Crossidiomyces difficile Bacterium Growth Inhibitor

Abstract

Provided is a Clostridiodes difficile growth inhibitor. A Clostridiodes difficile growth inhibitor containing killed Enterococcus faecalis. Also provided are a parenteral pharmaceutical preparation, a bactericide, a disinfectant, an antibacterial agent, a sanitizer, or a detergent for inhibiting growth of Clostridiodes difficile.

Description

Technical Field

[0001] The present invention relates to an inhibitor for inhibiting the growth of Clostridioides difficile bacteria.

Background Art

[0002] Clostridioides (Clostridium) difficile infection (CDI) often occurs due to the disruption of the gastrointestinal flora caused by the use of antibacterial drugs. Most cases involve the use of antibacterial drugs, and furthermore, factors such as aging and the presence of underlying diseases are risk factors.

[0003] Most cases of CDI are gastrointestinal infections, and the symptoms are often diarrhea and abdominal pain, but it may also be accompanied by fever and leukocytosis. In severe cases, it can lead to death due to toxic megacolon, ileus, and gastrointestinal perforation.

[0004] "Carriers" who carry Clostridioides difficile in the gastrointestinal tract but do not show symptoms are recognized. Such "carriers" are often found in hospitalized patients who frequently use antibacterial drugs. Clostridioides difficile is excreted together with the feces of CDI patients and asymptomatic carriers, contaminating the environment including the fingers of medical staff, posing a problem as a healthcare-associated infection. Therefore, it is a frequently observed infectious disease in medical settings where many elderly patients who require excretory care such as diaper changes are hospitalized and infection control including appropriate use of antibacterial drugs is not sufficiently implemented. However, in medical institutions where antibacterial drugs are misused or infection control is inadequate, due to the low level of interest and knowledge regarding CDI, this disease is often not appropriately diagnosed, and the incidence rate appears low on the surface, which is a problem. Thus, in medical institutions and elderly care facilities, CDI is in an environment where it is likely to become chronic, and the need to take infection prevention measures has been debated.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide an agent for inhibiting the growth of Clostridioides difficile.

Means for Solving the Problems

[0007] As a result of intensive efforts, the present inventors have found that heat-killed Enterococcus faecalis can inhibit the growth of Clostridioides difficile, and have thus completed the present invention.

[0008] The gist of the present invention is as follows. (1) An agent for inhibiting the growth of Clostridioides difficile, containing heat-killed Enterococcus faecalis. (2) A parenteral pharmaceutical preparation for inhibiting the growth of Clostridioides difficile, containing heat-killed Enterococcus faecalis. (3) A bactericide, disinfectant, antibacterial agent, germicide or cleaning agent for inhibiting the growth of Clostridioides difficile, containing heat-killed Enterococcus faecalis. (4) A topical pharmaceutical product, containing heat-killed Enterococcus faecalis. (5) A sanitary product, containing heat-killed Enterococcus faecalis.

Effects of the Invention

[0009] According to the present invention, the growth of Clostridioides difficile can be inhibited. This specification incorporates the contents described in the specification and / or drawings of Japanese Patent Application No. 2020-43740, which is the basis of the priority of the present application.

Brief Description of the Drawings

[0010]

Figure 1

Modes for Carrying Out the Invention

[0011] Hereinafter, embodiments of the present invention will be described in more detail.

[0012] The present invention provides a Clostridioides difficile growth inhibitor containing heat-killed Enterococcus faecalis. The present invention also provides a method for inhibiting the growth of Clostridioides difficile using heat-killed Enterococcus faecalis. Further, the present invention provides a medicament for use in a method for inhibiting the growth of Clostridioides difficile containing heat-killed Enterococcus faecalis.

[0013] Enterococcus faecalis is known as a lactic acid coccus having biological response modifier (BRM) activity (Journal of Pharmacy 112:919-925 1992; Journal of Pharmacy 113:396-399 1992; Veterinary Clinical Medicine 3:11-20 1994). Enterococcus faecalis EF-2001 strain can be obtained from Nippon Bell Co., Ltd. (2-14-3, Nagata-cho, Chiyoda-ku, Tokyo).

[0014] Enterococcus Faecalis-2001 strain can be collected from the feces of normal humans and has the following properties.

[0015] It is a Gram-positive coccus. Colony morphology (tryptose soya agar medium, cultured for 24 hours): diameter 1.0 mm, smooth, round, white colony. Bacterial morphology: spherical to oval (1.0×1.5 μm), often forms chains in liquid medium. Non-spore-forming. Facultatively anaerobic. Ferments glucose to produce lactic acid (final pH 4.3). Non-gas-producing. Catalase negative. Grows at 10 - 45°C (optimum 37°C). Grows in pH 9.6, 6.5% NaCl, 40% bile. Positive for 0.04% potassium tellurite. Positive for 0.01% tetrazolium. Positive for 0.1% methylene blue milk. Hydrolyzes arginine. Ferments amygdalin, cellobiose, fructose, galactose, glucose, glycerol, lactose, maltose, mannose, mannitol, ribose, salicin, sucrose, melibiose, sorbitol to produce acid. Resistant to 60°C for 30 minutes. Digests casein and gelatin. Decarboxylates tyrosine to tyramine. Lancefield antigen group D. GC% 35.0±1.0% Enterococcus faecalis is often a dead bacterium, and the bacterial cells may be subjected to treatments such as disruption treatment (homogenization treatment, enzyme treatment, ultrasonic treatment, etc.), heating, drying (lyophilization, spray drying, etc.). Heating treatment can turn live bacteria into dead bacteria. The dead bacteria of Enterococcus faecalis can be expected to have an intestinal immune activation effect. The particle size of the bacterial cells is preferably 0.05 μm to 50 μm, more preferably 0.08 - 20 μm, and even more preferably 0.1 - 10 μm. After mixing the bacterial cells with a diluent, a paste can be added to make them granular. The diluent and the paste are preferably selected from materials permitted for addition to foods and pharmaceuticals.

[0016] The heat-killed Enterococcus faecalis acts to suppress the growth of Clostridioides difficile. By suppressing the growth of Clostridioides difficile, Clostridioides difficile infections can be prevented and / or treated. The heat-killed Enterococcus faecalis can be used in medicine as an active ingredient of a parenteral preparation. Further, the heat-killed Enterococcus faecalis can be used in pharmaceuticals and quasi-drugs as an active ingredient of a bactericide. The heat-killed Enterococcus faecalis can be used for sterilizing medical devices and for external use (on the skin of humans and animals) as an active ingredient of a disinfectant. The heat-killed Enterococcus faecalis can be used in sanitary products as an active ingredient of an antibacterial agent or a disinfectant. Furthermore, by using the heat-killed Enterococcus faecalis for cleaning the body, instruments, equipment, facilities, etc., the growth of Clostridioides difficile can be suppressed. Therefore, the present invention provides a bactericide, disinfectant, antibacterial agent, disinfectant or cleaning agent for suppressing the growth of Clostridioides difficile, which contains the heat-killed Enterococcus faecalis. The present invention also provides an external medicine containing the heat-killed Enterococcus faecalis. Furthermore, the present invention provides a sanitary product containing the heat-killed Enterococcus faecalis.

[0017] In the present invention, in order to perform sterilization, disinfection, disinfection or cleaning using the heat-killed Enterococcus faecalis, the heat-killed Enterococcus faecalis may be applied to the surface (skin, mucosa, etc.) of the body of humans and non-human animals. Alternatively, the heat-killed Enterococcus faecalis may be applied to the surface of instruments, equipment, facilities, etc. that can be contacted by the body of humans and non-human animals, or may be sprayed or dispersed in the space where humans and non-human animals breathe.

[0018] A bactericide, disinfectant, antibacterial agent, germicide or detergent containing heat-killed Enterococcus faecalis may be formulated into dosage forms such as sprays (aerosols, pump sprays), patches (aqueous poultices, oily plasters, etc.), ointments, creams, gels, solid external preparations, liquid external preparations (liniments, lotions, etc.) and used as external pharmaceuticals (external pharmaceutical compositions), quasi-drugs or hygiene products. As the base of the preparation, oily components such as hydrocarbons (petrolatum, liquid paraffin, etc.), higher fatty acids and their esters (adipic acid, myristic acid, palmitic acid, stearic acid, their esters, etc.), waxes (beeswax, lanolin, etc.), higher alcohols (cetanol, stearyl alcohol, etc.), water, polyhydric alcohols (glycerin, 1,3-propanediol, propylene glycol, etc.), aqueous components such as lower alcohols (ethanol, isopropanol, etc.), surfactants (glyceryl stearate, sorbitan fatty acid esters, polyoxyethylene alcohol ethers, carboxylates, sulfate esters, etc.), preservatives (parahydroxybenzoic acid esters, etc.), antioxidants (sodium bisulfite, ascorbic acid, etc.), pH adjusters (citric acid hydrate, lactic acid, acetic acid, etc.) may be used.

[0019] The content of heat-killed Enterococcus faecalis in the preparation varies depending on the type of the preparation, but is usually 0.001 to 100% by mass, preferably 0.01 to 100% by mass.

[0020] The amount of dead Enterococcus faecalis used only needs to be an amount that can confirm the growth inhibitory effect on Clostridioides difficile, and it varies depending on the dosage form, application site, age, weight, presence or absence and type of underlying diseases of the patient, etc. For example, in the case of adults, the dosage per application, when converted to the amount of dead Enterococcus faecalis, is at a concentration of about 0.01 to 500 mg / mL, preferably about 0.05 to 300 mg / mL, more preferably about 0.1 to 100 mg / mL on the skin or nasal and oral mucosa, and it may be used once to several times a day (for example, about 2, 3, 4, 5 times). When applying dead Enterococcus faecalis to something other than humans and non-human animals (for example, applying it to the surfaces and spaces of instruments, equipment, facilities, etc.), it can be used in the same amount as described above.

[0021] Dead Enterococcus faecalis may be added to gauze, absorbent cotton, alcohol cotton, cotton swabs, bandages, masks, gloves, band-aids, surgical tapes, cleaning supplies (towels, sheets, etc.), wet tissue cloths, moist towelettes, soaps, excretory coagulants, etc.

[0022] The parenteral pharmaceutical preparation containing dead Enterococcus faecalis can be formulated into dosage forms such as external preparations [spray agents (aerosol agents, pump spray agents), patches (aqueous type cataplasms, oily type plasters, etc.), ointments, creams, gels, external solid preparations, external liquid preparations (liniments, lotions, etc.)], injections, infusions, ophthalmic agents (eye drops, eye washes, eye ointments, etc.), suppositories, etc., and can be used as pharmaceuticals. As the base of external preparations (such as sprays), hydrocarbon compounds (such as petrolatum and liquid paraffin), higher fatty acids and their esters (such as adipic acid, myristic acid, palmitic acid, stearic acid, and their esters), waxes (such as beeswax and lanolin), higher alcohols (such as cetanol and stearyl alcohol), etc., oily components, water, polyhydric alcohols (such as glycerin, 1,3 - propanediol, propylene glycol, etc.), lower alcohols (such as ethanol and isopropanol), etc., aqueous components, surfactants (such as glycerin stearate, sorbitan fatty acid esters, polyoxyethylene alcohol ethers, carboxylates, sulfate esters, etc.), preservatives (such as paraoxybenzoic acid esters, etc.), antioxidants (such as sodium bisulfite and ascorbic acid), pH adjusters (such as citric acid hydrate, lactic acid, acetic acid, etc.), etc. may be used. The external preparation is preferably applied to the skin, nasal cavity or oral mucosa. Injections and infusions can be formulated using solvents [aqueous solvents (such as distilled water, physiological saline, Ringer's solution, etc.), non - aqueous solvents (such as ethanol, propylene glycol, vegetable oil, etc.)], solubilizing agents (such as glutamic acid, aspartic acid, polysorbate 80, etc.), preservatives (such as benzalkonium chloride and benzethonium chloride, etc.), stabilizers (such as sulfites and sodium pyrosulfite, etc.), emulsifiers and suspending agents (such as lecithin and aluminum monostearate, etc.), buffers (acids, alkalis, citrate salts, etc.), coloring agents, etc. The preparation is preferably sterilized in the final step of production or manufactured by aseptic operation methods. Also, a sterile freeze - dried product can be manufactured and dissolved in sterile distilled water for injection or other solvents before use. Injections and infusions are applied into the body through the skin or through the skin or mucosa. Application through the nasal cavity may also be possible. Administration methods include intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, and intranasal administration. The eye drops can be formulated using a solvent [aqueous solvent (sterile purified water, physiological saline, etc.), non-aqueous solvent (ethanol, propylene glycol, vegetable oil, etc.)], buffer solution, isotonic agent, preservative (methyl paraben, ethyl paraben, benzalkonium chloride, benzethonium chloride, etc.), thickening agent (methyl cellulose, chondroitin sulfate, etc.), suspending agent (nonionic surfactant, etc.), coloring agent, etc. The eye wash can be prepared using physiological saline solution, boric acid solution, benzethonium chloride, buffer solution, etc. The eye ointment can be formulated using paraffin, liquid paraffin, etc. as a base, and adding a preservative, stabilizer, etc. as necessary. The suppository can be formulated using a base [oily base (cocoa butter, palm oil, palm kernel oil, etc.), water-soluble base (glycerogelatin, macrogol, etc.)], surfactant (lecithin, cholesterol, etc.), etc.

[0023] The content of the heat-killed Enterococcus faecalis in the pharmaceutical preparation varies depending on the type of preparation, but is usually 0.001 to 100% by mass, preferably 0.01 to 100% by mass.

[0024] The dosage of the heat-killed Enterococcus faecalis may be an amount that can confirm the growth inhibitory effect on Clostridioides difficile, and varies depending on the dosage form, application site, age, weight, presence or absence and type of underlying diseases of the patient, etc. For example, in the case of adults, the dosage per administration, in terms of the amount of heat-killed Enterococcus faecalis, is about 0.01 to 500 mg / mL, preferably about 0.05 to 300 mg / mL, more preferably about 0.1 to 100 mg / mL, and it is advisable to administer once to several times a day (for example, about 2, 3, 4, 5 times).

[0025] In addition, by coating or kneading the antibacterial agent of the present invention onto products, antibacterial towels, antibacterial plastic tableware products, antibacterial toys, antibacterial PC-related products, antibacterial stationery, etc. can also be manufactured.

Example

[0026] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. [Example 1] Confirmation test of the activity of lactic acid bacteria EF-2001 strain against Clostridioides difficile Abstract The activity of lactic acid bacteria against Clostridioides difficile was investigated. A low concentration (1.63 mg / mL) or high concentration (163 mg / mL) solution of lactic acid bacteria was added at a ratio of 0.65 mL per 1 mL of inoculum, and Clostridioides difficile was cultured anaerobically under the conditions of lactic acid bacteria concentrations of 0.64 mg / mL or 64 mg / mL. The viable cell count was measured at the start of culture and after 12, 24, and 48 hours of culture. At both low (0.64 mg / mL) and high (64 mg / mL) concentrations of lactic acid bacteria, a significant decrease in the viable cell count of C. difficile was observed compared to the control group at 12, 24, and 48 hours after incubation. This suggests that lactic acid bacteria have an inhibitory effect on the growth of C. difficile. Materials and Methods 1. Test Substance 1.1. Test Substance Name: Lactic acid bacteria powder EF-2001 (Nippon Belm Co., Ltd.) (heat-sterilized E. faecalis, diameter 500 nm = 0.5 μm) Properties: Yellowish brown powder Storage conditions: Room temperature (18.0 to 28.0°C), dark and moisture-proof Storage location: Storage room in the test substance storage room of the test facility 2. Administered specimens 2.1. Preparation of test substances Weighed the required amount of lactic acid bacteria powder EF-2001 (electronic balance: XP205DR, METTLER TOLEDO Co., Ltd.), and prepared it to be 500 mg / mL with GAM broth (refer to 15.3.4). Diluted this 500 mg / mL solution with GAM broth to 163 mg / mL and 1.63 mg / mL. Since the lactic acid bacteria powder precipitated, it was stirred well and suspended. Prepared it immediately before use. 3. Pathogenic microorganisms 3.1. Strains used Clostridioides (Clostridium) difficile (ATCC43255, hereinafter C. difficile) 3.2. Storage conditions Frozen and stored in an ultra-low temperature freezer (set temperature: -80°C, MDF-394AT, Sanyo Electric Co., Ltd.) until use. 3.3. Reagents (1) GAM agar medium (Nissui Pharmaceutical Co., Ltd.) (2) GAM broth medium (Nissui Pharmaceutical Co., Ltd.) (3) Physiological saline (Otsuka Pharmaceutical Factory, Inc.) 3.4. Preparation of GAM broth Weighed 29.5 g of GAM broth powder, suspended it in 500 mL of injection water (Otsuka Pharmaceutical Factory, Inc.), and then treated it with an autoclave (LSX-500, Tomy Seiko Co., Ltd.) (115°C, 15 minutes). After preparation, it was stored under refrigerated conditions. 3.5. Pre-culture Thawed the preserved strain of C. difficile, inoculated it on GAM agar medium, placed it in an anaerobic jar filled with a deoxidizer, and cultured it in a thermostat (ILE800, Yamato Scientific Co., Ltd.) set at 37°C for 5 days. After culturing, picked colonies, added them to GAM broth medium, placed it in an anaerobic jar filled with a deoxidizer, and cultured it in a thermostat set at 37°C for 2 days. The cultured solution after culturing was used as the inoculum stock solution. 3.6. Preparation of inoculum solution and viable cell count measurement The inoculum stock solution diluted 10-fold with GAM broth was used as the inoculum solution. For the viable cell count, a portion of the inoculum solution was collected, appropriately diluted with physiological saline, spread on GAM agar medium, placed in an anaerobic jar filled with a deoxidizer, and cultured in an incubator set at 37°C for 5 days. The number of colonies after culture was measured using a handy colony counter (CC-1, AS ONE Corporation), and the viable cell count per 1 mL of the inoculum solution was calculated. As a result, the concentration of the inoculum solution was 2.8 × 105 CFU / mL. The remaining inoculum solution after use was autoclaved (LSX-500, TOMY SEIKO CO., LTD.) (121°C, 15 minutes) and then discarded. 4. Test method 4.1. Growth inhibition test 4.1.1. Test group composition TIFF0007705844000001.tif31143 The indicated concentration is the final concentration after mixing. *: GAM broth was added. 4.1.2. Test method To 1 mL of the inoculum solution in a test tube, the prepared test samples (1.63 mg / mL solution and 163 mg / mL solution) were added at a ratio of 0.65 mL each, placed in an anaerobic jar filled with a deoxidizer, and cultured in an incubator set at 37°C. At the start of culture and after 12, 24, and 48 hours of culture, the test tubes were taken out respectively. A portion of the culture solution was collected, appropriately diluted, and the undiluted and diluted culture solutions were spread on CCFA medium (Nippon Becton Dickinson KK), and then anaerobically cultured in an incubator set at 37°C for 5 days. The number of colonies after culture was measured using a handy colony counter, and the viable cell count was calculated. As a control, GAM broth was added instead of the test sample. The number of samples was 5. 5. Summary of results The mean value and standard error of the viable cell count were calculated. For the significance test of the viable cell count, the Wilcoxon rank sum test was used for the control group vs the test sample group. The hazard ratio was considered significant at 5%, and was presented separately as less than 5% and less than 1%. For the statistical analysis, a commercially available statistical program (SAS system: SAS Institute Japan) was used. Test Results The test results are shown in Table 1, Appendix 1-1 to 1-3, and Figure 1. The viable cell count of Clostridioides difficile in the control group was 360.0 ± 75.9 (×10 3 CFU / mL) at the start of culture, 3680.0 ± 239.6 (×10 3 CFU / mL) after 12 hours of culture, 3880.0 ± 475.8 (×10 3 CFU / mL) after 24 hours of culture, and 33020.0 ± 2267.5 (×10 3 CFU / mL) after 48 hours of culture. The viable cell count of Clostridioides difficile in the low-concentration group was 306.0 ± 22.7 (×10 3 CFU / mL) at the start of culture, 33.2 ± 4.9 (×10 3 CFU / mL) after 12 hours of culture, 27.1 ± 6.7 (×10 3 CFU / mL) after 24 hours of culture, and 15.7 ± 4.9 (×10 3 CFU / mL) after 48 hours of culture. A significant decrease in the viable cell count was observed at 12 hours, 24 hours, and 48 hours after culture compared with the control group. The viable cell count of Clostridioides difficile in the high-concentration group was 300.0 ± 22.1 (×10 3 CFU / mL) at the start of culture, 12.0 ± 1.1 (×10 3 CFU / mL) after 12 hours of culture, 8.4 ± 3.4 (×10 3 CFU / mL) after 24 hours of culture, and 5.7 ± 3.2 (×10 3 CFU / mL) after 48 hours of culture. A significant decrease in the viable cell count was observed at 12 hours, 24 hours, and 48 hours after culture compared with the control group. Discussion The activity of lactic acid bacteria against Clostridioides difficile was investigated by measuring the viable cell count over time. At both low (0.64 mg / mL) and high (64 mg / mL) concentrations of lactic acid bacteria, a significant decrease in the viable cell count of C. difficile was observed compared to the control group at 12, 24, and 48 hours after incubation. This suggests that lactic acid bacteria have an inhibitory effect on the growth of C. difficile.

[0027] (Table 1) JPEG0007705844000002.jpg49170

[0028] (Appendix 1-1~1-3) JPEG0007705844000003.jpg220153

[0029] [Product example 1] Disinfectant liquid, antibacterial spray, disinfectant wipes A disinfectant solution was prepared according to the following recipe. (Prescription) Lactic acid bacteria powder EF-2001 (Nippon Belm Co., Ltd.) (heat-sterilized E. faecalis, diameter 500 nm = 0.5 μm) 0.6 mass% Glycerin 0.05% by mass Capric acid monoglyceride 0.50% by mass Ethanol 50% by weight Distilled water remaining Total amount 100% by mass The above solution was filled into a commercially available pump-type spray container to produce an antibacterial spray, and the above solution was soaked into gauze or tissue to produce a disinfecting sheet. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Industrial Applicability]

[0030] The present invention can be used for the prevention and / or treatment of infectious diseases caused by Clostridioides difficile, sterilization, disinfection, decontamination, cleaning, etc.

Claims

1. A parenteral pharmaceutical preparation for inhibiting the growth of Clostridioides difficile bacteria, containing heat-killed bacteria of Enterococcus faecalis EF-2001 strain.

2. A bactericide, disinfectant, antiseptic or cleaning agent for inhibiting the growth of Clostridioides difficile bacteria, containing heat-killed bacteria of Enterococcus faecalis EF-2001 strain.

3. A topical pharmaceutical for inhibiting the growth of Clostridioides difficile bacteria, containing heat-killed bacteria of Enterococcus faecalis EF-2001 strain.

4. A sanitary product for inhibiting the growth of Clostridioides difficile bacteria, containing heat-killed bacteria of Enterococcus faecalis EF-2001 strain.

5. An antibacterial product for inhibiting the growth of Clostridioides difficile bacteria, coated with or kneaded with an antibacterial agent containing heat-killed bacteria of Enterococcus faecalis EF-2001 strain.

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