Sterilization method and composition used in sterilization method

Abstract

This method for sterilizing bacteria contained in a biofilm includes: readying a composition containing an organic compound having a standard redox potential (pH7) of -0.7 V to -0.2 V, and water; bringing the composition, and the biofilm and a conductor, into contact with each other; and applying, to the conductor, a potential that exceeds the lower limit of the potential window and that is equal to or below -0.4 V based on a silver / silver chloride electrode.

Description

Sterilization method and composition used in the sterilization method

[0001] The present invention relates to a sterilization method and a composition for use in the sterilization method.

[0002] Currently, biofilms produced by bacterial communities are a global problem in the management of infectious diseases in healthcare settings.

[0003] Chemical methods using antibiotics and the like are widely known for killing bacteria. However, the emergence of drug-resistant bacteria due to the inappropriate use of antibiotics is a problem. Therefore, sterilization techniques other than chemical methods have been researched for many years. For example, Non-Patent Documents 1 to 4 describe the application of a high potential (for example, about 7 V) to a conductor (electrical conductor) such as stainless steel or titanium to produce a sterilization product called H ２ A method for removing biofilms formed on conductor surfaces by the generation of ions and negative-negative surface repulsion has been reported.

[0004] Current Opinion in Solid State and Materials Science, Vol. 25, Issue 4, August 2021, 100926.Applied Sciences, 2022, Vol. 12, Issue 13, 6320.Bioelectrochemistry 121 (2018) p.84-94.Colloids and Surfaces B: Biointerfaces, Vol. 117, 1, May 2014, p.152-157.

[0005] However, although the techniques disclosed in Non-Patent Documents 1 to 4 do not pose the problem of drug-resistant bacteria, they require sterilization under harsh conditions that cause electrolysis of solvents, etc. Sterilization under such harsh conditions requires a lot of energy consumption, making it costly, and there is also the risk of side reactions generating active chemical species that have adverse effects on the human body.

[0006] Therefore, an objective of the present invention is to provide a method for sterilizing bacteria contained in biofilms that can be performed more efficiently under milder conditions and that reduces adverse effects on the human body, and a composition to be used in said sterilization method.

[0007] As a result of extensive research into achieving the above object, the present inventors have found that the above object can be achieved by the following configuration.

[0008] [1] A method for sterilizing bacteria contained in a biofilm, the method comprising: preparing a composition containing water and an organic compound having a standard oxidation-reduction potential (pH 7) of -0.7 V to -0.2 V; bringing the composition into contact with the biofilm and a conductor; and applying a potential to the conductor that exceeds the lower limit of the potential window and is -0.4 V or less vs. a silver / silver chloride electrode. [2] The sterilization method described in [1], wherein the potential applied to the conductor is -1.2 V to -0.8 V. [3] The sterilization method described in [1] or [2], wherein the organic compound is a compound having a dipyridine structure. [4] The sterilization method described in [3], wherein the organic compound is methyl viologen. [5] The sterilization method described in any of [1] to [4], wherein the composition further contains a cation. [6] The sterilization method described in any of [1] to [5], wherein the composition further contains silver ions. [7] The sterilization method described in any of [1] to [6], wherein the biofilm is formed on the conductor. [8] The sterilization method according to any one of [1] to [6], wherein the biofilm is formed on a substrate different from the conductor. [9] The sterilization method according to [8], wherein the substrate is an insulator.

[10] The sterilization method according to [9], wherein the substrate is polytetrafluoroethylene (PTFE).

[11] The sterilization method according to any one of [7] to

[10] , wherein the conductor on which the biofilm is formed or the substrate is a medical device that is implanted in a living body or a medical device that is used beyond or in direct contact with a mucous membrane of a living body.

[12] The sterilization method according to any one of [1] to

[11] , wherein the bacterium is at least one species selected from the group consisting of Klebsiella pneumoniae, Staphylococcus epidermidis, and Pseudomonas aeruginosa.

[13] The sterilization method according to any one of [1] to

[11] , wherein the bacterium is a gram-negative bacillus.

[14] The sterilization method according to

[13] , wherein the gram-negative bacillus is a bacterium belonging to the family Enterobacteriaceae.

[15] A composition used in the sterilization method according to any one of [1] to

[14] , comprising the organic compound having a standard oxidation-reduction potential of -0.7 V to -0.2 V and water.

[16] The composition according to

[15] , wherein the organic compound is a compound having a dipyridine structure.

[17] The composition according to

[16] , wherein the organic compound is methyl viologen.

[18] The composition according to any one of

[15] to

[17] , further comprising a cation.

[19] The composition according to any one of

[15] to

[18] , further comprising a silver ion.

[0009] The present invention provides a method for sterilizing bacteria contained in biofilms, which can be carried out efficiently under mild conditions and which reduces adverse effects on the human body, and a composition to be used in the sterilization method.

[0010] 1 is a flowchart illustrating the sterilization method of the present embodiment. 2 is a schematic diagram illustrating the presumed mechanism of the sterilization method of the present embodiment. 3 is a schematic diagram illustrating the presumed mechanism of the sterilization method of a modified example of the present embodiment. 4 is a time-current curve (CA diagram) obtained in Experiment 1. 5 is a time-current curve (CA diagram) obtained in Experiment 2-1. 6 is a diagram illustrating the evaluation results (number of colonies) of the sterilization effect in Experiments 1, 2-1, and 3-1. 7 is a diagram illustrating the evaluation results (amount of bacteria reduction) of the sterilization effect in Experiments 1 and 2-1. 8 is a diagram illustrating the evaluation results (number of colonies) of the sterilization effect in Experiments 2-1 to 2-4 and Experiments 4-1 to 4-4. 9 is a diagram illustrating the evaluation results (amount of bacteria reduction) of the sterilization effect in Experiments 2-1 to 2-4. 10 is a diagram illustrating the evaluation results (amount of bacteria reduction) of the sterilization effect in Experiments 5-1 to 5-2. 11 is a diagram illustrating the evaluation results (amount of bacteria reduction) of the sterilization effect in Experiments 6-1 to 6-3. 12 is a diagram illustrating the evaluation results (amount of bacteria reduction) of the sterilization effect in Experiments 7-1 to 7-2. 1 is a diagram showing the evaluation results (number of colonies) of the sterilization effect in Experiments 8-1 to 8-9. FIG. 2 is a diagram showing the evaluation results (reduction in bacteria) of the sterilization effect in Experiments 8-1 to 8-8.

[0011] The present invention will be described in detail below. The following description of the constituent elements may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. In other words, "A to B" means A or more and B or less.

[0012] The sterilization method of this embodiment will be described according to the flowchart shown in Figure 1. The sterilization method of this embodiment includes the following steps S1 to S3: Step S1: preparing a composition (hereinafter referred to as "sterilization composition") containing an organic compound (hereinafter referred to as "specific organic compound") having a standard oxidation-reduction potential (pH 7) of -0.7 V to -0.2 V and water, Step S2: bringing the sterilization composition into contact with a biofilm and a conductor, and Step S3: applying a potential within a predetermined range to the conductor.

[0013] 2 is a schematic diagram illustrating the presumed mechanism of the sterilization method of this embodiment. A biofilm 10 containing bacterial cells (bacteria) 1 is attached to a conductor 2. In the process in which the bacterial cells 1 decompose organic compounds to obtain energy (particularly in anaerobic conditions), the NADPH used in the decomposition of the organic compounds is decomposed. ＋ NADH is produced from NADH, and electrons are transferred from NADH to conductor 2, an electron acceptor, via the electron transport chain (arrow 4), and NAD ＋ and is used to obtain energy again.

[0014] The present inventors surprisingly discovered that applying a negative potential to conductor 2 in a composition containing specific organic compound 5 reduces the activity of bacterial cells 1 in a biofilm 10 and ultimately sterilizes them, leading to the present invention. Furthermore, a weak negative potential within the potential window is sufficient for applying to conductor 2. This enables sterilization under mild conditions without electrolysis of the solvent (water). Application of a negative potential to conductor 2 limits the transfer of electrons from bacterial cells 1 to conductor 2 (arrow 4). In particular, application of a potential of −0.4 V or less relative to a silver / silver chloride electrode results in the transfer of electrons from conductor 2 to bacterial cells 1 (arrow 3). The transfer of electrons from conductor 2 to bacterial cells 1 inhibits the cells' energy acquisition, further reducing their activity and ultimately killing (sterilizing) the bacteria. It is speculated that specific organic compound 5 acts as an electron mediator, improving the efficiency of electron transfer between conductor 2 and bacterial cells 1. This allows for smooth electron transfer (arrow 3) from the conductor 2 to the bacterial cells 1, enabling efficient sterilization even with a weak applied potential. This principle of thermodynamically suppressing the metabolism of bacteria 1 is innovative and is expected to have a continuous effect on drug-resistant bacteria lurking in the biofilm 10. Note that the mechanism explained above is speculative and does not affect the scope of the present invention in any way.

[0015] Each step of the sterilization method of this embodiment will be described in detail below. In this specification, "sterilization" includes not only the complete annihilation of bacteria in a biofilm, but also the killing of a portion of the bacteria in the biofilm (i.e., reducing the number of bacteria in the biofilm).

[0016] [Step S1] Step S1: First, a sterilizing composition containing an organic compound (specific organic compound) having a standard oxidation-reduction potential (pH 7) of −0.7 V to −0.2 V and water is prepared.

[0017] The standard oxidation-reduction potential (pH 7) of the specific organic compound is not particularly limited as long as it is in the range of −0.7 V to −0.2 V, but from the viewpoint of obtaining a higher bactericidal effect, it is preferably −0.6 V to −0.3 V, and more preferably −0.6 V to −0.4 V.

[0018] The specific organic compound is not particularly limited as long as it is an organic compound having a standard oxidation-reduction potential within the above range, but for example, compounds containing a nitrogen-containing aromatic heterocycle, especially compounds having a dipyridine structure, are preferred. Specific compounds include the compounds shown in Table 1 and their derivatives. Among them, from the viewpoint of obtaining a higher bactericidal effect, methyl viologen and its derivatives are preferred, and methyl viologen is more preferred. The specific organic compound may be composed of a single compound or two or more compounds.

[0019] The standard oxidation-reduction potential of a specific organic compound can be measured directly by electrochemical measurement using a potential sweep with a three-electrode system, and was also determined by referring to the values ​​described in the following documents 1 to 3. Reference 1: Ryo Nakagawa and Yuta Nishina, “Simulating the redox potentials of unexplored phenazine derivatives as electron mediators for biofuel cells” J. Phys. Energy 3 (2021) 034008 Reference 2: Mary Lou Fultz and Richard A. Durst, “Mediator compounds for the electrochemical study of biological redox systems: a compilation” Analytica Chimica Acta, 140 (1982) 1-18. Elsevier Scientific Publishing Company Reference 3: Octavio Reyes-Salas et al. “Titrimetric and Polarographic Determination of Carminic Acid and its Quantification in Cochineal (Dactylopius coccus) Extracts” J. Mex. Chem. Soc. 2011, 55(2), 89-93

[0020]

[0021] The content of the specific organic compound in the sterilizing composition is not particularly limited, but from the viewpoint of obtaining a higher sterilizing effect, it is, for example, 1 μM (M = mol / L) to 1000 μM, preferably 10 μM to 200 μM.

[0022] The water contained in the sterilizing composition is not particularly limited and may be pure water, distilled water, ion-exchanged water, etc. The content of water in the sterilizing composition is not particularly limited, but is, for example, 90.0% by mass to 99.9% by mass when the entire composition is taken as 100% by mass.

[0023] The sterilizing composition may further contain positive ions (cations). The combined use of a specific organic compound and a cation produces a synergistic effect, further enhancing the sterilizing effect. While the mechanism is unclear, it is speculated as follows: In a composition containing a specific organic compound 5, applying a negative potential to the conductor 2 promotes the transfer of electrons to the bacteria 1 (arrow 3 in Figure 2). If the sterilizing composition contains a cation, the cation flows into the bacterial cell 1. This causes an osmotic abnormality within the bacterial cell 1, reducing the activity of the bacteria 1 and ultimately resulting in sterilization. Furthermore, if the cation has the property of damaging DNA or enzymes within the bacteria 1, the sterilizing effect can be further enhanced. The mechanism described above is speculative and does not affect the scope of the present invention in any way.

[0024] The cation is not particularly limited, and examples thereof include cations containing metals such as metal ions or complex ions, or organic cations generated from drugs, etc. Examples of metals contained in metal ions and metal-containing cations include silver (Ag), copper (Cu), cobalt (Co), aluminum (Al), nickel (Ni), zinc (Zn), molybdenum (Mo), vanadium (V), zirconium (Zr), tungsten (W), palladium (Pd), platinum (Pt), etc. From the viewpoint of further enhancing the bactericidal effect, silver ions (Ag ＋ ), copper ions (Cu ＋ , Cu ２＋ ) is preferred, and silver (Ag ＋) is more preferable. The cation may be a so-called cation derived from a physiological electrolyte. Examples of the cation derived from a physiological electrolyte include sodium ions (Na ＋ ), potassium ions (K ＋ ), calcium ions (Ca ２＋ ), magnesium ions (Mg ２＋ As the positive ions (cations), one type of cation may be used, or two or more types of cations may be used.

[0025] The sterilizing composition may further contain an anion (anion) that serves as a counterion of the cation. The anion is not particularly limited, and examples thereof include halide ions such as fluorine, chlorine, bromine, and iodine. The cation may be an ion derived from a salt consisting of a cation and an anion. Preferred salts include metal halides such as silver chloride and copper chloride. Cations can be introduced into the sterilizing composition by dispersing or dissolving (including partially dissolving) these salts in the sterilizing composition.

[0026] The concentration of the cation in the sterilizing composition is not particularly limited and can be adjusted as appropriate within the range in which the effects of this embodiment are achieved. From the viewpoint of obtaining a higher sterilizing effect, for example, the concentration of the cation in the sterilizing composition is preferably 1 μM to 1 M, more preferably 10 μM to 200 mM. The concentration of the anion in the sterilizing composition is not particularly limited and, like the concentration of the cation, from the viewpoint of obtaining a higher sterilizing effect, is preferably 1 μM to 1 M, more preferably 10 μM to 200 mM.

[0027] The sterilization composition may consist solely of the specific organic compound and water, or may contain other components in addition to the specific organic compound and water as long as the effects of this embodiment are achieved. Examples of other components include the cations and anions described above, as well as electron mediators other than the specific organic compound, electron source compounds, buffers, and coagulants. The electron source compounds are compounds used in bacterial metabolism, and include, but are not limited to, organic compounds (amino acids, sugars, organic acids, etc.). Examples of buffers include, but are not limited to, borates, bicarbonates, Tris-HCl, citrates, phosphates, succinates, phosphates, and acetates. Examples of coagulants include agar, gelatin, and agar, with agar being preferred.

[0028] The sterilizing composition can be prepared by uniformly mixing the specific organic compound, water, and, if necessary, other components by a general-purpose method.

[0029] The sterilization composition may also be prepared by adding a predetermined amount of a specific organic compound to a composition containing a general-purpose medium (liquid medium, solid medium), a buffer solution, physiological saline, etc. (hereinafter also referred to as "medium composition"). Commercially available medium, buffer solution, physiological saline, etc. may be used. As the medium, for example, Lysogeny broth (LB medium) is used. The medium composition contains water and the other components described above. Therefore, the sterilization composition of this embodiment may be a mixture of a specific organic compound and the medium composition. Note that the medium composition does not need to contain a buffer solution or physiological saline. The water content in the sterilization composition takes into account the water contained in the medium composition.

[0030] [Step S2] Step S2: Next, the sterilizing composition is brought into contact with the biofilm and the conductor.

[0031] The bacteria contained in the biofilm, i.e., the bacteria to be sterilized in this embodiment, are not particularly limited and may be, for example, either gram-positive or gram-negative bacteria. For example, the sterilization method of this embodiment is effective against Klebsiella pneumoniae (gram-negative bacteria), Pseudomonas aeruginosa (gram-negative bacteria), and Staphylococcus epidermidis (gram-positive bacteria), which are designated by the U.S. Food and Drug Administration (FDA) as bacteria that have a significant impact on endoscope contamination.

[0032] Furthermore, in the sterilization method of this embodiment, if the bacteria contained in the biofilm are Gram-negative bacilli, particularly Gram-negative bacilli belonging to the family Enterobacteriaceae, electron transfer (arrow 3 in FIG. 2 ) becomes more efficient, resulting in a more excellent sterilization effect. Examples of bacteria belonging to the family Enterobacteriaceae include the aforementioned Klebsiella pneumoniae (Klebsiella spp.), Enterobacter spp., Escherichia spp., Salmonella spp., Serratia spp., Shigella spp., and Yersinia spp.

[0033] A biofilm is a higher-order structure formed by bacteria attached to a solid (substrate) surface, and is covered with, for example, polysaccharides produced by the bacteria. As shown in Figure 2, a biofilm 10 may be formed on a conductor 2 (an example of a substrate).

[0034] The conductor is not particularly limited in material, shape, and size as long as it can transfer electrons to and from the biofilm. Examples of the material include metal materials such as silver, copper, aluminum, nickel, iron, and alloys containing these metals (e.g., stainless steel (SUS)), and carbon materials such as amorphous carbon, graphite, and carbon nanotubes.

[0035] Examples of conductors include medical instruments that are implanted in a living body (such as medical implants), medical instruments that are used beyond the mucous membrane of a living body (such as forceps), and medical instruments that are used in direct contact with the mucous membrane of a living body (such as endoscopes). If such instruments are used as conductors, the sterilization method of this embodiment can also be used as a method for cleaning and washing medical instruments, medical implants, etc.

[0036] The method for contacting the sterilizing composition with the biofilm and conductor is not particularly limited, but for example, when the sterilizing composition is liquid, a method of immersing the biofilm and conductor in the sterilizing composition, or a method of dripping the sterilizing composition onto the biofilm and conductor can be used. Furthermore, when the sterilizing composition is solid (for example, when the sterilizing composition is a solid medium), a method of directly contacting the sterilizing composition with the conductor with or without pressure can be used. When the conductor is a flat plate, direct contact typically means contacting the surface on which the biofilm is formed with the sterilizing composition.

[0037] [Step S3] Step S3: Next, a potential of −0.4 V or less is applied to the conductor, exceeding the lower limit of the potential window, relative to a silver / silver chloride electrode.

[0038] The inventors discovered that, regardless of the type of bacteria, when the sterilizing composition of this embodiment is used, applying a weak potential of −0.4 V or less promotes electron transfer from the conductor 2 to the bacterial cell 1 (arrow 3 in FIG. 2 ), inhibiting the cell's energy acquisition and ultimately sterilizing the bacteria. From the perspective of achieving a higher sterilizing effect, it is preferable to apply a potential of preferably −0.6 V or less, more preferably −0.8 V or less, and even more preferably −0.9 V or less to the conductor. Furthermore, the lower limit of the negative potential applied to the conductor is not particularly limited as long as it exceeds the lower limit of the potential window. The lower limit of the potential window is determined by the components of the sterilizing composition, the pH of the sterilizing composition, the material of the electrode, and other factors, and is clear to those skilled in the art. Applying a potential exceeding the lower limit of the potential window enables sterilization under mild conditions that do not cause electrolysis of the solvent (water). The sterilization method of this embodiment is low-cost due to reduced energy consumption, and also suppresses side reactions that may generate reactive chemical species that are harmful to the human body. Furthermore, if a high potential is applied to cause water electrolysis, reactions such as hydrogen generation may take precedence, potentially suppressing electron transfer from the conductor 2 to the bacterial cells 1 (arrow 3 in Figure 2). In this embodiment, the applied potential is weak, so hydrogen generation does not occur, and the metabolism of the bacteria 1 can be efficiently suppressed and sterilized. The lower limit of the potential applied to the conductor is, for example, -1.4 V or higher, preferably -1.2 V or higher, and more preferably -1.1 V or higher, based on a silver / silver chloride electrode.

[0039] The method of applying a potential to the conductor is not particularly limited. For example, a method may be used in which a working electrode, a reference electrode, and a counter electrode are brought into contact with a sterilization composition, and the working electrode is then brought into contact with the conductor. The electrode set (working electrode, counter electrode, and reference electrode) is connected to a potentiostat, and the potentiostat is controlled to apply a predetermined potential to the working electrode. The materials for the working electrode, counter electrode, and reference electrode are not particularly limited, and known working electrodes, counter electrodes, and reference electrodes for electrochemical measurements can be used. The reference electrode is preferably a silver / silver chloride electrode. Alternatively, instead of preparing a separate working electrode, a conductor on which a biofilm has formed may be used as the working electrode.

[0040] The sterilization composition may be liquid or solid. If it is liquid, the electrode set (working electrode, counter electrode, and reference electrode) may be immersed in the sterilization composition. Alternatively, the sterilization composition may be dripped onto the electrode set manufactured by printed electronics or the like so that it comes into contact with the electrode set. If the sterilization composition is solid, the working electrode, counter electrode, and reference electrode may be directly contacted with the sterilization composition, as with the conductor.

[0041] This step (step S3) may be performed under anaerobic conditions not containing oxygen (for example, a nitrogen atmosphere) or under aerobic conditions containing oxygen (for example, in the atmosphere). Since the atmosphere is not limited to anaerobic conditions, the sterilization method of this embodiment can be performed with simpler equipment.

[0042] The temperature (sterilization temperature) during this step (step S3) is not particularly limited and may be, for example, 0° C. to 100° C. or room temperature. The time for applying the potential (sterilization time) is also not particularly limited and may be adjusted appropriately depending on the type of specific organic compound, the type of bacteria, the type and size of the conductor, etc., and may be, for example, 30 minutes to 24 hours.

[0043] According to the sterilization method of the present embodiment described above, simply applying a weak electric potential to the conductor can reduce the activity of bacteria in a biofilm, ultimately killing (killing) all or part of the bacteria in the biofilm. Therefore, a continuous effect on drug-resistant bacteria lurking in the biofilm can be expected. Furthermore, by using the sterilization method of the present embodiment, the need for antibacterial agents is eliminated or the amount of antibacterial agent used can be reduced, thereby suppressing the emergence of new drug-resistant bacteria.

[0044] Furthermore, when the conductor is a medical instrument or a medical implant, even if mechanical cleaning is difficult due to factors such as a complex shape, the sterilization method of this embodiment can kill bacteria in biofilms, and is expected to be effective in preventing bacterial infection. Furthermore, when the conductor is a medical instrument that is implanted in a living body (e.g., various implants such as dental implants) or a medical instrument that is used beyond or in direct contact with the mucous membrane of a living body (e.g., forceps, endoscopes, etc.), biofilms may form in small areas that cannot be reached by mechanical cleaning. The sterilization method of this embodiment can also kill (sterilize) bacteria in such biofilms.

[0045] <Modification> In the above-described embodiment, as shown in Fig. 2, the biofilm 10 is formed on the conductor 2, but the present invention is not limited to this. For example, as shown in Fig. 3, the biofilm 10 may be formed on a substrate 12 that is different from the conductor 2. The sterilization method described in this modification is similar to the sterilization method described in the above-described embodiment, except that the biofilm 10 is formed on the substrate 12, and achieves similar effects. Description of similar content will be omitted.

[0046] In this modification, the specific organic compound 5 also increases the efficiency of electron transfer (arrow 3 in FIG. 3 ) from the conductor 2 to the bacterial cells 1. Therefore, by applying a negative potential to the conductor 2, it is possible to kill (sterilize) the bacteria 1 in the biofilm 10 on the substrate 12 without applying a potential directly to the substrate 12.

[0047] The material of the substrate 12 is not particularly limited, but since there is no need to apply a potential, an insulating material such as a resin (plastic) can be used. Examples of resins include polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), polycarbonate (PC), polyvinyl chloride (PVC), polypropylene (PP), polyethylene (PE), and polystyrene (PS).

[0048] Examples of the substrate 12 include medical instruments that are implanted in a living body (such as medical implants), medical instruments that are used beyond the mucous membrane of a living body (such as forceps), and medical instruments that are used in direct contact with the mucous membrane of a living body (such as endoscopes). If such materials are used as the substrate 12, the sterilization method of this modified example can also be used as a method for cleaning and washing medical instruments, medical implants, etc.

[0049] In this modification, the conductor 2 may be, for example, the electrode (working electrode) itself.

[0050] The conductor 2 and the substrate 12 may be in contact or not in contact as shown in Figure 3. The shortest distance between the conductor 2 and the substrate 12 may be, for example, 0 m (contact state) to 1 m. If the shortest distance between the conductor 2 and the substrate 12 is within the above range, the bacteria 1 in the biofilm 10 can be efficiently killed (sterilized).

[0051] The sterilization method of this modification is capable of sterilizing biofilms formed on an insulator (substrate 12) such as PTFE, to which an electric potential cannot be applied, and therefore can be applied to the sterilization and cleaning of medical instruments made of an insulator.

[0052] The present invention will be described in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the examples shown below.

[0053] [Experiment 1] In this experiment, a stainless steel (SUS304) tube was used as a conductor, and Klebsiella pneumoniae (KP) in a biofilm formed on the SUS tube was sterilized. Methyl viologen (MV) was used as the specific organic compound.

[0054] (1) Biofilm formation Klebsiella pneumoniae (KP) was added to TSB medium (tryptone soy broth medium), and the cell optical density OD ６００ｎｍ= 1.0. A SUS tube was immersed in this medium and cultured at 37°C for 24 hours. As a result, a biofilm containing Klebsiella pneumoniae (KP) was formed on the surface of the SUS tube.

[0055] (2) Preparation of Sterilizing Composition Methyl viologen (MV) was added to a defined medium (DM) to a concentration of 100 μM and mixed uniformly. The standard oxidation-reduction potential (pH 7) of methyl viologen is −0.440 V, as shown in Table 1.

[0056] (3) Potential Application A three-electrode electrochemical cell was prepared, consisting of a titanium (Ti) wire as the working electrode, a platinum (Pt) wire as the counter electrode, and a silver / silver chloride electrode as the reference electrode. The SUS tube on which the biofilm had formed was electrically connected to the working electrode (titanium wire). MV-containing medium (DM) was poured into the three-electrode electrochemical cell, and the MV-containing medium (DM) was contacted with the electrode set (working electrode, counter electrode, and reference electrode) and the SUS tube on which the biofilm had formed. Chronoamperometry (CA) measurements were performed at room temperature under anaerobic conditions (nitrogen atmosphere) with the working electrode potential set to -1.0 V (vs. Ag / AgCl). A potential was applied to the SUS tube via the working electrode. Figure 4A shows the time-current curve (CA diagram).

[0057] [Experiment 2-1] In this experiment, the same experiment as in Experiment 1 was carried out except that the potential was applied under aerobic conditions (in the atmosphere). Figure 4B shows the time-current curve (CA diagram).

[0058] [Experiment 2-2] to [Experiment 2-4] In Experiments 2-2 to 2-4, Klebsiella pneumoniae (KPt), Staphylococcus epidermidis (SE), and Pseudomonas aeruginosa (PA) were used instead of Klebsiella pneumoniae (KP), respectively, and the experiments were otherwise performed in the same manner as Experiment 2-1. That is, in Experiments 2-2 to 2-4, a potential was applied under aerobic conditions (in the atmosphere) as in Experiment 2-1.

[0059] [Experiment 3-1] In this experiment, methyl viologen (MV) was not used, and no potential was applied. First, a biofilm containing KP was formed on a SUS tube using the same method as in Experiment 1. The SUS tube on which the biofilm had formed was washed three times with phosphate buffered saline (PBS), and then cultured in DM (without MV) at room temperature for 1 hour without applying a potential.

[0060] [Experiment 3-2] to [Experiment 3-4] In Experiments 3-2 to 3-4, Klebsiella pneumoniae (KPt), Staphylococcus epidermidis (SE), and Pseudomonas aeruginosa (PA) were used instead of Klebsiella pneumoniae (KP), respectively, and the other experiments were performed in the same manner as Experiment 3-1. That is, in Experiments 3-2 to 3-4, methyl viologen (MV) was not used and no potential was applied.

[0061] [Experiment 4-1] In this experiment, methyl viologen (MV) was used, but no potential was applied. First, a biofilm containing KP was formed on a SUS tube using the same method as in Experiment 1. The SUS tube on which the biofilm had formed was washed three times with PBS, and then cultured for 1 hour at room temperature in DM containing 100 μM methyl viologen (MV) without applying a potential.

[0062] [Experiment 4-2] to [Experiment 4-4] In Experiments 4-2 to 4-4, Klebsiella pneumoniae (KPt), Staphylococcus epidermidis (SE), and Pseudomonas aeruginosa (PA) were used instead of Klebsiella pneumoniae (KP), respectively, and the other experiments were performed in the same manner as Experiment 4-1. That is, in Experiments 4-2 to 4-4, a specific organic compound (MV) was used, but no potential was applied.

[0063] [Method for evaluating bactericidal effect] In each experiment, the SUS tube was removed from the DM (liquid medium) and washed three times with PBS (pH = 7.2). The washed SUS tube and 1 mL of PBS were placed in a container, and the biofilm was detached from the SUS tube by vigorously shaking it with a vortex shaker (30 seconds), sonicating it (7 minutes), and then vigorously shaking it again (30 seconds). The PBS containing the detached biofilm was diluted 100-fold. Two samples were prepared by spreading 100 μL of the diluted biofilm-containing PBS on a TSB-agar medium plate and culturing them overnight at 37 °C. After culturing, the number of colonies formed in each of the two samples was counted and the average value was calculated. The number of colonies (colony forming units (CFU (Colony Forming Unit) / mL)) for each experiment was calculated from this average value.

[0064] [Evaluation Results] <Effect of Atmosphere on Bactericidal Effect> FIG. 4C shows the results (number of colonies) of Experiment 1 (with MV, with potential application under anaerobic conditions), Experiment 2-1 (with MV, with potential application under aerobic conditions), and Experiment 3-1 (control, without MV, without potential application).

[0065] Compared to Experiment 3-1 (control), the number of bacterial colonies was reduced in Experiments 1 and 2-1. This indicates that applying a potential to a SUS tube in a composition containing MVs can kill bacteria in a biofilm, regardless of the atmosphere during potential application. In particular, Experiment 2-1, in which a potential was applied under aerobic conditions, dramatically reduced the number of bacteria.

[0066] To more clearly compare the results of Experiments 1 and 2-1, Figure 4D shows the bacterial reduction in each experiment. The bacterial reduction is the difference between the colony count in Experiment 3-1 (control) and the colony count in each experiment, as shown in Figure 4C. As shown in Figure 4D, under aerobic conditions (Experiment 2-1), a bactericidal effect of more than 100 times was obtained compared to under anaerobic conditions (Experiment 1).

[0067] <Bactericidal Effect on Biofilms Formed by Various Bacteria> Table 2 shows the bactericidal efficiency in Experiments 2-1 to 2-4 (with MV, with potential application under aerobic conditions), each of which used a different type of bacteria. The bactericidal efficiency is expressed by the following formula, where "X" is the number of colonies in each of Experiments 2-1 to 2-4, and "Y" is the number of colonies in each of Experiments 3-1 to 3-4 (control, without MV, without potential application), which used the same corresponding inoculum: Bactericidal Efficiency (%) = {(Y - X) / Y)} × 100

[0068]

[0069] As shown in Table 2, in all of Experiments 2-1 to 2-4, a high sterilization efficiency of 95% or more was obtained in a short sterilization time of 1 hour, regardless of the type of bacteria.

[0070] 5A shows the results of Experiments 2-1 to 2-4 and the corresponding Experiments 4-1 to 4-4 (control, with MV, no applied potential) using various bacteria. Regardless of the type of bacteria, the number of bacterial colonies was significantly reduced in all of Experiments 2-1 to 2-4 compared to Experiments 4-1 to 4-4 (control). These results confirmed that simply immersing biofilms in a composition containing MVs does not produce a bactericidal effect, but that applying a potential to the SUS tube produces a bactericidal effect.

[0071] To more clearly compare the results of Experiments 2-1 to 2-4, Figure 5B shows the bacterial reduction in each of Experiments 2-1 to 2-4. The bacterial reduction is the difference between the colony counts in Experiments 4-1 to 4-4 (control) shown in Figure 5A and the corresponding colony counts in Experiments 2-1 to 2-4. As shown in Figure 5B, a greater bactericidal effect was obtained against Gram-negative bacteria (KP, KPt, PA) than against Gram-positive bacteria (SE).

[0072] [Experiment 5-1] This experiment was conducted in the same manner as Experiment 2-1, except that an insulating PTFE tube was used instead of a stainless steel tube. Specifically, in this experiment, a biofilm containing Klebsiella pneumoniae (KP) was formed in a PTFE tube, and a potential was applied to a working electrode (titanium wire) in contact with the PTFE tube in a DM (liquid medium) containing methyl viologen (MV) under aerobic conditions (in the atmosphere). In this experiment, the working electrode (titanium wire) itself served as the "conductor" to which the potential was applied.

[0073] [Experiment 5-2] This experiment was conducted in the same manner as Experiment 5-1, except that no potential was applied. That is, in this experiment, a PTFE tube with a biofilm containing Klebsiella pneumoniae (KP) was cultured in DM containing tilviologen (MV) at room temperature for 1 hour without applying a potential.

[0074] [Experiment 5-3] In this experiment, first, a biofilm containing KP was formed on a PTFE tube in the same manner as in Experiment 5-1. No further manipulations were performed in this experiment.

[0075] [Evaluation of bactericidal effect] For Experiments 5-1 and 5-2, the number of colonies (colony forming units (CFU / mL)) for each experiment was determined using the same method as in the above-mentioned "Method for evaluating bactericidal effect." In Experiment 5-3, the PTFE tubes on which the biofilms had formed were removed from the TSB medium, and the number of colonies (colony forming units (CFU / mL)) for each experiment was determined using the same method as in the above-mentioned "Method for evaluating bactericidal effect."

[0076] Figure 6 shows the amount of bacterial reduction in each of Experiments 5-1 and 5-2. The amount of bacterial reduction is the difference between the number of colonies in Experiment 5-3 (control) and the number of colonies in each experiment. As shown in Figure 6, Experiment 5-1 (with MV, with potential application) had a much greater amount of bacterial reduction than Experiment 5-2 (with MV, without potential application). These results confirmed that even when the substrate on which a biofilm is formed is an insulator (PTFE), applying a potential to a conductor in the sterilizing composition can provide a high sterilization effect.

[0077] [Experiment 6-1] This experiment was conducted in the same manner as Experiment 5-1, except that Pseudomonas aeruginosa (PA) was used instead of Klebsiella pneumoniae (KP). That is, in this experiment, Pseudomonas aeruginosa (PA) in a biofilm formed on a PTFE tube was sterilized in DM (liquid medium) containing methyl viologen (MV).

[0078] [Experiment 6-2] In this experiment, DM (liquid medium) containing 20 μM silver chloride together with 100 μM methyl viologen (MV) was used as the sterilizing composition. Otherwise, the experiment was carried out in the same manner as in Experiment 6-1.

[0079] [Experiment 6-3] In this experiment, the sterilization composition used was a liquid medium (DM) containing 20 μM silver chloride (AgCl) instead of methyl viologen (MV). Otherwise, the experiment was carried out in the same manner as in Experiment 6-1.

[0080] [Experiment 6-4] In this experiment, methyl viologen (MV) and silver chloride were not used, and no potential was applied. Otherwise, the experiment was conducted in the same manner as in Experiment 6-1. That is, in this experiment, a PTFE tube with a biofilm containing Pseudomonas aeruginosa (PA) was cultured in DM at room temperature for 1 hour without applying a potential.

[0081] [Experiment 7-1] This experiment was conducted in the same manner as Experiment 5-1, except that Staphylococcus epidermidis (SE) was used instead of Klebsiella pneumoniae (KP). That is, in this experiment, Staphylococcus epidermidis (SE) in a biofilm formed on a PTFE tube was sterilized in DM (liquid medium) containing methyl viologen (MV).

[0082] [Experiment 7-2] In this experiment, DM (liquid medium) containing 20 μM silver chloride together with 100 μM methyl viologen (MV) was used as the sterilizing composition. Otherwise, the experiment was carried out in the same manner as in Experiment 7-1.

[0083] [Experiment 7-3] In this experiment, methyl viologen (MV) and silver chloride were not used, and no potential was applied. Otherwise, the experiment was conducted in the same manner as in Experiment 7-1. That is, in this experiment, a PTFE tube with a biofilm containing Staphylococcus epidermidis (SE) was cultured in DM at room temperature for 1 hour without applying a potential.

[0084] [Evaluation of Bactericidal Effect] For Experiments 6-1 to 6-4 and 7-1 to 7-3, the number of colonies (colony forming units (CFU / mL)) was determined in each experiment using the same method as in the above-mentioned "Method for evaluating bactericidal effect."

[0085] Figure 7A shows the amount of bacterial reduction in each of Experiments 6-1 to 6-3 using Pseudomonas aeruginosa. The amount of bacterial reduction is the difference obtained by subtracting the number of colonies in each experiment from the number of colonies in Experiment 6-4 (control). As can be seen from Figure 7A, the amount of bacterial reduction was greater in both Experiment 6-1 (MV only) and Experiment 6-2 (combined use of MV and AgCl) compared to Experiment 6-3 (AgCl only). Furthermore, the amount of bacterial reduction was even greater in Experiment 6-2 (combined use of MV and AgCl) compared to Experiment 6-1 (MV only). From these results, it was confirmed that the sterilizing composition also has a high sterilizing effect against Pseudomonas aeruginosa (PA), and furthermore, the combination of MV and AgCl in the sterilizing composition ＋ It was confirmed that the combined use of these two compounds produced a synergistic effect, resulting in a higher bactericidal effect.

[0086] Figure 7B shows the amount of bacterial reduction in each of Experiments 7-1 and 7-2 using Staphylococcus epidermidis (SE). The amount of bacterial reduction is the difference obtained by subtracting the number of colonies in each experiment from the number of colonies in Experiment 7-3 (control). As can be seen from the results of Experiment 7-1 shown in Figure 7B, a bactericidal effect was confirmed against Staphylococcus epidermidis (SE) on an insulating substrate (PTFE) (Experiment 7-1), similar to that for Klebsiella pneumoniae (KP) on an insulating substrate (PTFE) (Figure 6, Experiment 5-1) and Pseudomonas aeruginosa (PA) (Figure 7A, Experiment 6-1). Furthermore, Experiment 7-2 (combined use of MV and AgCl) showed a greater amount of bacterial reduction compared to Experiment 7-1 (MV only). This result also demonstrates the effectiveness of MV and AgCl in the sterilizing composition. ＋ It was confirmed that a higher bactericidal effect could be obtained by using them together.

[0087] [Experiment 8-1] In this experiment, the same experiment as in Experiment 2-1 was carried out, except that carminic acid (CAMA) was used instead of methyl viologen (MV). That is, Klebsiella pneumoniae (KP) in a biofilm formed on the surface of a SUS tube was sterilized under aerobic conditions (in the atmosphere). However, the biofilm was prepared by adding Klebsiella pneumoniae (KP) to a TSB medium and measuring the cell optical density (OD ６００ｎｍ = 0.01, and a SUS tube was immersed in the medium and cultured at 37°C for 3 days. The standard redox potential of carminic acid (pH 7) is -0.519 V, as shown in Table 1.

[0088] [Experiment 8-2] This experiment was carried out in the same manner as Experiment 8-1, except that anthraquinone-2-sulfonate (AQS) was used instead of carminic acid (CAMA). The standard redox potential (pH 7) of anthraquinone-2-sulfonate is −0.225 V, as shown in Table 1.

[0089] [Experiment 8-3] This experiment was conducted in the same manner as Experiment 8-1, except that 1,1'-diheptyl-4,4'-bipyridinium (DBPDB) was used instead of carminic acid (CAMA). The standard redox potential (pH 7) of 1,1'-diheptyl-4,4'-bipyridinium is −0.48 V, as shown in Table 1.

[0090] [Experiment 8-4] This experiment was carried out in the same manner as Experiment 8-1, except that benzyl viologen (BV) was used instead of carminic acid (CAMA). Note that the standard redox potential (pH 7) of benzyl viologen is −0.358 V, as shown in Table 1.

[0091] [Experiment 8-5] to [Experiment 8-8] Experiments 8-5 to 8-8 correspond to Experiments 8-1 to 8-4, respectively, and used the same specific organic compounds as in Experiments 8-1 to 8-4, but no potential was applied.

[0092] [Experiment 8-9] In this experiment (control), no specific organic compounds were used, and no potential was applied. A biofilm containing KP was formed on a SUS tube using the same method as in Experiment 8-1. The SUS tube on which the biofilm had formed was then cultured in DM (containing no specific organic compounds) at room temperature for 1 hour without applying a potential.

[0093] [Method for evaluating bactericidal effect] In each experiment, the SUS tube was removed from the DM (liquid medium) and washed three times with PBS (pH = 7.2). The washed SUS tube and 1 mL of PBS were placed in a container, and the biofilm was detached from the SUS tube by vigorously shaking it with a vortex shaker (30 seconds), sonicating it (7 minutes), and then vigorously shaking it again (30 seconds). The PBS containing the detached biofilm was diluted 100-fold. Three samples were prepared by spreading 100 μL of the diluted biofilm-containing PBS on TSB-agar medium plates and culturing them overnight at 37 °C. After culturing, the number of colonies formed in each of the three samples was counted and the average value was calculated. The number of colonies (colony forming units (CFU (Colony Forming Unit) / mL)) for each experiment was calculated from this average value.

[0094] [Evaluation Results] Figure 8A shows the colony counts for Experiments 8-1 to 8-9. Figure 8B shows the bacterial reduction in each of Experiments 8-1 to 8-8. The bacterial reduction is the difference obtained by subtracting the colony count for each experiment from the colony count for Experiment 8-9 (control) shown in Figure 8A. As shown in Figures 8A and 8B, Experiments 8-1 to 8-4 (with specific organic compound and applied potential) showed a greater bacterial reduction than Experiments 8-5 to 8-8 (with specific organic compound and no applied potential), confirming that a higher sterilization effect was obtained.

[0095] The sterilization method of the present invention can be used for washing and cleaning medical instruments and medical implants to prevent infections caused by them.

[0096] 1 Bacterial cell (bacteria) 2 Conductor 5 Specific organic compound 10 Biofilm 12 Substrate

Claims

1. A method for sterilizing bacteria contained in a biofilm, comprising the steps of: A composition is provided that includes an organic compound having a standard oxidation-reduction potential (pH 7) of −0.7 V to −0.2 V and water; contacting the biofilm and a conductor with the composition; applying a potential to the conductor that is greater than the lower limit of the potential window and is equal to or less than −0.4 V versus a silver / silver chloride electrode.

2. The sterilization method according to claim 1, wherein the potential applied to the conductor is −1.2 V to −0.8 V.

3. The sterilization method according to claim 1 , wherein the organic compound is a compound having a dipyridine structure.

4. The sterilization method according to claim 3, wherein the organic compound is methyl viologen.

5. The method of claim 1 , wherein the composition further comprises a cation.

6. The method of claim 1 , wherein the composition further comprises silver ions.

7. The sterilization method according to claim 1 , wherein the biofilm is formed on the conductor.

8. The sterilization method according to claim 1 , wherein the biofilm is formed on a substrate other than the conductor.

9. The sterilization method according to claim 8 , wherein the substrate is an insulator.

10. The sterilization method according to claim 9, wherein the substrate is polytetrafluoroethylene (PTFE).

11. The sterilization method according to claim 8, wherein the conductor or the substrate on which the biofilm is formed is a medical device that is implanted in a living body, or a medical device that is used across or in direct contact with a mucous membrane of a living body.

12. The sterilization method according to claim 1, wherein the bacteria is at least one selected from the group consisting of Klebsiella pneumoniae, Staphylococcus epidermidis, and Pseudomonas aeruginosa.

13. The method for sterilizing according to claim 1, wherein the bacteria is a gram-negative bacillus.

14. The sterilization method according to claim 13, wherein the gram-negative bacillus is a bacterium belonging to the family Enterobacteriaceae.

15. The organic compound has a standard redox potential of −0.7 V to −0.2 V; The composition used in the sterilization method according to any one of claims 1 to 14, comprising the water.

16. The composition according to claim 15 , wherein the organic compound is a compound having a dipyridine structure.

17. 17. The composition of claim 16, wherein the organic compound is methyl viologen.

18. The composition of claim 15 , wherein the composition further comprises a cation.

19. The composition of claim 15, wherein the composition further comprises silver ions.