Method for agglutinating Gram-positive bacteria

Abstract

The present invention aims to provide a method for determining the presence or absence of Gram-positive bacteria and for isolating Gram-positive bacteria in a short time and using a simple method. [Solution] A method for agglutinating Gram-positive bacteria, comprising contacting Gram-positive bacteria with a polythiophene compound containing repeating units represented by the following formula (1), thereby agglutinating the Gram-positive bacteria. (In the formula, X represents a divalent hydrocarbon group having 2 to 10 carbon atoms, and R 1 , R 2 , and R 3 Each of these independently represents a monovalent hydrocarbon group with 1 to 6 carbon atoms, and n represents an integer from 5 to 20. TIFF2026119695000027.tif47170

Description

Technical Field

[0001] The present invention relates to a method for aggregating Gram-positive bacteria.

Background Art

[0002] As an alternative material for nucleic acid staining reagents, π-conjugated polymers with excellent optical properties and sensitivity have attracted attention. Among them, it has been reported that cationic polythiophene compounds react with phosphate compounds (Non-Patent Document 1).

[0003] Further, as a method for identifying bacteria, the Gram staining method has been conventionally used. However, the Gram staining method requires time for culturing bacteria before staining, and it was not possible to simply identify bacteria in a short time. Further, the Gram staining method only identifies bacteria, and it was impossible to separate only Gram-positive bacteria from a system in which Gram-positive bacteria and Gram-negative bacteria coexist.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a method for determining the presence or absence of Gram-positive bacteria and separating Gram-positive bacteria by a short-time and simple method.

Means for Solving the Problems

[0006] The inventors of the present invention have discovered that phosphonilated polythiophene compounds exhibit a specific reaction with Gram-positive bacteria, thereby completing the present invention. Furthermore, the inventors have discovered that some phosphonilated polythiophene compounds also exhibit a specific reaction with Gram-negative bacteria, thereby completing the present invention.

[0007] The present invention provides the following [1] to

[10] . [1] The following formula (1): [ka] (In the formula, X represents a divalent hydrocarbon group having 2 to 10 carbon atoms, and R 1 , R 2 , and R 3 A method for agglutinating Gram-positive bacteria, comprising contacting Gram-positive bacteria with a polythiophene compound containing repeating units represented by (1 to 6 carbon atoms, and n being an integer from 5 to 20) to agglutinate the Gram-positive bacteria. [2] X represents an alkylene group with 2 to 10 carbon atoms, R 1 , R 2 , and R 3 The method according to [1], wherein each of the elements independently represents an alkyl group or phenyl group having 1 to 6 carbon atoms, and n represents an integer from 10 to 15. [3] The method according to [1] or [2], wherein the Gram-positive bacteria are one or more species selected from the group consisting of Staphylococcus and Bacillus bacteria. [4] The number of Gram-positive bacteria per 0.1 mg of polythiophene compound (number of Gram-positive bacteria / 0.1 mg of polythiophene compound) is 1.0 × 10⁻⁶ 2 ~1.0×10 9 The method described in any one of the following items [1] to [3], wherein the cells are cells. [5] A method for inhibiting the growth of Gram-positive bacteria, wherein the growth of Gram-positive bacteria is suppressed by agglutination of Gram-positive bacteria, as described in any one of items [1] to [4]. [6] Methods for isolating Gram-positive bacteria, including the following: (I) The following formula (1): [Chemical formula] (In the formula, X represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R 1 , R 2 , and R 3 each independently represent a monovalent hydrocarbon group having 1 to 6 carbon atoms, and n represents an integer of 5 to 20.) Mixing a polythiophene compound containing a repeating unit represented by the formula with a sample containing Gram-positive bacteria to form aggregates of Gram-positive bacteria in the sample; (II) centrifuging the sample in which aggregates of Gram-positive bacteria have formed to form a precipitate of Gram-positive bacteria in the sample; and (III) recovering the precipitate of Gram-positive bacteria formed in the sample. [7] The method according to [6], wherein the sample containing Gram-positive bacteria is a mixed sample of Gram-positive bacteria and Gram-negative bacteria. [8] The method according to [7], wherein the Gram-positive bacteria are one or more selected from the group consisting of Staphylococcus bacteria and Bacillus bacteria, and the Gram-negative bacteria are one or more selected from the group consisting of Escherichia bacteria and Pseudomonas bacteria. [9] The method according to any one of [6] to [8], wherein in (I), aggregates of Gram-positive bacteria are formed by contacting the polythiophene compound with Gram-positive bacteria for 5 hours or less.

[10] The following formula (2): [Chemical formula] (In the formula, X represents a divalent hydrocarbon group having 2 to 10 carbon atoms, and n represents an integer of 5 to 20.) A method for fibroticizing Gram-negative bacteria, comprising contacting a polythiophene compound containing a repeating sequence represented by the formula with Gram-negative bacteria to fibroticize Gram-negative bacteria. [Advantages of the Invention]

[0008] According to the present invention, Gram-positive bacteria can be specifically agglutinated in a short time and using a simple method, making it possible to determine the presence or absence of Gram-positive bacteria. Furthermore, the method for agglutinating Gram-positive bacteria according to the present invention can be used as a method for isolating Gram-positive bacteria and a method for inhibiting the growth of Gram-positive bacteria. Moreover, according to the method for isolating Gram-positive bacteria according to the present invention, Gram-positive bacteria can be isolated in a living state by agglutination, rather than by antibacterial or sterilization. Furthermore, according to the present invention, Gram-negative bacteria can be specifically fibrillated using a simple method, which is also effective in determining the presence or absence of Gram-negative bacteria. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a graph showing the identification results of PBBT (Lot. 191204) by 1H NMR measurement in an example of the production of a polythiophene compound. [Figure 2] Figure 2 is a graph showing the results of PTH identification by 1H NMR measurement in an example of the production of a polythiophene compound. [Figure 3] Figure 3 is a graph showing the time change in OD600 of various bacteria in culture media with and without PTB in Test Example 1. " / PTB" indicates culture media with PTB added (the same applies in subsequent figures). (a) represents S. aureus, (b) represents E. coli, (c) represents B. subtilis, and (d) represents P. aeruginosa. The average of three measurements is plotted, and the standard deviation is shown as an error bar. [Figure 4] Figure 4 is a graph showing the time change of OD600 for each initial concentration of S. aureus suspension in culture media with PTB added (S4 / PTB, S6 / PTB, S8 / PTB) and culture media without PTB (S. aureus) in Test Example 2. In the figure, "S4", "S6", and "S8" refer to bacterial suspensions with initial concentrations of 1.0 × 10⁴ cells / mL, 1.0 × 10⁶ cells / mL, and 1.0 × 10⁸ cells / mL, respectively (the same applies to subsequent figures). S. aureus refers to a bacterial suspension without PTB added, with an initial concentration of 1.0 × 10⁴ cells / mL. The average of three measurements is plotted, and the standard deviation is shown as an error bar. [Figure 5] Figure 5 is a graph showing the results of (a) UV-vis absorption and (b) fluorescence spectra for each initial concentration of S. aureus suspension in Test Example 2. [Figure 6] Figure 6 shows (a) particle size distribution and (b) zeta potential for each initial concentration of S. aureus suspension in culture media with PTB added (S4 / PTB, S6 / PTB, S8 / PTB) and culture media without PTB (S. aureus) in Test Example 2. In (b), the average of three measurements is plotted, and the standard deviation is shown as error bars. [Figure 7] Figure 7 shows the fluorescence microscopy results of S. aureus in the PBT-added culture medium at different initial concentrations of bacterial suspension ((a) S4 / PTB, (b) S6 / PTB, (c) S8 / PTB) in Test Example 2. From left to right, the images are fluorescence, DIC, and superimposed. [Figure 8] Figure 8 shows photographic images of the S4 / PTB culture medium observed under a fluorescence microscope at (a) 1 hour, (b) 6 hours, and (c) 24 hours after the start of culture in Test Example 3. From left to right, the images show the fluorescence image, DIC image, and superimposed image. [Figure 9] Figure 9 shows photographic images of the S8 / PTB culture medium observed under a fluorescence microscope at (a) 1 hour, (b) 6 hours, and (c) 24 hours after the start of culture in Test Example 3. From left to right, the images show the fluorescence image, DIC image, and superimposed image. [Figure 10] Figure 10 is a photographic image showing the results of fluorescence microscopy observations of a mixed sample of S. aureus and E. coli in Test Example 4, after centrifugation 24 hours after the start of culture. The images show (a) the mixed sample before centrifugation, (b) the supernatant after centrifugation, and (c) the precipitate after centrifugation. From left to right, the images show the fluorescence image, DIC image, and superimposed image, respectively. [Figure 11] Figure 11 shows images of agar plates of (a) B. subtilis and (b) E. coli from Test Example 5, after incubating for 24 hours with various polythiophene compounds (hereinafter referred to as PT compounds). Disks 1, 2, 3, and 4 were inoculated with PTB, Et-PTB, Ph-PTB, and PTH, respectively. [Figure 12]Figure 12 is a graph showing the time change in OD600 of S. aureus when PTB, Et-PTB, Ph-PTB, and PTH were added in Test Example 6-1. The blank represents the medium without added PT compounds. The average of three measurements is plotted, and the standard deviation is shown as an error bar. [Figure 13] Figure 13 is a graph showing the OD600 values ​​of S. aureus 48 hours after the start of static culture, according to the initial concentrations of the bacterial suspension (S4, S6, S8) in culture media with various PT compounds added (PTB, Et-PTB, Ph-PTB, PTH) and culture media without added compounds (Blank) in Test Example 6-2. [Figure 14] Figure 14 is a graph showing the OD600 values ​​of E. coli 48 hours after the start of static culture, according to the initial concentrations of bacterial suspension (S4, S6, S8) in culture media with various PT compounds added (PTB, Et-PTB, Ph-PTB, PTH) and culture media without added compounds (Blank) in Test Example 7. [Figure 15] Figure 15 is a photographic image showing the results of fluorescence microscopy observation of E. coli in Test Example 8, with and without the addition of various PT compounds ((b) PTB, (c) Et-PTB). From left to right, the images show the fluorescence image, the DIC image, and the superimposed image. [Figure 16] Figure 16 is a photographic image showing the results of observing the culture medium in Figure 15(c) in Test Example 8 at a higher magnification. From left to right, the images are fluorescence, bright-field, and superimposed. [Figure 17] Figure 17 shows photographic images of fluorescence microscopy observations of E. coli / Et-PTB culture in Test Example 8, at (a) 1h, (b) 3h, (c) 6h, and (d) 24h after the start of culture. From left to right, the images show the fluorescence image, DIC image, and superimposed image. [Modes for carrying out the invention]

[0010] <Method for agglutinating Gram-positive bacteria> The present invention provides a method for agglutinating Gram-positive bacteria, which involves contacting Gram-positive bacteria with a polythiophene compound to cause agglutination of the Gram-positive bacteria.

[0011] -Polythiophene compounds- Polythiophene compounds contain repeating units represented by the following formula (1).

[0012] [ka]

[0013] In formula (1), X represents a divalent hydrocarbon group. The divalent hydrocarbon group may be saturated or unsaturated. The divalent hydrocarbon group may also be linear, branched, or cyclic. The number of carbon atoms in the divalent hydrocarbon group is preferably 2 to 10, more preferably 3 to 7. This allows for the aggregation of Gram-positive bacteria and the suppression of their growth.

[0014] In certain embodiments, the divalent hydrocarbon group may be a divalent linear hydrocarbon group. Examples of divalent linear hydrocarbon groups include linear alkylenes, linear alkenylenes, or linear alkylenes, with linear alkylenes being preferred.

[0015] Examples of linear alkylenes include those having 1 to 10 carbon atoms. Examples of linear alkylenes having 1 to 10 carbon atoms include methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, n-heptalene, n-octylene, n-nonalene, and n-decalene. Linear alkylenes having 2 to 10 carbon atoms are preferred, and linear alkylenes having 3 to 7 carbon atoms are more preferred.

[0016] In formula (1), R 1 , R 2 , and R 3Each of these independently represents a monovalent hydrocarbon group. The monovalent hydrocarbon group may be saturated or unsaturated. The monovalent hydrocarbon group may also be linear, branched, or cyclic. The number of carbon atoms in the monovalent hydrocarbon group is, for example, 1 to 12, preferably 1 to 10, more preferably 1 to 8, and particularly preferably 1 to 6. This allows for the aggregation of Gram-positive bacteria and the suppression of their growth.

[0017] Examples of monovalent hydrocarbon groups include monovalent linear hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups.

[0018] A monovalent linear hydrocarbon group refers to a hydrocarbon group composed solely of a linear structure, and whose main chain does not contain a cyclic structure. However, the linear structure may be linear or branched. Examples of monovalent linear hydrocarbon groups include alkyl, alkenyl, and alkynyl groups. Alkyl, alkenyl, and alkynyl groups may be linear or branched.

[0019] As alkyls, alkyls having 1 to 12 carbon atoms are preferred, alkyls having 1 to 6 carbon atoms are more preferred, and alkyls having 1 to 4 carbon atoms are even more preferred. Examples of alkyls having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0020] As for the alkenyl, alkenyls having 2 to 12 carbon atoms are preferred, alkenyls having 2 to 6 carbon atoms are more preferred, and alkenyls having 2 to 4 carbon atoms are even more preferred. Examples of alkenyls having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0021] As for the alkynyl, alkynyls having 2 to 12 carbon atoms are preferred, alkynyls having 2 to 6 carbon atoms are more preferred, and alkynyls having 2 to 4 carbon atoms are even more preferred. Examples of alkynyls having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0022] Alkyl groups are preferred as monovalent chain hydrocarbon groups.

[0023] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as its ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon may be monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons; it may contain a chain-like structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be monocyclic or polycyclic.

[0024] As for cycloalkyls, cycloalkyls having 3 to 12 carbon atoms are preferred, cycloalkyls having 3 to 6 carbon atoms are more preferred, and cycloalkyls having 5 to 6 carbon atoms are even more preferred. Examples of cycloalkyls having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0025] As the cycloalkenyl, cycloalkenyls having 3 to 12 carbon atoms are preferred, cycloalkenyls having 3 to 6 carbon atoms are more preferred, and cycloalkenyls having 5 to 6 carbon atoms are even more preferred. Examples of cycloalkenyls having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0026] As for cycloalkynyls, cycloalkynyls having 3 to 12 carbon atoms are preferred, cycloalkynyls having 3 to 6 carbon atoms are more preferred, and cycloalkynyls having 5 to 6 carbon atoms are even more preferred. Examples of cycloalkynyls having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0027] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferred.

[0028] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to consist solely of an aromatic ring; it may also contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be monocyclic or polycyclic. Preferred monovalent aromatic hydrocarbon groups are aryl groups having 6 to 12 carbon atoms, more preferably aryl groups having 6 to 10 carbon atoms, and even more preferably aryl groups having 6 carbon atoms. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0029] Phenyl is preferred as the monovalent aromatic hydrocarbon group.

[0030] Among these, alkyl, cycloalkyl, and aryl groups are preferred as monovalent hydrocarbon groups.

[0031] In formula (1), n ​​is preferably an integer between 5 and 20, and more preferably an integer between 10 and 15.

[0032] P in equation (1) + The counterion for is not particularly limited as long as it is an anion. The counterion is preferably a halogen (e.g., fluorine, bromine, chlorine, iodine) ion, and more preferably a bromide ion.

[0033] Each repeating unit represented by formula (1) may have other structural units or elements bonded to its ends. The other structural units or elements are not particularly limited as long as they do not hinder the effects of the present invention, and are preferably nonhydrophilic and nonionic. Examples of other elements include hydrogen or halogen atoms (e.g., fluorine atoms, bromine atoms, chlorine or iodine atoms), which are usually hydrogen atoms or bromine atoms, respectively. Examples of other structural units include polymers or combinations of monomer units different from the repeating units represented by formula (1). The mass of the other structural units may be, for example, less than 20% by mass, less than 10% by mass, or less than 5% by mass, relative to the total mass of the polythiophene compound.

[0034] -Method for producing polythiophene compounds- The polythiophene compounds of the present invention can be synthesized using known methods. For example, the polythiophene compounds can be produced according to the synthesis scheme represented by the following formula (10). The production method shown in the following synthesis scheme is a representative method, and the method for producing the polythiophene compounds is not limited thereto. [ka]

[0035] In the formula, X, R1, R2, R3, and n are the same as defined above. Y1 to Y7 are preferably halogen ions (e.g., fluorine, bromine, chlorine, iodine), and more preferably bromide ions. Y3 is common to Y1 or Y2. Y5 is common to the halogen in the hydrogen halide reacted with the compound represented by formula (6) in step C, or to Y1 or Y2 in the compound represented by formula (3) in step D. Y6 and Y7 are common to the halogen in the halogenating agent in step E.

[0036] (Process A) Step A is a step in which a compound represented by formula (2) (p-methoxyphenol) is reacted with a compound represented by formula (3) to obtain a compound represented by formula (4). The reaction in step A is preferably carried out in the presence of a base. As the base, a carbonate such as potassium carbonate is preferred. The solvent is not particularly limited, but organic solvents such as acetone are preferred. These may be used individually or as a mixture of two or more solvents. When the reaction in step A is carried out in the presence of a base, it is preferable to use a crown ether. Examples of crown ethers include 12-crown-4, 15-crown-5, and 18-crown-6, which can be selected depending on the base used. For example, when potassium carbonate is used as the base, 12-crown-4 is preferred. The reaction temperature in step A is preferably 40 to 80°C, more preferably 45 to 70°C, and even more preferably 50 to 60°C. The reaction time is preferably 20 to 70 hours, more preferably 30 to 60 hours, and even more preferably 45 to 50 hours.

[0037] (Process B) Step B is a step in which a compound represented by formula (4) is reacted with a compound represented by formula (5) to obtain a compound represented by formula (6). The reaction in step B is preferably carried out using a nickel catalyst reagent. As a nickel catalyst reagent, for example, [1,3-bis(diphenylposphino)propane]nickel(II) dichloride (Ni(dppp)Cl2) is preferred. The reaction in step B is preferably carried out in the presence of magnesium and iodine. The solvent is not particularly limited, but organic solvents such as tetrahydrofuran are preferred. These may be used individually or as a mixture of two or more solvents. The reaction temperature in step B is preferably 40 to 80°C, more preferably 50 to 70°C, and even more preferably 55 to 65°C. The reaction time is preferably 5 to 50 hours, more preferably 10 to 30 hours, and even more preferably 12 to 25 hours.

[0038] (Process C) Step C is a step in which the compound represented by formula (6) is reacted with acetic anhydride and hydrogen halide to obtain the compound represented by formula (7). Examples of halogens include fluorine, bromine, chlorine, and iodine, with bromine being preferred. The reaction temperature in step C is preferably 70 to 130°C, more preferably 80 to 120°C, and even more preferably 90 to 110°C. The reaction time is preferably 10 to 40 hours, more preferably 15 to 30 hours, and even more preferably 20 to 25 hours.

[0039] (Process D) The compound represented by formula (7) can also be obtained by step D. Step D is a step in which the compound represented by formula (3) is reacted with the compound represented by formula (5) to obtain the compound represented by formula (7). The solvent is not particularly limited, but organic solvents such as tetrahydrofuran and hexane are preferred. These may be used individually or as a mixture of two or more solvents. Preferably, the solvents are tetrahydrofuran and hexane, and more preferably a mixture of tetrahydrofuran and hexane. The reaction temperature in step D is preferably room temperature, specifically 15-30°C or 10-25°C. The reaction time is preferably 0.5-5 hours, more preferably 1-3 hours, and even more preferably 1.5-2.5 hours. The compound represented by formula (5) may be reacted with alkyllithium prior to its reaction with the compound represented by formula (3). Examples of alkyllithium include butyllithium such as n-butyllithium, sec-butyllithium, and tert-butyllithium, phenyllithium, and methyllithium, with butyllithium being preferred and n-butyllithium being more preferred. The reaction temperature with alkyllithium is preferably -10 to -60°C, more preferably -20 to -50°C. The reaction time is preferably 1 to 30 minutes, more preferably 5 to 15 minutes.

[0040] (Process E) Step E is a step in which a halogenating agent is reacted with the compound represented by formula (7) to obtain the compound represented by formula (8). Examples of halogenating agents include fluorinating agents, brominating agents, chlorinating agents, and iodinating agents, with brominating agents being preferred. For example, N-bromosuccinimide (NBS) is preferred as a brominating agent. As a solvent, organic solvents such as N,N-dimethylformamide (DMF) are preferred. The reaction in step E is preferably carried out in an ice bath or at room temperature. Specifically, the temperature in the ice bath is, for example, 0 to 5°C or 0 to 1°C. Specifically, the room temperature in the ice bath is, for example, 15 to 30°C or 10 to 25°C. The reaction time is preferably 5 to 30 hours, more preferably 10 to 25 hours, and even more preferably 15 to 20 hours.

[0041] (Process F) Step F is a step in which a polymerization reaction of the compound represented by formula (8) is carried out to obtain a compound containing repeating units represented by formula (9). The polymerization reaction is preferably carried out using a nickel catalyst. As a nickel catalyst, for example, [1,3-bis(diphenylposphino)propane]nickel(II) dichloride (Ni(dppp)Cl2) is preferred. Polymerization reactions using nickel catalysts include, for example, isopropylmagnesium chloride-lithium chloride complex ( i PrMgCl·LiCl) is preferred. The solvent is not particularly limited, but organic solvents such as tetrahydrofuran are preferred. These may be used individually or as a mixture of two or more solvents. The reaction temperature is preferably 10 to 35°C, and more preferably room temperature (e.g., 20 to 25°C). The reaction time is preferably 1 to 5 hours, and more preferably 1.5 to 4 hours. In step F, the polymerization reaction results in both ends of the compound containing the repeating unit represented by formula (9) being typically Y. 6 , Y 7 , or hydrogen, or both ends are Y 6, Y 7 , or hydrogen. The compound containing the repeating unit represented by formula (9) may have other elements or structures at each end, as long as they do not hinder the effects of the present invention, and specific examples thereof are the same as those of the ends of the repeating unit represented by formula (1) described above.

[0042] (Process G) Step G is a step in which a compound containing a repeating unit represented by formula (9) is reacted with a trivalent organophosphorus compound (R3P) to obtain a compound containing a repeating unit represented by formula (1). The organophosphorus compound is not particularly limited as long as it can be reacted with a compound containing the repeating unit represented by formula (9) to obtain a compound containing the repeating unit represented by formula (1) described above, and in R3P, R is the same as R1, R2, and R3 described above. Of these, the organophosphorus compounds are preferably trimethylphosphine, triethylphosphine, and triphenylphosphine. The solvent is not particularly limited, but examples include organic solvents such as tetrahydrofuran, dimethylformaldehyde, and dimethyl sulfoxide. These may be used individually or as a mixture of two or more solvents. Of these solvents, tetrahydrofuran, dimethylformaldehyde, and dimethyl sulfoxide are preferred, and a mixture of tetrahydrofuran, dimethylformaldehyde, and dimethyl sulfoxide is more preferred. The reaction temperature is preferably 60 to 120°C, more preferably 70 to 110°C, and even more preferably 75 to 100°C. The reaction time is preferably 10 to 70 hours, more preferably 20 to 60 hours, and even more preferably 30 to 50 hours.

[0043] The compounds obtained in each step may be purified by treatments such as distillation, vacuum distillation, column chromatography, and recrystallization; reagents may be removed by treatments such as evaporator and filtration; washing with purified water, sodium chloride, etc.; extraction with purified water, sodium bicarbonate, sodium chloride, hexane, etc.; and drying with magnesium sulfate, etc., one or more times.

[0044] -Gram-positive bacteria- The bacteria targeted can be any Gram-positive bacteria, and are not particularly limited. Examples of Gram-positive bacteria include bacteria of the genera Staphylococcus, Bacillus, Enterococcus, and Streptococcus. Examples of Staphylococcus bacteria include Staphylococcus aureus, Staphylococcus epidermis, and Staphylococcus saprophyticus. Examples of Bacillus bacteria include Bacillus subtilis, Bacillus subtilis var. natto, Bacillus anthracis, and Bacillus thuringiensis. Examples of Enterococcus bacteria include Enterococcus faecalis, vancomycin-resistant Enterococcus faecalis, and Enterococcus faecium. Examples of Streptococcus bacteria include Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes. Of these, Staphylococcus and Bacillus bacteria are preferred, more preferably Staphylococcus aureus and Bacillus natto, and even more preferably Staphylococcus aureus.

[0045] -contact- Contact of polythiophene compounds with Gram-positive bacteria can be carried out, for example, by directly contacting the polythiophene compound with Gram-positive bacteria or by mixing it with a sample containing Gram-positive bacteria. Contact may be carried out with a single type of Gram-positive bacteria, with two or more types present in a system, or with a system containing both Gram-positive and Gram-negative bacteria. The agglutination method for Gram-positive bacteria of the present invention can specifically agglutinate Gram-positive bacteria even in systems containing both Gram-positive and Gram-negative bacteria. Specific examples of Gram-negative bacteria that may be mixed with Gram-positive bacteria in the present invention are described in the section <Method for Isolating Gram-Positive Bacteria>.

[0046] The number of Gram-positive bacteria relative to the polythiophene compound when the polythiophene compound is brought into contact with Gram-positive bacteria is not particularly limited. For example, the number of Gram-positive bacteria per 0.1 mg of polythiophene compound (number of Gram-positive bacteria / 0.1 mg of polythiophene compound) is preferably 1.0 × 10⁶. 9 The size is smaller than cells, more preferably 1.0 × 10⁻⁶ 7 The size is less than or equal to 1.0 × 10⁻¹ cells, and more preferably 1.0 × 10⁻¹ 5 The number of cells is less than or equal to 1.0 × 10⁻⁶. The lower limit is preferably 1.0 × 10⁻⁶. 2 More than 1.0 × 10⁻¹⁶ cells, more preferably 1.0 × 10⁻¹⁶ cells. 3 The number of cells is greater than or equal to 10. Therefore, the ratio (number of Gram-positive bacteria / 0.1 mg of polythiophene compound) is preferably 1.0 × 10. 2 ~1.0×10 9 These are cells, more preferably 1.0 × 10⁶ 3 ~1.0×10 7 The cells are, more preferably 1.0 × 10⁶ 3 ~1.0×10 5 These are cells. This allows for the aggregation of Gram-positive bacteria in a shorter time and more easily, and further inhibits the growth of Gram-positive bacteria.

[0047] The time for contacting the polythiophene compound with Gram-positive bacteria is not particularly limited, as long as it is sufficient for aggregates of the Gram-positive bacteria to form. Preferably, it is 20 minutes or more, more preferably 30 minutes or more, and even more preferably 50 minutes or more.

[0048] Contact may be carried out by bacterial culture. The culture medium may be a solid medium or a liquid medium, but a liquid medium is preferred from the viewpoint of facilitating the formation of aggregates. The culture method can be selected according to the target bacterial species and is not particularly limited as long as it is a method in which the polythiophene compound comes into contact with Gram-positive bacteria. For example, when targeting aerobic bacteria or facultative anaerobic bacteria, aeration culture is used, and shaking culture is preferred. When performing shaking culture, the shaking speed is preferably 50 to 200 rpm, and more preferably 110 to 190 rpm. The culture temperature is preferably the optimal temperature for the target bacteria, usually 20 to 40°C, and preferably 30 to 38°C.

[0049] <Method for inhibiting the growth of Gram-positive bacteria> Since the aggregation of Gram-positive bacteria can suppress the growth of Gram-positive bacteria, the method for agglutinating Gram-positive bacteria of the present invention can be used to suppress the growth of Gram-positive bacteria. In other words, the method for suppressing the growth of Gram-positive bacteria of the present invention includes contacting Gram-positive bacteria with a polythiophene compound to suppress their growth.

[0050] The conditions for the contact between the polythiophene compound, the target Gram-positive bacteria, and the polythiophene compound and Gram-positive bacteria are the same as those described in section <Method for agglutination of Gram-positive bacteria>.

[0051] <Method for determining the presence or absence of Gram-positive bacteria> The polythiophene compounds described above can specifically agglutinate Gram-positive bacteria and can therefore be used to determine the presence or absence of Gram-positive bacteria in a sample. More specifically, the method for determining the presence or absence of Gram-positive bacteria according to the present invention includes mixing a polythiophene compound with a sample that may contain Gram-positive bacteria, and confirming the formation of aggregates in the sample mixed with the polythiophene compound.

[0052] The conditions for polythiophene compounds and specific examples of Gram-positive bacteria to be targeted are as described in the section <Method for agglutination of Gram-positive bacteria>.

[0053] -mixture- (sample) The sample may or may not contain Gram-positive bacteria, and Gram-positive bacteria may be mixed with Gram-negative bacteria. Specific examples of Gram-negative bacteria that may be mixed with Gram-positive bacteria in this invention are described in the section <Method for Isolating Gram-Positive Bacteria>.

[0054] The number of Gram-positive bacteria in the sample relative to the polythiophene compound is not particularly limited and is, for example, the same as the conditions described in section <Method for agglutination of Gram-positive bacteria>.

[0055] (Contact time) The polythiophene compound is mixed with a sample that may contain Gram-positive bacteria, and the time for contact with the Gram-positive bacteria present in the sample is the same as the conditions described in section <Method for agglutination of Gram-positive bacteria>.

[0056] When using a polythiophene compound containing repeating units represented by the following formula (2), the time for contacting the polythiophene compound with Gram-positive bacteria should be sufficient to produce aggregates of Gram-positive bacteria, and not to cause fibrosis of Gram-negative bacteria as described below. Specifically, it is preferably 5 hours or less, more preferably 4 hours or less, and even more preferably 2 hours or less. The lower limit is preferably 20 minutes or more, more preferably 30 minutes or more, and even more preferably 50 minutes or more. Therefore, the time for contacting the polythiophene compound containing repeating units represented by the following formula (2) with Gram-positive bacteria is preferably 20 minutes to 5 hours, more preferably 30 minutes to 4 hours, and even more preferably 50 minutes to 2 hours.

[0057] Even if a compound containing repeating units represented by the following formula (2) is used as the polythiophene compound, if the formation of aggregates is confirmed by visual observation such as microscopic observation described below, the aggregation of Gram-positive bacteria can be distinguished from the fibrillation of Gram-negative bacteria. Therefore, the contact time only needs to be sufficient for aggregates to be formed and is not particularly limited. That is, it is preferably 20 minutes or more, more preferably 30 minutes or more, and even more preferably 50 minutes or more.

[0058] -Confirmation of aggregate formation- The formation of aggregates can be confirmed, for example, by visual inspection such as microscopic observation, by checking for the presence or absence of precipitates in the sample after centrifugation, or by checking for a decrease in the turbidity of the sample.

[0059] Microscopes such as fluorescence microscopy and confocal laser microscopy can be used. If aggregate formation is observed, it can be determined that Gram-positive bacteria are present in the sample.

[0060] Centrifugation can usually be performed at 700-1500 rpm, preferably 900-1200 rpm. The centrifugation time is usually 30 seconds to 8 minutes, preferably 1 to 6 minutes. If sediment is observed in the centrifuged sample, it can be determined that Gram-positive bacteria are present in the sample.

[0061] The turbidity of the sample can be confirmed, for example, by absorbance measurement. Absorbance is usually measured at 400-700 nm, preferably 500-650 nm. If the turbidity of the sample after mixing with the polythiophene compound decreases compared to the sample before mixing with the polythiophene compound, it can be determined that Gram-positive bacteria are present in the sample.

[0062] Of these methods, confirmation is preferably made by microscopic observation or by the presence or absence of precipitates in the sample after centrifugation. Furthermore, a centrifugation sample in which the presence of Gram-positive bacteria has been confirmed can be used in the Gram-positive bacteria isolation method described later.

[0063] <Method for isolating Gram-positive bacteria> The method for isolating Gram-positive bacteria according to the present invention includes the following (I) to (III). (I) Mix a polythiophene compound with a sample containing Gram-positive bacteria to generate aggregates of Gram-positive bacteria in the sample. (II) The sample in which aggregates of Gram-positive bacteria have been formed is centrifuged to produce a precipitate of Gram-positive bacteria in the sample. (III) Collect the precipitate of Gram-positive bacteria generated in the sample.

[0064] The polythiophene compounds are the same as those described in the section <Method for agglutination of Gram-positive bacteria>. Furthermore, the Gram-positive bacteria to be targeted are as described in the section <Agglutination Method for Gram-Positive Bacteria>. In addition, the amount of Gram-positive bacteria-containing sample used should be such that the number of Gram-positive bacteria per 0.1 mg of polythiophene compound satisfies the conditions described in the section <Agglutination Method for Gram-Positive Bacteria>.

[0065] - Sample containing Gram-positive bacteria - Furthermore, the sample containing Gram-positive bacteria may be a mixed sample of Gram-positive and Gram-negative bacteria. According to the method for isolating Gram-positive bacteria of the present invention, even in a system containing a mixture of Gram-positive and Gram-negative bacteria, Gram-positive bacteria can be specifically isolated alive without sterilization.

[0066] Gram-negative bacteria are not particularly limited, but examples include bacteria of the genera Escherichia, Pseudomonas, Acinetobacter, and Klebsiella. Examples of Escherichia bacteria include Escherichia coli, Escherichia albertii, and Escherichia blattae. Examples of Pseudomonas bacteria include Pseudomonas aeruginosa, Pseudomonas aeruginosa, and Pseudomonas fluorescens. Examples of bacteria of the genus Acinetobacter include Acinetobacter baumannii, Acinetobacter junii, and Acinetobacter boissieri. Examples of bacteria of the genus Klebsiella include Klebsiella pneumonia, Klebsiella granulomatis, and Klebsiella oxytoca. Of these, bacteria of the genus Escherichia and Pseudomonas are preferred, more preferably Escherichia coli and Pseudomonas, and even more preferably Pseudomonas.

[0067] In (I), the formation of agglutinations of Gram-positive bacteria is preferably carried out by contacting the polythiophene compound with Gram-positive bacteria for a predetermined time. The contact conditions, such as contact time and contact method, are the same as those described in section <Method for agglutination of Gram-positive bacteria>.

[0068] In (II), centrifugation should be carried out under the same conditions as described in item <Method for determining the presence or absence of Gram-positive bacteria>.

[0069] In (III), the recovery of the Gram-positive bacterial precipitate can be carried out by conventional methods; for example, only the supernatant of the Gram-positive bacterial-containing sample after centrifugation may be removed. The recovered Gram-positive bacterial precipitate may be concentrated or diluted to adjust its concentration, frozen to prepare a preserved sample, or used as is for bacterial identification tests, etc.

[0070] <Method for inducing fibrillation of Gram-negative bacteria> The present invention provides a method for fibrillating Gram-negative bacteria, which involves contacting Gram-negative bacteria with a polythiophene compound to induce fibrillation of the Gram-negative bacteria.

[0071] -Polythiophene compounds- Polythiophene compounds contain repeating units represented by the following formula (2).

[0072] [ka]

[0073] X and n in equation (2) are the same as those in equation (1).

[0074] P in equation (2) + The counterion for is the same as that in equation (1).

[0075] Specific examples of Gram-negative bacteria to be targeted are as described in the section <Methods for Isolating Gram-Positive Bacteria>.

[0076] The method for producing a polythiophene compound containing the repeating unit represented by formula (2) is not particularly limited, but for example, in the method for producing a polythiophene compound of steps A to G described above, it can be produced by using triethylphosphene as the trivalent organophosphorus compound used in step G.

[0077] -contact- Contact of polythiophene compounds with Gram-negative bacteria can be carried out, for example, by directly contacting the polythiophene compound with Gram-negative bacteria or by mixing it with a sample containing Gram-negative bacteria. Contact may be carried out with a single species of Gram-negative bacteria, with a system containing two or more species, or with a system containing both Gram-negative and Gram-positive bacteria.

[0078] When a polythiophene compound is brought into contact with Gram-negative bacteria, the number of Gram-negative bacteria per 0.1 mg of the polythiophene compound ((number of Gram-negative bacteria) / (0.1 mg of polythiophene compound)) is preferably 1.0 × 10⁶. 9 The size is smaller than cells, more preferably 1.0 × 10⁻⁶ 7 The size is less than or equal to 1.0 × 10⁻¹ cells, and more preferably 1.0 × 10⁻¹ 5 The number of cells is less than or equal to 1.0 × 10⁻⁶. The lower limit is preferably 1.0 × 10⁻⁶. 2 More than 1.0 × 10⁻¹⁶ cells, more preferably 1.0 × 10⁻¹⁶ cells. 3 The number of cells is greater than or equal to 10. Therefore, (number of Gram-negative bacteria) / (0.1 mg polythiophene compound) is preferably 1.0 × 10 2 ~1.0×10 9 These are cells, more preferably 1.0 × 10⁶ 3 ~1.0×10 7 The cells are, more preferably 1.0 × 10⁶ 3 ~1.0×10 5 These are cells.

[0079] The time for which the polythiophene compound is in contact with Gram-negative bacteria is not particularly limited, as long as it is sufficient to induce fibrosis in the Gram-negative bacteria. Preferably, it is more than 5 hours, more preferably 5.5 hours or more, and even more preferably 10 hours or more. The upper limit is preferably 48 hours or less, more preferably 30 hours or less, and even more preferably 25 hours or less. Therefore, the time for which the polythiophene compound is in contact with Gram-positive bacteria is preferably more than 5 hours, more preferably 5.5 hours or more, even more preferably 5.5 to 48 hours, and even more preferably 10 to 30 hours, or 10 to 25 hours.

[0080] Contact may be carried out by bacterial culture. The culture medium may be a solid medium or a liquid medium, but a liquid medium is preferred from the viewpoint of facilitating fibrosis. The culture method can be selected according to the target bacterial species and is not particularly limited as long as it is a method of contact between the polythiophene compound and Gram-negative bacteria. For example, when targeting aerobic bacteria or facultative anaerobes, aeration culture is used, and shaking culture is preferred. When performing shaking culture, the shaking speed is preferably 50 to 200 rpm, and more preferably 110 to 190 rpm. The culture temperature may be the optimal temperature for the bacteria, usually 20 to 40°C, and preferably 30 to 38°C.

[0081] <Method for determining the presence or absence of Gram-negative bacteria> The above-mentioned polythiophene compounds can specifically cause fibrosis in Gram-negative bacteria and can therefore be used to determine the presence or absence of Gram-negative bacteria in a sample. More specifically, the method for determining the presence or absence of Gram-negative bacteria according to the present invention includes mixing the above-mentioned polythiophene compound with a sample that may contain Gram-negative bacteria, and confirming the occurrence of fibrosis in the sample mixed with the above-mentioned polythiophene compound.

[0082] The conditions for polythiophene compounds and specific examples of target Gram-negative bacteria are as described in the section <Method for Fibrosis of Gram-Negative Bacteria>.

[0083] The sample may or may not contain Gram-negative bacteria, and Gram-negative bacteria may be mixed with Gram-positive bacteria. Specific examples of Gram-positive bacteria are described in the section <Method for Agglutination of Gram-Positive Bacteria>.

[0084] When mixing a polythiophene compound with a sample that may contain Gram-negative bacteria, the number of Gram-negative bacteria per 0.1 mg of polythiophene compound is the same as the conditions described in section <Method for fibrosis of Gram-negative bacteria>. If the concentration of Gram-positive bacteria in a sample is unknown, one possible approach is to prepare multiple serial dilutions of the sample from 5 to 20 times, mix a polythiophene compound under similar conditions at each dilution, and check whether aggregates are formed at any of the dilution ratios.

[0085] The occurrence of fibrosis can be visually confirmed by microscopic observation, as described below. Therefore, the time for which the polythiophene compound is mixed with a sample that may contain Gram-negative bacteria and exposed to the Gram-negative bacteria in the sample is not particularly limited, as long as it is sufficient to induce fibrosis in the Gram-negative bacteria. More specifically, it is the same as the conditions described in the section <Method for inducing fibrosis in Gram-negative bacteria>.

[0086] Fibrosis can be confirmed by visual inspection, such as microscopic observation. Specific examples of microscopy are described in the section <Method for Determining the Presence or Absence of Gram-Positive Bacteria>. If fibrosis is observed, it can be determined that Gram-negative bacteria are present in the sample.

[0087] <Use of the invention> As described above, the agglutination method for Gram-positive bacteria of the present invention can specifically and quickly and easily agglutinate Gram-positive bacteria. Furthermore, the fibrillation method for Gram-negative bacteria of the present invention can specifically and quickly and easily fibrillate Gram-negative bacteria. Therefore, it is useful as a quick and easy method for identifying bacteria in various fields. For example, it could be used as a means of instantaneous identification of bacteria in medical settings. Sepsis is a disease in which bacteria multiply rapidly and can be fatal. In a time-sensitive situation, there is no time for bacterial culture, and it is necessary to immediately identify and administer an antibiotic suitable for the bacteria. Providing information for antibiotic selection in a short time and with a simple method, and improving accuracy, is extremely important. In addition, for example, in dairy product factories, dairy products are a nutrient source for bacteria, so bacterial screening is always performed before processing. This invention makes it easier to screen for bacterial species that vary depending on the environment. In the field of bacteriology, it is necessary to differentiate and identify various bacteria from soil, and a simple bacterial identification method like the one of this invention is extremely useful. Furthermore, the agglutination method for Gram-positive bacteria of the present invention can be applied to methods for separating Gram-positive bacteria, and since Gram-positive bacteria can be separated while alive, it becomes possible to perform detailed detection after instantaneous identification of bacteria in medical settings such as those described above. [Examples]

[0088] The present invention will be described below with reference to examples. The following examples are not intended to limit the present invention. In the following, Poly[3-(4-trimethylphosphinobutyl)thiophene bromide] will be referred to as PTB, Poly[3-(4-triethylphosphinobutyl)thiophene bromide] as Et-PTB, Poly[3-(4-triphenylphosphinobutyl)thiophene bromide] as Ph-PTB, and Poly[3-(6-trimethylphosphinohexyl)thiophene bromide] as PTH.

[0089] <Polythiophene compounds> The structural formulas of the polythiophene compounds (hereinafter also referred to as "PT compounds") used in this study are shown in the following formulas (11) to (14) (formula (11): PTB, formula (12): Et-PTB, formula (13): Ph-PTB, formula (14): PTH). [ka] [ka] [ka] [ka]

[0090] <Examples of Polythiophene Compound Production> PTB, Et-PTB, and Ph-PTB were prepared according to the synthetic scheme represented by formula (15). PTH was prepared in the same manner as the synthetic scheme represented by formula (15), except that 1,6-dibromohexane was used instead of 1,4-dibromobutane, as described below. In the examples of the preparation of polythiophene compounds, 1-(4-Bromobutyl)-4-methoxybenzene is denoted as BBMB, 3-[4-(4-Methoxyphenoxy)]thiophene as MPBT, 3-(4-Bomobutyl)thiophene as BBT, 2,5-Dibromo-3-(4-bromobutyl)thiophene as DBrBBT, and Poly[3-(4-bromobutyl)thiophene] as PBBT. [ka]

[0091] (Reagents used in the production of polythiophene compounds, etc.) This document describes the reagents used in the production of polythiophene compounds and their purification methods. • 3-bromothiofen (Tokyo Chemical Industries, Ltd., 97%) A commercially available product purified by vacuum distillation was used (bp 40℃, 1.0kPa). • 1,4-Dibromobutane (Tokyo Chemical Industries, Ltd., 95%) We used a commercially available product purified by vacuum distillation (bp 100℃, 0.7kPa). • 1,6-Dibromohexane (Tokyo Chemical Industries, Ltd., > 97.0%) n-Hexane (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade (Purity: 96%)) The product used was purified by atmospheric distillation (bp 70℃). Tetrahydrofuran (THF) (Fujifilm Wako Pure Chemical Industries, Ltd., 99.5%) We used commercially available products purified using a solvent purification system (Mitsuwa Rikagaku Kogyo Co., Ltd., Glass Contour). n-Hexane (Fujifilm Wako Pure Chemical Industries, Ltd., ultra-dehydrated) We used commercially available products purified using a solvent purification system (Mitsuwa Rikagaku Kogyo Co., Ltd., Glass Contour).

[0092] The following reagents were used as purchased. • p-Methoxyphenol (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) • Potassium carbonate (K2CO3) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) · 1.4.7.10.13.16-Hexaoxacyclooctadecane (18-crown-6) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) Acetone (Kanto Chemical Co., Ltd.) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) • Sodium bicarbonate (NaHCO3) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) • Sodium chloride (NaCl) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) Magnesium sulfate (MgSO4) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) • Magnesium powder (Fujifilm Wako Pure Chemical Industries, Ltd., special grade) • [1,3-Bis(diphenylphosphino)propane]dichloronickel(II)(Ni(dppp)C) l2 (SIGMA-ALDRICH) Iodine (I2) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) • Hydrogen chloride (HCl) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) Magnesium sulfate (Mg) (Anhydrous) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) • Methanol (Kanto Chemical Co., Ltd.) • Ethyl acetate (Kanto Chemical Co., Ltd.) • Hydrobromic acid (HBr) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade) • Acetic anhydride (Fujifilm Wako Pure Chemical Industries, Ltd., special grade) n-butyllithium, 2.6Mn-hexane solution (Kanto Chemical Co., Ltd.) • N,N-dimethylformamide (DMF) (Fujifilm Wako Pure Chemical Industries, Ltd., dehydrating solvent for synthesis) • N-bromosuccinimide (NBS) (Fujifilm Wako Pure Chemical Industries, Ltd., 98%) • Diethyl ether (Kanto Chemical Co., Ltd., purity >99.5%) Chloroform (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade (Purity: 99%)) Isopropylmagnesium chloride-lithium chloride complex (iPrMgCl·LiCl) (1.3M THF solution) (SIGMA-ALDRICH) • Hydrochloric acid (dilute hydrochloric acid) (Kanto Chemical Co., Ltd., Grade 1) • Dimethyl sulfoxide (DMSO) (Fujifilm Wako Pure Chemical Industries, Ltd., Special Grade (Purity: 99%)) Trimethylphosphine (P(Me)3) (SIGMA-ALDRICH, 1.0M in THF) Triethylphosphine (P(CH2CH3)3) (SIGMA-ALDRICH, 1.0M in THF) Triphenylphosphine (P(Ph)3) (SIGMA-ALDRICH)

[0093] (Manufacture of PBBT) First, BBMB was synthesized. p-methoxyphenol, K2CO3, 18-crown-6, 1,4-dibromobutane, and acetone were placed in a three-necked round-bottom flask and heated under reflux at 56°C for 48 hours under an N2 atmosphere to obtain a turbid solution. The precipitated white precipitate was removed by gravity filtration, and the acetone was removed by evaporator to obtain a turbid solution. The obtained solution was dissolved in n-hexane, and then extracted three times each with saturated NaHCO3 aqueous solution, purified water, and saturated NaCl aqueous solution using a separatory funnel to obtain a colorless, transparent solution. The obtained solution was dried overnight over MgSO4, and after removing MgSO4 by gravity filtration, n-hexane was removed by evaporation. Unreacted 1,4-dibromobutane was removed by vacuum distillation at 80°C, and the solution was purified by recrystallization using methanol. The initial charge, yield, and yield are shown in Table 1.

[0094] [Table 1]

[0095] MPBT was synthesized. In a glove box under an Ar atmosphere, Mg, THF, and I2 were placed in a 100 mL round-bottom flask and stirred at room temperature for 1 hour. BBMB was dissolved in THF in a sample vial and gradually added to the 100 mL round-bottom flask, stirring for 1 hour to obtain a clear gray solution. The supernatant of the obtained solution was transferred to a 100 mL two-neck round-bottom flask. Furthermore, Ni(dppp)Cl2, 3-bromothiophene, and THF were added to a 100 mL three-neck round-bottom flask. This was removed from the glove box while connected to a condenser, and the supernatant solution from the 100 mL two-neck round-bottom flask was added dropwise to the 100 mL three-neck round-bottom flask while cooling with ice. After heating and stirring at 60°C for 15 hours, the solution was quenched by adding it dropwise to 1 M HCl, and the resulting solution was extracted 3 times with ethyl acetate, 8 times with purified water, and 3 times with saturated NaCl aqueous solution to obtain a brown solution. The obtained solution was dried overnight over MgSO4, and after removing MgSO4 by natural filtration, ethyl acetate was removed by distillation using an evaporator. The resulting MPBT was purified by recrystallization using methanol for Lot. 190802 and by column chromatography (Wakogel C-300) using a mixed solvent of n-hexane and ethyl acetate (v / v=9:1) for Lot. 191030 (Rf=0.40). The charge, yield, and yield are shown in Table 2.

[0096] [Table 2]

[0097] BBT synthesis (Method 1) was carried out. In an ice bath, HBr and acetic anhydride were added to a 300 mL three-necked round-bottom flask. MPBT was then added at room temperature, and reflux was carried out at 100°C for 24 hours under an N2 atmosphere. After that, the reaction was stopped by pouring the reaction solution into purified water, and after extraction with n-hexane, the reaction solution was neutralized with saturated NaHCO3 aqueous solution. After washing with purified water and saturated NaCl aqueous solution, the mixture was dried overnight over MgSO4. MgSO4 was removed by gravity filtration, and the solvent was removed by evaporation using an evaporator. The resulting yellowish-brown transparent liquid was purified by column chromatography (Wakogel: C-300, FC-40, developing solvent: n-hexane) (Rf = 0.25). The charge, yield, and yield are shown in Table 3.

[0098] [Table 3]

[0099] BBT synthesis (Method 2) was carried out. 3-bromothiophene was weighed into a three-necked round-bottom flask in a glove box under an Ar atmosphere, and n-hexane was added. Then, the flask was removed from the glove box, and n-butyllithium was added dropwise to the reaction solution, and the mixture was stirred at -40°C for 10 minutes. 10 mL of THF was then added dropwise, and the mixture was stirred at the same temperature for 1 hour. After stirring for 1 hour, 1,4-dibromobutane was added dropwise at 10°C, and the mixture was stirred at room temperature for 2 hours. After eluten on n-hexane, the mixture was quenched with methanol and dilute hydrochloric acid. This was extracted with ethyl acetate, washed with purified water and saturated NaCl aqueous solution, and dried overnight over MgSO4. MgSO4 was removed by gravity filtration, and the solvent was removed by evaporation. The resulting yellow transparent liquid was purified by column chromatography (Wakogel: FC-40) using n-hexane as the solvent (Rf=0.25). The initial charge, yield, and yield are shown in Table 4.

[0100] [Table 4]

[0101] DBrBBT was synthesized. BBT (obtained by Method 2) was weighed into a 50 mL three-necked round-bottom flask, and 3 mL of DMF was added. NBS was dissolved in 3 mL of DMF in a 9 mL sample vial. The NBS solution was added dropwise from a syringe to the reaction solution in the 50 mL three-necked round-bottom flask while the mixture was stirred under an N2 atmosphere in an ice bath for 17 hours. The reaction was stopped by pouring the clear yellow reaction solution into purified water. Extraction was performed with n-hexane, washed with purified water and saturated NaCl aqueous solution, and then dried overnight over MgSO4. The resulting yellow liquid was purified by column chromatography (Wakogel: FC-40, developing solvent: n-hexane) (Rf = 0.50). The initial charge, yield, and yield are shown in Table 5.

[0102] [Table 5]

[0103] DBrBBT was synthesized. BBT was weighed into a 50 mL three-necked round-bottom flask and DMF was added. Under an N2 atmosphere and in an ice bath, NBS DMF solution was added dropwise from a syringe over 30 minutes. The mixture was then stirred at room temperature for 18 hours. After the reaction was complete, the resulting yellow, transparent reaction solution was poured into purified water to stop the reaction. Extraction was performed with n-hexane, washed with purified water and saturated NaCl aqueous solution, and then dried overnight over MgSO4. The resulting yellow liquid was purified by column chromatography (Wakogel: FC-40, developing solvent: n-hexane) (Rf = 0.50). The initial charge, yield, and yield are shown in Table 6.

[0104] [Table 6]

[0105] PBBT was synthesized. In a glove box under an Ar atmosphere, DBrBBT (obtained by Method 2) was dissolved in THF in a 50 mL round-bottom flask, and iPrMgCl·LiCl solution was added dropwise from a syringe, followed by stirring for 2 hours. In addition, a catalyst solution was prepared by adding 10 mL of THF to Ni(dppp)Cl2 in a 30 mL round-bottom flask. The prepared Ni(dppp)Cl2 solution was added from a syringe and stirred at room temperature for 3 hours. After the reaction was complete, the mixture was quenched by developing it in a mixed solution of 300 mL of methanol and 5 mL of dilute hydrochloric acid, and the crude organism was recovered by suction filtration. This was re-precipitation with n-hexane and then suction filtration was performed to obtain a reddish-purple solid. The amount charged, yield, and yield are shown in Table 7.

[0106] [Table 7]

[0107] (Identification of PBBT) Gel permeation chromatography (GPC) measurement A 1 mg / mL THF solution of the polymer was prepared using HPLC-grade THF and filtered using Sunprep (Millipore Millex®-FG (hydrophobic), 0.2 μm). Measurements were performed using a high-speed GPC instrument, HLC-8320GPC(TOSOH). Measurement conditions Column: TOSOH TSKgel SuperMultiporeHZ-M Detector: RI detector RID-10A (TOSOH) Oven temperature: 40℃ Flow rate: 1.0mL / min

[0108] 1 H NMR measurement A 10 g / L sample solution was prepared by dissolving 10 mg of the sample in 1 mL of chloroform-d1. The solution was measured using AVANCE NEO (BRUKER). Measurement conditions Resonance frequency: 400MHz Total number of times: 16 Measurement temperature: 25℃

[0109] The obtained PBBT was a reddish-purple solid, with yields of 31% in Lot. 191204 and 24% in Lot. 201207. The identification results are shown below. Table 8 shows the number-average molar mass (Mn), weight-average molar mass (Mw), molar mass dispersion (Mw / Mn), and degree of polymerization n, obtained from GPC using THF as the solvent. 1 The assignment of each peak in the 1H NMR spectrum is shown in Figure 1. The integration ratios were consistent with the proton ratios determined from the structural formula. The head-to-tail ratios determined from the β-protons were 77% for Lot. 191204 and 80% for Lot. 191204. These measurement results confirmed the synthesis and purification of PBBT.

[0110] [Table 8]

[0111] (Manufacturing Example 1: PBT Manufacturing) PBT was synthesized. The synthesis procedure is as follows: PBBT (Lot. 191204) was placed in a 20 mL three-neck round-bottom flask, and 2.0 mL of THF was added to dissolve it. After adding 3.0 mL of DMF, 1.0 mL of P(Me)3THF solution was added via syringe, and the mixture was heated and stirred at 80°C under an N2 atmosphere for 24 hours using an aluminum block. 3.0 mL of DMSO was then added, and the mixture was heated and stirred at the same temperature for another 24 hours. The resulting reaction solution was re-precipitated in 150 mL x 2 of diethyl ether, filtered using a membrane filter, and a purple solid was obtained. This was dissolved in a small amount of methanol and collected by casting onto a Teflon® sheet. The initial charge, yield, and yield are shown in Table 9.

[0112] [Table 9]

[0113] (Manufacturing Example 2: Manufacturing and Identification of PTH) PTH was produced. First, PTH was produced in the same manner as in Production Example 1, except that 1,6-dibromohexane was used instead of 1,4-dibromobutane when producing BBMB. The obtained PTH was identified in the same manner as described above in the item (Identification of PBBT). 1 The assignment of each peak in the 1H NMR spectrum is shown in Figure 2. The integration ratios matched the proton ratios determined from the structural formula. These measurement results confirmed the synthesis and purification of PTH.

[0114] (Manufacturing Example 3: Manufacturing of Et-PBT) Et-PBT was synthesized. The synthesis procedure is shown below. The procedure was the same as in Production Example 1, except that 1.3 mL of P(CH2CH3)3THF solution was used instead of 1.0 mL of P(Me)3THF solution, and Et-PBT was recovered. The input amount, yield, and yield are shown in Table 10.

[0115] [Table 10]

[0116] (Manufacturing Example 4: Manufacturing of Ph-PBT) Ph-PBT was synthesized. The synthesis procedure is shown below. The synthesis was carried out in the same manner as in Production Example 1, except that a 50 mL three-neck round-bottom flask was used instead of a 20 mL three-neck round-bottom flask, PBBT was dissolved in 3.5 mL of THF instead of 2.0 mL of THF, P(Me)3 pre-dissolved in 0.5 mL of THF in a sample bottle was used instead of 1.0 mL of P(Me)3THF solution, and heating and stirring was performed for 48 hours instead of 24 hours. Ph-PBT was recovered. The amount charged, yield, and yield are shown in Table 11.

[0117] [Table 11]

[0118] Table 12 shows the physical properties of the PTB, Et-PTB, Ph-PTB, and PTH manufactured as described above.

[0119] [Table 12]

[0120] <Reagents and other materials used in the experiment> The reagents used in the experiment and their purification methods are described.

[0121] Tris-HCl buffer Tris-HCl buffer was prepared as follows: A 0.05 M Tris-HCl buffer solution with a pH of 7.40 was prepared by dissolving 0.97 g of Trisma® base (Sigma-Aldrich, >99.9%) and 6.61 g of Trisma® hydrochloride (Sigma-Aldrich, >99.0%) in 1.0 L of purified water.

[0122] LB medium The LB liquid medium was prepared as follows: 10g of Tryptone USP (Solabia Biokar Diagnostics), 5g of Bacto® Yeast Extract (Thermo Fisher Scientific), and 10g of sodium chloride (Fujifilm Wako Pure Chemical Industries, special grade, >99.5%) were dissolved in 1.0L of purified water and transferred to a bottle. The mixture was sterilized using an autoclave BS-305 (TOMY) (120°C, 20 minutes).

[0123] LB agar medium was prepared as follows: 15 g of agar (Fujifilm Wako Pure Chemical Industries, reagent grade) was added to 1 L of LB liquid medium, and the agar was dissolved by heating and stirring at 60°C. The mixture was sterilized using an autoclave (120°C, 20 minutes), and 20 mL was poured into petri dishes. The mixture was left to stand overnight in an IC402 (Yamato) incubator (37°C).

[0124] • Bacterial suspension The concentrations of various bacterial suspensions were adjusted as follows. This study used Gram-positive bacteria such as Staphylococcus aureus IAM1011 (S. aureus) and Bacillus subtilis var. natto (B. subtilis var. natto), and Gram-negative bacteria such as Escherichia coli K12W3110 (E. coli) and Pseudomonas aeruginosa ATCC27853 (P. aeruginosa). Glycerol stocks of the bacteria were scraped with a platinum loop, spread onto agar plates, and incubated statically in an incubator (37°C) for 18 hours. Colonies were taken with a toothpick and spread onto new agar plates, which were used as master plates. Colonies from the master plates were taken with a toothpick, transferred to 3 mL of liquid medium in a 15 mL tube, and incubated overnight with shaking using a constant temperature shaking incubator BR-42FL (TAITEC) (37°C, 180 rpm). Optical density (OD) at 600 nm was measured. 600 The concentration was adjusted by measuring the ) using a UV-Vis spectrophotometer UV-1200V (SHIMADZU) and diluting the suspension with liquid culture medium. The following indicators were used to adjust the concentration of the suspension.

[0125] • S. aureus: OD 600 =1.0, CFU=4.5×10 8 cells / mL ·B. subtilis:OD 600 =1.0, CFU=1.0×10 9 cells / mL · E. coli:OD 600 =1.0, CFU=1.0×10 9 cells / mL ·P. aeruginosa:OD 600 =1.0, CFU=3.7×10 8 cells / mL

[0126] <Various measurements>

[0127] (UV-visible absorption measurement) A disposable cell with a path length of 1.0 cm (BrandTech) was used, and a quartz cell with a path length of 1.0 cm was used for other applications. Measurements were taken using V-770 (JASCO) under the following conditions.

[0128] Measurement mode: absorbance Scanning speed: Medium Slit width: 2.0 nm

[0129] (Fluorescence measurement) A disposable cell with a path length of 1.0 cm (BrandTech) was used, and a quartz cell with a path length of 1.0 cm was used for other applications. Measurements were taken using an F-7100 (HITACHI) under the following conditions.

[0130] Excitation wavelength: Maximum absorption wavelength Fluorescence onset wavelength: Excitation wavelength + 5 nm Fluorescence termination wavelength: 800nm Scanning speed: 240 nm / min Photomal voltage: 400V

[0131] (X-ray diffraction (XRD) measurement) Measurements were taken using SmartLab (Rigaku) ​​under the following conditions.

[0132] Light source: CuK alpha, 45kV, 200mA Divergence slit: 5° Scattering slit: 5° Light-receiving slit: 20mm Scanning range: 1.5° < θ < 40° Scanning speed: 4° / min

[0133] (Dynamic Light Scattering (DLS) measurement) A disposable cell (BrandTech) with a path length of 1.0 cm was used for particle size measurement, and a flow cell was used for zeta potential measurement. The measurements were performed using an ELSZ-2000ZS (Otsuka Electronics) under the following conditions.

[0134] Particle size measurement Number of measurements: 25 Measurement temperature: 25℃ Measurement solvent: water

[0135] Zeta potential measurement Number of measurements: 25 Measurement temperature: 25℃ Measurement solvent: water

[0136] (Optical density (OD) measurement) A disposable cell with a path length of 1.0 cm was used. Measurements were taken using UV-1200V (SHIMADZU). The OD (Oxygen Demand) of the 96-well plate was measured using the MTP-300 (CORONA) device.

[0137] (Scanning transmission electron microscope (STEM) observation) A 200-mesh carbon-supported film (NisshinEM) was used. Measurements were taken using a SU-8000 (HITACHI) under the following conditions.

[0138] Acceleration voltage: 10.0kV Working distance (WD): 8.0 mm Probe current: high

[0139] (Fluorescence microscope observation) Measurements were taken using the following filter set with an Axiovert200M (Zeiss). 43HE DsRED(489043-9901-000)λex=550nm,λem=570nm

[0140] (Confocal laser microscopy observation) Measurements were taken using the following filter set with an LSM700 (Zeiss). Filter Set 43(000000-1114-101)λex=545nm, λem=570nm

[0141] <Test Examples 1-4> Bacterial Recognition of PTB <Test Example 1: Inhibitory effect of PTB on the growth of Gram-positive bacteria> The inhibitory effect of PTB on Gram-positive bacterial growth was investigated by contacting four bacterial species: S. aureus, E. coli, B. subtilis, and P. aeruginosa. 5 mL of liquid culture medium was placed in a 15 mL tube, and the initial concentration of each bacterial suspension was set to 1.0 × 10⁻⁶. 4The cells / mL was prepared. 100 μL of PTB Tris-HCl buffer (1.0 g / L) was added to each bacterial suspension (i.e., the number of bacteria per 0.1 mg of polythiophene compound was 1.0 × 10⁶). 4 PBT was added to form cells. The same process was followed below.) Then, the cells were cultured with shaking (37°C, 180 rpm, 24 hours) and OD 600 The time evolution of [the substance] was measured. The results are shown in Figure 3.

[0142] As shown in Figure 3, all bacteria grew similarly in the medium without PTB. However, in the medium with PTB added, different growth curves were obtained depending on the type of bacteria. In the medium with PTB added to the Gram-negative bacteria E. coli or P. aeruginosa, OD 600 The amount increased and showed similar results to the system without PTB. On the other hand, in the medium to which PTB was added to Gram-positive bacteria S. aureus or B. subtilis, OD 600 No increase was observed. This suggests that PTB specifically inhibited the growth of S. aureus and B. subtilis. Therefore, it is suggested that PTB exhibits a specific growth inhibitory effect on Gram-positive bacteria, that cell wall structure is an important factor in PTB's bacterial recognition, and that it is possible to distinguish between Gram-positive and Gram-negative bacteria.

[0143] <Test Example 2: Dependence of the initial concentration of the S. aureus suspension on the inhibitory effect of PT compounds on the growth of Gram-positive bacteria> The inhibitory effect of PTB on Gram-positive bacterial growth was investigated by culturing S. aureus suspensions at different initial concentrations after contacting S. aureus with PTB. Only S. aureus was used as the bacteria, and the initial concentration was 1.0 × 10⁶. 4 In addition to being cells / mL, 1.0 × 10 6 cells / mL, and 1.0 × 10 8 Except for using a S. aureus suspension of cells / mL, the procedure was the same as in Test Example 1, OD 600The time evolution was measured. After 24 hours, UV-vis absorption, fluorescence measurement, and fluorescence microscopy observation were performed. The culture medium components were removed from the bacterial suspension by centrifugation (3000 rpm, 5 minutes), and the particle size and zeta potential of the sample dispersed in Tris-HCl buffer were measured. OD 600 The measurement results are shown in Figure 4, the UV-vis absorption and fluorescence spectrum measurements in Figure 5, the particle size and zeta potential measurements in Figure 6, and the fluorescence microscope observation results in Figure 7.

[0144] Figure 4 shows that the inhibitory effect of PTB on the growth of Gram-positive bacteria differs depending on the initial concentration of the bacterial suspension. In Figure 4, S4 / PTB showed OD 600 The value was always approximately 0. On the other hand, in S6 / PTB and S8 / PTB, the OD of S. aureus in the system without PTB was 600 The value was smaller than that, and although the proliferation of S. aureus was suppressed, OD 600 It increased.

[0145] In the case of S4 / PTB, the solution remained clear after culturing, and as shown in Figure 5(a), absorption originating from PTB (absorbance around 0.1) with a maximum at 410 nm was observed. In the case of S6 / PTB and S8 / PTB, the absorbance increased (absorbance around 0.2 and 0.4, respectively), and turbidity was observed after culturing. As shown in Figure 5(b), the fluorescence intensity at 485 nm was 64 for S4 / PTB, but decreased to 34 and 12 for S6 / PTB and S8 / PTB. Differences in the growth rate of S. aureus were observed depending on the initial concentration of the bacterial suspension, and PTB quenching occurred accordingly. It was suggested that the inhibitory effect on the growth of Gram-positive bacteria can be adjusted by conditions such as the initial concentration of the bacterial suspension, the ratio of the initial concentration of the bacterial suspension to the added PT compound, and the structure of the PT compound.

[0146] As shown in Figure 6(a), the particle size distribution is narrow when only S. aureus is present, with a maximum value of 10. 3 The size is on the order of nm, indicating that the size of S. aureus is approximately 1 μm. S8 / PTB also has a maximum value of 10 3 It is on the order of nm, approximately 10 3There are many aggregates of nm size, and it is thought that 1 to several S. aureus units are clustered together. In S6 / PTB, there are approximately 10 3 nm and approximately 10 5 It takes a maximum value at nm, and a part of S. aureus is 10 5 It appears that aggregates of approximately nanometer size are being formed. 5 ~10 6 The range is between nm, and most are 10 5 It appears that the bacteria exist as large aggregates larger than nanometers. Furthermore, zeta potential measurements in Figure 6(b) revealed that there are differences in the surface charge of the bacteria depending on the concentration. A larger absolute value of the zeta potential indicates stronger repulsion between particles and higher dispersion stability. In the case of S. aureus alone, the surface of S. aureus was negatively charged. The zeta potential of S8 / PTB was close to that of S. aureus alone, while the zeta potentials of S6 / PTB and S4 / PTB were closer to equipotential values.

[0147] As shown in Figure 7, S. aureus aggregated in S4 / PTB and S6 / PTB, and large aggregates of about 100 μm were observed. On the other hand, in S8 / PTB, although most S. aureus was dispersed, aggregates were observed in some areas. Dispersed bacteria are referred to as the "free state" in this paper. Since almost no free S. aureus was observed around the aggregates in S4 / PTB, it is thought that S. aureus that grew in this system formed large aggregates due to the action of PTB. These observations reflect the particle size distribution and zeta potential measurements shown in Figure 6.

[0148] <Test Example 3: Aggregation process of S. aureus> In Test Example 3, S. aureus culture solutions were collected at 1 hour, 6 hours, and 24 hours after the start of shaking culture from systems with initial S. aureus suspension concentrations of S4 / PTB and S8 / PTB, and the aggregation process was observed using a fluorescence microscope. The results of the S4 / PTB system are shown in Figure 8, and the results of the S8 / PTB system are shown in Figure 9.

[0149] As shown in Figure 8, in S4 / PTB, it was observed that the size of the aggregates gradually increased over time.

[0150] From the above results, the following relationship between S. aureus growth and surface charge change can be inferred in S4 / PTB. The surface of S. aureus is negatively charged due to phosphate groups. In the case of S4 / PTB, at the start of culture, the number of PTB molecules is relatively large relative to the number of bacteria, so it is thought that positively charged PTB surrounds the bacterial surface. As shown in Test Example 5, PTB does not show antibacterial activity against S. aureus, and the bacteria grow even if PTB is adsorbed on the bacterial surface. As growth progresses, the number of PTB molecules relative to the number of bacteria decreases, so the phosphate groups on the bacterial surface are exposed. At this time, since both positive and negative charges are present on the surface of S. aureus, the zeta potential shows a value close to equipotential, -6.9mV. Because it has both positive and negative charges, it is thought that an attractive force is generated between it and neighboring bacteria due to electrostatic interactions, and aggregates are formed.

[0151] As shown in Figure 9, in S8 / PTB, the number of S. aureus increased with cultivation, and most S. aureus existed in a free state, but some S. aureus were observed to form aggregates.

[0152] Based on the above results, the following relationship between S. aureus proliferation and surface charge change can be inferred in S8 / PTB. As described above, the surface of S. aureus is negatively charged due to phosphate groups. In the case of S8 / PTB, the number of PTB molecules was relatively small relative to the number of bacteria, and the zeta potential after 24 hours of culture was smaller than in the case of S4 / PTB. This indicates that there are few positively charged PTB molecules on the bacterial surface, and negative charges are dominant. It is thought that a repulsive force acts between neighboring bacteria, causing them to disperse without agglutinating.

[0153] <Test Example 4: Isolation of Bacteria Using PTB> The selectivity of Gram-positive bacteria by adding PTB to a mixture of S. aureus and E. coli and culturing it was evaluated. Instead of preparing separate bacterial suspensions for each of the four bacterial species including S. aureus, the initial concentrations of S. aureus and E. coli in the sample were 1.0 × 10⁶ each. 4 A mixed sample containing cells / mL was prepared, and S. aureus and E. coli were cultured with shaking in the same manner as in Test Example 1, except that a mixed sample of S. aureus and E. coli was used. After 24 hours, the supernatant and precipitate were separated by centrifugation (1000 rpm, 5 minutes) using a centrifuge LC-121 (TOMY). Slides of the mixed sample before centrifugation, supernatant, and precipitate were prepared and observed under a fluorescence microscope. The results are shown in Figure 10.

[0154] Figure 10(a) shows that in the mixed sample before centrifugation, aggregated cocci were observed among a large amount of dispersed bacilli. Furthermore, the aggregated areas emitted fluorescence, indicating the presence of PTB at these locations. Since S. aureus is a cocci and E. coli is a bacilli, it is thought that S. aureus aggregated due to PTB, while E. coli proliferated without agglutination. Figure 10(b) shows that dispersed E. coli (bacilli) were observed in the supernatant after centrifugation. Figure 10(c) shows that aggregated S. aureus (cocci) were observed in the precipitate after centrifugation. From the above, it is suggested that, according to the present invention, it is possible to selectively isolate Gram-positive bacteria from a mixed sample of Gram-positive and Gram-negative bacteria by using a PT compound.

[0155] <Test Examples 5-6> Influence of Phosphoniumized Polythiophene Structure on Gram-Positive Bacterial Growth Inhibitory Effect To investigate the effect of the structure of PT compounds on the growth effect of Gram-positive bacteria, four PT compounds with different side chain structures (PTB, Et-PTB, Ph-PTB, and PTH) were used.

[0156] <Test Example 5: Antibacterial activity of phosphoniated polythiophene> The antibacterial activity of PT compounds against B. subtilis and E. coli was investigated using disk diffusion. Approximately 1 × 10⁻⁶8 200 μL of a bacterial suspension of B. subtilis or E. coli at cells / mL was uniformly applied to an LB agar medium using a cotton swab. Four disks (filter paper with a diameter of 6 mm) were placed on the medium at equal intervals, and 2 μL of a Tris-HCl buffer solution (1.0 g / L) of the PT compound was dropped onto the disks. It was left standing in an incubator IC402 (Yamato) at 37 °C for 24 hours. The results are shown in Fig. 11.

[0157] From Fig. 11, no inhibition zones were observed on the LB agar medium for both B. subtilis and E. coli. From Test Examples 1 to 4, it is considered that the PT compound has an effect of inhibiting the growth of bacteria, but has no antibacterial property and no effect of killing bacteria.

[0158] <Test Example 6: Behavior of Phosphoniumated Polythiophene against S. aureus> (Test Example 6-1) The growth inhibitory effect of the PT compound against S. aureus was compared. As the PT compound, in addition to PTB, Et-PTB, Ph-PTB, and PTH were each used. In the same manner as in Test Example 1, except for this, S. aureus was cultured with shaking, and the time change of OD 600 was measured. The results are shown in Fig. 12.

[0159] (Test Example 6-2) To further compare the strength of the growth inhibitory effect, the PT compound was added to bacterial suspensions of S. aureus with different initial concentrations, and the time change of OD 600 was measured. The total volume of the liquid contained in each well of a 96-well plate was set to 150 μL. Using a liquid medium, bacterial suspensions with initial concentrations of 1.0×10 4 cells / mL, 1.0×10 6 cells / mL, and 1.0×10 8 cells / mL were prepared, and 5 μL of a Tris-HCl buffer solution (1.0 g / L) of the PT compound was added to each of them. The plate was statically cultured in an incubator at 37 °C, and OD 600 was measured 48 hours after the start of culture. The results are shown in Fig. 13.

[0160] (Results of Test Example 6-1) From Figure 12, in all cases where the PT compound was added, the OD was lower than in the case without the PT compound (Blank), indicating that the growth of S. aureus was suppressed. Also, from the order of the OD values being lower for PTB≈Et-PTB, Ph-PTB, and PTH, it was suggested that there was a difference in the strength of the growth inhibitory effect against S. aureus depending on the structure of the PT compound. 600 が低い値をとり、S.aureusの増殖が抑制されていた。また、PTB≒Et-PTB、Ph-PTB、PTHの順にOD 600 の値が低かったことから、PT化合物の構造によってS.aureusに対する増殖抑制効果の強さに差があることが示唆された。

[0161] (Results of Test Example 6-2) From Figure 13, when comparing the strength of the growth inhibitory effect of the four PT compounds against S. aureus, it was found that from the strongest, it was Et-PTB>PTB>Ph-PTB>PTH. The growth inhibitory effect against S. aureus depends on the structure of the PT compound. From the comparison between PTB and PTH, it was suggested that the shorter the side chain, the stronger the growth inhibitory effect, and from the comparison between PTB, Et-PTB, and Ph-PTB, the smaller the substituent of the phosphonium group, the stronger the growth inhibitory effect.

[0162] <Test Examples 7-8> Behavior of Polythiophene Compounds Against E. coli <Test Example 7: Growth Inhibitory Effect of Polythiophene Compounds Against E. coli To compare the growth inhibitory effect of PT compounds against E. coli, PT compounds (PTB, Et-PTB, Ph-PTB, and PTH) were added to bacterial suspensions of E. coli with different initial concentrations, and the time change of OD 600 was measured. Except for using E. coli instead of S. aureus, in the same manner as in Test Example 6-2, E. coli was statically cultured, and the OD 600 was measured 48 hours after the start of culture. The results are shown in Figure 14.

[0163] From Figure 14, when PTB, Ph-PTB, or PTH was added, in all initial concentrations of the bacterial suspension, similar to the case without the PT compound (Blank), the OD 600The amount increased. These PT compounds were shown to have no growth inhibitory effect on E. coli. On the other hand, when Et-PTB was added, compared to Blank, OD increased. 600 The low levels of Et-PTB demonstrated that it has an inhibitory effect on the growth of E. coli. Furthermore, no difference in the growth inhibitory effect of Et-PTB was observed depending on the initial concentration of the bacterial suspension.

[0164] <Test Example 8: Fibrosis of E. coli by Et-PTB> Fluorescence microscopy observations (results shown in Figure 15) and confocal laser microscopy observations (results shown in Figure 16) were performed on E. coli, E. coli / PTB, and E. coli / Et-PTB 24 hours after the start of culture in Test Example 7. For E. coli / Et-PTB, fluorescence microscopy observations were also performed at 1h, 3h, 6h, and 24h after the start of culture (results shown in Figure 17).

[0165] Figures 15(a) and (b) show that while some fluorescence originating from PTB was observed in the PTB-added area, E. coli proliferated and dispersed normally, similar to the case without PT compound addition (Blank). On the other hand, Figure 15(c) shows that the addition of Et-PTB induced fibrillation in E. coli. Figure 14 shows the OD of E. coli / Et-PTB. 600 The reason for the low value is thought to be that E. coli does not disperse due to fibrosis. As seen in Figure 16, when the sample from Figure 15(c) was observed at a higher magnification, it was observed that the entire fibrous E. coli did not appear red, but rather that Et-PTB was present locally. This suggests that Et-PTB adsorbs to specific sites on the bacterial surface of E. coli.

[0166] Figure 17 shows that E. coli, which are of normal size at the start of culture, elongate as the culture time progresses. At 3 hours from the start of culture, a small number of fibrous E. coli were observed, but most were in a free state. After 6 hours from the start of culture, a large amount of E. coli fibrosis was observed. Furthermore, in Test Example 3, which was cultured under the same conditions as this test except for the bacterial species, S4 / PTB aggregation was observed at 1 hour from the start of culture, indicating that there is a time difference between E. coli fibrosis and S. aureus aggregation formation. From this, it is suggested that, in the present invention, even when using Et-PTB, by adjusting the culture time, it is possible to effectively determine the presence or absence of Gram-positive or Gram-negative bacteria and isolate Gram-positive bacteria.

Claims

1. The following formula (1): 【Chemistry 1】 (In the formula, X represents a divalent hydrocarbon group having 2 to 10 carbon atoms. R １ , R ２ , and R ３ Each of these independently represents a monovalent hydrocarbon group having 1 to 6 carbon atoms. n represents an integer from 5 to 20. The method involves contacting a polythiophene compound containing repeating units represented by () with Gram-positive bacteria to cause agglutination of the Gram-positive bacteria. A method for agglutinating Gram-positive bacteria.

2. X represents an alkylene group with 2 to 10 carbon atoms. R １ , R ２ , and R ３ Each independently represents an alkyl group or phenyl group having 1 to 6 carbon atoms. The method according to claim 1, wherein n represents an integer between 10 and 15.

3. The method according to claim 1 or 2, wherein the Gram-positive bacteria are one or more species selected from the group consisting of Staphylococcus and Bacillus bacteria.

4. The number of Gram-positive bacteria per 0.1 mg of polythiophene compound (number of Gram-positive bacteria / 0.1 mg of polythiophene compound) is 1.0 × 10⁻⁶. ２ ~1.0 x 10 ９ The method according to claim 1 or 2, wherein the cells are cells.

5. The method according to claim 1 or 2, wherein the growth of Gram-positive bacteria is suppressed by agglutination of Gram-positive bacteria.

6. Methods for isolating Gram-positive bacteria, including the following: (I) The following formula (1): 【Chemistry 2】 (In the formula, X represents a divalent hydrocarbon group having 2 to 10 carbon atoms. R １ , R ２ , and R ３ each independently represents a monovalent hydrocarbon group having 1 to 6 carbon atoms. n represents an integer from 5 to 20.) A polythiophene compound containing repeating units represented by ( ) is mixed with a Gram-positive bacteria-containing sample to generate agglutinations of Gram-positive bacteria in the sample; (II) Centrifuging the sample in which aggregates of Gram-positive bacteria have been formed to generate a precipitate of Gram-positive bacteria in the sample; and (III) Collect the precipitate of Gram-positive bacteria generated in the sample.

7. The method according to claim 6, wherein the sample containing Gram-positive bacteria is a mixed sample of Gram-positive and Gram-negative bacteria.

8. Gram-positive bacteria are one or more species selected from the group consisting of Staphylococcus and Bacillus species. The method according to claim 7, wherein the Gram-negative bacteria are one or more species selected from the group consisting of bacteria of the genus Escherichia and bacteria of the genus Pseudomonas.

9. The method according to any one of claims 6 to 8, wherein, in (I), a polythiophene compound is brought into contact with Gram-positive bacteria for 5 hours or less to produce agglutinations of Gram-positive bacteria.

10. The following formula (2): 【Transformation 3】 (In the formula, X represents a divalent hydrocarbon group having 2 to 10 carbon atoms. n represents an integer from 5 to 20.) The method involves contacting Gram-negative bacteria with a polythiophene compound containing a repeating sequence represented by ( ), thereby causing the Gram-negative bacteria to become fibrillated. Methods for inducing fibrosis in Gram-negative bacteria.

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