WO2023287025A1

WO2023287025A1 - Cutibacterium avidum strain, culture medium derived therefrom, and antibacterial use thereof

Info

Publication number: WO2023287025A1
Application number: PCT/KR2022/008319
Authority: WO
Inventors: 이주혁; 오병섭; 강세원; 김지선; 박승환; 이정숙
Original assignee: 한국생명공학연구원
Priority date: 2021-07-12
Filing date: 2022-06-13
Publication date: 2023-01-19

Abstract

The present invention relates to a deposited strain, a lysate thereof, a culture medium, an extract of the culture medium, and antibacterial uses thereof. A strain and a culture medium derived therefrom, according to an aspect of the present invention, can be effectively used in the prevention, amelioration, or treatment of bacterial infections.

Description

Cutibacterium avidum strain, culture solution derived therefrom and antibacterial use thereof

It relates to deposited strains, their lysates, cultures, extracts of cultures, and antibacterial uses thereof.

The microbiome refers to microorganisms existing in a specific environment and their entire genetic information, and refers to the collection of genomes that represent the entire genetic information of a single organism. Therefore, the human microbiome refers to microorganisms living inside and outside the human body and their entire genetic information. The human body lives in a symbiotic relationship with many microorganisms, and in particular, the largest number of microorganisms in the body exists in the intestine as an optimal environment for microorganisms to consume nutrients and form a systematic community. Intestinal microbes supply nutrients that cannot be produced by the host's own enzymes alone, and are closely related to the host's metabolism and immune system, while preventing various diseases such as irritable bowel syndrome, obesity, atopy, depression, rheumatoid arthritis, autism spectrum disorder, and dementia. It has been reported to be associated with In recent years, the imbalance of the intestinal microbiota has deteriorated due to Western eating habits and indiscriminate use of antibiotics, leading to deterioration of intestinal health. there is.

Accordingly, there is a need to develop materials for the improvement, prevention, or treatment of diseases using human intestinal-derived microorganisms.

One aspect is to provide a Cutibacterium avidum strain belonging to the genus Cutibacterium ( Cutibacterium sp. ) deposited under accession number KCTC 14558BP.

Another aspect is to provide a lysate or culture medium derived from the strain.

Another aspect is to provide a pharmaceutical composition for preventing or treating bacterial infection comprising the strain, a lysate of the strain, a culture thereof, or a mixture thereof as an active ingredient.

Another aspect is to provide a health functional food for preventing or improving bacterial infection comprising the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient.

Another aspect is to provide a cosmetic composition comprising the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient.

Another aspect is to provide a method for preventing or treating a bacterial infection or inflammatory disease by administering the strain, a lysate, a culture medium, or a mixture thereof of the strain to a subject in need thereof.

Another aspect is to provide a strain, a lysate of the strain, a culture medium, or a mixture thereof for use in preparing a composition for preventing or treating bacterial infections or inflammatory diseases.

One aspect provides a Cutibacterium avidum strain belonging to the genus Cutibacterium ( Cutibacterium sp. ) deposited under accession number KCTC 14558BP.

In one embodiment, the strain may be a strain containing 16s rRNA of SEQ ID NO: 1.

Another aspect provides a lysate or culture medium derived from the strain.

Another aspect provides a pharmaceutical composition for preventing or treating bacterial infections comprising the strain, a lysate of the strain, a culture thereof, or a mixture thereof as an active ingredient.

In one embodiment, the bacterial infection may be Clostridium difficile infection (CDI).

In one embodiment, inflammatory bowel disease (inflammatory bowel diseases, IBD) comprising the strain, a lysate of the strain, a culture thereof, or a mixture thereof as an active ingredient; irritable bowel syndrome; enteritis; Crohn's disease; Behcet's disease; Or it may be to provide a pharmaceutical composition for preventing or treating ulcerative colitis (ulcerative colitis).

Another aspect provides a health functional food for preventing or improving bacterial infection comprising the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient.

In one embodiment, a health functional food for improving intestinal health by inhibiting Clostridium difficile or inhibiting inflammation, comprising the strain, a lysate, a culture medium, or a mixture thereof of the strain as an active ingredient may be providing

In one embodiment, inflammatory bowel disease (inflammatory bowel diseases, IBD) comprising the strain, a lysate of the strain, a culture thereof, or a mixture thereof as an active ingredient; irritable bowel syndrome; enteritis; Crohn's disease; Behcet's disease; Or it may be to provide a health functional food for preventing or improving ulcerative colitis (ulcerative colitis).

Another aspect provides a cosmetic composition comprising the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient.

Another aspect provides a method for preventing or treating a bacterial infection or inflammatory disease by administering the strain, a lysate of the strain, a culture medium, or a mixture thereof to a subject in need thereof.

Another aspect provides a strain, a lysate of the strain, a culture medium, or a mixture thereof for use in preparing a composition for preventing or treating bacterial infections or inflammatory diseases.

The strain may be derived from a human body, in particular from a human feces sample, but is not limited thereto.

The strain may be a live strain or an attenuated strain (killed strain). As used herein, the term "attenuated" means modified to reduce the pathogenicity of the strain. Attenuation can be done for the purpose of reducing toxicity and other side effects when the strain is administered to a subject. Attenuated strains can be prepared through various methods known in the art. For example, attenuation can be achieved by deleting or disrupting virulence factors that allow the strain to survive in the host cell. The deletion and destruction are well known in the art, and may be performed by methods such as homologous recombination, chemical mutagenesis, irradiation mutagenesis, or transposon mutagenesis.

The strain may have antibacterial activity.

The strain may inhibit the growth of bacteria (eg , C. difficile ). In addition, the strain reduces inflammatory factors induced by C. difficile , for example, pro-inflammatory cytokines (eg, TNF, or CCL2), or increases anti-inflammatory cytokines (IL-10) it could be

Another aspect of the Cutibacterium genus ( Cutibacterium sp .) Belonging to Cutibacterium avidum ( Cutibacterium avidum ) Provides a lysate, a culture medium, an extract of the culture medium, or a mixture thereof.

The strain is as described above.

In this specification, the term "lysate" may mean a product obtained by disrupting the cell wall of the strain itself by chemical or physical force.

As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and may supply nutrients so that Bailonella seminalis can grow and survive in vitro. It may mean the entire medium including the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time in a medium that can be used. In addition, the culture solution may mean a culture solution obtained by removing the cells from the cell culture solution obtained by culturing the strain. On the other hand, the liquid from which the cells are removed from the culture solution is also called "supernatant", and the culture solution is left still for a certain period of time to take only the liquid of the upper layer except for the part sunk in the lower layer, remove the cells through filtration, or centrifuge the culture solution to It can be obtained by removing the precipitate and taking only the upper liquid. The "cell" refers to the strain itself of the present invention, and includes the strain itself isolated and selected from a fecal sample or the strain isolated from the culture medium by culturing the strain. The cells can be obtained by centrifuging the culture solution and taking the part that has settled in the lower layer, or can be obtained by removing the upper liquid with the lower part of the culture medium that has settled for a certain period of time.

The culture solution may include a culture solution itself obtained by culturing the strain, a concentrate thereof, or a lyophilized product or a culture supernatant obtained by removing the strain from the culture solution, a concentrate thereof, or a lyophilisate.

The culture medium is obtained by culturing Cutibacterium avidum in an appropriate medium (eg, Reinforced Clostridial Medium medium) at any temperature above 10 ° C or below 40 ° C for a certain period of time, for example, 4 to 50 hours can

In one embodiment, the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture medium to remove the strain.

In another embodiment, the concentrate may be obtained by concentrating the supernatant obtained after filtering the strain culture medium itself, or the culture medium using a centrifugal separation or filter.

The culture medium and culture conditions for culturing the Cutibacterium avidum can be appropriately selected or modified by those skilled in the art.

As used herein, the term "culture medium extract" refers to an extract obtained from the culture medium or a concentrate thereof, and may include an extract, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a purified product obtained by purifying the same, or a fraction obtained by fractionating the same. there is.

Another aspect provides a disease improvement, prevention or treatment use of a Cutibacterium avidum strain, a lysate derived from the strain, a culture medium, or an extract of the culture medium.

In this specification, the term "treat" may mean that inflammation is cured in a shorter time compared to natural healing. The treatment may include amelioration or alleviation of inflammation or amelioration or alleviation of a bacterial infection. In addition, the treatment may refer to healing or recovery of symptoms caused by inflammation or bacterial infection.

Uses of the strain may include preventing, ameliorating, or treating inflammatory diseases, or preventing, ameliorating, or treating bacterial infections (antibacterial activity), or preventing or improving intestinal health.

Specifically, the inflammatory diseases include inflammatory bowel diseases (IBD); irritable bowel syndrome; Behcet's disease; enteritis, Crohn's disease; ulcerative colitis; It may be any one selected from the group consisting of.

In addition, the improvement of intestinal health may be helpful for proliferation of beneficial bacteria in the intestine and suppression of harmful bacteria, help for intestinal health by regulating immunity, or help for smooth bowel movement.

As used herein, the term "antimicrobial agent" refers to inhibiting, reducing or preventing the growth of bacteria, inhibiting or reducing the ability of bacteria to cause an infection in a subject, or inhibiting or reducing the ability of bacteria to proliferate or remain infectious in the environment Refers to a substance that can be reduced.

Specifically, examples of the bacterial infections may include infections caused by gram-positive bacteria or gram-negative bacteria. Specifically, the bacterial infection is Clostridium , Helicobacter, Helicobactor , Escherichia , Salmonella , Staphylococcus , Streptococcus , Haemophilus ), Klebsiella , Moraxella , Enterobacter , Proteus , Serratia , Pseudomonas , Acinetobacter , Citrobacter ), Stenotrophomonas , Bacteroides , Prevotella , and Fusobacterium . More specifically, the bacterial infection is Clostridium difficile infection (CDI), or Clostridium difficile associated disease (CDAD: Clostridium difficile associated disease), for example, Clostridium difficile associated diarrhea (Clostridium difficile associated disease) diarrhea) may be included.

Another aspect provides a composition for preventing or treating a bacterial infection comprising a cutibacterium avidum strain, a lysate of the strain, a culture medium, an extract of the culture medium, or a mixture thereof as an active ingredient.

The composition is 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, based on the total weight of the composition. %, 0.00001% to 10%, 0.00001% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20%, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10% by weight, or 0.1% to 5% by weight of a strain, a lysate thereof, a culture medium, or an extract of a culture medium thereof.

In the present specification, the term "included as an active ingredient" means that the strain of the present specification, a lysate of the strain, a culture medium, or an extract of the culture medium is added to the extent that the above-mentioned effects can be exhibited, and drug delivery and stabilization It means that various components are added as subcomponents for the purpose of formulation in various forms.

In another embodiment, the composition can be a pharmaceutical composition.

The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated for oral or parenteral administration. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or combinations thereof. Parenteral dosage forms may be injections.

The composition may be a health functional food composition.

The health functional food composition may be used alone or in combination with the strain or its culture medium or other food or food component, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of health functional food. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The health food composition may also contain nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverages, fruit flesh for preparing vegetable beverages, or a combination thereof.

The composition may be a cosmetic composition.

The cosmetic composition may have, for example, softening lotion, nutrient lotion, massage cream, nutrient cream, essence, pack, gel, ampoule, or skin-adhesive cosmetic formulation.

Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions other than the composition as active ingredients, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and flavors. can include

In addition, the composition may be a composition for external application for skin.

In the present specification, the external skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The external skin preparation is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, water-based components, oil-based components, powder components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or combinations thereof and may be suitably blended as needed. The external skin preparations include metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridin, and calin's fruit hot water extracts, various herbal medicines, tocopherol acetate, glycylitnic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose , saccharides such as trehalose, etc. can also be blended appropriately.

Another aspect also provides a method for preventing, ameliorating, or treating a condition in a subject comprising treating or administering to a subject in need thereof an effective amount of the above composition.

The condition of the subject may be a condition related to inflammation.

The administration may be administered by a method known in the art. The administration is directly administered to a subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. It can be. The administration may be administered systemically or locally.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject in need of an improvement effect of a condition related to inflammation.

The administration of the composition according to one embodiment is 0.00001 mg to 1,000 mg per day per subject, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be variously prescribed depending on factors such as administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art take these factors into consideration. Thus, the dosage can be appropriately adjusted. The number of administrations can be once a day or twice or more within the range of clinically acceptable side effects, and the administration site can be administered to one or more than two sites, daily or every 2 to 5 days, total The number of administration days may be administered from 1 day to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For non-human animals, the same dosage per kg as for humans is used, or the above dosage is converted by the volume ratio (eg, average value) of the organ (heart, etc.) between the target animal and the human. A single dose can be administered.

According to the strain according to one aspect and the culture solution derived therefrom, there is an effect that can be usefully used for prevention, improvement, or treatment of bacterial infection.

Figure 1 is a diagram showing the phylogenetic tree analysis of C. avidum KGMB02810 strain isolated from feces of healthy people according to one embodiment.

Figure 2 is a diagram showing the identification of C. avidum KGMB02810 strain isolated from feces of a healthy person according to one embodiment.

Figure 3 is a graph measuring the concentration-dependent antibacterial activity of the culture medium (CaCFS) of C. avidum strains according to one embodiment; CaCFS: C. avidum cell free supernatant

Figure 4 is a photograph showing the antibacterial activity of live bacteria of C. avidum strain; CaCFS: C. avidum cell free supernatant

Figure 5 is a graph comparing the antibacterial activity against C. difficile of cultures of other strains (KGMB09337, KCTC 5339) of C. avidum species according to one embodiment.

Figure 6 is a graph showing the effect of the culture medium (CaCFS) of C. avidum strains on beneficial microorganisms in the intestine according to one embodiment.

Figure 7 is a graph measuring the degree of antibacterial activity according to the incubation time of the culture solution (CaCFS) of C. avidum strains according to one embodiment.

Figure 8 is a graph measuring the degree of antibacterial activity according to the culture temperature of the culture solution (CaCFS) of C. avidum strains according to one embodiment.

Figure 9 is a graph measuring the antibacterial activity after proteolytic enzyme treatment in order to identify the active ingredient of the C. avidum strain culture medium (CaCFS) according to one embodiment.

10 is a graph in which antibacterial activity is measured after separating only the proteins in CaCFS in order to identify the active ingredient of the C. avidum strain culture medium (CaCFS) according to one embodiment.

Figure 11 is a graph measuring the antibacterial activity after treatment with ethyl acetate in order to identify the active ingredient of the C. avidum strain culture medium (CaCFS) according to one embodiment.

Figure 12 is a graph showing the survival rates of a group orally administered with a C. avidum strain (KGMB02810) and a control group according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group

13 is a graph showing changes in body weight of a group orally administered with a C. avidum strain (KGMB02810) according to one embodiment and a control group; NC: control group, CD: negative control group, Ca: experimental group

14 is a graph showing the amount of C. difficile in feces of a group orally administered with a C. avidum strain (KGMB02810) according to one embodiment and a control group; NC: control group, CD: negative control group, Ca: experimental group

15 is a graph showing the amounts of TcdA and TcdB, which are toxins of C. difficile, in feces of a group orally administered with a C. avidum strain (KGMB02810) and a control group according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group

Figure 16 is a graph showing the effect of reducing inflammatory cytokines in a CDI mouse model of C. avidum strain culture medium (CaCFS) according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group

17 is a photograph of intestinal tissue in a CDI mouse model of a culture medium (CaCFS) of a C. avidum strain according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group

Figure 18 is a photograph showing the effect of preventing long-length shrinkage in a CDI mouse model of a culture solution (CaCFS) of a C. avidum strain according to one embodiment; NC: control group, CD: negative control group, Van: positive control group, Ca: experimental group

19 is a graph obtained by analyzing the species richness of microorganisms in feces of a CDI mouse model according to one embodiment through next-generation sequencing (NGS); NC: control group, CD: negative control group, Ca: experimental group

20 is a hierarchical heat map comparing the correlation between intestinal microbes at the family level using metagenome data of the fecal intestinal microbiome of a CDI mouse model according to an embodiment and the number of beneficial bacteria in the intestine Here is a graph measuring relative abundance; NC: control group, CD: negative control group, Ca: experimental group

21 is a graph showing changes in C. difficile after co-cultivation of C. difficile and CaPPT by Gram staining, SEM, and TEM in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment. It is a photograph observed with; RCMPPT: negative control, CaPPT: experimental group (protein in CaCFS), Vancomycin: positive control

22 is a photograph confirming leakage of cellular contents of C. difficile after co-cultivation of C. difficile and CaPPT in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment. to be; RCMPPT: negative control group, CaPPT: experimental group (protein in CaCFS)

Figure 23 is a graph measuring the amount of leaked nucleic acid in the supernatant after co-cultivation of C. difficile and CaPPT in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment. ; RCMPPT: negative control group, CaPPT: experimental group (protein in CaCFS)

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

실시예 1. C. avidum 균주의 분리 및 동정Example 1. Isolation and identification of C. avidum strains

In order to isolate and identify strains from feces of healthy adults, intestinal microorganisms were isolated using an obligate anaerobic microorganism isolation technique in the intestine.

Referring to Figure 1, in order to identify the intestinal microorganism Cutibacterium avidum KGMB02810 isolated from healthy feces, the 16S rRNA sequence was amplified through PCR, and SEQ ID NO: 1 16S rRNA with a length of 1,459 bp As a result of comparing the sequence with the EzBioCloud database (http://www.ezbiocloud.net), it was 100% consistent with Cutibacterium avidum ATCC 25577T (=KCTC 5339T), and for analysis of bacteria and phylogeny within the same genus, Neighbor-joining algorithm was applied. Based on this, it was repeated 1000 times.

Referring to Figure 2, C. avidum KGMB02810 strain, C. avidum KGMB09337 strain, and C. avidum ATCC 25577T strain share 1,983 genes through whole-genome sequencing, and strain identification through each strain-specific gene identification Could.

실시예 2. C. avidum 균주의 배양액(CaCFS)의 항균활성 확인Example 2. Confirmation of antibacterial activity of C. avidum strain culture medium (CaCFS)

To evaluate the antibacterial activity against C. difficile , a system using a liquid culture method was established, and the culture medium was cultured after isolating C. avidum KGMB02810 from healthy Korean feces, and then cultured in RCM (Reinforced Clostridial Medium) medium in an anaerobic chamber at 37 ° C for 30 hours. cultured. The culture medium was subjected to centrifugation and filter-filtration to obtain a cell free supernatant (CFS). For antibacterial activity screening, after diluting C. difficile grown at OD=1 to OD=0.2, mixing 500ul of the diluted bacterial solution with 500μl of the diluted culture medium (10~30%, v/v) according to each concentration, and incubating at 37 ℃ for 4 Time incubation, and antibacterial activity measured the reduced OD value of the experimental group compared to the control group, the results are shown in Figure 3.

In addition, to confirm that the antibacterial activity against C. difficile is limited to the supernatant (CFS) of the culture medium, 10 μl of heat-treated C. avidum and live C. avidum were applied to a plate coated with 100 μl of C. difficile with an OD of 0.5. After dropping it on top, it was incubated at 37 ° C. for 10 hours, and the results are shown in FIG. 4 .

As shown in Figure 3, as a result of the screening, it was confirmed that the C. avidum culture medium (CaCFS) had a concentration-dependent antibacterial activity against C. difficile. As shown in Figure 4, the antibacterial activity against C. difficile was not shown in heat-treated C. avidum, and when the generation of a clear zone was observed in live C. avidum, live C. avidum also showed antibacterial activity against C. difficile. confirmed to have.

실시예 3. C. avidum 종의 다른 균주 (KGMB09337, KCTC 5339) 배양액의 C. difficile에 대한 항균 활성Example 3. Antibacterial activity of cultures of other strains of C. avidum species (KGMB09337, KCTC 5339) against C. difficile

In order to confirm whether the antibacterial activity of C. avidum KGMB02810 strain against C. difficile is specific, cultures of other strains (KGMB09337, KCTC5339) were prepared in the manner described in Example 2 above, and they were used to treat C. difficile. The antibacterial activity ability was evaluated. Similarly, for antibacterial activity screening, after diluting C. difficile grown at OD = 1 to OD = 0.2, mixing 500 μl of the diluted bacterial solution and each 500 μl, incubating at 37 ° C for 4 hours, the antibacterial activity was measured by measuring absorbance. As a result, is shown in Figure 5. In addition, it was measured whether CaCFS, which has antibacterial activity against C. difficile, has antibacterial activity against beneficial intestinal microorganisms (7 types of lactic acid bacteria), and the results are shown in FIG. 6.

Figure 5 is a graph showing the antibacterial activity against C. difficile of cultures of other strains (KGMB09337, KCTC 5339) of C. avidum species according to one embodiment.

As shown in Figure 5, the culture solution of C. avidum KGMB09337 and KCTC 5339 strains did not show antibacterial activity. In view of this, the antimicrobial activity of C. avidum against C. difficile is strain-specific, and as shown in FIG. showed no antibacterial activity.

실시예 4. CaCFS 속의 항 C. difficile 물질 검토 (1)Example 4. Examination of anti-C. difficile substances in CaCFS (1)

In order to know the characteristics of the C. difficile antibacterial active substance, it was confirmed in the following way.

First, in order to check how the antimicrobial activity of CaCFS changes according to C. avidum culture time, C. avidum was prepared at regular intervals (24, 26, 30, 34, 38, 42, 46 hours), and then An antibacterial test was conducted in the manner described in Example 2 above. In order to confirm the heat sensitivity of CaCFS, an antibacterial test was conducted after giving CaCFS heat between 30 and 90 ° C for a certain period of time (0 to 80 minutes), and the results are shown in FIGS. 7 and 8 .

As shown in Figure 7, it was confirmed that the antibacterial activity of CaCFS was the highest when C. avidum was cultured for 26-30 hours. As shown in Figure 8, the antibacterial substance of CaCFS almost disappeared even if heat of 60 ℃ or more was applied for only 5 minutes, and the heat of 40 ℃ or less did not lose activity during the experimental period. That is, it was confirmed that the material was sensitive to heat.

실시예 5. CaCFS 속의 항 C. difficile 물질 검토 (2)Example 5. Examination of anti-C. difficile substances in CaCFS (2)

In order to confirm whether the heat-sensitive antibacterial substance is a protein, several enzymes (Phosphatase, Catalase, a-amylase, Proteinase K, Pepsin, Papain, Chymotrypsin, Trypsin) were reacted at a concentration of 1 mg/ml for 1 hour at 37°C. , antibacterial experiments were conducted. In addition, ammonium sulfate protein precipitation was used to obtain proteins in CaCFS. For the protein precipitation method, 40% (w/v) Ammonium sulfate was added to 1 L of CaCFS, stirred at 4 °C for 1 hour, and then centrifuged. After that, the supernatant was removed, the precipitate was dissolved in 10 ml of 20 mM sodium phosphate buffer, and dialysis was performed at 4° C. for 24 hours to remove salt. The thus-obtained CaCFS genus proteins (CaPPT) were measured for their antibacterial activity in the manner described in Example 2. through liquid culture. In order to confirm the antibacterial activity of CaCFS with an organic solvent extract, CaCFS was extracted with ethyl acetate. Ethyl acetate was added in the same amount as 500ml CaCFS, stirred at room temperature for 30 minutes, and left as it was to separate the aqueous layer and the organic layer. Then, only the organic layer was separated and collected, and ethyl acetate in the solution was completely evaporated using a rotary evaporator, and the remaining pellet was weighed and dissolved in 20 mM sodium phosphate buffer at a concentration of 40 mg/ml. . In addition, in order to remove the microbial infection in the solution, after going through a filter-filtration process, an antibacterial test was conducted at a predetermined concentration, and the results are shown in FIGS. 9, 10, and 11.

As shown in Figure 9, the antibacterial activity of CaCFS was reduced by more than 40% by the proteolytic enzymes Pepsin and Trypsin. As shown in FIG. 10, it was confirmed that CaPPT has antibacterial activity similar to that of CaCFS. As shown in FIG. 11, the organic matter in the CaCFS fraction did not show antibacterial activity.

Through the above results, the substance exhibiting antibacterial activity against C. difficile was identified as a protein.

실시예 6. CDI 마우스 모델에 대한 효과Example 6. Effect on CDI mouse model

To confirm the ability of C. avidum KGMB02810 strain to inhibit CDI disease in vivo, antibiotic drinking water (Knanamycin 400mg, Vancomycin 45mg, Colistin 28.3mg, Gentamycin 35mg, Metronidazole 215mg in 1L DW) was supplied free of charge for 3 days, and then sterilized drinking water was used. replaced One day later, Clindamycin was intraperitoneally injected into C57BL/6 mice at a concentration of 10 mg/kg. After oral administration of 1 X 10 ⁶ CFU of C.difficile to the negative control group (CD group) and experimental group (Ca group) mice, 30 minutes later, the experimental group (Ca group) received 1 X 10 ⁹ CFU per mouse. 100 μl of C. avidum KGMB02810 was orally administered daily, and 100 μl of physiological saline was orally administered daily to the control group (NC group) and negative control group (CD group). And the remaining groups (NC, CD groups) were orally administered with 100 μl of PBS. The degree of C. difficile infection was measured daily by weight, fecal condition, and movement, and the amount of TcdA and TcdB, which are toxins of C. difficile in feces, was measured simultaneously using a C. difficile toxin detection ELISA kit, and the results are shown in FIG. 12 and FIG. 13, Fig. 14, and Fig. 15.

As inflammatory cytokine indicators for C. difficile infection, the amount of IL-6 and TNF-alpha was measured using an ELISA kit after obtaining mouse serum. Analysis was performed, and the results are shown in FIGS. 16 and 17.

In order to confirm the ability of the C. avidum KGMB02810 strain to inhibit CDI disease, mice were sacrificed and the length of the large intestine was measured, and the results are shown in FIG. 18 .

As shown in FIG. 12, the survival rate of the group orally administered with C. avidum (Ca group) was significantly higher than that of the C. difficile control group (CD group) by 70%. As shown in FIG. 13, the body weight decreased less than that of the negative control group (CD group), but increased the same or more than that of the C. difficile non-inoculated group (NC group) from the 5th day. As shown in Figure 14, the amount of C. difficile inside the feces was reduced 100 times in the group administered with C. avidum KGMB02810 compared to the negative control group (CD group). As shown in FIG. 15, the amount of C. difficile toxin in the feces of the experimental group (Ca group) was reduced by more than 3 times compared to that of the negative control group (CD group).

As shown in Figure 16, it was confirmed that both inflammatory cytokines (IL-6, TNF-alpha) were significantly reduced compared to the negative control group (CD group). As shown in FIG. 17, in the intestine of the negative control group (CD group), intestinal epithelial cells were eliminated, and immune cells such as neutrophils infiltrated into the lower epithelial layer. In addition, swelling of the submucosa was observed. However, the experimental group (Ca group) did not show or relieved these symptoms, so the treatment effect on C. difficile infection was confirmed once again.

As shown in Figure 18, although it was shortened by about 15% compared to the C. difficile non-inoculated group (NC group), the group orally administered with C. avidum KGMB02810 (Ca group) and the group orally administered with Vancomycin (Van group) It was confirmed that the shortening of colon length was remarkably restored.

실시예 7. CDI 마우스 모델의 장내 마이크로바이옴 분석Example 7. Intestinal microbiome analysis of CDI mouse model

Through next-generation sequencing (NGS) technology, metagenome analysis was performed on the fecal intestinal microbiome in the colon, and the results are shown in FIG. 19 .

Using the metagenome data, the mutual relationship between each group and the intestinal microbes was shown as a hierarchical heat map at the family level, and lactic acid bacteria (Bifidobacterium sp. and Lactobacillus sp.) The relative abundance of was confirmed, and the results are shown in FIG. 20.

As shown in FIG. 19, it was confirmed that the diversity and abundance of species in the C. difficile control group (CD group) decreased compared to the C. difficile non-inoculated group (NC group) in the analysis of taxonomic diversity (Alpha diversity) within the sample. It was confirmed that diversity and abundance increased when C. avidum KGMB02810 was orally administered (Ca group).

As shown in FIG. 20, the positive clusters (Lactobacillaceae, Ruminococcaceae, Lachnospiraceae, Muribaculaceae) and negative clusters of the C. difficile non-inoculated group (NC group) and the group orally administered with C. avidum KGMB02810 (Ca group) are similar, It was confirmed that it was clearly distinguished from the C. difficile control group (CD group). Compared to the C. difficile non-inoculated group (NC group), the abundance of the C. difficile control group (CD group) decreased noticeably, but the abundance of the group orally administered with C. avidum KGMB02810 (Ca group) recovered. Confirmed.

Through the above results, it was confirmed that the administration of the C. avidum KGMB02810 strain has the effect of restoring intestinal function damage caused by C. difficile infection and restoring the intestinal microbiome.

실시예 8. 배양액(CaCFS)의 항균 활성 기전 분석Example 8. Analysis of antibacterial activity mechanism of culture medium (CaCFS)

In order to confirm the mechanism of action of the antibacterial activity against C. difficile represented by the C. avidum KGMB02810 strain, CaPPT was co-cultured with C. difficile for 10 minutes and then morphological analysis was performed, and the results are shown in FIGS. 21 and 22 shown in

In order to verify the above results once more, after treating C. difficile with CaPPT, the amount of nucleic acid in the supernatant was measured for absorbance at a wavelength of 260 nm, and the results are shown in FIG. 23 .

As shown in FIGS. 21 and 22, it was confirmed that the cell wall staining of C. difficile treated with CaPPT became faint in the Gram staining results, and in the results of scanning electron microscopy (SEM), C. difficile treated with CaPPT. It was confirmed that the outer cell wall was destroyed. In addition, transmission electron microscopy (TEM) results confirmed that the peptidoglycan layer of C. difficile treated with CaPPT was fragmented and collapsed. Additionally, in order to measure the leakage of cellular contents of C. difficile due to CaPPT treatment, after treating C. difficile with CaPPT, nucleic acid was stained with Hoechst 33342, and cell membrane was stained with 4-64FX. Then, as a result of observation under a fluorescence microscope, it was confirmed that the nucleic acid of C. difficile leaked out of the cell when CaPPT was treated.

As shown in FIG. 23, it was confirmed that the absorbance value significantly increased when CaPPT was treated compared to C. difficile treated with RCMPPT.

Through the above results, it was confirmed that the antibacterial active protein against C. difficile produced by the C. avidum KGMB02810 strain has a mechanism of action that destroys the cell wall of C. difficile.

[Accession number]

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14558BP

Entrusted date: 20210429

Claims

Deposited under Accession No. KCTC 14558BP, Cutibacterium genus ( Cutibacterium sp. ) Belonging to Cutibacterium avidum ( Cutibacterium avidum ) strain.
The strain according to claim 1, wherein the strain comprises the 16s rRNA of SEQ ID NO: 1.
A lysate of the strain of claim 1.
A culture solution of the strain of claim 1.
A pharmaceutical composition for preventing or treating a bacterial infection comprising the strain of claim 1, a lysate of the strain, a culture medium thereof, or a mixture thereof as an active ingredient.
The pharmaceutical composition according to claim 5, wherein the bacterial infection is Clostridium difficile infection (CDI).
Inflammatory bowel diseases (IBD) comprising the strain of claim 1, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient; irritable bowel syndrome; enteritis, Crohn's disease; Behcet's disease; Or a pharmaceutical composition for preventing or treating ulcerative colitis (ulcerative colitis).
A food composition for preventing or improving bacterial infection comprising the strain of claim 1, a lysate of the strain, a culture medium thereof, or a mixture thereof as an active ingredient.
The food composition according to claim 8, wherein the bacterial infection is Clostridium difficile infection (CDI).
A food composition for improving intestinal health by inhibiting Clostridium difficile or inhibiting inflammation, comprising the strain of claim 1, a lysate of the strain, a culture medium thereof, or a mixture thereof as an active ingredient.
Inflammatory bowel diseases (IBD) comprising the strain of claim 1, a lysate of the strain, a culture medium thereof, or a mixture thereof as an active ingredient; irritable bowel syndrome; enteritis, Crohn's disease; Behcet's disease; Or a food composition for preventing or improving ulcerative colitis (ulcerative colitis).
A cosmetic composition comprising the strain of claim 1, a lysate of the strain, a culture medium thereof, or a mixture thereof as an active ingredient.
A method for preventing or treating a bacterial infection comprising administering the strain of claim 1, a lysate of the strain, a culture thereof, or a mixture thereof to a subject in need thereof.
A method for preventing or treating an inflammatory disease by administering the strain of claim 1, a lysate of the strain, a culture thereof, or a mixture thereof to a subject in need thereof.
Use of the strain of claim 1, a lysate of the strain, a culture medium thereof, or a mixture thereof for preparing a composition for preventing or treating bacterial infection.
Use of the strain of claim 1, a lysate of the strain, a culture medium thereof, or a mixture thereof for preparing a composition for preventing or treating inflammatory diseases.