. In step 3, the inhibition circle could be favorably observed in terms of size, number, etc., and bacteria A could be harvested from the center of the inhibition circle (Figures 4 to 6).
On the other hand, when a conventional nutrient medium such as YME agar medium was used, the growth rate significantly differed depending on the strain, resulting in the formation of large colonies, which inhibited the formation of colonies by other strains (not shown). Furthermore, when the nutrient concentration was simply lowered, the types of colonies that appeared were limited.

<Step 2 and Step 3>
In order to know 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" 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 antibacterial activity will be lost due to the 10-fold dilution with blood immediately after injection.

In actual screening, it is not easy to obtain many 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, which led to the present invention, which involves collecting antibiotic-producing bacteria that form inhibition zones from soil.

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 "colonies of soil bacterium A on soil R agar solid medium" and incubated, inhibition zones were found for all bacteria (Figures 4 to 6).
From the center of the inhibition circle, the "bacteria A forming the inhibition circle" could be suitably collected (bacteria A may be a mixture).

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

<Step 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 zones, showed MIC values of 1/8 or less.
As for fungi, 147 strains (samples), which accounted for 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).

<Step 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 determined and compared with those of Staphylococcus aureus (MSSA1).
As a result, it was found that none of the 11 samples contained resistant MRSA strains (Tables 1 and 2).

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

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 (Tables 1 and 3).
From the above, it was found that the present invention is an excellent method for screening antibacterial agents and antibiotics for all gram-positive bacteria.

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 (Tables 1, 6, and FIG. 1(c)).
This result shows that the discovery efficiency is 1000 times higher than that of the conventional method of searching from a compound library and 10 times higher than that of the conventional method of searching from natural products (Figures 1(a)(b)(c)). The main reason for this is that the introduction of the inhibition zone formation method in the present invention has made it easier to obtain many samples with high antibacterial activity.

<<Pseudomonas aeruginosa>>
The antibacterial activity, bacterial species, and butanol solubility of the therapeutically active substances selected in step 8 of the samples that showed therapeutic effects against Pseudomonas aeruginosa PAO1 are shown in Table 4.
In the silkworm infection model (step 8), 18 samples were obtained that showed a therapeutic effect against Pseudomonas aeruginosa (Table 4).
All of the 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 indicates that the therapeutic activity of these samples is due to the immune activation effect of silkworms.

Table 5 shows the acetone extractable fraction, butanol soluble fraction, and activity yield for the 10 samples that showed therapeutic effects against Pseudomonas aeruginosa in the silkworm infection model (step 8).

Of the 329 strains that formed inhibition zones against E. coli W3110, 27 samples were obtained with MIC values of 1/8 or less, and 18 samples were obtained that had anti-Pseudomonas aeruginosa therapeutic effects in silkworms (Table 4, FIG. 1(c)).
Until now, it has not been possible to discover substances that are effective against gram-negative bacteria using conventional methods (FIG. 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.

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

<Step 9>
<< Staphylococcus aureus >>
The therapeutic effect of the butanol-soluble fraction was tested for 10 of the 11 samples in which the MRSA strain was determined not to exhibit resistance. As a result, it was found that the therapeutically effective substance was dissolved 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").

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

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 shows that the present invention is effective in discovering antibacterial agents and antibiotics.

Samples No. t5-2, t5-3, and t5-18 have the same absorption pattern in HPLC analysis, and are produced by the same species of bacteria (Streptomyces capoamus), suggesting that they are substances derived from the same producing bacteria. All of these may be new antibacterial agents and antibiotics.

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 in a silkworm infection model was examined.
As a result, therapeutic activity was found in the fraction eluted at 18 to 19 minutes. Furthermore, this fraction was analyzed by LC-MS to determine the exact mass of each peak that appeared, and it was found that the mass matched with WAP-8294 A1 and WAP-8294 A2, which have already been reported as antibiotics that show therapeutic effects. The fact that known excellent antibiotics could be discovered demonstrated that the present invention is effective in the discovery of antibacterial agents and antibiotics.

<<Pseudomonas aeruginosa>>
The therapeutic effect of the butanol-transferable fraction was tested for the 18 samples that showed a therapeutic effect, and therapeutic effects were suggested for 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, w17-10) (Table 1, item "Butanol-transferable").

Example 2
Example of carrying out steps a and b with fungi
As shown in the results of Example 1, when the 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 circle, steps 4, 5, and 6 were carried out as described above, and the number of bacteria that were confirmed to have formed an inhibition circle (after step 6) was counted.

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

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 (MIC value is 1/10 or less) is present in the sample up to step 7, the sample may also contain immunosuppressant substances or toxic substances that cause the silkworms to die, and therefore the true antibacterial therapeutic effect in the silkworm infection model may not be seen.

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 silkworms were cultured in liquid medium until they reached full growth, 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.

That is, when the fungus B in Example 1 was Candida fungus, 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 of the silkworms died.

If Candida was not injected, the mice remained alive on both the first and second days (Nos. A and C).
On the other hand, when Candida fungus was injected, the mice died on the second day regardless of whether or not they received a sample injection (No. B, D), whereas 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 at the same time. In other words, it was thought that the natural immune suppressants in the sample suppressed the silkworms' natural immunity, and therefore the silkworms may have died due to Candida fungus.

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, particularly against fungi. We therefore conducted the following study.

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

In Table 9 below, 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 above. As the bacterium B, a bacterium of the genus Candida (Candida albicans, ATCC10231) was used.

<<Selection of Bacterium A against Bacterium B (Candida)>>
As mentioned above, in the case of Candida fungus, the number of samples was 232 after step 7, but 0 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 called "#1" to "#10" respectively (see Table 10).

<<<Condition 1>>>
In step 7, the acetone-extracted sample had high anti-Candida activity (low MIC value (high 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.

<<Step 7: Preparation of acetone extract fraction>>
Glycerol stocks of the bacteria selected under the above two conditions were spread on 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 each containing 20 mL of YME liquid medium, and cultured with shaking at 30° C. and 200 rpm for 4 days.
After the liquid culture, the same amount of acetone was added, and the mixture was centrifuged at 8,000 rpm for 5 minutes. The centrifuged supernatant was dried and dissolved in the above-mentioned Milli-Q water in an amount of 1/20 of the liquid medium. The pH was then adjusted to 7.

<<Step a: Preparation of butanol-soluble fraction>>
The same amount of butanol as that of the acetone extract was added, and the mixture was centrifuged at 8,000 rpm for 5 minutes to obtain a centrifugal supernatant. The time required for this step was within 10 minutes per sample.
The resulting centrifugal supernatant was dried for 12 hours using a centrifugal evaporator, and then dissolved in the same amount of physiological saline as before the addition of butanol.

<<Step 7: Measuring MIC>>
Candida was cultured in Sabouraud liquid medium for 2 days. The culture medium was centrifuged to collect the bacteria, and the bacterial concentration was adjusted to OD ₅₃₀ =0.5 with physiological saline.
The resulting bacterial solution with OD ₅₃₀ =0.5 was diluted 1/1000 with RPMI1640-MOPS and dispensed in 100 μL portions 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 to perform serial dilution.
Thereafter, the mixture was cultured at 37° C. for 2 days, and the minimum concentration at which the proliferation of the bacteria was inhibited was determined as the MIC value.

<<Step b: Fractionation using 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, followed by elution 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.

<<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 bacteria were collected. The culture medium was suspended in 10 times saline, and 50 μL of the suspension was intravascularly injected into 5th instar silkworm larvae that had been fed for one day. Immediately after that, 50 μL of the sample diluted with saline was intravascularly injected into each larva.
The survival and death of the silkworms was judged one and two days after the injection.

In evaluating therapeutic effects (tests) using silkworms, in order to expect a therapeutic effect, it is necessary for the MIC value of the sample to be 1/10 or less. This is because when 50 μL of a sample is injected into the blood of a silkworm (body weight 2 g), it is immediately diluted to 1/10. In this study, the antibacterial therapeutic effect of a sample with a MIC of 1/10 or less was examined in silkworms.

[Results of Example 2]
<Step 7: Antibacterial Activity Test>
Table 10 shows the MIC values (dilution ratios) 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).

As can be seen from Table 10, of the 10 samples #1 to #10, 6 samples (Nos. 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 antibacterial activity tests were carried out. As a result, the high concentration MeOH elution fractions of all six samples showed antibacterial activity with "MIC value <1/10" (see Table 10).

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

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

More than half of the silkworms died one day after injection of the acetone extract fraction (1/1 (undiluted), 1/10 diluted). That is, as described above, when the sample that had not been subjected to 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 "samples subjected to step a: butanol dissolution and/or step b: column fractionation" and then injected with Candida, more than half were still alive one day later (survival rate "2/3" or "3/3"), as can be seen from Table 11. This suggests that natural immune suppressants or toxic substances were removed by carrying out step a or step b.

Furthermore, among the samples and those 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 injection 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 the case of No. D in Table 8, in which neither step a nor step b was performed, more than half of the mice died on the first day, so naturally more than half of the mice died on the second day (see Table 8).
In Table 11, "AmpB" represents "amphotericin B," an antifungal drug that is widely used against fungal infections and was used as a positive control.

When Bacterium 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), but by performing steps a and b, the number of samples in which a therapeutic effect was confirmed in step 8 was 1 (#1) out of 10 selected samples (see Table 12).

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

Even if 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, it was possible to simultaneously search for many strains of Bacterium A and samples from Bacterium A.

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 (ratio of about 1/10) (see Table 12). This suggests that of the 222 samples not yet examined in Example 2, there may be about 22 samples (about 1/10) that have a therapeutic effect.

<Step 9: Isolating and obtaining antibacterial agent>
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 or not.

The "substance obtained in step a (butanol transfer, etc.) and/or the substance adsorbed and eluted in step b (ODS column, etc.)" can be further purified easily in step 9, and the novelty of the substance as an antibacterial agent can be easily determined. In other words, such substances can be purified by HPLC using a C18 column, etc., and the novelty can be determined based on the molecular weight determined by LC/MS.

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. " In addition, since it is known that the therapeutic effects of antibiotics are consistent between silkworms and mice, it is believed that antibiotics that have a therapeutic effect on silkworms in the present invention will also have a therapeutic effect on mice (and therefore humans).
Furthermore, some of the results obtained using the screening method of the present invention were "expected to have a therapeutic effect against resistant bacteria," which suggests that they may be novel antibiotics that have not yet been used clinically.

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

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 (10)

A method for discovering an antibacterial agent, comprising all of the following steps 1, 2, 3, 7 and 8:
Step 1: A soil bacteria colony formation step in which the supernatant of a soil suspension is spread and cultured on a sterilized " poorly nutrient solid medium containing agar and a soil extract, which is the supernatant of soil that has been sterilized by adding water to the soil and mixing it" to form colonies of bacteria A contained in the soil. Step 2: 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 that is the target of the antibacterial treatment, and adding the bacteria B to the medium to prepare a stratification liquid, and pouring the stratification liquid onto the solid medium on which the bacteria A colonies have been formed in step 1 above, and cooling it. Step 3: A purification step of bacterium A, in which an inhibition circle formed around the colony is found on the surface of the bacterium B, which is the target of antibacterial treatment, grown all over the surface of the solid layer medium, and bacterium A is selected and picked from the colony of bacterium A present at approximately the center of the inhibition circle, and purified to only bacterium A. Step 7: 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, and water is added to prepare multiple samples with different concentrations of bacterium A , and the antibacterial activity against the bacterium B, which is the target of antibacterial treatment, is measured for each sample , and a "sample derived from bacterium 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. 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. 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. 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 layer solution by adding the bacteria B to the medium for the bacteria B to be treated with antibacterial agents and 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 liquid agar medium for the bacteria B to be treated with antibacterial agents, and pouring the layer solution onto the solid medium in which the bacteria A was purely cultured in step 4 and cooling it to form a layer solid medium. Step 6: A step of confirming that an inhibition circle is formed around the pure cultured bacteria A on the surface of the layer solid medium formed on the pure cultured bacteria A, which is the target of antibacterial agents, and which has grown all over the surface of the layer solid medium. 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: Dissolving in a poorly water-soluble or water-insoluble organic solvent Step b: Column fractionation The method for screening an antibacterial agent according to any one of claims 1 to 4 , wherein the step 7 is carried out after carrying out the following step a and/or step b:
Step a: Dissolving in a poorly water-soluble or water-insoluble organic solvent Step b: Column fractionation 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. 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. 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. 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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