KR102613646B1 - Novel bacillus velezensis with antivacterial activity for antibiotics resistant enterobacteia - Google Patents

Abstract

The present invention relates to a novel Bacillus velezensis strain and its use. The novel Bacillus velezensis of the present invention is resistant not only to pathogenic enteric bacteria but also to antibiotic-resistant enteric bacteria, especially carbapenems. It can effectively prevent and treat intestinal bacteria, and can be used for various purposes such as therapeutic agents and antibacterial agents.

Description

Novel Bacillus velegensis strain with antibacterial activity against antibiotic-resistant enteric bacteria {NOVEL BACILLUS VELEZENSIS WITH ANTIVACTERIAL ACTIVITY FOR ANTIBIOTICS RESISTANT ENTEROBACTEIA}

The present invention relates to a novel Bacillus velezensis that has antibacterial activity against antibiotic-resistant enteric bacteria and its use.

Antimicrobial resistance is when pathogens that were eliminated by antibiotic treatment become resistant and can no longer be treated with the same antibiotic. According to a report published in 2022, there were 4.95 million deaths related to antibiotic resistance in 2019. Among them, 1.27 million people died directly due to antibiotic resistance. Moreover, since resistance to carbapenems, the most desirable drugs of last resort to treat multidrug resistant bacterial infections, was reported in Enterobacteriaceae strains in the early 1990s, carbapenemase-producing Carbapenemase-producing Enterobacteriaceae (CRE) is spreading around the world, causing a crisis in global health care.

Meanwhile, the species that causes CRE the most is Klebsiella pneumoniae, which caused more than 55,700 deaths in 2019. As the use of carbapenems in clinical practice increases, the evolution of carbapenem-resistant bacteria is a significant concern. Although standards for treating CRE infections are being developed, problems such as resistance, treatment failure, and toxicity still remain, and the development of effective anti-CRE drugs is urgently needed.

Republic of Korea Patent Publication No. 10-2020-0126261

Accordingly, the inventor of the present invention confirmed that the novel Bacillus velezensis strain is effective for infection and treatment of not only pathogenic enteric bacteria but also antibiotic-resistant enteric bacteria, and completed the present invention.

Accordingly, the purpose of the present invention is to provide a novel Bacillus velezensis strain, a pharmaceutical composition, an antibacterial composition, and a food composition for preventing or treating diseases caused by pathogenic enteric bacteria containing the strain. .

In order to achieve the above object, the present invention provides a novel Bacillus velezensis strain deposited under the deposit number KCTC 15186BP.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by pathogenic intestinal bacteria, comprising the above-mentioned strain, its lysate, or its culture medium as an active ingredient.

In addition, the present invention provides an antibacterial composition against antibiotic-resistant enteric bacteria, comprising the above strain, its lysate, or its culture medium as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving diseases caused by pathogenic intestinal bacteria, comprising the above-mentioned strain, its lysate, or its culture fluid as an active ingredient.

The novel Bacillus velezensis strain of the present invention has antibacterial activity not only against pathogenic enteric bacteria but also against antibiotic-resistant enteric bacteria, so it can provide a new therapeutic agent that can solve the problem of antibiotic resistance.

Figure 1 shows the results of bacterial growth and pH measurements over time in food stage media. Probiotic strains were inoculated and cultured to check growth (A) and pH (B) for 2 hours.
Figure 2 shows the effect of B. velezensis on clinical isolates of CRKP in a prophylactic infection mouse model. (A) Body weight and (B) disease severity during the experimental period. Disease severity was 1, healthy; 2, slightly painful; 3, moderate pain; 4, Severe pain; 5, assessed as death. (C) Survival rate during the experiment. (D) CRKP treated group (left image), CRKP + B. velezensis treated group (right image). Statistical significance compared to the control was analyzed using unpaired Student's t-test (*** p <0.001; ** p <0.01; * p < 0.05).
Figure 3 shows the effect of B. velezensis on clinical isolates of CRKP in a therapeutic infection mouse model. (A) Body weight and (B) disease severity during the experimental period. Disease severity was 1, healthy; 2, slightly painful; 3, moderate pain; 4, Severe pain; 5, assessed as death. (C) Survival rate during the experiment. (D) CRKP treated group (left image), CRKP + B. velezensis treated group (right image). Statistical significance compared to control was analyzed using unpaired Student's t-test (*** p <0.001; ** p <0.01; * p < 0.05).
Figure 4 shows the results of a 2-week repeated oral administration toxicity test of B. velezensis in guinea pigs. Guinea pigs were weighed on days 1, 3, 5, 7, 9, 10, and 14. Candidate probiotics were administered at 2×10 ⁸ CFU/mouse/day at 200 μl per animal once daily for 2 weeks.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. Additionally, when describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

The present invention relates to a novel Bacillus velezensis strain deposited with deposit number KCTC 15186BP.

The Bacillus belegensis may be a strain derived from bokbunja vinegar, but is not limited thereto.

The Bacillus belegensis may have antibacterial activity against pathogenic enteric bacteria, and the enteric pathogenic bacteria include Klebsiella pneumoniae , Vibrio cholerae, Salmonella gallinarum, and Salmonella. Salmonella enteritidis, Salmonella typhimurium, Salmonella cholerasuis, Salmonella derby, Staphylococcus aureus, enterotoxigenic E. coli , Listeria monocytogenes, Campylobacter jejuni, Shigella flexneri, Salmonella enterica, Clostridium difficile, vancomycin-resistant Enterica It may include, but is not limited to, vancomycin resistant enterococcus, Clostridium perfringens, etc.

The Bacillus belegensis may have antibacterial activity against antibiotic-resistant enteric bacteria, the antibiotic-resistant enteric bacteria may be Klebsiella pneumoniae , and the antibiotic may be a carbapenem. , but is not limited to this.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of diseases caused by pathogenic intestinal bacteria, comprising the Bacillus belegensis strain, its lysate, or its culture medium as an active ingredient.

In addition, it relates to an antibacterial composition against antibiotic-resistant enteric bacteria, comprising the Bacillus belegensis strain, its lysate, or its culture medium as an active ingredient.

The diseases caused by pathogenic intestinal bacteria include food poisoning, typhoid fever, peritonitis, ulcerative colitis, Crohn's disease, intestinal Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, neuropathic enteritis syndrome, small intestinal microbial overgrowth, enteral-feeding diarrhea, or ischemic disease. It may be enteritis, but is not limited to this.

The antibiotic-resistant enteric bacteria may be Klebsiella pneumoniae , but are not limited thereto.

The term “culture medium” used in the present invention is obtained after seeding the target microorganism in a medium containing elements suitable for the growth of microorganisms and culturing it for a certain period of time, and is a concept that includes microorganisms and medium.

The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. The auxiliary agent may be any one known in the art without limitation, but the effect may be increased by further including, for example, Freund's complete auxiliary agent or incomplete auxiliary agent.

The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .

When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected taking into account the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ㎍/ml, it may be toxic to the human body.

In addition, the present invention provides a food composition for preventing or improving diseases caused by pathogenic enteric bacteria, comprising the Bacillus belegensis, its lysate, or its culture fluid as an active ingredient.

In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition to the extract as an active ingredient, the food composition contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating tuberculosis. In the present invention, 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and refers to food that is related to the structure and function of the human body. It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, the health functional food in the form of a tablet is made by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or The mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention and excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agent. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[Example]

<Example 1> CRKP

CRKP (carbapenem-resistant Klebsiella pneumoniae) was obtained from the Pathogenic Resource Bank of Soonchunhyang University Hospital (Tajdozian et al. 2021), cultured in MacConkey broth (BD Difco, USA), and incubated at 37°C for 18 hours. Then, bacterial growth was measured using a spectrophotometer (DR 1900, HACH, USA), and the optical density at 600 nm (OD600 nm) was adjusted to 1.0. Then, the number of bacteria was counted using colony-forming unit (CFU) assay. For subsequent use, stock cultures in 60% glycerol were prepared and stored at -80°C.

<Example 2>

Isolation and identification of probiotic strains from naturally fermented vinegar

Candidate probiotics were obtained from Gochang bokbunja vinegar obtained from Gochang, Jeolla-do and fermented for 14 days. Candidate probiotics were transferred to MRS agar (BD Difco, USA) plates and incubated at 37°C for 24 hours. Then, single colonies were selected and subcultured in MRS broth (BD Difco, USA). After overnight incubation in a shaking incubator at 37°C under aerobic conditions, bacterial growth was measured with a spectrophotometer. For subsequent use, stocks in 60% glycerol were prepared and stored at -80°C. The probiotic strain was requested from a specialized company (Biofact Co, Daejeon, Korea) for 16S rRNA sequencing and species confirmation.

biochemical testing

Biochemical tests were performed using the API 50CH kit (bioMerieux, France) to compare the carbohydrate expression patterns of the probiotic strains with other reference strains. After culturing the candidate probiotic strains on MRS agar plates, single colonies were selected and mixed with 1 ml suspension medium. Then, the turbidity of the suspension was determined using 2 McFarland and 0.1 ml of the suspension was diluted in 10 ml of CHB medium (bioMerieux, France). Then, the strips were removed from the API kit and placed on a tray. Finally, the prepared bacterial suspension was transferred to the wells of the strip and incubated at 37 °C for 48 hours. Afterwards, the color change of each well was observed and an identification tag was written as (+/-).

Quantitative RT-PCR assay

qRT-PCR was performed to confirm the isolated strain. Genomic DNA was extracted from the isolated candidate probiotic strains using the QIAamp DNA Mini Kit (QIAGEN, Germany). Genomic DNA was also extracted from B. velezensis KCTC 13417, Bacillus amyloliquefaciens KCTC 3002, Bacillus subtilis KCTC 3135 , and Bacillus lichenoformis KCTC 1659. The above strains were obtained from the Korean Collection for Type Cultures (KCTC) as reference microorganisms for distinguishing closely related species of the Bacillus genus. Then, the extracted DNA was diluted and qRT-PCR was performed using species-specific primers. Primers for the macrolactin gene were used to distinguish B. velezensis and B. amyloliquefaciens . The primers used are shown in Table 1. qRT-PCR was performed in a final volume of 20 μl consisting of 10 μl of SYBER Green Supermix (BIO-RAD, USA), 4 μl nuclease-free water, 5 μl of genomic DNA, and 0.5 μl each of forward and reverse primers. Amplification was performed according to the following thermal cycling steps: an initial DNA denaturation step at 95 °C for 3 min, 39 cycles at 95 °C for 10 s for denaturation, 55 °C for 10 s for annealing, and 72 °C for 30 s for extension. .

result

Identification based on 16S rRNA gene sequencing

16S rRNA gene sequencing analysis was performed on the isolates (Table 2), and the results were compared with sequences in the National Center for Biotechnology Information (NCBI) database. 16S rRNA gene sequencing of the probiotic strains identified B. velezensis strain FZB42, B. velezensis strain CBMB205, B. amyloliquefaciens strain NBRC 15535, and B. subtilis subsp. It had 99% sequence homology with the 16S rRNA gene sequence of the subtilis strain, and also showed 98% sequence homology with B. licheniformis strain DSM 13 and B. licheniformis strain BCRC 11702. These findings suggest that the isolated strain belongs to the genus Bacillus .

Carbon usage assay of isolated probiotic strains by API kit

To confirm and characterize the phenotype of the probiotic strain, biochemical characterization was performed using the API 50CH Biochemical Kit. The carbon source usage patterns of the isolated probiotic strains were compared with B. velezensis KCTC 13417, B. amyloliquefaciens KCTC3002, and B. lichenoformis KCTC 1659, and the results are shown in Table 3. The candidate strain showed a carbon source usage pattern more similar to B. velezensis than to B. licheniformis and B. amyloliquefaciens , but had some differences from KCTC B. velezensis .

Identification of isolated probiotic strains by qRT-PCR assay

Candidate probiotic strains were identified by performing qRT-PCR assays using species-specific primer sets (Table 4), and the resulting threshold cycles (Ct values) were analyzed. It was found that the B. velezenesis specific primer was specific to both B. velezensis KCTC 13417 and the candidate probiotic strain of the present invention, but other closely related species were not detected. The amplification results of B. amyloliquefaciens KCTC 3002 , B.velezensis KCTC 13417, and candidate probiotic strains were observed using B. amyloliquefaciens-specific primers, but the primers did not work specifically. Therefore, to distinguish B. amyloliquefaciens and B. velezensis , PCR was performed using macrolactin-specific primers. Macrolactin is a gene cluster detected only in B. velezensis . After performing PCR using macrolactin-specific primers, it was confirmed that the amplification cycle of B. velezensis KCTC 13417 and the candidate probiotic strain was similar, but not similar to that of B. amyloliquefaciens KCTC 3002. These results clearly demonstrate that the candidate probiotic strain is B. velezensis . Species-specific primers for B. lichenoformis and B. subtilis amplified B. lichenoformis KCTC 1659 and B. subtilis KCTC 3135, respectively, but not for other strains. Based on these results, it was confirmed that the isolated probiotic strain was B. velezensis , and the Bacillus velezensis strain (KCTC 15186BP) was deposited as a patented microorganism at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on November 9, 2022. It was carried out.

<Example 3>

using laboratory-scale fermenters based on food-grade media. B. velezensis culture of

Food-grade medium (FGM) was used to cultivate the strains of the present invention, which have anti-CRE therapeutic potential. Since the strain of the present invention grows well in FGM, it was used for culture. Additionally, FGM is harmless to humans and is based on MRS medium, consisting of glucose, yeast peptone, Tween 80, and magnesium sulfate (Table 5). The pH of the optimized FGM was 7.5, and the pH was adjusted to 6.3 using 6 N HCl. Before growing in the fermenter system, strains were cultured in 30 ml FGM. After incubation at 37°C for 18 hours, the OD and pH of the culture broth were checked with a spectrophotometer and pH meter (METTLER TOLEDO, Switzerland) every 2 hours during the incubation period. After confirming bacterial growth and pH, probiotic strains were grown in a laboratory-scale fermentor system (FMT-ST-S07, Fermentec, South Korea). The fermenter was first filled with 2L of FGM. Then, the fermenter filled with FGM was autoclaved at 121°C for 15 minutes. Then, once the medium reached the optimal temperature, 1.0% of the probiotic culture was aseptically added to the fermenter system and incubated at 37 °C for 10 h using the fermentor impeller. After incubation, cultures were recovered in sterile containers, centrifuged, washed, and resuspended in culture supernatant at 1.5 × 10 ⁹ CFU/200 μl for each treatment model mouse. FGM-cultured probiotics were used in the treatment model, whereas MRS-cultured probiotics were used in the prevention model and toxicity experiments.

result

Probiotic strains were cultured in FGM and bacterial growth and pH were checked every 2 hours (Figure 1). Figure 1a shows the growth curve of candidate probiotic strains. Its OD reached 0.8 after 10 hours of incubation. The pH of the broth culture was also checked, and it was confirmed that the pH decreased after 2 hours of incubation, and the decrease continued slowly until 18 hours (Figure 1b). Based on these results, the strain of the present invention was cultured in a laboratory-scale fermentor system.

<Example 4>

Effects of Bacillus belegensis in a CRKP-infected mouse model

To evaluate the efficacy of probiotics in a CRKP-infected mouse model, 9-week-old female BALB/c mice (Dooyeol Biotech, Korea) were used as prevention and treatment models. The prevention model is intended to confirm the preventive effect of administering probiotics before infection, and the treatment model is intended to confirm the treatment effect by administering probiotics after infection. At the time of probiotic treatment in both models, the probiotic-treated group received B. velezensis . However, the infected and control groups were administered distilled water orally for the same treatment conditions. In both models, oral administration of sodium bicarbonate (NaHCO ₃ ) for gastrointestinal neutralization increased the intestinal arrival of live pathogens, thereby increasing the severity of the disease in the animals. Additionally, neutropenia was induced by intraperitoneal injection of cyclophosmide 3 days before infection. Fluorouracil (5-FU) was also administered intraperitoneally to induce additional immunosuppression. Disease severity score, body weight, and survival rate of both models were determined during the experimental period.

The preventive effect was confirmed in BALB/c mice infected with CRKP185 of B. velezensis, a candidate probiotic strain. The probiotic strain was administered at 200 μl of 1.5 × 10 ⁹ CFU/mouse daily as a single dose by oral gavage 3 days before infection. 450 mg/kg (200 μl) cyclophosmide (SigmaAldrich, USA) was administered intraperitoneally 3 days before infection. On day ³ after inducing neutropenia, administer via oral administration at a dose of ^6.7 Infection was induced twice. NaHCO ₃ (0.2M, 200 μl) (SigmaAldrich, USA) was administered along with the infection. 5-FU (50 mg/kg, 200 μl) (Sigma Aldrich, USA) was administered intraperitoneally on days 13 and 15 after induction of infection.

The therapeutic potential of B. velezensis , a candidate probiotic strain, was studied in CRKP-infected BALB/c mice. In the therapeutic mouse model, 200 μl of cyclophosmide (450 mg/kg) was injected intraperitoneally 3 days prior to infection. Then, after pretreatment with NaHCO ₃ (0.2M, 200 μl), infection was induced by oral administration of 200 μl CRKP at 9 × 10 ⁹ CFU/mouse on days 0, 2, and 6. The next day after infection, mice were treated with B. velezensis twice at 1.5×10 ⁹ CFU/200μl/mouse. Then, 5-FU (50 mg/kg, 200 μl) was injected intraperitoneally as a single dose on days 10 and 14.

result

In CRKP-infected mice B. velezensis Evaluation of the preventive effect of.

The preventive effect of the candidate strains was evaluated (Figure 2). During the experimental period, the body weight of untreated mice decreased significantly compared to mice treated with B.velezensis (Figure 2a). On days 18 and 20, the weight loss rate for the treated group was 8.4%, compared to 35.8% for the untreated group. Probiotic treatment groups and disease severity scores were also confirmed from days 0 to 21 (Figure 2b). In the group that was not treated with probiotics, the severity score steadily increased during the experiment and then increased sharply in the second to third weeks, but the increase rate was lower in the probiotic-treated group. The survival rates of the two groups of mice are shown in Figure 2c. The untreated group showed a survival rate of 60% on day 20, and all mice died on day 21, whereas 80% of mice in the treated group survived until the end of the experiment. Figure 2d shows images of different groups of mice. At the end of the experiment, it was found that the infected group not treated with probiotics all died (left image), but the mice in the probiotic-treated group were healthy (right image). These results indicate that the candidate probiotic strain showed a preventive effect against CRKP infection in a mouse model.

In CRKP-infected mice B. velezensis Evaluation of treatment effectiveness.

The therapeutic effect of B. velezensis in lethal CRKP-infected mice was evaluated (Figure 3). On days 13 and 18, the untreated group lost 29.5% of body weight, while the treated group showed very little weight loss (Figure 3A). Disease severity was measured in the probiotic-treated and infected groups on days 0 and 20 of the experiment (Figure 3b). Differences in disease scores between groups were observed. The survival rate of mice is shown in Figure 3c. The survival rate of the untreated group decreased to 50.2%, whereas all mice in the treated group survived. Figure 3D shows images after CRKP infection, where the infected group was diseased or eventually died (left image), while all mice in the probiotic-treated group were healthy (right image).

<Example 5>

Evaluation of repeated oral acute toxicity of probiotics in guinea pigs.

A 2-week repeated oral acute toxicity evaluation of probiotics was conducted in guinea pigs. It was administered orally to adult male guinea pigs (body weight range from 1,000 to 1,280 g), and sterile water was used as an excipient. In the treatment group, probiotics (2×10 ⁸ CFU/ml/200 μl) were administered once daily and clinical symptoms, mortality, and body weight changes were observed throughout the entire period.

As a result, no significant change in body weight was observed in the group (Figure 4). Additionally, no unusual clinical symptoms or deaths were observed in guinea pigs treated with B. velezensis (Table 6).

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center KCTC15186BP 20221109

Claims (12)

A new Bacillus velezensis strain derived from Bokbunja vinegar deposited with accession number KCTC 15186BP. delete According to clause 1,
The Bacillus belegensis strain has antibacterial activity against pathogenic enteric bacteria. According to clause 3,
The pathogenic enteric bacteria include Klebsiella pneumoniae , Vibrio cholerae, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Salmonella cholerasuis, Salmonella derby, Staphylococcus aureus, enterotoxigenic E. coli, Listeria monocytogenes, Campylobacter jejuni ), Shigella flexneri, Salmonella enterica, Clostridium difficile, vancomycin resistant enterococcus and Clostridium perfringens , a strain selected from the group consisting of. According to clause 1,
The Bacillus belegensis is a strain that has antibacterial activity against antibiotic-resistant enteric bacteria. According to clause 5,
The antibiotic-resistant intestinal bacteria are strains that reduce Klebsiella pneumoniae . According to clause 5,
The antibiotic is a carbapenem. A pharmaceutical composition for the prevention or treatment of diseases caused by pathogenic intestinal bacteria, comprising the Bacillus belegensis strain (KCTC 15186BP) of claim 1, its lysate, or its culture medium as an active ingredient. According to clause 8,
The diseases caused by pathogenic intestinal bacteria include food poisoning, typhoid fever, peritonitis, ulcerative colitis, Crohn's disease, intestinal Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, neuropathic enteritis syndrome, small intestinal microbial overgrowth, intestinal feeding diarrhea, and ischemic disease. A composition selected from the group consisting of enteritis. An antibacterial composition against antibiotic-resistant enteric bacteria, comprising the Bacillus belegensis (KCTC 15186BP) of claim 1, its lysate, or its culture medium as an active ingredient. According to clause 10,
The composition, wherein the antibiotic-resistant enteric bacteria are Klebsiella pneumoniae . A food composition for preventing or improving diseases caused by pathogenic intestinal bacteria, comprising the Bacillus velegensis (KCTC 15186BP) of claim 1, its lysate, or its culture fluid as an active ingredient.