Novel bacillus subtilis strain and method for preventing, treating, or alleviating tuberculosis using same

Abstract

The present invention relates to a novel Bacillus subtilis strain and a method for preventing, treating, or alleviating tuberculosis using same. More specifically, the present invention relates to a Bacillus subtilis PMC 205 strain extracted from kimchi and a use thereof, for example, an antibacterial composition having antibacterial activity against bacteria belonging to the genus Mycobacterium, a pharmaceutical composition or a food composition with antibacterial activity against bacteria belonging to the genus Mycobacterium for alleviating, treating, or preventing tuberculosis.

Description

Novel Bacillus subtilis strain and method for preventing, treating or improving tuberculosis comprising the same

[0001] The present invention relates to a novel Bacillus subtilis strain and a method for preventing, treating, or ameliorating tuberculosis comprising the same. More specifically, the present invention relates to a Bacillus subtilis strain PMC 205 extracted from kimchi and its uses, for example, an antibacterial composition having antibacterial activity against bacteria belonging to the genus Mycobacterium, and a pharmaceutical composition or food composition having antibacterial activity against bacteria belonging to the genus Mycobacterium for ameliorating, treating, or preventing tuberculosis.

[0002]

[0003] Tuberculosis is an infectious disease caused by the bacteria Mycobacterium tuberculosis. Tuberculosis remains a major public health problem worldwide, causing approximately 1.8 million deaths annually. Drug-resistant tuberculosis, including multidrug-resistant (MDR), extensively drug-resistant (XDR), and completely drug-resistant (TDR) strains, continues to pose a threat to public health. Antibiotic-resistant tuberculosis is less effective when treated with existing antibiotics. Furthermore, existing treatments, coupled with long-term, high-dose medications, cause serious side effects.

[0004] Therefore, to overcome these problems, there is an urgent need to develop a new type of anti-tuberculosis drug that is different from existing compound-based antibiotics.

[0005]

[0006] Prior literature

[0007] Non-patent literature 1. WHO. Global tuberculosis report 2021. Geneva: World Health Organization; 2021.

[0008] Non-patent document 2. Singh Dewhare S. Drug resistant tuberculosis: Current scenario and impending challenges. Indian J Tuberc. 2022 Apr;69(2):227-233.

[0009] Non-patent literature 3. Black TA, Buchwald UK. The pipeline of new molecules and regimens against drug-resistant tuberculosis. J Clin Tuberc Other Mycobact Dis. 2021 Dec;25:100285.

[0010]

[0011] The present invention aims to provide a Bacillus subtilis PMC 205 strain.

[0012] The present invention also aims to provide an antibacterial composition comprising Bacillus subtilis PMC 205 strain.

[0013] The present invention also aims to provide a composition for preventing, treating, or improving tuberculosis comprising Bacillus subtilis PMC 205 strain.

[0014]

[0015] According to the first implementation example,

[0016] The present invention provides a Bacillus subtilis PMC 205 strain or a culture solution thereof having antibacterial activity against bacteria belonging to the genus Mycobacterium.

[0017] According to the present invention, the Bacillus subtilis PMC 205 strain may have the deposit number KCTC 15188BP extracted from kimchi.

[0018] According to the present invention, the bacteria belonging to the genus Mycobacterium are Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium One or more may be selected from the group consisting of M. leprae, M. malmoense, M. marinum, M. microti, M. phlei, M. scrofulaceum, M. smegmatis, M. triplex, M. tuberculosis, M. ulcerans, and M. xenopi.

[0019]

[0020] According to the second implementation example,

[0021] The present invention aims to provide an antibacterial composition having antibacterial activity against bacteria belonging to the genus Mycobacterium, comprising Bacillus subtilis PMC 205 strain or a culture solution thereof.

[0022] According to the present invention, the Bacillus subtilis PMC 205 strain may have the deposit number KCTC 15188BP extracted from kimchi.

[0023] According to the present invention, the bacteria belonging to the genus Mycobacterium are Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium One or more may be selected from the group consisting of M. leprae, M. malmoense, M. marinum, M. microti, M. phlei, M. scrofulaceum, M. smegmatis, M. triplex, M. tuberculosis, M. ulcerans, and M. xenopi.

[0024] In the present invention, the antibacterial composition can be used in a living body.

[0025] In the present invention, the antibacterial composition can be used in an in vitro environment.

[0026]

[0027] According to the third implementation example,

[0028] The present invention provides a pharmaceutical composition for treating or preventing tuberculosis having antibacterial activity against bacteria belonging to the genus Mycobacterium, comprising Bacillus subtilis PMC 205 strain or a culture solution thereof as an active ingredient.

[0029] According to the present invention, the Bacillus subtilis PMC 205 strain may have the deposit number KCTC 15188BP extracted from kimchi.

[0030] According to the present invention, the bacteria belonging to the genus Mycobacterium are Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium One or more may be selected from the group consisting of M. leprae, M. malmoense, M. marinum, M. microti, M. phlei, M. scrofulaceum, M. smegmatis, M. triplex, M. tuberculosis, M. ulcerans, and M. xenopi.

[0031] In the present invention, the tuberculosis may be at least one selected from the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymphatic tuberculosis, pulmonary tuberculosis, breast tuberculosis, and spinal tuberculosis.

[0032] In the present invention, the tuberculosis may be latent tuberculosis.

[0033] In the present invention, the pharmaceutical composition comprises rifampicin, rifapentine, isoniazid, pyrazinamide, ethambutol, streptomycin, fluoroquinolone, kanamycin, cycloserine, prothionamide, levofloxacin, moxifloxacin, ofloxacin, rifabutin, capeomycin, amikacin, ciprofloxacin, protionamide, ethionamide, cycloserine, It may further comprise at least one selected from the group consisting of thioacetazone, clofazimine, amoxicillin / clavulanate, a derivative of dianomidiphenylsulphone, clarithromycin, azithromycin, and linezolid.

[0034]

[0035] According to the fourth implementation example,

[0036] The present invention aims to provide a food composition for preventing or improving tuberculosis having antibacterial activity against bacteria belonging to the genus Mycobacterium, which comprises Bacillus subtilis PMC 205 strain or a culture solution thereof as an active ingredient.

[0037] According to the present invention, the Bacillus subtilis PMC 205 strain may have the deposit number KCTC 15188BP extracted from kimchi.

[0038] According to the present invention, the bacteria belonging to the genus Mycobacterium are Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium One or more may be selected from the group consisting of M. leprae, M. malmoense, M. marinum, M. microti, M. phlei, M. scrofulaceum, M. smegmatis, M. triplex, M. tuberculosis, M. ulcerans, and M. xenopi.

[0039] In the present invention, the tuberculosis may be at least one selected from the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymphatic tuberculosis, pulmonary tuberculosis, breast tuberculosis, and spinal tuberculosis.

[0040] In the present invention, the tuberculosis may be latent tuberculosis.

[0041]

[0042] The present invention provides a Bacillus subtilis PMC 205 strain, which has antibacterial activity. More specifically, the strain has antibacterial activity against Mycobacterium tuberculosis. Accordingly, the strain can be used to treat, prevent, or improve tuberculosis.

[0043] At this time, the strain has the advantage of being free of drug resistance and side effects caused by long-term, high-dose drug treatment compared to compound-based drug treatment.

[0044]

[0045] Figure 1 shows the genetic characteristics and in vitro anti-tuberculosis efficacy of Bacillus subtilis PMC205 according to the present invention, showing (A) the results of high-throughput genome sequencing, (B) the results according to COG (cluster of orthologous genes) classification clusters, and (C) the results of Ortho ANI (average nucleotide identity).

[0046] Figure 2 shows the results of the antimycobacterial activity evaluation of Bacillus subtilis PMC205 according to the present invention.

[0047] FIG. 3 shows an efficacy evaluation of Bacillus subtilis PMC205 according to the present invention in a lethal XDR pulmonary tuberculosis mouse model, showing (A) survival rate, (B) body weight, (C) disease severity score, and (D) CFU / lung results.

[0048] Figure 4 shows the characteristics of the pulmonary microbiome in the treatment of M. tuberculosis with Bacillus subtilis PMC 205 according to the present invention, and shows the results of analyzing the pulmonary microbiome based on (A) cytokine content and (B) 16S rRNA gene.

[0049] Figure 5 shows the efficacy evaluation of Bacillus subtilis PMC 205 according to the present invention on intrapulmonary M. tuberculosis in a latent tuberculosis mouse model, showing (A) body weight and (B) CFU / lung results.

[0050]

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0052] The term "latent tuberculosis" as used herein refers to inactive tuberculosis, meaning tuberculosis in a dormant state after a primary tuberculosis infection, and means tuberculosis bacteria infecting a host without the onset of disease symptoms.

[0053] The term "antibacterial" as used herein refers to killing microorganisms or inhibiting their growth. In one embodiment, the term may be used herein to mean killing specific bacteria or inhibiting their growth. As a specific example, the term may be used herein to mean killing or inhibiting the growth of bacteria belonging to the genus Mycobacterium. In more specific embodiments, the present specification provides Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, M. avium complex (MAC), M. bovis, M. chelonae, M. fortuitum, M. gordonae, M. haemophilum, M. intracellulare, M. kansasii, M. lentiflavum, M. leprae, It may be used to mean killing or inhibiting the growth of one or more bacteria selected from the group consisting of Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

[0054] The term "prevention" as used herein may include, without limitation, any act of blocking, suppressing or delaying symptoms caused by tuberculosis by using the composition of the present invention containing Bacillus subtilis PMC 205 strain as an active ingredient.

[0055] The terms “improvement” and “treatment” used herein may include, without limitation, any act of improving or benefiting symptoms caused by tuberculosis by using the composition of the present invention comprising Bacillus subtilis PMC 205 strain as an active ingredient.

[0056]

[0057]

[0058] *Below, the specific contents of the invention are disclosed.

[0059]

[0060] I. Strains

[0061] Strains obtained from fermented foods

[0062] The present invention provides a Bacillus subtilis PMC 205 strain. The Bacillus subtilis PMC 205 strain can be obtained from a fermented food. According to one exemplary embodiment, the Bacillus subtilis PMC 205 strain can have the KCTC 15188BP deposit number, which is extracted from kimchi.

[0063] Non-cytotoxic strains

[0064] Antimicrobial compositions for use in living and in vitro environments

[0065] The present invention provides an antibacterial composition comprising Bacillus subtilis PMC 205 strain. Since the strain is non-cytotoxic, it can be included in compositions used in vivo and in vitro.

[0066] The above antimicrobial composition may be an antimicrobial composition administered to a living organism. In this case, the organism refers to a living organism, and the organism includes mammals, including humans. That is, in one embodiment, the present invention provides an antimicrobial composition having antimicrobial activity against unwanted microorganisms present in mammals, including humans. In a specific embodiment, the present invention provides an antimicrobial composition having antimicrobial activity against pathogenic bacteria present in mammals, including humans.

[0067] The antimicrobial composition comprising the Bacillus subtilis PMC 205 strain may be an antimicrobial composition having antimicrobial activity against unwanted microorganisms existing in an in vitro environment. In a specific embodiment, the antimicrobial composition comprising the Bacillus subtilis PMC 205 strain provides an antimicrobial composition having antimicrobial activity against pathogenic bacteria existing in an in vitro environment. Here, the 'in vitro environment' may refer to the surface of a material requiring cleaning, as an example. In this case, 'cleaning' may refer to removing unwanted microorganisms. In this case, 'removal' may refer to killing some of the bacteria, inhibiting the growth of the bacteria, or reducing the number of the bacteria.

[0068] The surface of the above material may include, but is not limited to, the surface of household items such as furniture, tableware, and home appliances, and includes all surfaces of materials requiring cleaning. That is, the present invention provides an antibacterial composition having antibacterial activity against pathogenic microorganisms existing in an in vitro environment.

[0069] Antibacterial methods for use in living and in vitro environments

[0070] The present invention also provides an antibacterial method using an antibacterial composition comprising Bacillus subtilis PMC 205 strain.

[0071] The above antibacterial method may include administering the antibacterial composition to a living organism. The organism refers to a living organism, and the organism includes mammals, including humans. That is, the present invention provides an antibacterial method comprising administering the composition to a mammal, including humans.

[0072] The above antibacterial method may also include treating the antibacterial composition in vitro. The term "in vitro" refers to the surface of a material requiring cleaning, and the present invention provides an antibacterial method for treating the surface of a material requiring cleaning with the antibacterial composition.

[0073] The form of the strain included in the composition

[0074] The present invention provides a Bacillus subtilis PMC 205 strain and an antibacterial composition comprising the strain, a pharmaceutical composition for treating, preventing, and improving tuberculosis and inflammatory diseases, and a food composition. In one embodiment, the composition may comprise the Bacillus subtilis PMC 205 strain in the form of live cells, dead cells, or a culture solution thereof. The culture solution may be a culture solution comprising the Bacillus subtilis PMC 205 strain in the present invention, or may include a lysate, centrifuged supernatant or pellet, a concentrate, a dried product, etc.

[0075]

[0076] II. Antibacterial composition

[0077] The present invention provides an antibacterial composition comprising Bacillus subtilis PMC 205 strain. The antibacterial composition may have antibacterial activity against bacteria belonging to the genus Mycobacterium. In a specific embodiment, the antibacterial composition may be used to treat Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, It may have antibacterial activity against one or more bacteria selected from the group consisting of Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi. In a more specific embodiment, the antibacterial composition may have antibacterial activity against Mycobacterium tuberculosis.

[0078]

[0079] III. Composition for improving, treating or preventing tuberculosis

[0080] Pharmaceutical composition for the treatment or prevention of tuberculosis

[0081] The present invention provides a pharmaceutical composition for treating or preventing tuberculosis comprising Bacillus subtilis PMC 205 strain.

[0082] The term “tuberculosis” as used herein refers to an infectious disease transmitted by Mycobacterium tuberculosis. For example, the tuberculosis may be selected from the group consisting of, but is not limited to, ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymph node tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymph node tuberculosis, pulmonary tuberculosis, breast tuberculosis, and spinal tuberculosis.

[0083] In the pharmaceutical composition for treating or preventing tuberculosis according to the present invention, the pharmaceutical composition may further comprise a carrier. For example, the pharmaceutically acceptable carrier is one commonly used in the preparation of formulations, and may include, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition may further comprise a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0084] In the pharmaceutical composition for treating or preventing tuberculosis according to the present invention, the pharmaceutical composition may be prepared by mixing one or more diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants commonly used in this technical field. For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. Such solid preparations are prepared by mixing the strain of the present invention or its culture medium with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, and gelatin.

[0085] In the pharmaceutical composition for treating or preventing tuberculosis according to the present invention, the pharmaceutical composition may further include a substance that can improve the preventive or therapeutic effect of the disease. For example, the pharmaceutical composition may comprise rifampicin, rifapentine, isoniazid, pyrazinamide, ethambutol, streptomycin, fluoroquinolone, kanamycin, cycloserine, prothionamide, levofloxacin, moxifloxacin, ofloxacin, rifabutin, capeomycin, amikacin, ciprofloxacin, protionamide, ethionamide, cycloserine, The pharmaceutical composition may further comprise at least one selected from the group consisting of thioacetazone, clofazimine, amoxicillin / clavulanate, a derivative of dianomidiphenylsulphone, clarithromycin, azithromycin, and linezolid. In one exemplary embodiment, the pharmaceutical composition may further comprise rifamficin in the Bacillus subtilis PMC 205 strain.

[0086] Tuberculosis treatment methods

[0087] The present invention provides a method for treating tuberculosis, comprising administering to a subject a composition comprising Bacillus subtilis PMC 205 strain. In this case, the subject may refer to a mammal, including a human.

[0088] In the method for treating tuberculosis according to the present invention, the composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and weight, the degree of the disease, the drug form, the route of administration, and the time, but may be appropriately selected by a person skilled in the art.

[0089] In the method for treating tuberculosis according to the present invention, the composition is administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to consider all of the above factors and administer an amount that achieves the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art. In this case, the composition of the present invention and rifampicin can be administered together. When rifampicin is administered together, the disease prevention and treatment effect of the composition of the present invention can be enhanced. Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.1 to 100 mg per kg of body weight, preferably 0.5 to 10 mg, administered daily or every other day, or divided into 1 to 3 doses per day. However, the dosage may increase or decrease depending on the route of administration, severity of obesity, sex, body weight, age, etc., and thus the above dosage does not limit the scope of the present invention in any way.

[0090] Food composition

[0091] The present invention provides a food composition for preventing or improving tuberculosis, comprising a Bacillus subtilis PMC 205 strain. In this case, the food composition comprises a health functional food composition, and the health functional food is not particularly limited as long as it comprises the Bacillus subtilis PMC 205 strain, but preferably, the strain may be included in an amount of 0.01 to 20 wt% based on the total weight of the health functional food composition.

[0092] In the food composition for preventing or improving tuberculosis according to the present invention, the food composition is not particularly limited in terms of the type of food as long as it is commonly manufactured and / or sold. For example, it includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and can be used in the form of pills, powders, granules, infusions, tablets, capsules, or drinks, and includes all health functional foods in the conventional sense.

[0093] In the food composition for preventing or improving tuberculosis according to the present invention, the food composition has no particular limitation on the liquid component except that it contains the strain as described above, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical beverage. Examples of the natural carbohydrates include typical sugars such as monosaccharides, for example, glucose, fructose, etc.; disaccharides, for example, maltose, sucrose, etc.; polysaccharides, for example, dextrin, cyclodextrin; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. In addition to the flavoring agents described above, natural flavoring agents (thaumatin, stevia extracts (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

[0094] In the food composition for preventing or improving tuberculosis according to the present invention, the food composition may contain, in addition to the above-described additional ingredients, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0095]

[0096] Hereinafter, the present invention will be described in detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0097]

[0098] <Experimental Method>

[0099] 1. Isolation, identification, and genetic characterization of probiotic strains from kimchi.

[0100] Probiotic strains were isolated from kimchi. The liquid portion of the kimchi was streaked onto MRS agar (de Man, Rogosa, and Sharp, 288210, BD difco, USA) plates using a loop. The plates were incubated at 37°C in an aerobic incubator (general incubator, N-Biotek, Korea). After incubation, colonies were cultured in MRS broth (288130, BD difco, USA) and stored as 15% glycerol stocks at -80°C. The strains were identified by 16s rRNA gene sequencing.

[0101] 16s rRNA gene sequencing was performed at Biofact (Korea) and compared with the GenBank database using BLAST (basic local alignment search tool) at the National Center for Biotechnology Information (NCBI). For whole genome sequencing of the strain, gDNA was extracted from the strain and sequenced using PacBio RSII (PacBio, USA) at CJ Bioscience (Korea), and then analyzed using the Ezbiocloud server. The genome information of the strain Bacillus subtilis PMC205 was deposited on the NCBI website under the accession number PRJNA865470. The probiotic strain was cultured in food-grade medium (20 g / l glucose, 25 g / l yeast peptone standard F, 1 g / l Tween 80, 0.2 g / l magnesium sulfate, pH 6.3).

[0102]

[0103] 2. In vitro antifungal activity test on macrophages

[0104] Murine Raw 264.7 macrophages that reached confluence were infected with Mycobacterium tuberculosis and treated with the probiotics or drugs according to the present invention, cultured for 3 days, and then CFU analysis was performed. The H37Rv and XDR strains of Mycobacterium tuberculosis used in this study were purchased from the American Type Culture Collection (ATCC, USA) and the Korean Mycobacterium Resource Center (KMRC, Korea), respectively.

[0105]

[0106] 3. In vivo evaluation of the anti-tuberculosis efficacy of probiotics

[0107] The in vivo antituberculosis effects of the probiotics of the present invention were evaluated in XDR pulmonary tuberculosis mouse models and latent tuberculosis mouse models. First, 6-week-old Balb / c mice were infected with a high-dose pulmonary inhalation of the XDR strain using a nebulizer, and then the probiotic strain was administered by pulmonary inhalation three times a day, starting one week after infection. In the latent pulmonary tuberculosis mouse model, mice were infected with a low-dose XDR strain, and one month later, the probiotic strain was administered by pulmonary inhalation once a day, twice a week for eight months. Rifampicin was used as a control drug and administered orally at a dose of 30 mg / kg. Saline was used as a vehicle in these animal experiments. For further analysis, lungs were extracted, and M. tuberculosis was quantitatively analyzed by CFU and real-time PCR. Primers used were KY18 (forward, 5'-CACATGCAAGTCGAACGGAAAGG-3'; reverse, 5'-CCTTTCCGTTCGACTTGCATGTG-3') and KY75 (forward, 5'-GCCCGTATCGCCCGCACGCTCACA-3'; reverse, 5'-TGTGAGCGTGCGGGCGATACGGGC-3'). Perfused lungs were used to investigate the lung microenvironment in an XDR pulmonary tuberculosis mouse model. Microbiome and metabolite analyses were performed according to previously reported methods. Lung cytokines were analyzed using a Mouse ELISA Kit (Thermo Fisher Scientific, USA), and hematoxylin and eosin (H&E) staining analysis was performed at KPNT (Korea Pathology Technical Center, Korea).

[0108]

[0109] <Result>

[0110] 1. Analysis of genetic characteristics of probiotic strains

[0111] A probiotic strain isolated from baekchu kimchi, a traditional fermented food, was identified based on the 16s rRNA gene sequence and then confirmed by whole genome analysis. As a result, the genome of the isolated probiotic strain was composed of a 4,096,449 bp single circular chromosome with 4,036 coding sequences (CDS) (Fig. 1A), and a total of 3,691 predicted proteins were grouped according to clusters of Orthologous Groups (COG) functional classification (Fig. 1B). Similarity analysis using the Ortho ANI (Orthologous Average Nucleotide Identity) method showed high similarity to B. subtilis, whereas similarity to other Bacillus species was low. Therefore, it was ultimately confirmed that the probiotic strain isolated from baekchu kimchi was a B. subtilis strain (Fig. 1C). In addition, the above-mentioned isolated Bacillus subtilis strain was designated as PMC 205 as a new strain genetically different from previously reported strains.

[0112]

[0113] 2. Evaluation of antimycobacterial activity of Bacillus subtilis PMC 205

[0114] The antimycobacterial activity of Bacillus subtilis PMC 205 according to the present invention was confirmed using an in vitro macrophage infection model. Raw 264.7 cells were infected with the H37Rv strain, and the PMC 205 extract was treated for 3 days, after which the colony-forming unit (CFU) was quantified. As a result, 2.3 × 10 6 It was confirmed that when treated with PMC 205 extract equivalent to CFU / ml or more, the growth of M. tuberculosis was significantly inhibited compared to the untreated control group, which was similar to the effect when treated with 1 μg / ml of rifampicin (RIF) (Fig. 2).

[0115]

[0116] 3. Evaluation of the efficacy of Bacillus subtilis PMC 205 in a mouse model of lethal XDR pulmonary tuberculosis.

[0117] The in vivo efficacy of Bacillus subtilis PMC 205 according to the present invention was evaluated using an XDR pulmonary tuberculosis mouse model. 6-week-old Balb / c mice were infected with a high-dose pulmonary inhalation of the XDR strain using a nebulizer, and one week after infection, Bacillus subtilis PMC 205 was administered once a day, three times a week in the same manner, and the mortality rate was observed. As a result, the infected control mice began to die from the fifth week and all died by the 12th week. While the survival rate was 40% when administered 30 mg / kg of RIF, all mice administered Bacillus subtilis PMC 205 according to the present invention survived at the end of the experiment. Interestingly, it was confirmed that no anti-tuberculosis effect was observed when Lactobacillus, Leuconostoc, and Pediococcus, which were tested under the same conditions, were individually treated (Fig. 3A). In addition, it was confirmed that when treated with Lactobacillus, Leuconostoc, and Pediococcus strains, or RIF, a decrease in body weight of mice was observed, whereas when administered with Bacillus subtilis PMC 205 according to the present invention, no significant decrease in body weight was observed (Fig. 3B). In addition, in terms of disease severity scores, when Bacillus subtilis PMC 205 according to the present invention was administered, the same results as in uninfected mice were observed, unlike when treated with Lactobacillus, Leuconostoc, and Pediococcus strains, or RIF (Fig. 3C), and when further analysis was performed on the extracted lungs, it was confirmed that in the lungs of mice administered Bacillus subtilis PMC 205 according to the present invention, M. tuberculosis was significantly reduced at Log 3.6 and Log 2.6 at 6 and 10 weeks, respectively, compared to mice administered Lactobacillus, Leuconostoc, and Pediococcus strains, or RIF (Fig. 3D).

[0118]

[0119] 4. Characterization of the intrapulmonary microbiome in the treatment of M. tuberculosis with Bacillus subtilis PMC 205.

[0120] In order to investigate the characteristics of the pulmonary microbiome in the treatment of M. tuberculosis with Bacillus subtilis PMC 205 according to the present invention, cytokines in the lungs extracted from a lethal XDR pulmonary tuberculosis animal model were quantified. As a result, all proinflammatory cytokines tested (IFN-γ, TNF-α, IL-6, and IL-10) increased after M. tuberculosis infection, but in mice administered Bacillus subtilis PMC 205 according to the present invention, the cytokines were significantly decreased (Fig. 4A). Analysis of the lung microbiome based on the 16S rRNA gene showed that in mice infected with M. tuberculosis, the genus Mycobacterium increased in the lungs and the species diversity decreased, whereas in mice administered Bacillus subtilis PMC 205 according to the present invention, the mycobacteria decreased and the species diversity increased again, confirming that the normal bacterial flora was restored (Fig. 4B).

[0121]

[0122] 5. Evaluation of the efficacy of Bacillus subtilis PMC 205 against M. tuberculosis in the lungs in a mouse model of latent tuberculosis.

[0123] The anti-tuberculosis effect of Bacillus subtilis PMC 205 according to the present invention was also confirmed in a mouse model of latent pulmonary tuberculosis. M. tuberculosis was inhaled into the lungs of mice at a low dose, and after one month, Bacillus subtilis PMC 205 was administered by pulmonary inhalation once a day, twice a week for 8 months. As a result, no weight loss was observed in the mice administered Bacillus subtilis PMC 205 according to the present invention (Fig. 5A), and as a result of analyzing the extracted lungs, it was confirmed that in the mice administered Bacillus subtilis PMC 205 according to the present invention, M. tuberculosis was reduced by approximately log 1.3, which was similar to the effect of mice administered 30 mg / kg / day RIF (Fig. 5B).

[0124]

[0125] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0126]

[0127] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0128] Accession number: KCTC15188BP

[0129] Date of Consignment: November 9, 2022

[0130]

[0131] The present invention provides a Bacillus subtilis PMC 205 strain, which exhibits antibacterial activity. More specifically, the strain exhibits antibacterial activity against Mycobacterium tuberculosis. Accordingly, the strain is expected to be useful in treating, preventing, or ameliorating tuberculosis.

[0132]

[0133] agtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgccgaaggtgggacagatgattg

Claims

1. Bacillus subtilis PMC 205 strain or its culture solution having antibacterial activity against bacteria belonging to the genus Mycobacterium. Bacillus subtilis PMC 205 strain or a culture solution thereof, characterized in that the strain has the deposit number KCTC 15188BP extracted from kimchi.

2. In paragraph 1, The bacteria belonging to the genus Mycobacterium are Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium A Bacillus subtilis PMC 205 strain or a culture thereof, characterized in that at least one is selected from the group consisting of M. leprae, M. malmoense, M. marinum, M. microti, M. phlei, M. scrofulaceum, M. smegmatis, M. triplex, M. tuberculosis, M. ulcerans, and M. xenopi.

3. An antibacterial composition having antibacterial activity against bacteria belonging to the genus Mycobacterium, comprising the Bacillus subtilis PMC 205 strain according to paragraph 1 or 2 or a culture solution thereof.

4. In paragraph 3, An antimicrobial composition characterized in that the above antimicrobial composition is used in a living body.

5. A pharmaceutical composition for the treatment or prevention of tuberculosis having antibacterial activity against bacteria belonging to the genus Mycobacterium, comprising the Bacillus subtilis PMC 205 strain or a culture solution thereof according to paragraph 1 or 2 as an active ingredient.

6. In paragraph 5, A pharmaceutical composition characterized in that the above tuberculosis is at least one selected from the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymph node tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymph node tuberculosis, pulmonary tuberculosis, breast tuberculosis, and spinal tuberculosis.

7. In paragraph 5, A pharmaceutical composition characterized in that the above tuberculosis is latent tuberculosis.

8. In paragraph 5, The pharmaceutical composition comprises rifampicin, rifapentine, isoniazid, pyrazinamide, ethambutol, streptomycin, fluoroquinolone, kanamycin, cycloserine, prothionamide, levofloxacin, moxifloxacin, ofloxacin, rifabutin, capeomycin, amikacin, ciprofloxacin, protionamide, ethionamide, cycloserine, thioacetazone, A pharmaceutical composition characterized in that it further comprises at least one selected from the group consisting of clofazimine, amoxicillin / clavulanate, a derivative of dianomidiphenylsulphone, clarithromycin, azithromycin, and linezolid.

9. A food composition for preventing or improving tuberculosis having antibacterial activity against bacteria belonging to the genus Mycobacterium, comprising the Bacillus subtilis PMC 205 strain or a culture solution thereof according to paragraph 1 or 2 as an active ingredient.

10. In paragraph 9, A food composition characterized in that the above tuberculosis is at least one selected from the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymph node tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymph node tuberculosis, pulmonary tuberculosis, breast tuberculosis, and spinal tuberculosis.

11. In paragraph 9, A food composition characterized in that the above tuberculosis is latent tuberculosis.

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