Composition for improving drug sensitivity of biofilm, and antimicrobial composition for microorganisms in biofilm

Abstract

To provide a technique that enables improvement of drug sensitivity with respect to microorganisms in a biofilm.SOLUTION: The present invention can provide a composition for improving drug sensitivity to microorganisms in a biofilm, the composition containing a redox molecule. Further, the present invention can provide an antimicrobial composition for microorganisms in a biofilm, the antimicrobial composition containing an antimicrobial component and being used in combination with a composition for improving drug sensitivity to microorganisms in the biofilm.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for increasing the drug sensitivity of a biofilm, an antimicrobial composition against microorganisms in a biofilm, and the like. [Background technology]

[0002] In recent years, research into biofilm inhibition has been conducted. Generally, microorganisms (e.g., filamentous fungi, bacteria, microalgae, etc.) form biofilms using substances (e.g., extracellular matrix such as polysaccharides, proteins, and DNA) produced extracellularly. The biofilms contain the microorganisms that produced the biofilms, as well as other microorganisms (e.g., other species, other filamentous fungi, bacteria, and in some cases, viruses), and various microbial products. Furthermore, the microorganisms are thought to be distributed three-dimensionally within the biofilm, repeatedly multiplying and producing extracellular substances. For example, plaque, a mass of bacteria that forms in the oral cavity and adheres to teeth and gums, is considered a type of biofilm and is one of the direct causes of periodontal disease, including gingivitis and periodontitis. Furthermore, when biofilms develop in wounds (e.g., injuries (cuts, bites, etc.), burns, bedsores, etc.), they are thought to adversely affect wound treatment (e.g., antimicrobial treatment).

[0003] Furthermore, biofilms are often highly adhesive and structurally strong, making them difficult to physically remove. At the same time, it is known that the metabolic activity and properties of microorganisms in biofilms change, significantly reducing their susceptibility to antimicrobial components such as antibacterial agents and surfactants. For this reason, even if antimicrobial components are used against microorganisms in biofilms, it is considered difficult for them to exert a sufficient antimicrobial effect on the microorganisms in the biofilms.

[0004] For these reasons, technologies that inhibit biofilm formation or disperse biofilms using additives other than antimicrobial components are being investigated.

[0005] For example, techniques for suppressing biofilms include a method using D-amino acids (Non-Patent Document 1), a method using quorum sensing (communication between microorganisms) (Non-Patent Document 2), a method using nanoparticles or nanopreparations (Non-Patent Document 3), and photodynamic therapy (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-109978 [Patent Document 2] Japanese Patent Application Laid-Open No. 2004-208526 [Patent Document 3] Japanese Patent Application Publication No. 10-236914 [Patent Document 4] Special Publication No. 2007-503917 [Non-patent literature]

[0007] [Non-Patent Document 1] AI Hochbaum et al, J. Bacteriol., 2011, 193, 5616-5622. [Non-patent document 2] J. Lonn-Stensrud et al, J Antimicrob Chemother. 2009, 63, 309-316. [Non-patent document 3] RY Pelgrift, et al, Adv Drug Deliv Rev. 2013, 65, 1803-1815. [Non-patent document 4] Carpenter BL, et al. Molecules. 2015, 20, 10604-10621. [Non-patent document 5] Carolina FG, et al. npj Biofilms. Microbio., 2020, 6, 1-11. [Non-patent document 6] Chenhui Yang et al., Nature Communications, 2020,11, 1-11. [Non-Patent Document 7] CK Stover et al., Nature, 2000,406, 959-964. Summary of the Invention [Problem to be solved by the invention]

[0008] Currently, methods for controlling drug susceptibility in biofilms have not been fully established. The presence of microorganisms in biofilms reduces their drug susceptibility, and the efficacy of drugs against microorganisms in biofilms is reduced. Therefore, a main object of the present invention is to provide a technique for improving the drug susceptibility of biofilms. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have discovered a new component that can improve the drug sensitivity of biofilms, and have completed the present invention.

[0010] The present invention can provide a composition containing a redox molecule for increasing the drug susceptibility of a biofilm. The present invention also provides an antimicrobial composition for use against microorganisms in a biofilm, which contains an antimicrobial component and is used in combination with the composition for improving the drug susceptibility of the biofilm.

[0011] The redox molecule may be an organic redox molecule. The redox molecule may be one or more compounds selected from the group consisting of phenazine-based compounds, phenothiazine-based compounds, anthraquinone-based compounds, triphenylmethane-based compounds, and xanthene-based compounds. The redox molecule may be one or more selected from the group consisting of safranine, methylene blue, anthraquinone sulfonic acid, brilliant blue FCF, and rose bengal. The microorganisms in the biofilm may be bacteria. The drug may be an antibiotic. The antibiotic may be an aminoglycoside compound. The composition may be an oral composition or a topical composition for skin. The antimicrobial composition may be an oral preparation or an external preparation for skin. [Effects of the Invention]

[0012] The present invention can provide a technique for improving the drug susceptibility of biofilms. [Brief explanation of the drawings]

[0013] [Figure 1] This shows the antibacterial activity test using the test substance safranin and the antibiotic kanamycin (1 μg / mL) against a planktonic bacterium (Shewanella oneidensis MR-1) that is not present in a biofilm. The horizontal axis is the incubation period (hours), and the vertical axis is OD600, which is used to measure the number of bacteria. [Figure 2] This shows an antibacterial activity test using the test substance safranin and the antibiotic kanamycin (0.1 μg / mL) against a planktonic bacterium (Shewanella oneidensis MR-1) that is not present in a biofilm. The horizontal axis is the incubation period (hours), and the vertical axis is OD600, which is used to measure the number of bacteria. DETAILED DESCRIPTION OF THE INVENTION

[0014] Preferred embodiments for carrying out the present invention will be described below. Note that the embodiments described below are examples of typical embodiments of the present invention, and the scope of the present invention should not be construed as being narrow. In this specification, percentages are expressed by mass unless otherwise specified. Furthermore, the upper limit (or less) and the lower limit (or more) of each numerical range (to) can be combined arbitrarily as desired. Furthermore, in the explanations of "1.", "2.", and each of these items below, overlapping explanations of each technical feature, each configuration, each definition, each term, each method, etc., such as components for improving biofilm drug sensitivity (heteroatom-containing aromatic compounds, redox molecules, pigments), antimicrobial components, and combinations thereof, dosage, dosage, and method of use, may be omitted as appropriate. The explanations of "1.", "2.", and each of these items can be applied to any of the technologies or embodiments of "1.", "2.", and each of these items, etc., and each technical feature can be adopted as appropriate in each of the technologies or embodiments.

[0015] 1. About this technology Currently, there is no established method for controlling drug susceptibility in a biofilm state. Therefore, the main objective of the present inventors was to provide a technology for improving the drug susceptibility of biofilms and an antimicrobial technology for microorganisms in biofilms using this technology.

[0016] As an example, the current situation regarding the relationship between infectious disease control and biofilms is as follows: The emergence of multidrug-resistant bacteria has made many infectious diseases difficult to treat, and approximately 14% (7.7 million) of global deaths in 2019 were caused by infections with pathogenic microorganisms. Therefore, the development of technology to improve the antibiotic susceptibility of pathogenic bacteria is an urgent issue. However, most existing technologies target planktonic microorganisms, and no method has been established for controlling antibiotic susceptibility in biofilms, the main form of pathogenic microorganisms in the human body. However, if the objectives of this technology are achieved, it is expected to be useful in the future treatment of infectious diseases, even against pathogenic microorganisms that are attracting global attention due to their high antibiotic resistance and tendency to become chronic and recur.

[0017] To date, efforts have been made to develop drugs and materials other than antibiotics to kill biofilms.

[0018] For example, Non-Patent Document 3 describes the dispersion and sterilization of biofilms using nanoparticles. However, there are issues regarding cost and effects on the human body, so components that are lower in cost and, based on experience, safer components are preferred.

[0019] For example, Patent Document 1 (JP 2017-109978 A) proposes a liquid drug comprising a radical-generating catalyst and at least one selected from the group consisting of a halous acid, a halous acid ion, and a halous acid salt, wherein the radical-generating catalyst comprises an ammonium salt (excluding peroxodisulfate) represented by chemical formula (XI) described in claim 1, and the Lewis acidity of the radical-generating catalyst is 0.4 eV or higher and is not acidic. However, Patent Document 1 does not provide sufficient information about components that can improve the drug sensitivity of microorganisms in biofilms.

[0020] As a result of extensive research, the present inventors have discovered a previously unforeseen technology that can improve the drug susceptibility of biofilms, even though most existing technologies are targeted at planktonic microorganisms. That is, as a result of extensive research, the present inventors have newly discovered a component that can improve the drug susceptibility of biofilms (hereinafter also referred to as a "biofilm drug susceptibility improving component"). The present inventors have thus concluded that by improving the drug susceptibility of biofilms, it is possible to more effectively reduce the antibiotic concentration used, or to more effectively reduce the antibiotic concentration that was previously considered necessary. Potential benefits of reducing the antibiotic concentration used include reducing the environmental pollution impact of antibiotics and reducing usage costs.

[0021] In this specification, "improving the drug susceptibility of biofilms" includes, for example, improving the drug susceptibility of microorganisms within a biofilm, improving or enhancing reduced drug susceptibility due to a biofilm, and increasing, improving or enhancing the drug susceptibility of something that has had reduced drug susceptibility due to a biofilm, even in the presence of a biofilm; and it is sufficient if at least one of these can be achieved.

[0022] Furthermore, the inventors believe that the present technology has the potential to be applied to multiple species, not just one, since it exerts antimicrobial effects against multiple microorganisms and multiple antimicrobial components. The biofilm drug sensitivity improving component can be an inorganic compound or an organic compound. Generally, inorganic compounds refer to compounds composed of elements other than carbon, such as carbon allotropes (e.g., graphite), carbon oxides (e.g., carbon dioxide), carbon sulfides (e.g., carbon disulfide), metal carbonates (e.g., calcium carbonate), and metal cyanides (e.g., potassium cyanide).

[0023] The present invention can provide a composition for improving the drug sensitivity of biofilms, comprising the biofilm drug sensitivity improving component. The composition may be a composition for improving the drug sensitivity of microorganisms in a biofilm. The drug sensitivity improving component is preferably one or more selected from redox molecules, aromatic compounds containing heteroatoms in the molecule, and dyes. From the viewpoint of improving sensitivity, the biofilm drug sensitivity improving component is preferably a compound having redox activity, more preferably an organic compound having redox molecule activity, and from the viewpoint of ease of visibility, preferably a compound having color development properties. Furthermore, the present invention is preferably an antimicrobial composition for use against microorganisms in biofilms, which contains an antimicrobial component and is used in combination with the component for improving biofilm drug sensitivity.

[0024] The redox molecule is preferably one or more selected from the group consisting of phenazine compounds (e.g., safranine), phenothiazine compounds (e.g., methylene blue), anthraquinone compounds (e.g., anthraquinone sulfonic acid), triphenylmethane compounds (e.g., brilliant blue FCF), and xanthene compounds (e.g., rose bengal).

[0025] The microorganism is preferably a filamentous fungus and / or a bacterium, more preferably a bacterium, and even more preferably a gram-negative bacterium. In the case of a bacterium, a pathogenic bacterium is preferred. The drug is preferably an antibiotic, more preferably an aminoglycoside compound. The composition is preferably an oral composition or a topical composition for skin application.

[0026] 1-1. Biofilm drug sensitivity improving ingredients The biofilm drug sensitivity enhancing component used in the present invention may be, for example, a redox molecule, a dye, or a cyclic organic compound which may contain a heteroatom in the molecule, and may be one or more selected from these.

[0027] The component for improving biofilm drug sensitivity is preferably a compound having oxidation-reduction activity and / or color-developing properties, and more preferably a compound having at least oxidation-reduction activity. Among the above redox molecules, organic redox molecules are preferred. Among the above pigments, organic pigments are preferred. The pigment may also be a dye component.

[0028] The component for improving the drug sensitivity of biofilms may be either an organic compound or an inorganic compound, but is preferably an organic compound.

[0029] The biofilm drug sensitivity improving component may be a cyclic organic compound (hereinafter also referred to as "cyclic organic compound") that may contain a heteroatom in the molecule, and among these, a compound (hereinafter also referred to as "aromatic compound") that has at least an aromatic ring skeleton (e.g., a simple aromatic skeleton) in the molecule is preferred, and more preferably an organic compound that contains a heteroatom in the molecule and has at least an aromatic ring skeleton (e.g., a simple aromatic skeleton) in the molecule (hereinafter also referred to as "heteroatom-containing aromatic compound"). The heteroatom is not particularly limited, but examples thereof include a nitrogen atom, a sulfur atom, an oxygen atom, and a halogen atom such as a bromine atom or a chlorine atom, and one or more types selected from these can be used. The aromatic compound is not particularly limited, but examples thereof include simple aromatic compounds, aromatic monocyclic compounds, aromatic polycyclic compounds, and fused ring compounds, and one or more selected from these may be used.

[0030] Furthermore, the biofilm drug sensitivity improving component preferably has, in its molecule, one or more selected from an aromatic monocyclic hydrocarbon skeleton, a heterocyclic skeleton that may contain a heteroatom (e.g., a heterocyclic skeleton containing a heteroatom (e.g., a heteromonocyclic skeleton containing a heteroatom)), a cyclic ketone skeleton (e.g., a cyclic skeleton having one or two ketone structures (>C=O)), a sulfo group (-SO3H; also referred to as a "sulfonic acid group"), and salts thereof. The biofilm drug sensitivity improving component may also have an appropriate combination of these skeletons, or a skeleton combining other aromatic rings or substituents, etc., such combined skeletons include, but are not limited to, a fused ring skeleton, a fused complex ring skeleton, a quinone skeleton, and a triphenylmethane skeleton.

[0031] The heteroatom in the heteromonocyclic skeleton that may contain or contains a heteroatom may be one or more selected from a nitrogen atom, an oxygen atom, and a sulfur atom, and the number of heteroatoms in the heterocycle is preferably 1 or 2. Examples of the heterocyclic skeleton containing a heteroatom include a cyclic amine skeleton and a pyran skeleton. Examples of the cyclic amine skeleton include an aromatic amine skeleton and a heterocyclic amine skeleton (such as a pyridine skeleton or a pyrazine skeleton), and may also be a cyclic amine skeleton containing other heteroatoms (such as one or two oxygen atoms and / or sulfur atoms) in the ring (such as a thiazine skeleton). Examples of the cyclic ketone skeleton include a quinone skeleton and a cyclohexadienone skeleton, and examples of the quinone skeleton include a benzoquinone skeleton (ortho, para).

[0032] In organic compounds containing heteroatoms in the molecule, the term "within the molecule" is not particularly limited, but more specific examples include within a ring, within a functional group, or within a substituent, and may be a combination of one or more selected from these. More specific examples include, but are not limited to, heterocyclic compounds containing heteroatoms in the ring; aromatic hydrocarbon compounds or heterocyclic compounds having heteroatoms such as nitrogen atoms, oxygen atoms, sulfur atoms, or halogen atoms in the substituents. Furthermore, the heteroatom-containing aromatic compound may have within the molecule a hydrocarbon group that has one or two or none of heteroatoms, functional groups, or substituents, as long as the effects of the present invention can be achieved.

[0033] The aromatic compound is not particularly limited and may be a compound in which multiple aromatic skeletons are bonded directly or indirectly, and may contain heteroatoms in the molecule. For example, a direct bond between one carbon atom of an aromatic skeleton and one carbon atom of another aromatic skeleton may be a single bond. Examples of indirect bonds include bonds via a carbon atom that may have a functional group, a single bond, a double bond, etc.; bonds via a hydrocarbon group that may have a heteroatom, a functional group, or a substituent, etc. Examples of compounds in which multiple aromatic skeletons are bonded directly or indirectly include bonds between multiple benzene rings; bonds between a tricyclic compound and one or more benzene rings; etc. Examples of bonding modes of multiple compounds include bonds via carbon atoms (e.g., Ph-C-Ph, Ph=C-Ph, etc.), bonds via single bonds (e.g., Ph-Ph, etc.), and combinations thereof.

[0034] The "hydrocarbon group" in this specification is not particularly limited, and examples thereof include chain (straight-chain or branched-chain) and cyclic (alicyclic or aromatic) groups, and any of these may be used. The chain group is not particularly limited, and examples thereof include alkyl groups (e.g., having 1 to 6 carbon atoms (e.g., methyl, ethyl, propyl, butyl, etc.)). The cyclic group includes aromatic monocyclic hydrocarbon groups (e.g., phenyl, benzyl, etc.). The substitution may be with a "functional group" described below. One or more of these groups may be selected.

[0035] The "heteroatom" used herein is not particularly limited, and may be, for example, one or more selected from an oxygen atom, a nitrogen atom, a sulfur atom, a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), and the like.

[0036] The "functional group" in this specification is not particularly limited, and may be, for example, one or more selected from a hydroxy group, an aldehyde group, a carboxy group, a carbonyl group, a ketone group, an ether bond, an ester bond (e.g., an ester of carboxylic acid, phosphoric acid, sulfuric acid, nitric acid, or carbonate), an azo group, an amino group (-NHH), an amine group (-NHR, -NRR'), an amide bond, a sulfonic acid group, a thiol group, a vinyl group, and a phosphate group. In the present invention, R and R' may be the same or different. R and R' are not particularly limited, and examples thereof include substituted or unsubstituted hydrocarbon groups, and the hydrocarbon groups may be the "hydrocarbon groups" described above. The amine group may be cyclic.

[0037] In another preferred embodiment of the present invention, the biofilm drug sensitivity improving component is preferably a compound containing at least a heteroatom and at least one or more aromatic ring skeletons in the molecule. The aromatic ring skeleton may be a simple aromatic ring skeleton that may contain a heteroatom in the ring. Examples of the simple aromatic ring skeleton include, but are not limited to, compounds having one or more types selected from aromatic hydrocarbon skeletons (e.g., monocyclic (benzene ring skeleton) and polycyclic (naphthalene skeleton, etc.)), heterocyclic skeletons (monocyclic and polycyclic), and polycyclic skeletons such as bicyclic and tricyclic (e.g., fused polycyclic hydrocarbon skeletons, fused heterocyclic rings, etc.). Preferred heterocyclic rings include, for example, heterocyclic rings containing one or a combination of oxygen, nitrogen, or sulfur atoms in the ring.

[0038] In another embodiment of the present invention, the aromatic compounds may be compounds having multiple phenyl groups bonded thereto (single bonds, double bonds, etc.) and having one or more substituents containing a heteroatom. The aromatic compounds may also be heterocyclic compounds containing a heteroatom.

[0039] The biofilm drug sensitivity enhancing component may be a compound for enhancing the drug sensitivity of microorganisms in a biofilm, and may be used in a method for enhancing the drug sensitivity of microorganisms in a biofilm. The biofilm drug sensitivity enhancing component may be in the form of an acid salt, a basic salt, or a normal salt.

[0040] The biofilm drug sensitivity enhancing component used in the present invention can be a redox molecule, a dye, or a heteroatom-containing aromatic compound that is used in pharmaceuticals, cosmetics, topical skin preparations, or foods and is available as a raw material or commercially available product, or can be appropriately used. Also, a component that can be produced by a known production method can be used.

[0041] Furthermore, examples of salts that can be used in the present invention include alkali metal salts (such as Na and K) and alkaline earth metal salts (such as Ca and Mg), and one or more salts selected from these can be used.

[0042] The biofilm drug sensitivity improving component used in the present invention may be a compound having antibacterial activity against planktonic bacteria, or may be a compound having no antibacterial activity against planktonic bacteria. Preferably, it may be a compound having no antibacterial activity against planktonic bacteria. The "no antibacterial activity against planktonic bacteria" can be determined by referring to Test Example 2 described below. A bacterial strain (e.g., MR-1 strain) was inoculated into an LB medium liquid culture containing 10 μM of the test substance, and a control LB medium liquid culture inoculated with the same bacterial strain (e.g., MR-1 strain) without the test substance was cultured for one day (room temperature, 30°C). The number of bacteria in each culture was measured by turbidity (OD600nm). If the turbidity (OD600nm) with the test substance / the turbidity (OD600nm) with the control was 0.7 or higher, the test substance had no antibacterial activity against airborne bacteria. A value of 1.0 or higher indicates that the test substance exerted no antibacterial effect, and the closer the value is to 0, the stronger the antibacterial effect.

[0043] 1-1-1. Cyclic organic compounds The cyclic organic compound used in the present invention is not particularly limited, and may be either a cyclic organic compound that does not contain a heteroatom in the molecule or a cyclic organic compound that contains a heteroatom in the molecule. Among the cyclic organic compounds, if they have redox activity, they may be used as organic redox molecules, or if they impart color to an object by absorbing and emitting visible light, they may be used as dyes. The cyclic organic compound is preferably an aromatic compound.

[0044] Examples of the cyclic organic compound include cyclic compounds having a monocyclic hydrocarbon skeleton (alicyclic hydrocarbon skeleton, aromatic monocyclic hydrocarbon skeleton) or a condensed polycyclic hydrocarbon skeleton (e.g., anthracene skeleton) in the molecule. The condensed polycyclic hydrocarbon is not particularly limited, but examples include bicyclic compounds such as indene, naphthalene, azulene, and heptalene; tricyclic compounds such as biphenylene, fluorene, phenalene, phenanthrolene, and anthracene; and tetracyclic compounds such as naphthalene, etc., with 2 to 9 rings. Among these, aromatic organic compounds are preferred, and the aromatic compound preferably has at least an aromatic monocyclic hydrocarbon skeleton in the molecule. Compounds (e.g., anthracene) that do not contain heteroatoms in the ring but have a condensed polycyclic hydrocarbon skeleton (containing at least an aromatic monocyclic hydrocarbon skeleton in the polycyclic ring) are preferred.

[0045] Examples of cyclic organic compounds that do not contain heteroatoms include tricyclic compounds with an anthracene skeleton, such as anthracene. Such cyclic organic compounds may have a substituent or a functional group.

[0046] Examples of cyclic aromatic compounds containing heteroatoms in the molecule include cyclic aromatic compounds that do not contain heteroatoms in the ring but contain heteroatoms in substituents or functional groups. Examples include cyclic compounds having a benzene skeleton that do not contain heteroatoms in the ring but contain heteroatoms in substituents or functional groups (e.g., phenol, aniline, etc.). More specifically, examples include cyclic organic compounds such as phenol that contain oxygen atoms or hydroxy groups in substituents or functional groups, and aniline that contain nitrogen atoms, amino groups, or amine groups in substituents or functional groups. Other examples include cyclic organic compounds having an anthracene skeleton that does not contain heteroatoms in the ring but contains heteroatoms in substituents or functional groups.

[0047] Among the aromatic compounds, preferred are aromatic compounds containing a heteroatom in the molecule, and the heteroatom-containing aromatic compound is not particularly limited, and examples thereof include the above-mentioned aromatic compounds containing a heteroatom in the molecule. The heteroatom-containing aromatic compound may be a commercially available product generally used in pharmaceuticals, foods, etc., or may be one obtained by a known production method.

[0048] The heteroatom-containing aromatic compound is not particularly limited, and examples thereof include phenazine compounds, phenothiazine compounds, quinone compounds (e.g., naphthoquinone compounds, anthraquinone compounds), triphenylmethane compounds, xanthene compounds, diazine compounds, thiazine compounds, oxazine compounds, phenanthridine compounds, azo compounds, lactone compounds, sultone compounds, indigoid compounds, cyanine compounds, oxonol compounds, styryl compounds, porphyrin compounds, thioxanthene compounds, squarylium compounds, croconium compounds, azulenium compounds, dithiol metal salt compounds, naphthoquinone compounds, indophenol compounds, coumarin compounds, ketocoumarin compounds, pyrylium salt compounds, thiopyrylium salts, thiazole compounds, quinoline compounds, benzophenone compounds, thiobenzophenone compounds, and mixtures thereof. One or more compounds selected from these compounds may be used. These may be commercially available products generally used in pharmaceuticals, foods, etc., or products obtained by known production methods.

[0049] Examples of the phenazine compounds include safranine, neutral red, and Janus green B. The phenazine compounds are heterocyclic compounds such as phenazine (C ), which contains a structure in which two benzene ring skeletons are connected to a central pyrazine ring skeleton by sharing one carbon-carbon bond. 12It is more preferable that the phenazine compound is a compound having at least a hydroxyl group (H8N2) skeleton in the molecule. The phenazine compound is preferably a tricyclic fused ring compound. The substituents possessed by the phenazine compound are not particularly limited, and examples thereof include one or more selected from linear 1-3 alkyl groups (e.g., methyl group, ethyl group), phenyl group, amino group, amine group (R, R' are alkyl groups, etc.), azo group (-N=N-), etc.

[0050] Examples of the phenothiazine compound include methylene blue, toluidine blue O, thionine, and azure A. The phenothiazine compound is preferably a heterocyclic compound having a structure in which two benzene ring skeletons are connected to a central thiazine (CHNS; [1,4]thiazine) skeleton by sharing a carbon-carbon bond with each other. The thiazine skeleton is preferably a heterocyclic skeleton having a six-membered ring containing one sulfur atom and one nitrogen atom. The substituents possessed by the phenothiazine compound are not particularly limited, and examples thereof include one or more selected from linear 1-3 alkyl groups (e.g., methyl, ethyl), amino groups, and amine groups (R and R' are alkyl groups, etc.).

[0051] The quinone compound is not particularly limited, and examples thereof include benzoquinone compounds, naphthoquinone compounds, and anthraquinone compounds, and one or more of these can be used. The quinone compound is preferably a compound having a structure in which the CH groups on two carbon atoms in the molecule of an aromatic hydrocarbon skeleton, a heterocyclic skeleton, or a fused-ring polycyclic skeleton are each replaced with a C=O group, and may contain a heteroatom within the ring.

[0052] The number of ring members in the monocyclic moiety present in the quinone compound is not particularly limited, but for example, the monocyclic moiety may be any of a 4-, 5-, 6-, 7-, or 8-membered ring, with a 6-membered ring (benzene ring) being preferred. The C=O moiety of the quinone compound may be, for example, a p-benzoquinone (1,4-benzoquinone) skeleton or an o-benzoquinone (1,3-benzoquinone) skeleton, with a p-benzoquinone skeleton being preferred.

[0053] More preferred embodiments of the quinone compounds include, for example, benzoquinone compounds such as p-benzoquinone, methyl-p-benzoquinone, 2,5-dimethyl-1,4-benzoquinone, methoxybenzoquinone, and 2,5-dihydroxy-1,4-benzoquinone; naphthoquinone compounds (preferably 1,4-naphthoquinone compounds) such as 2-hydroxy-1,4-naphthoquinone, 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, and 2,3-dichloro-1,4-naphthoquinone; and anthraquinone compounds such as anthraquinonesulfonic acid, anthraquinone, 2-methylanthraquinone, 2-tert-butylanthraquinone, 1-chloroanthraquinone, 1,4-dihydroxyanthraquinone, and 1-nitroanthraquinone.

[0054] The anthraquinone compounds are not particularly limited, and examples thereof include diaminodianthraquinone (tricyclic; e.g., 2,6-diaminoanthraquinone), anthrapyrimidine, flavanthrone (octacyclic; two heterocycles with one N atom), anthanthrone, indanthrone (heptacyclic; two heterocycles), pyranthrone (octacyclic), violanthrone (nopecyclic), and anthraquinonesulfonic acid (tricyclic; e.g., anthraquinone-2-sulfonic acid, anthraquinone-2,6-disulfonic acid).

[0055] The anthraquinone compound is preferably a compound having a tricyclic or higher polycyclic skeleton (a condensed polycyclic hydrocarbon skeleton or a condensed heterocyclic skeleton) and having two oxygen atoms (preferably two ketones with >C=O) in the molecule. The anthraquinone compound may not have an anthraquinone skeleton in the molecule, but may be a compound having two anthrone skeletons in the molecule, such as pyranthrone (octacyclic) or violanthrone (ninacyclic). The anthraquinone-based compound is more preferably anthraquinone (chemical formula C 14 It is a compound that has at least a hydroxyl group (H8O2) skeleton in its molecule.

[0056] The anthraquinone compound may contain, in its molecule, one or more groups selected from, for example, a nitrogen atom, an amino or amine group (-NH2, -NHR, -NRR'), sulfonic acid, a halogeno group such as bromine or chlorine (-Cl, -F, -Br, -I, etc.), an alkyl group such as methyl or ethyl, and a hydroxyl group, and these compounds may be nitrogen-containing heterocycles. The substituents possessed by the anthraquinone compound are not particularly limited and may be one or more groups selected from, for example, a sulfonic acid group, an amino or amine group, a hydroxyl group, an alkyl group (such as a methyl group), etc., with a sulfonic acid group being preferred.

[0057] Among the anthraquinone compounds, anthraquinone sulfonic acid is preferred, and it preferably has one or two sulfonic acid groups. The anthraquinone sulfonic acid is preferably one that has oxidation-reduction activity, but may have weak color development properties.

[0058] The anthraquinone sulfonic acid is not particularly limited, but may be one or more selected from anthraquinone-2-sulfonic acid, anthraquinone-2,6-disulfonic acid, anthraquinone-1-sulfonic acid, 1-amino-4-bromoanthraquinone-2-sulfonic acid, anthraquinone-1,8-disulfonic acid, alizarin astrol, nuclear fast red, and salts thereof (sodium, potassium, etc.).

[0059] Examples of the triphenylmethane compounds include brilliant blue FCF, ethyl violet, Coomassie brilliant blue (CBB), bromochlorophenol blue, tetrabromophenol blue, bromophenol blue, and bromocresol purple. The triphenylmethane compound preferably has at least an aromatic hydrocarbon skeleton having three phenyl groups on the same carbon. Alternatively, the triphenylmethane compound may be a compound having at least a triphenylmethane ((Ph)C- (C may form a single bond with the substituent R or H, and C may form a double bond with any one of the Ph) skeletons). The compound preferably contains one or more atoms selected from a nitrogen atom, a sulfur atom, an oxygen atom, and a halogen atom (a chlorine atom, a bromine atom) in the molecule. These atoms may be contained as substituents or functional groups in the triphenylmethane skeleton. The substituents possessed by the triphenylmethane compound are not particularly limited, and include one or more atoms selected from an amine group (R and R' are alkyl groups (e.g., methyl, ethyl, etc.), aromatic groups, etc.), a benzyl group, a phenyl group, a sulfonic acid group, etc., which may have a substituent (e.g., an amine group, a sulfonic acid group), etc.

[0060] Examples of the xanthene compounds include rose bengal (tricyclic compound), fluorescein (tricyclic compound), rhodamine (tricyclic compound), and pyronine Y (tricyclic compound). The xanthene compounds are preferably compounds having at least a xanthene skeleton, and the xanthene skeleton is preferably a compound in which the central benzene ring of anthracene is replaced with a pyran ring, which is a six-membered heterocyclic compound (or ether compound) containing one oxygen atom. The xanthene compounds are preferably tricyclic or tetracyclic fused ring compounds. The substituents possessed by the xanthene compounds are not particularly limited, and examples thereof include one or more selected from the group consisting of an amino group, an amine group (R and R' are alkyl groups (e.g., methyl, ethyl, etc.), aromatic groups, benzyl groups, phenyl groups, and sulfonic acid groups, each optionally having a substituent (e.g., an amine group, sulfonic acid group, halogeno group, or carboxy group).

[0061] Examples of the oxazine-based compound include brilliant cresyl blue (a tricyclic compound), nile blue (a tetracyclic compound), gallocyanine (a tricyclic compound), and basic blue 3 (a tricyclic compound). The oxazine-based compound is preferably a compound having at least an oxazine skeleton, and the oxazine skeleton is preferably a six-membered heterocyclic compound containing one oxygen atom, one nitrogen atom, and two double bonds. The oxazine-based compound is preferably a tricyclic or tetracyclic fused ring compound.

[0062] Examples of the phenanthridine compound include ethidium bromide (tricyclic compound), etc. The phenanthridine compound is more preferably a compound having at least a phenanthridine skeleton, which is a heterocyclic compound containing one nitrogen atom, and more preferably a tricyclic fused ring compound.

[0063] Examples of the azo compounds include Bismarck Brown, New Coccine, and Basic Red 29. The azo compounds are preferably compounds having an azo group (-N=N-) in the molecule (chemical structure), more preferably compounds having one azo group between aromatic compounds, and the aromatic compounds may be either aromatic hydrocarbons or heteroaromatic compounds (preferably nitrogen-containing heterocyclic compounds), preferably having one or two azo groups in the molecule, and preferably aromatic compounds bonded to each other via the azo groups.

[0064] The indigoid compounds include compounds having indigo or a structure similar thereto, such as indigo carmine. The indigoid compounds are preferably compounds having at least an indigo skeleton structure. The indigoid compounds preferably have, within their chemical structure (intramolecularly), two heterocyclic compounds having an indole skeleton in which a benzene ring containing one nitrogen atom and a pyrrole ring are fused together.

[0065] The aromatic compounds containing a heteroatom in the molecule are preferably one or more selected from the group consisting of phenazine compounds, phenothiazine compounds, anthraquinone compounds, triphenylmethane compounds, and xanthene compounds. Among these, phenazine compounds are preferred, and among the phenazine compounds, safranine is more preferred.

[0066] Of the aromatic compounds containing a heteroatom in the molecule, one or more selected from the group consisting of safranine, methylene blue, anthraquinone sulfonic acid, brilliant blue FCF, and rose bengal are preferred.

[0067] Among the aromatic compounds (preferably phenazines) containing a heteroatom in the molecule, safranine (chemical formula C 20 H 19 N4 + ,Cl - ; also known as Basic Red 2, Safranin O; CAS Registry Number 477-73-6) is preferred.

[0068] Among the aromatic compounds (preferably phenothiazines) containing a heteroatom in the molecule, methylene blue (chemical formula C 16 H 18 N3SCl; IUPAC name 3,7-bis(dimethylamino)phenothiazinium chloride; also known as methylthioninium; CAS number 61-73-4) is preferred.

[0069] Among the aromatic compounds containing a heteroatom in the molecule (preferably anthraquinone-based compounds), anthraquinone sulfonic acids having an anthraquinone skeleton in the molecule and one or more sulfonic acid groups are preferred, and among these, compounds having one or two sulfonic acid groups as substituents are preferred, and among these, one or more selected from anthraquinone-2-sulfonic acid, anthraquinone-2,6-disulfonic acid, and salts thereof are preferred.

[0070] Among the aromatic compounds (preferably triphenylmethane compounds) containing a heteroatom in the molecule, Brilliant Blue FCF (C 37 H 34 N2Na2O9S3; CAS 3844-45-9) is preferred.

[0071] Among the aromatic compounds (preferably xanthene compounds) containing a heteroatom in the molecule, Rose Bengal (C 20 H2Cl4I4Na2O5; CAS 632-69-9) is preferred.

[0072] The present invention can use one or more components selected from the above-mentioned organic compounds of the same type or a combination of organic compounds of different types.

[0073] 1-1-2. Redox molecules The redox molecule used in the present invention is not particularly limited, but is preferably a compound having redox activity. It can be a commercially available product generally used in pharmaceuticals, foods, etc., or can be obtained by a known production method. In this case, it is preferable to select a substance having redox activity.

[0074] The term "redox molecule" as used herein refers to a molecule that has the ability to donate and accept electrons. When examining the redox activity of a compound, electrochemical measurement methods, more specifically, cyclic voltammetry, can be used.

[0075] The redox molecule may be either an inorganic compound or an organic compound, but is preferably an organic compound. The redox molecule may also be a cyclic organic compound (preferably a heteroatom-containing aromatic compound), and the heteroatom contained in the molecule is not particularly limited, but may include, for example, one or more types selected from oxygen atoms, nitrogen atoms, sulfur atoms, and halogen atoms. More preferred groups are not particularly limited, but preferably include, for example, one or more types selected from ketone groups, amino groups, amine groups, sulfonic acid groups, carboxy groups, and halogeno groups.

[0076] The redox molecule used in the present invention is preferably a compound having redox activity selected from the compounds described above in "1-1-1." For example, a "phenazine compound" may be changed to a "phenazine redox molecule," or may be a phenazine compound having redox activity.

[0077] Furthermore, suitable organic compounds for the redox molecules used in the present invention can be appropriately selected from the compounds listed in "1-1-1." above, but may also be compounds other than those listed in "1-1-1." above.

[0078] The redox molecule is not particularly limited, and examples thereof include phenazine-based redox molecules, phenothiazine-based redox molecules, quinone-based redox molecules (e.g., naphthoquinone-based redox molecules, anthraquinone-based redox molecules), triphenylmethane-based redox molecules, xanthene-based redox molecules, diazine-based redox molecules, thiazine-based redox molecules, oxazine-based redox molecules, phenanthridine-based redox molecules, azo-based redox molecules, lactone-based redox molecules, sultone-based redox molecules, indigoid-based redox molecules, cyanine-based redox molecules, and oxonol-based redox molecules. Examples include styryl-based redox molecules, porphyrin-based redox molecules, thioxanthene-based redox molecules, squarylium-based redox molecules, croconium-based redox molecules, azulenium-based redox molecules, dithiol metal salt-based redox molecules, naphthoquinone-based redox molecules, indophenol-based redox molecules, coumarin-based redox molecules, ketocoumarin-based redox molecules, pyrylium salt-based redox molecules, thiopyrylium salts, thiazole-based redox molecules, quinoline-based redox molecules, benzophenone-based redox molecules, and thiobenzophenone-based redox molecules, as well as mixtures thereof. One or more types selected from these redox molecules or compounds can be used.

[0079] The redox molecule is preferably one or more selected from the group consisting of phenazine-based compounds, phenothiazine-based compounds, quinone-based compounds (e.g., anthraquinone-based compounds), triphenylmethane-based compounds, and xanthene-based compounds.

[0080] The redox molecule is preferably one or more selected from the group consisting of safranine, methylene blue, anthraquinone sulfonic acid (preferably anthraquinone-2-sulfonic acid or anthraquinone-2,6-disulfonic acid), brilliant blue FCF, and rose bengal.

[0081] 1-1-3. Pigment

[0082] The dye used in the present invention is not particularly limited and may be either acidic or basic. The dye used in the present invention is preferably a substance that has color by absorbing and emitting visible light. The dye used in the present invention may be either an inorganic substance or an organic substance, but an organic dye is preferred. A dye for dyeing may be selected from the dyes used in the present invention. The dyes used in the present invention that are dyeable may be used as dyes or dye components.

[0083] The dye used in the present invention is not particularly limited, but may be selected from the above-mentioned "1-1-1." For example, a "phenazine-based compound" may be replaced with a "phenazine-based dye." The organic dye used in the present invention is not particularly limited, but examples thereof include phenazine-based dyes, phenothiazine-based dyes, quinone-based dyes (e.g., anthraquinone-based dyes), triphenylmethane-based dyes, xanthene-based dyes, and mixtures thereof. One or more selected from these dyes may be used.

[0084] Among the dyes, a basic organic dye, an acidic organic dye, or a combination thereof is preferred. Examples of basic organic dyes include compounds containing a nitrogen atom, an amino group, or the like in the molecule, such as basic phenazine dyes and basic thiazine dyes. Examples of more suitable groups for acidic organic dyes include, but are not limited to, compounds containing a carboxy group, a sulfonic acid group, a phosphonic acid group, a halogeno group, or the like in the molecule, such as acidic quinoline dyes.

[0085] Of the above dyes, tricyclic or tetracyclic fused ring compounds are preferred, and tricyclic fused ring compounds are more preferred.

[0086] The dye is preferably one or more compounds selected from the group consisting of phenazine-based compounds, phenothiazine-based compounds, anthraquinone-based compounds, triphenylmethane-based compounds, and xanthene-based compounds.

[0087] The dye is preferably one or more selected from the group consisting of safranine, methylene blue, anthraquinone sulfonic acid (preferably anthraquinone-2-sulfonic acid, anthraquinone-2,6-disulfonic acid), brilliant blue FCF, and rose bengal.

[0088] 1-2.Biofilm The biofilm used in the present invention is not particularly limited, but examples include biofilms containing bacteria, filamentous fungi, microalgae, etc., and one or more species selected from these can be used. The microorganism to which the present invention is applied is preferably a microorganism that forms a biofilm. Microorganisms present in a biofilm are likely to have reduced drug susceptibility due to their presence in the biofilm, but application of the present invention can improve this reduced drug susceptibility. Therefore, the microorganisms targeted by the present invention are more preferably pathogenic microorganisms, and even more preferably infectious pathogenic microorganisms that cause infectious diseases, and may also be applied to drug-resistant bacteria.

[0089] Examples of pathogenic and infectious pathogenic bacteria (e.g., bacteria and filamentous fungi) to which the present invention is applicable include, for example, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Escherichia coli, periodontal disease bacteria, skin bacteria, acid-fast bacteria such as Mycobacterium tuberculosis, and fungi such as Cryptococcus and Candida, and it is preferable to apply the present invention to one or more species selected from these. Examples of periodontal disease bacteria or oral bacteria include Actinobacillus actinomycetemcomitans (AA bacteria), Profilomonas gingivalis (PG bacteria), Prevotella intermedia (PI bacteria), Streptococcus mutans, and spirochetes. Skin bacteria include Staphylococcus epidemicus (staphylococcus), Staphylococcus aureus (Staphylococcus aureus), Propionebacterium acnes (acne bacteria), other streptococci, and Pseudomonas bacteria (e.g., Pseudomonas aeruginosa).

[0090] The inventors hypothesize that the inside of a biofilm is anaerobic, suppressing the physiological activity of microorganisms, but believe that even in such a case, the use of the components of the present technology can activate the activity of the microorganisms in the biofilm, making it much easier to exert the drug effect. In other words, the inventors believe that the present technology has been able to effectively exert an antimicrobial effect against microorganisms in the anaerobic environment of a biofilm, which was not sufficiently effective with conventional technologies.

[0091] The microorganisms to which the present invention is applied are preferably bacteria and / or filamentous fungi. The bacteria and filamentous fungi to which the present invention is applied are preferably those capable of forming biofilms. The bacteria and filamentous fungi (preferably bacteria) are preferably those present in, for example, oral plaque and wound biofilms. Examples of such wounds include chronic wounds, more specifically ulcers, bedsores, etc. Causes of chronic wounds include, for example, chronic diseases, diabetes, blood flow disorders, nutritional disorders, infections, pressure, edema, etc., and one or more of these may be selected.

[0092] The present invention can improve drug sensitivity against bacteria in oral plaque or wound plaque, thereby exerting antibacterial activity. Therefore, the present invention can effectively prevent, treat, or improve periodontal disease and / or wounds, which have been difficult to prevent, treat, or improve due to the presence of biofilms. Furthermore, the present invention can effectively provide one or more types of care selected from periodontal disease care, wound care, and bedsore care.

[0093] The microorganism to which the present invention is applied is more preferably a bacterium. The applicable bacterium may be either a gram-negative bacterium or a gram-positive bacterium, with gram-negative bacteria being preferred. The microorganism may be either anaerobic or aerobic, with aerobic microorganisms being more effective in exerting the effects of the present invention.

[0094] The bacterium is preferably a non-spore-forming bacterium. The bacterium may be either aerobic or anaerobic. The bacterium is preferably a pathogenic bacterium. Among the bacteria, bacteria of the genus Shewanella and Pseudomonas can be mentioned, and one or more selected from these can be used. Bacteria of the genus Shewanella and Pseudomonas are preferred. For example, one or more selected from Pseudomonas aeruginosa (also known as Pseudomonas aeruginosa) and Shewanella oneidensis are preferred.

[0095] 1-3. Drugs or antimicrobial ingredients The drug used in the present invention is not particularly limited, and is preferably a component having antimicrobial activity or a drug containing such an active ingredient. The drug may be a composition. The drug may be an antimicrobial component. Antimicrobials include, but are not limited to, antibacterial agents (antifungal, antibacterial, etc.). The antimicrobial component may be an antimicrobial component that is generally used in pharmaceuticals, foods, etc., or that will be available in the future. The drug may be one that inhibits the growth and proliferation of microorganisms, or one that can kill microorganisms. The drug used in the present invention is preferably an antibiotic.

[0096] The components used as the drugs or antimicrobial components are not particularly limited, but include aminoglycosides, β-lactams, macrolides, quinolones (new quinolones), lincomycins, rifamycin compounds, tetracycline compounds, amphenicol compounds, sulfonamide compounds, trimethoprim compounds, polymyxin compounds, pleuromutilin compounds, glycopeptide compounds, imidazole compounds, and nitrofuran compounds, and one or more selected from these can be used.

[0097] The properties of the drug or antimicrobial component are not particularly limited, but include bacteriostatic properties (the property of suppressing the growth rate of growing microorganisms) and bactericidal properties (the property of killing growing microorganisms), and it is preferable that the drug or antimicrobial component has either or both of these properties, and the component may be a compound.

[0098] Examples of the bacteriostatic component include macrolides, tetracyclines, lincomycins, chloramphenicols, etc. Examples of the bactericidal component include penicillins, cephalosporins, fosfomycins, quinolones (new quinolones), aminoglycosides, etc. One or more selected from these can be used.

[0099] Examples of the drug or antimicrobial component generally include cell wall synthesis inhibitors (penicillins, cephalosporins, fosfomycins, glycopeptides, etc.), cell membrane function inhibitors (polyenes, peptides, etc.), protein synthesis inhibitors (tetracyclines, macrolides, aminoglycosides, etc.), nucleic acid synthesis inhibitors (quinolones, pyridonecarboxylic acids, etc.), and folic acid synthesis inhibitors (sulfonamides, etc.). One or more selected from these may be used.

[0100] The drug or antimicrobial component is not particularly limited, but examples include antibiotics and antibacterial agents, and one or more selected from these may be used.

[0101] The aminoglycoside compounds are not particularly limited, but examples thereof include gentamicin, kanamycin, streptomycin, tobramycin, neomycin, amikacin, dibekacin, arbekacin, and apramycin. Among the aminoglycoside compounds, gentamicin and / or kanamycin are preferred. The aminoglycoside compounds are preferably glycoside antibiotics containing amino sugars or aminocyclitols.

[0102] Examples of β-lactam compounds include, but are not limited to, penicillin compounds such as benzylpenicillin, phenoxymethylpenicillin, cloxacillin, oxacillin, nafcillin, methicillin, amoxicillin, ampicillin, ticarcillin, carbenicillin, piperacillin, azlocillin, mezlocillin, and mecillinam; cefazolin, cephalexin, cephalothin, cefaclor, cefuroxime, cefotiam, loracarbef, cefepime, and cefozoprevent. Examples of suitable β-lactam compounds include cephalosporins such as lan, cefpirome, ceftazidime, ceftaroline, ceftolozane, and ceftobiprole; cephamycins such as cefotetan, cefoxitin, and cefmetazole; penems such as faropenem and ritipenem; carbapenems such as ertapenem, doripenem, imipenem, meropenem, biapenem, and panipenem; and monobactams such as aztreonam, tigemonam, and carumonam. The β-lactam compound is preferably an antibiotic having a β-lactam ring as the core nucleus.

[0103] Examples of macrolide compounds include, but are not limited to, erythromycin, spiramycin, josamycin, clarithromycin, azithromycin, rokitamycin, telithromycin, solithromycin, etc. The macrolide compounds are preferably antibiotics that inhibit bacterial protein synthesis, in which a neutral sugar or an amino sugar is bound to a large molecular weight lactone ring (macrolide ring).

[0104] Lincomycin compounds are not particularly limited, but examples thereof include clindamycin and lincosamide. Streptogramin compounds are not particularly limited, but examples thereof include quinupristin / dalfopristin.

[0105] Examples of quinolone compounds include, but are not limited to, ofloxacin, ciprofloxacin, enoxacin, norfloxacin, sparfloxacin, grepofloxacin, levofloxacin, moxifloxacin, gemifloxacin, gatifloxacin, prulifloxacin, pazufloxacin, garenoxacin, sitafloxacin, tosufloxacin, delafloxacin, and nalidixic acid.

[0106] Rifamycin compounds include, but are not limited to, rifaximin, rifabutin, and rifampicin.

[0107] The tetracycline compounds are not particularly limited, but examples thereof include doxycycline, tetracycline, minocycline, tigecycline, and eravacycline.

[0108] The amphenicol compound is not particularly limited, but examples thereof include chloramphenicol, etc. The amphenicol compound is preferably an antibiotic having a phenylpropanoid skeleton. The sulfonamide compounds and trimethoprim compounds are not particularly limited, but examples thereof include trimethoprim, iclaprim, and sulfamethoxazole. The oxazolidinone compounds are not particularly limited, but examples thereof include linezolid and tedizolid, etc. The sulfonamide compounds are preferably synthetic antibacterial components having a skeleton based on sulfanilamide.

[0109] The polymyxin compound is not particularly limited, but examples thereof include colistin and polymyxin B. The polymyxin compound is preferably a cationic polypeptide antibacterial component. The pleuromutilin compound is not particularly limited, but examples thereof include tiamulin, etc. The pleuromutilin compound is preferably an antifungal component produced by fungi.

[0110] Glycopeptide compounds include, but are not limited to, vancomycin, teicoplanin, telavancin, dalbavancin, and oritavancin.

[0111] The imidazole compound is not particularly limited, but examples thereof include metronidazole, etc. The imidazole compound is preferably an antifungal component having an imidazole structure. The nitrofuran-based compound is not particularly limited, but examples thereof include nitrofurantoin, etc. The nitrofuran-based compound is preferably a furan-based synthetic antibacterial component.

[0112] The drug or antimicrobial component is preferably an aminoglycoside compound, such as one or more selected from kanamycin, gentamicin, and streptomycin. The aminoglycoside compound is preferably kanamycin and / or gentamicin.

[0113] 1-4.Applications The biofilm drug sensitivity enhancing component has the effect of enhancing the drug sensitivity of biofilms and the drug sensitivity of microorganisms within biofilms, and therefore the present invention can provide compositions for enhancing the drug sensitivity of biofilms containing the biofilm drug sensitivity enhancing component, as well as compositions for enhancing the drug sensitivity of microorganisms within biofilms and compositions for enhancing antimicrobial activity. Furthermore, the biofilm drug sensitivity enhancing component can enhance the drug sensitivity of microorganisms within biofilms and reduce the amount of antimicrobial component used, and therefore the composition of the present invention can be provided as a composition for reducing the amount of antimicrobial component used against microorganisms within biofilms. These compositions for enhancing the drug sensitivity of biofilms may be compounding agents or additives to be included in antimicrobial compositions, or may be compositions used in combination or as a combination product to enhance the antimicrobial effect of antimicrobial compositions.

[0114] The component for improving biofilm drug sensitivity used in the present invention can be contained in or used in a composition for improving biofilm drug sensitivity, an antimicrobial composition, a composition for preventing, ameliorating, or treating infectious diseases, etc., and can be used as an active ingredient in such compositions, etc. The composition may be an agent, or the agent may be a composition. Furthermore, the component for improving biofilm drug sensitivity can be used to produce a composition, etc.

[0115] Furthermore, the present invention can provide an antimicrobial composition for use against microorganisms in biofilms, in which the component for improving biofilm drug sensitivity, which contains an antimicrobial component, or a composition containing the same is used.

[0116] The present invention also provides use of the biofilm drug sensitivity improving component or a composition containing the same in a drug for improving the drug sensitivity of biofilms.The present invention also provides use of the biofilm drug sensitivity improving component or a composition containing the same, or use of the biofilm drug sensitivity improving component or a composition containing the same, in the manufacture of a drug for improving the drug sensitivity of biofilms.The drug may be an agent for enhancing antimicrobial activity or reducing the amount of antimicrobial component used, or a composition for preventing, ameliorating, or treating infectious diseases, or an agent used for that purpose.

[0117] The present invention provides the component for improving biofilm drug sensitivity, or a composition containing the component, or use thereof, for use in improving the drug sensitivity of biofilms. The purpose of the use may be to enhance the antimicrobial action of an antimicrobial composition against microorganisms in a biofilm, or to reduce the amount of the antimicrobial component used in the antimicrobial composition against microorganisms in a biofilm, or may be for the prevention, amelioration, or treatment of an infectious disease.

[0118] The present invention can provide a method for improving the drug sensitivity of a biofilm using the component for improving drug sensitivity for biofilms, a method for improving the drug sensitivity of microorganisms in a biofilm, a method for enhancing the antimicrobial action of an antimicrobial composition against microorganisms in a biofilm, or a method for reducing the amount of an antimicrobial component used in an antimicrobial composition against microorganisms in a biofilm, all of which use the component for improving drug sensitivity for biofilms. The present invention can also provide a method for improving antimicrobial activity, a method for reducing the amount of microbial component used, or a method for preventing, ameliorating, or treating infectious diseases by using an antimicrobial component in combination with the biofilm drug sensitivity improving component.

[0119] The present invention also provides an antimicrobial composition for use against microorganisms in biofilms, containing the component for improving biofilm drug sensitivity and an antimicrobial component.The present invention also provides a combination or combined product of an antimicrobial composition comprising the component for improving biofilm drug sensitivity or a formulation containing the same, and an antimicrobial component for microorganisms in biofilms or a formulation containing the same.These components may be enclosed separately or together in a container, packaging bag, or the like.The combination may be a kit or set, each of which may be a product or commercial product.

[0120] A preferred method of use of the present invention is to provide an antimicrobial method against microorganisms in a biofilm, in which the biofilm drug sensitivity improving component is used in combination with an antimicrobial component.These components may be used in combination at the same time or at different times, and it is preferable that the components are applied to the same location.

[0121] Suitable methods of using the present invention include using the biofilm drug sensitivity improving component in combination with the antimicrobial component at the application site, or using the biofilm drug sensitivity improving component at the application site in advance before using the antimicrobial component at the application site. For example, the biofilm drug sensitivity enhancing component may be applied to the application site for a predetermined period of time, followed by application of an antimicrobial component to the application site where the biofilm drug sensitivity enhancing component has been applied. The predetermined period is not particularly limited, but a suitable lower limit is preferably 1 hour or more, more preferably 3 hours or more, even more preferably 6 hours or more, more preferably 12 hours or more, and even more preferably 18 hours or more. A suitable upper limit is preferably 48 hours or less, more preferably 42 hours or less, even more preferably 38 hours or less, more preferably 30 hours or less, 28 hours, or 24 hours or less. After the predetermined period of use, the biofilm drug sensitivity enhancing component may be removed from the application site, or the biofilm drug sensitivity enhancing component may be further applied to the application site with or without removing the biofilm drug sensitivity enhancing component. It is preferable to apply an antimicrobial component to the application site containing the biofilm drug sensitivity enhancing component after the predetermined period of time, and to use these components in combination. Also, for example, a mixture of a drug sensitivity improving component and an antimicrobial component may be used at the application site. Each component or mixture used may be used once or multiple times.

[0122] A more preferred embodiment is to use the biofilm drug sensitivity improving component in combination with an antimicrobial component on areas where oral biofilms or wound biofilms are present (e.g., teeth, gums, bedsores, etc.), or to use the biofilm drug sensitivity improving component in advance on the application area. Examples of application to the application site include, but are not limited to, contact, spreading, spraying, washing, and coating.

[0123] The pH in the preferred method of use of the present invention is not particularly limited, but a preferred lower limit is pH 4 or higher, pH 5 or higher, or pH 6 or higher, and a preferred upper limit is pH 10 or lower, pH 9 or lower, or pH 8 or lower. The pH may be the pH of the composition.

[0124] Furthermore, the temperature in the preferred method of use of the present invention is not particularly limited, but it is preferable that the effect can be exerted at approximately room temperature, for example, 4 to 60°C. The preferred lower limit is preferably 10°C or higher, more preferably 20°C or higher, and the preferred upper limit is preferably 50°C or lower, more preferably 40°C or lower.

[0125] The amounts of the component for improving biofilm drug sensitivity and the antimicrobial component used are not particularly limited as long as they are sufficient to obtain the effects of the present invention, and can be adjusted appropriately depending on the condition, weight, sex, age, and other factors of the subject. Furthermore, the components can be used according to any dosage and administration plan, and the amount may be divided into one or several doses per day, or may be used continuously for one day or more, or for several weeks or more to several months.

[0126] In a preferred method of use of the present invention, the ratio of the antimicrobial component to the component for improving biofilm drug sensitivity (amount of antimicrobial component used / amount of component for improving biofilm drug sensitivity) may be determined appropriately depending on the type of antimicrobial component, the type of component for improving biofilm drug sensitivity, etc.

[0127] For example, in a preferred method of use of the present invention, the ratio of the antimicrobial component to the biofilm drug sensitivity improving component (amount of antimicrobial component used / amount of biofilm drug sensitivity improving component used) is not particularly limited, but from the viewpoint of antimicrobial activity, a preferred lower limit of the antimicrobial component per 5 μg / mL of the biofilm drug sensitivity improving component is preferably 0.1 μg / mL or more, more preferably 0.5 μg / mL or more, even more preferably 1 μg / mL or more, more preferably 5 μg / mL or more, more preferably 10 μg / L or more, more preferably 20 μg / mL or more, more preferably 50 μg / mL or more, more preferably 100 μg / mL or more, more preferably 250, 500, 750, or 1000 μg / mL or more; and from the viewpoint of reducing the amount of the antimicrobial component used, a preferred upper limit is preferably 1000 mg / mL or less, more preferably 500, 250, or 100 mg / mL or less, even more preferably 50 or 10 mg / mL or less, and more preferably 5 or 1 mg / mL or less. The usage ratio may be the content ratio in the composition.

[0128] In addition, in a preferred method of use of the present invention, the ratio of the antimicrobial component to the component for improving biofilm drug sensitivity (amount of antimicrobial component used / amount of component for improving biofilm drug sensitivity) is not particularly limited, but from the viewpoint of exerting antimicrobial activity and reducing the amount of antimicrobial component used, the antimicrobial component is preferably not contained in an amount of 1000 μg / mL or more, more preferably not contained in an amount of 100 μg / mL or more, per 5 μg / mL of the component for improving biofilm drug sensitivity. The usage ratio may be the content ratio in the composition.

[0129] Furthermore, in a preferred method of use of the present invention, the ratio of the component for improving biofilm drug sensitivity to the antimicrobial component (amount of component for improving biofilm drug sensitivity used / amount of antimicrobial component used) is not particularly limited, but from the viewpoint of reducing the amount of antimicrobial component used, a suitable lower limit for the amount of component for improving biofilm drug sensitivity used per 100 μg / mL of antimicrobial component is preferably not more than 50 μg / mL, more preferably not more than 5 μg / mL, and even more preferably not more than 1 μg / mL. This usage ratio may also be the content ratio in the composition.

[0130] The amount of the biofilm drug sensitivity enhancer used in the preferred method of use of the present invention is not particularly limited, but from the viewpoint of improving biofilm drug sensitivity, the preferred lower limit is preferably 0.1 μg / mL or more, more preferably 0.5 μg / mL or more, even more preferably 1 μg / mL or more, more preferably 2.5 μg / mL or more, more preferably 5 μg / mL or more, more preferably 10 μg / mL or more, more preferably 25 μg / mL or more, and more preferably 50 μg / mL or more. The preferred upper limit is preferably 1000 μg / mL or less, more preferably 500 or 250 μg / mL or less, more preferably 100 μg / mL or less, and even more preferably 50 μg / mL or less, from the viewpoint of reducing the amount used. The amount of the redox molecule or heteroatom-containing aromatic compound used can be appropriately adjusted based on the amount of the biofilm drug sensitivity enhancer used, and a more preferred numerical range is preferably 0.1 μg / mL to 10,000 μg / mL, more preferably 1 μg / mL to 500 μg / mL. Furthermore, even when the amount of the redox molecule or heteroatom-containing aromatic compound used against bacteria (for example, Pseudomonas bacteria (e.g., Pseudomonas aeruginosa) or Shewanella bacteria) is, for example, 0.1 μg / mL to 1000 μg / mL, more preferably 2.5 μg / mL to 100 μg / mL, and even more preferably 2.5 μg / mL to 50 μg / mL, it is expected that the amount of the antimicrobial component (preferably an antibiotic) used can be reduced.

[0131] Furthermore, the amount of antimicrobial component (preferably an antibiotic) used in a preferred method of use of the present invention is not particularly limited, but the amount used can be reduced compared to general usage, and a preferred lower limit is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, more preferably 10 μg / mL or more, more preferably 20 μg / mL or more, more preferably 30 μg / mL or more, more preferably 40 μg / mL or more, more preferably 50 μg / mL or more, more preferably 60 μg / mL or more, more preferably 70 μg / mL or more, more preferably 80 μg / mL or more, more preferably 90 μg / mL or more, and even more preferably 100 μg / mL or more. Furthermore, a preferred upper limit, from the viewpoint of reducing the amount used, is preferably 10 mg / mL or less, more preferably 5 mg / mL or less, more preferably 2.5 mg / mL or less, more preferably 1000 μg / mL or less, more preferably 500 μg / mL or less, and even more preferably 250 or 100 μg / mL or less. Furthermore, the amount of the aminoglycoside compound used can be appropriately selected and combined with suitable upper and lower limits of the amount of the drug used described above, and the suitable numerical range is preferably 1 μg / mL to 1000 μg / mL, more preferably 10 μg / mL to 1000 μg / mL, and even more preferably 10 μg / mL to 100 μg / mL.

[0132] In the present invention, "prevention" refers to preventing or delaying the onset of symptoms or diseases in a subject, or reducing the risk of developing symptoms or diseases in a subject. In this technology, "improvement" refers to improving or maintaining a disease, symptom, or condition in a subject; preventing or delaying the worsening of a disease, symptom, or condition in a subject; or reversing, preventing, or delaying the progression of a disease, symptom, or condition in a subject.

[0133] In the present invention, the biofilm drug sensitivity improving component can be used, for example, in cosmetics, topical skin preparations, quasi-drugs, food and beverages, feed, and additives thereto, but is not particularly limited to these.

[0134] In addition to the biofilm drug sensitivity-enhancing component and the antimicrobial component, the present invention can optionally contain or incorporate components typically used in various formulations, such as cosmetics, quasi-drugs, and topical skin preparations, such as pharmaceuticals, as needed, provided that the effects of the present technology are not impaired. Examples of such components include preservatives, cell activators, antioxidants, moisturizers, UV protection agents, solvents (water, alcohols, etc.), oils, surfactants, thickeners, powders, chelating agents, pH adjusters, emulsifiers, stabilizers, colorants, glossing agents, flavoring agents, odorants, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure adjusters, and fragrances. One or more of these can be appropriately selected and used. The form of the composition of the present invention is not particularly limited, and may be any of liquid, paste, gel, solid, powder, and the like.

[0135] Furthermore, the compositions such as the drug sensitivity improving composition and antimicrobial composition may be used, for example, as cosmetics, topical skin preparations, quasi-drugs, foods and beverages, feeds, etc., but are not particularly limited thereto, and one or more of these may be selected. Among these, cosmetics, topical skin preparations, pharmaceuticals, quasi-drugs, etc. are preferred. In addition, the composition for improving the drug sensitivity of biofilms may be used as an additive or compounding agent for compositions, and may be a composition used to compound or add to compositions such as cosmetics, topical skin preparations, quasi-drugs, food and beverages, and feed. Examples of compositions of the present invention include sprays; oral care products such as mouthwashes (cleansing liquids, spray types, etc.) and toothpaste; and skin care products such as ointments, drug-containing sheets, and antibacterial agents (antibacterial liquids, spray types, etc.).

[0136] The composition of the present invention is preferably applied as an oral composition or a topical composition for skin application. By using the composition against periodontal disease bacteria present in oral plaque, the decrease in drug susceptibility caused by dental plaque can be suppressed, and the antimicrobial component's action and effect can be more effectively exerted. Furthermore, by using the composition against pathogenic microorganisms present in biofilms on wounds (preferably pressure ulcers), the decrease in drug susceptibility caused by biofilms can be suppressed, and the antimicrobial component's action and effect can be more effectively exerted. Furthermore, the component for improving drug susceptibility for biofilms can be impregnated into a wipe sheet to physically remove the biofilm, thereby more effectively exerting its antimicrobial action and other properties. Furthermore, the component for improving drug susceptibility for biofilms can also be brought into contact with biofilms, thereby effectively exerting its antimicrobial action and other properties. Therefore, the pain caused by wiping, such as from a wound (preferably a pressure ulcer), can be reduced. Furthermore, contacting the sheet with an area where a biofilm is likely to form can suppress microbial growth, and a preventive effect can also be expected.

[0137] 2. About this technology In addition, the present technology can also adopt the following configurations, but is not limited to the contents described here, and the contents described in "1." above can also be adopted as appropriate. [1] A composition for improving the drug susceptibility of a biofilm, comprising a component for improving the drug susceptibility of a biofilm (preferably one or more selected from the group consisting of a redox molecule, a heteroatom-containing aromatic compound, and a dye). The composition may be a composition for improving the drug susceptibility of microorganisms in a biofilm. [2] The composition according to [1], wherein the redox molecule is an organic redox molecule or the dye is an organic dye. [3] The composition according to [1] or [2], wherein the heteroatom-containing aromatic compound has redox activity and / or color development property. [4] The composition according to any one of [1] to [3], wherein the redox molecule is one or more compounds selected from the group consisting of phenazine compounds, phenothiazine compounds, anthraquinone compounds, triphenylmethane compounds, and xanthene compounds. [5] The composition according to any one of [1] to [4], wherein the redox molecule is one or more selected from the group consisting of safranine, methylene blue, anthraquinone sulfonic acid, brilliant blue FCF, and rose bengal. [6] The composition according to any one of [1] to [5], wherein the dye is one or more compounds selected from the group consisting of phenazine compounds, phenothiazine compounds, anthraquinone compounds, triphenylmethane compounds, and xanthene compounds. [7] The composition according to any one of [1] to [6], wherein the dye is one or more selected from the group consisting of safranine, methylene blue, anthraquinone sulfonic acid, brilliant blue FCF, and rose bengal. [8] The composition according to any one of [1] to [7], wherein the heteroatom-containing aromatic compound is one or more compounds selected from the group consisting of phenazine compounds, phenothiazine compounds, anthraquinone compounds, triphenylmethane compounds, and xanthene compounds. [9] The composition according to any one of [1] to [8], wherein the heteroatom-containing aromatic compound is one or more selected from the group consisting of safranine, methylene blue, anthraquinone sulfonic acid, brilliant blue FCF, and rose bengal.

[10] The composition according to any one of [1] to [9] above, wherein the microorganisms in the biofilm are bacteria and / or filamentous fungi, and preferably bacteria.

[11] The composition according to any one of [1] to

[10] above, wherein the drug is an antibiotic.

[12] The composition according to any one of [1] to

[11] above, wherein the drug is an aminoglycoside compound.

[13] The composition according to any one of [1] to

[12] above, which is an oral composition or a skin external composition.

[0138]

[14] An antimicrobial composition for use against microorganisms in biofilms, comprising an antimicrobial component and used in combination with a component for improving biofilm drug sensitivity described in any one of [1] to

[13] above or a composition containing the same.

[15] The composition according to

[14] , wherein the antimicrobial composition is an oral preparation or a topical preparation for skin.

[0139]

[16] A biofilm drug sensitivity enhancing component for enhancing drug sensitivity of microorganisms in a biofilm or for use in enhancing drug sensitivity.

[17] A biofilm drug sensitivity improving component for improving the efficacy of an antimicrobial component by improving the drug sensitivity of a biofilm, or for use in improving the efficacy.

[0140]

[18] A drug sensitivity enhancer for biofilms or a drug sensitivity enhancer component for biofilms for use in a drug sensitivity enhancer.

[19] A component for improving the drug sensitivity of biofilms, or a combination of a component for improving the drug sensitivity of biofilms and an antimicrobial component, for use in an antimicrobial composition or for use in an antimicrobial composition, which can improve the drug sensitivity of a biofilm in the antimicrobial composition.

[20] A biofilm drug sensitivity enhancer or a biofilm drug sensitivity enhancer component for producing a biofilm drug sensitivity enhancer.

[21] A component for improving biofilm drug sensitivity, or a combination of a component for improving biofilm drug sensitivity and an antimicrobial component, for producing an antimicrobial composition for improving biofilm drug sensitivity.

[22] A biofilm drug sensitivity enhancing component, or a combination of a biofilm drug sensitivity enhancing component and an antimicrobial component, for use in a combination of antimicrobial compositions that enhance the drug sensitivity of biofilms or for producing such a combination.

[23] A method for improving the drug sensitivity of a biofilm or a method for improving the drug sensitivity of microorganisms in a biofilm, using a component for improving drug sensitivity for a biofilm.

[24] An antimicrobial method in which an antimicrobial component is used to exert antimicrobial effects against microorganisms in a biofilm in a state or under conditions in which the drug sensitivity of the biofilm has been improved using a component for improving drug sensitivity for biofilms.

[25] An antimicrobial method for microorganisms in a biofilm, which uses a component for improving drug sensitivity for biofilms in combination with an antimicrobial component.

[0141]

[26] In any one of the above

[16] to

[25] , the component for improving biofilm drug sensitivity is preferably any one of the above [1] to

[13] .

[27] In any one of the above

[16] to

[26] , the microorganism is preferably the one described in the above

[10] .

[28] In any one of the above

[16] to

[27] , the drug is preferably the drug of the above

[11] or

[12] .

[29] In any one of the above

[16] to

[27] , it is preferable that the composition is used in the oral cavity or on the skin. [Example]

[0142] Hereinafter, the present technology will be described in further detail based on examples, test examples, etc. Note that the examples, etc. described below are examples of typical examples, etc. of the present technology, and the scope of the present technology should not be interpreted narrowly by these.

[0143] <Test Example 1: Antibiotic resistance test of colony biofilm> [Bacterial strain used] Strain used: Shewanella oneidensis MR-1 (Non-Patent Document 6: Nature Communications, 2020, 11, 1-11) (hereinafter also referred to as MR-1) Strain used: Pseudomonas aeruginosa PAO1 (Non-Patent Document 7: C.K. Stover et al., Nature, 2000, 406, 959-964) (hereinafter also referred to as PAO1) The culture conditions (medium, temperature, period) may be selected to suit the strain used, and the drugs used may be drugs generally recommended for the strain used. [Test substance (redox molecule)] Safranin (Wako Fujifilm)

[0144] <Method for testing the enhancement of drug susceptibility of colony biofilms by safranine> The following steps 1 to 4 were used to confirm whether the test substance would improve the drug susceptibility of bacteria in biofilms. Steps 1 and 2 can be omitted as appropriate. The incubation temperature was 30°C for the MR-1 strain and 37°C for the PAO1 strain.

[0145] 1. Grist (glycerol stock) preparation LB agar plates (obtained from: 63-6528-52 BD Difco™ LB broth mirror (Luria-Bertani), 63-6531-35 BD Bacto™ agar), incubation time 16 hours, incubation temperature 30°C (for Shewanella oneidensis MR-1), 37°C (for Pseudomonas aeruginosa PAO1).

[0146] 2. Preculture LB liquid medium 4mL, incubation time 16 hours, incubation temperature 30℃ (for MR-1), 37℃ (for PAO1), shaking incubation 190rpm

[0147] 3. Biofilm preparation and cultivation of biofilms in the presence of test substances and antibiotics Biofilms of each strain were prepared according to the following procedure, and the prepared biofilms were cultured in the presence of a test substance (safranine) and an antibiotic in combination. Control 1 (no test substance) was performed in the same manner as described below, except that safranine (0 μg / mL) in Table 1 was not added. Control 2 (no antibiotics) was performed in the same manner as described below, except that antibiotics (a) and (b) in Table 2 were not added (0 μg / mL). A combination of Controls 1 and 2 (no test substance and no antibiotics) was also performed.

[0148] Advance preparation Sterilized membrane filter (sterilized by UV irradiation for 10 minutes) 0.22μm MCE membrane (product number: GSWP04700) LB agar medium for biofilm creation (see Table 1 for composition) LB agar medium for antibiotic exposure (see Table 2 for composition): (a) Various concentrations of kanamycin were used for the MR-1 strain. (b) Various concentrations of gentamicin were used for the PAO1 strain. PBS (phosphate buffered saline) (also used to describe culture medium)

[0149] [Table 1]

[0150] [Table 2]

[0151] (1) After pre-culture, the bacterial strain was washed with PBS and diluted 5-fold with PBS to prepare 1 mL of bacterial solution. (2) The OD (600 nm) of the bacterial solution was measured, and the bacterial solution was diluted with PBS to obtain an adjusted OD (600 nm) value of 0.25. (3) A sterilized membrane filter was placed on the agar medium shown in Table 1. (4) 30 μL of the prepared bacterial solution was spotted on a filter placed on a safranine-containing agar medium, and the mixture was incubated at 30°C for 24 hours. As a result, multiple biofilms containing the bacterial strains were formed on the filters, and the filters with these biofilms were removed from the agar medium. Note that one colony can be counted as one biofilm. (5) The biofilm (including the bacterial strain) was transferred together with the filter onto the agar medium containing safranin and antibiotics listed in Table 2, and cultured at the specified temperature for 24 hours.

[0152] 4. Biofilm Recovery and Plating (1) After culturing the biofilm as described in "3." above, one biofilm was selected from the multiple biofilms on the filter, and this was designated as one colony. The entire colony was then scraped off with a toothpick and suspended in 1 mL of PBS. (2) The suspension containing one colony of the scraped strain was washed with PBS to remove any remaining antibiotics. The PBS solution containing one colony of the strain from which the antibiotics had been removed was diluted to a bacterial count of 10 4 , 10 5 , 10 6 A dilution series was made so that the concentration of CFU / mL was obtained, and PBS solutions containing the strains at each bacterial concentration were prepared. (3) 100 μL of the PBS solution containing the strain prepared in (2) above was plated on LB agar medium and cultured for 24 hours at 30°C for the MR-1 strain and 37°C for the PAO1 strain. (4) Colony count The smear method (colony method: CFU / mL) was used.

[0153] [Table 3]

[0154] [Table 4]

[0155] Shewanella oneidensis or Pseudomonas aeruginosa was cultured on LB agar containing 10 μM safranin to form a biofilm. A membrane filter was placed between the biofilm and the agar, allowing the biofilm to be transferred along with the membrane filter. The biofilm was then transferred onto LB agar containing antibiotics, and the number of viable bacteria was measured after 24 hours to evaluate the antibiotic resistance of the biofilm. The sensitivity of Shewanella oneidensis biofilms to kanamycin was increased by the addition of safranin. Specifically, 10 μg mL -1 Compared to the above kanamycin treatments, the number of viable bacteria was reduced more significantly when safranin was applied. -1 The above results showed improved sensitivity to gentamicin.

[0156] <Test Example 2: Antibacterial test of safranine against airborne bacteria and test to improve the antibacterial activity of antibiotics by safranine> The lack of antibacterial activity of safranine was confirmed using planktonic bacteria, which do not form biofilms. As in the above <Test Example 1>, kanamycin was used for the MR-1 strain, and gentamicin was used for the PAO1 strain. In Figure 1, LB represents the result of culturing in LB medium containing the MR-1 strain, and LB (10Km) represents the result of culturing in LB medium containing the MR-1 strain with kanamycin (1 μg / mL) added at the start of culturing. LB+SF represents the result of culturing in LB medium containing the MR-1 strain and safranine (10 μM). LB+SF (10Km) represents the result of culturing in LB medium containing the MR-1 strain and safranine with kanamycin (1 μg / mL) added at the start of culturing. 10Km refers to 1 μg / mL kanamycin. In Figure 2, LB represents the results of culturing in LB medium containing the MR-1 strain, LB (1.0 Km) represents the results of culturing in LB medium containing the PAO1 strain, to which kanamycin (0.1 μg / mL) was added 6 hours after the start of culturing, followed by further culturing. LB+SF represents the results of culturing in LB medium containing the PAO1 strain and safranine (10 μM). LB+SF (1.0 Km) represents the results of culturing in LB medium containing the MR-1 strain and safranine, to which kanamycin (0.1 μg / mL) was added 6 hours after the start of culturing, followed by further culturing. 1.0 Km refers to 0.1 μg / mL kanamycin. As described in the above <Test Example 1>, LB medium liquid culture was performed under the conditions of addition or absence of the test substance and addition or absence of the antibiotic, and the change in OD600 was confirmed over time to observe the change in the number of bacteria over time. The number of bacteria in each culture solution was measured by turbidity (OD600nm). The turbidity (OD600nm) when the test substance (safranine) was used / the turbidity (OD600nm) when the control was used was essentially 1 for the MR-1 strain, which was the same as the turbidity of the control, so safranine was determined to have no antibacterial activity.

[0157] When neither the MR-1 strain nor the PAO1 strain was used without the addition of antibiotics, there was little change in the number of bacteria over time, depending on whether safranine was present or not. As a result, no antibacterial effect of safranine against planktonic bacteria was observed. Furthermore, when neither the MR-1 strain nor the PAO1 strain was used with the addition of antibiotics, there was almost no difference in the effectiveness of the antibiotics depending on whether safranine was present or not. As a result, there was no evidence that safranine had the effect of improving the effectiveness of antibiotics against planktonic bacteria.

[0158] <Considerations on the results of Test Examples 1 and 2>

[0159] The results of Test Examples 1 and 2 show that the antibiotic susceptibility (kanamycin) of biofilms (colonies) formed by the Shewanella oneidensis MR-1 strain increased when safranine was added. The higher the safranine concentration, the greater the antibiotic susceptibility. This increase in antibiotic susceptibility was hardly observed when the bacteria were dispersed in a solution (suspended bacteria state). Furthermore, when Pseudomonas aeruginosa was used, the antibiotic susceptibility to gentamicin increased.

[0160] Safranin has been used as a dye to distinguish between gram-negative and gram-positive microorganisms (Patent Document 2: JP 2004-208526 A), as a dye to stain bacterial cells when removing oral biofilms, and as a technique to visualize the degree of removal (Patent Document 3: JP 10-236914 A), and as a photosensitizer during photodynamic therapy (Patent Document 4: JP 2007-503917 A).

[0161] However, there have been no reports on the effectiveness of improving the antibiotic susceptibility of biofilms. It has also been reported that the addition of copper and zinc ions is effective as a technique for improving the antibiotic susceptibility of biofilms (Non-Patent Document 5: Carolina FG, et al. npj Biofilms. Microbio. "Metal ions weaken the hydrophobicity and antibiotic resistance of Bacillus subtilis NCIB 3610 biofilms", 2020, 6, 1, 0604). However, this has only been confirmed to be effective for the highly hydrophobic Bacillus subtilis microbial species on the surface of biofilms. This technology has the advantage of being applicable to multiple species, not just one.

[0162] From the results of Test Example 2, it was found that safranine did not have the effect of improving the antibacterial effect and antibacterial component effect against suspended bacteria. In other words, it was considered that safranine does not have antibacterial activity. However, taking into consideration Test Example 1, the present inventors believed that safranine did not exert an antibacterial effect on bacteria, but rather had some effect on the biofilm, thereby improving the antibiotic susceptibility of bacteria present in the biofilm. Safranin is also known as a redox molecule, and is known as a redox indicator that switches between yellow (reduced form) and red (oxidized form). Safranin is also empirically known as a highly safe pigment component, and is known as a pigment that stains microorganisms (eukaryotic cell nuclei and bacterial cells) containing biofilms red. Therefore, when safranine is used, it has the advantage that the location where safranine is present can be visually identified.

[0163] Furthermore, the inventors considered that since safranine is a cyclic organic compound (preferably an aromatic compound containing a heteroatom in the molecule), a redox molecule, and a pigment component, compounds having one or more properties selected from these would also be able to improve the susceptibility of antibiotics to bacteria present in biofilms.

[0164] Therefore, based on Test Example 1, the present inventors prepared various redox molecules as Test Example 3, and performed a test to test the drug susceptibility of colony biofilms to be improved by the various redox molecules.

[0165] <Test Example 3: Antibiotic resistance test of colony biofilms (various redox molecules)> [Bacterial strain used] Strain used: Shewanella oneidensis MR-1 (hereinafter referred to as MR-1) The culture conditions (medium, temperature, period) may be selected to suit the strain used, and the drugs used may be drugs generally recommended for the strain used. [Test substances (various redox molecules)] Methylene blue (MB) (obtained from Wako Fujifilm) Sodium anthraquinone-2-sulfonate (AQS) (obtained from Tokyo Chemical Industry Co., Ltd.) Sodium anthraquinone-2,6-disulfonate (AQDS) (obtained from Tokyo Chemical Industry Co., Ltd.) Brilliant Blue FCF (BB) (Obtained from Wako Fujifilm) Rose Bengal (RB) (obtained from Tokyo Chemical Industry Co., Ltd.)

[0166] [Table 5]

[0167] [Table 6]

[0168] The procedures of Test Example 3 are described in simplified form in 1 to 4 below. The procedures of Test Example 3 were the same as those of Test Example 1 except for a slight difference in the culture time (4.(5) 30-hour culture).

[0169] 1. Grist raising LB agar plate, 16 hours, 30℃

[0170] 2. Preculture LB liquid medium 4mL, 16 hours, 30℃, 190rpm

[0171] 3. Biofilm Preparation Advance preparation Sterilized membrane filter (10 minutes UV irradiation) 0.22μm MCE membrane Part number: GSWP04700 LB agar medium for biofilm creation (see Table 5 for composition) LB agar medium for antibiotic exposure (see Table 6 for composition) PBS (also used to dilute culture medium) (1) Wash with PBS and prepare 1 mL of 5-fold dilution. (2) Measure the OD (600 nm) and adjust the dilution so that the value becomes 0.25. (3) Place a sterilized membrane filter on the agar medium. (4) 30 μL of the prepared bacterial solution was spotted on the filter and cultured for 24 hours. (5) The biofilm was transferred to an agar medium containing antibiotics along with the filter and cultured for 30 hours (agar medium containing the same redox molecules as those contained in the agar used for biofilm formation).

[0172] 4. Biofilm Recovery and Plating (1) Scrape off the biofilm with a bamboo skewer and suspend one colony in 1 mL of PBS. (2) After washing with PBS to remove residual antibiotics, 4 , 10 5 , 10 6 Prepare a dilution series and prepare a PBS solution. (3) 100 μL of the prepared PBS solution was plated on LB agar medium and cultured for 24 hours. (4) Colony count

[0173] [Table 7]

[0174] Based on these results, the inventors have provided a technology that reduces the required antibiotic concentration by improving the antibiotic sensitivity of biofilms using safranine, methylene blue, anthraquinone sulfonic acid, brilliant blue FCF, and rose bengal. Reducing antibiotic concentrations has the advantage of reducing the environmental pollution caused by antibiotics and reducing costs.

[0175] As described above, compounds having one or more properties selected from the following are considered to improve the antibiotic susceptibility of biofilms: they are cyclic organic compounds (preferably aromatic compounds containing heteroatoms in the molecule), they are redox molecules, and they are pigment components. The commonality among the chemical structures of the compounds used in Test Examples 1 to 3 is that they are organic compounds (preferably aromatic compounds containing heteroatoms) with a simple aromatic ring as the backbone, and therefore, it is considered that cyclic organic compounds (preferably aromatic compounds containing heteroatoms) improve the antibiotic susceptibility of biofilms.

[0176] Similarly to safranine, methylene blue (MB), brilliant blue FCF (BB), and rose bengal (RB) are also used as dyes. Considering that these dye-based compounds are empirically used as highly safe dyes, a technology can be provided to improve the drug sensitivity of microorganisms in biofilms with higher safety. Furthermore, the unique colors of the dyes can be used in various ways, such as to confirm the location of biofilms and their removal.

[0177] Furthermore, although anthraquinone-2-sodium sulfonate (AQS) and anthraquinone-2,6-sodium disulfonate (AQDS) have weak coloring properties, they are classified as redox molecules and cyclic organic compounds (preferably heteroatom-containing aromatic compounds) like other compounds, and these anthraquinone sulfonates can provide a technology for improving the drug sensitivity of microorganisms in biofilms.

[0178] For example, when sterilizing oral biofilms with antibacterial components, redox molecules or cyclic organic compounds (preferably heteroatom-containing aromatic compounds) can be used in combination, or teeth can be coated with redox molecules or cyclic organic compounds (preferably heteroatom-containing aromatic compounds) in advance. This allows oral biofilms to be removed highly efficiently with lower concentrations of antibacterial components than existing methods. Redox molecules or cyclic organic compounds (preferably heteroatom-containing aromatic compounds) have already been used to stain oral biofilms, so they are considered to be highly safe.

Claims

1. A composition for enhancing drug susceptibility of biofilms, comprising a redox molecule.

2. The composition of claim 1 , wherein the redox molecule is an organic redox molecule.

3. 3. The composition according to claim 1, wherein the redox molecule is one or more selected from the group consisting of phenazine-based compounds, phenothiazine-based compounds, anthraquinone-based compounds, triphenylmethane-based compounds, and xanthene-based compounds.

4. The composition according to claim 1 or 2, wherein the redox molecule is one or more selected from the group consisting of safranine, methylene blue, anthraquinone sulfonic acid, brilliant blue FCF, and rose bengal.

5. The composition of claim 1 or 2, wherein the microorganisms in the biofilm are bacteria.

6. The composition of claim 1 or 2, wherein the drug is an antibiotic.

7. The composition of claim 6 , wherein the antibiotic is an aminoglycoside compound.

8. The composition according to claim 1 or 2, which is an oral composition or a skin topical composition.

9. An antimicrobial composition for use against microorganisms in biofilms, comprising an antimicrobial component and used in combination with the composition of claim 1 or 2.

10. The composition according to claim 9, wherein the antimicrobial composition is an oral agent or a topical agent for skin.

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