WO2020055193A1

WO2020055193A1 - Microorganism with antibacterial activity for enteric pathogenic bacteria and pharmaceutical composition for preventing and treating enteric pathogenic bacteria induced disease using the same

Info

Publication number: WO2020055193A1
Application number: PCT/KR2019/011879
Authority: WO
Inventors: Sang Sun Yoon; Mi Young Yoon; Jin Sun You
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2018-09-14
Filing date: 2019-09-11
Publication date: 2020-03-19
Also published as: KR102269450B1; KR20200031543A; KR20210080303A; KR102363357B1; WO2020055193A9

Abstract

Provided are a pharmaceutical composition for preventing or treating enteric pathogenic bacteria-inducing diseases and food composition for improving enteric infection by enteric pathogenic bacteria, including a Bacteroides vulgatus microorganism with antibacterial activity for enteric pathogenic bacteria or a culture medium thereof, as an active ingredient.

Description

MICROORGANISM WITH ANTIBACTERIAL ACTIVITY FOR ENTERIC PATHOGENIC BACTERIA AND PHARMACEUTICAL COMPOSITION FOR PREVENTING AND TREATING ENTERIC PATHOGENIC BACTERIA INDUCED DISEASE USING THE SAME

The present disclosure relates to a microorganism with antibacterial activity for enteric pathogenic bacteria and a pharmaceutical composition for preventing or treating enteric pathogenic bacteria-inducing diseases including the same, and more particularly, to a microorganism that symbiosis with enteric pathogenic bacteria to inhibit growth or activity thereof, a pharmaceutical composition effective against diseases induced by infection of pathogenic bacteria by including the microorganism as an active ingredient, and a food composition for improving enteric infection by enteric pathogenic bacteria.

Enteric pathogenic bacteria include Salmonella, Cholera, Escherichia coli, and Typhoid, and the like, and these pathogenic bacteria may cause enteric diseases such as food poisoning and enteritis when infected in the intestines.

As a method of treating such infectious diseases, administration of antibiotics that kill or reduce causative pathogens may be proposed. Treatment based on antibiotic administration may provide an immediate therapeutic effect for infectious diseases in a short time. However, such treatment may cause antibiotic abuse, thereby increasing the frequency of the appearance of various antibiotic-resistant pathogens. For example, Salmonella typhimurium DT104 has been reported as a pathogen with a multiple antibacterial resistance pattern against antibiotics such as ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. Furthermore, antibiotic treatment causes changes in the enteric microbial community in humans and other mammals and may increase susceptibility of a host to infection by enteric pathogenic bacteria such as Shigella flexneri, Salmonella enterica, Clostridium difficile, and vancomycin-resistant Enterococcus.

For this reason, the development of a new therapeutic composition that effectively inhibits enteric pathogenic bacteria, has a therapeutic effect on infectious diseases caused by enteric pathogens, and reduces the use of antibiotics is continuously required.

The background art of the present disclosure has been prepared to more facilitate understanding of the present disclosure. It should not be understood that the matters described in the background art of the invention exist as prior arts.

The inventors of the present disclosure noticed that the antibiotic resistance of a host may be implemented through a number of factors, including inhibition of specific microbial species, changes in the metabolic environment and/or host responses as a result of a changed microbial community.

Furthermore, the inventors of the present disclosure further paid attention to the association between the onset of infectious enteric diseases by enteric pathogens and the enteric microbial community.

More specifically, in order to analyze the change in the enteric microbial community according to the antibiotic treatment, the inventors of the present disclosure observed a change in enteric microbial community after infecting Vibrio cholerae, one of the enteric pathogens in mice inoculated with a plurality of types of antibiotics.

As a result, the inventors of the present disclosure have observed species that increase in the enteric microbial community according to infection of Vibrio cholerae.

At this time, the inventors of the present disclosure confirmed that the microorganism of the species was co-cultured with Vibrio cholerae, as a result, the growth of Vibrio cholerae was inhibited.

Thus, the inventors of the present disclosure have found a new microorganism with enteric pathogen suppression ability and recognized that the microorganism may be used for the treatment and prevention of infection of the pathogen and thus enteric disease.

As a result, the inventors of the present disclosure have developed a pharmaceutical composition for the prevention or treatment of enteric pathogenic bacteria-inducing diseases including a microorganism with antibacterial activity for enteric pathogenic bacteria.

On the other hand, the inventors of the present disclosure identified newly discovered microorganism with antibacterial activity for enteric pathogenic bacteria and thus confirmed that the new microorganism belonged to the Bacteroides vulgatus species.

Therefore, an object to be solved by the present disclosure is to provide a microorganism of Bacteroides vulgatus VIC01 (Accession number: KCTC18713P) with antibacterial activity for enteric pathogenic bacteria and a pharmaceutical composition for preventing or treating enteric pathogenic bacteria-inducing diseases and a food composition for improving enteric infection by enteric pathogenic bacteria including the same.

The objects of the present disclosure are not limited to the aforementioned objects, and other objects, which are not mentioned above, will be apparent to a person having ordinary skill in the art from the following description.

In order to solve the problems as described above, the present disclosure provides a Bacteroides vulgatus VIC01 (Accession number: KCTC18713P) microorganism with antibacterial activity for enteric pathogenic bacteria.

At this time, in the present specification, the Bacteroides vulgatus microorganism, the microorganism with antibacterial activity for enteric pathogenic bacteria, the novel microorganism, and the microorganism of the present disclosure may be referred to as a Bacteroides vulgatus VIC01 (Accession number: KCTC18713P) microorganism.

The term "antibacterial activity" used in this specification may mean the activity or growth inhibition of enteric pathogenic bacteria, furthermore, the killing of pathogenic bacteria. However, it is not limited thereto, and in the present specification, "antibacterial activity" may mean inhibitory activity of all phenomena caused by enteric pathogenic bacteria.

According to a feature of the present disclosure, the enteric pathogenic bacteria may be at least one selected from the group consisting of Vibrio cholerae, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Salmonella choleraesuis, Salmonella derby, Staphylococcus aureus, enterotoxigenic E. coli, Listeria monocytogenes, Campylobacter jejuni, Shigella flexneri, Salmonella enterica, Clostridium difficile, vancomycin-resistant Enterococcus and Clostridium perfringens.

However, enteric pathogenic bacteria are not limited to those described above and may be various microorganisms causing enteric infection.

According to another feature of the invention, the Bacteroides vulgatus VIC01 (Accession number: KCTC18713P) microorganism may have a base sequence having at least 80% homology with a base sequence represented by SEQ ID NO: 1.

The term "homology" used in this specification may refer to the same degree as the base sequence disclosed in the present specification. Specifically, the comparison of homology may represent homology between two or more sequences in percentage (%). For example, through the comparison of homology, a microorganism having at least 80% homology with the base sequence represented by SEQ ID NO: 1, preferably a microorganism having at least 90% homology, and more preferably a microorganism having at least 95% homology may substantially have enteric pathogen inhibitory ability.

According to yet another feature of the present disclosure, the microorganism may be microorganisms producing short-chain fatty acids (SCFAs).

The term "short-chain fatty acid" used in the present specification refers to a fatty acid with one double bond.

At this time, the level of short-chain fatty acids may decrease in patients with enteric infection symptoms of pathogens such as diarrhea and may increase in patients who have returned to normal. That is, the level of short-chain fatty acids may be associated with enteric infectious pathogens, furthermore, diseases caused by these pathogens. For example, the short-chain fatty acids may provide an effective barrier against the invasion of pathogens. Furthermore, the short-chain fatty acids may suppress the activity for pathogens.

Meanwhile, according to yet another feature of the present disclosure, the short-chain fatty acid may include at least one selected from the group consisting of butyric acid, isobutyric acid, propionic acid, valeric acid, and isovaleric acid. However, the present disclosure is not limited thereto.

In order to solve the problems as described above, the present disclosure provides a pharmaceutical composition for preventing or treating enteric pathogenic bacteria-inducing diseases, including a Bacteroides vulgatus (Accession number: KCTC18713P) microorganism thereof with antibacterial activity for enteric pathogenic bacteria or a culture medium, as an active ingredient.

At this time, the Bacteroides vulgatus (Accession number: KCTC18713P) microorganism and the culture medium thereof may be excellent in bile resistance and acid resistance. In the case of administration, the Bacteroides vulgatus (Accession number: KCTC18713P) microorganism and the culture medium thereof are effective in preventing or treating diseases caused by enteric pathogenic bacteria by reaching the intestine to balance the enteric microbial community or substantially inhibit the activity of enteric pathogenic bacteria.

On the other hand, the term "culture medium" used in the present specification may refer to a liquid culture medium including the Bacteroides vulgatus (Accession number: KCTC18713P) microorganism of the present disclosure and a medium component necessary for its growth, or a culture filtrate which the Bacteroides vulgatus (Accession number: KCTC18713P) microorganism has been removed. In this case, the culture filtrate may include an active ingredient with antibacterial activity for enteric pathogenic bacteria secreted from the microorganism during the culture process.

The term "prevention" used in the present specification may mean all actions that inhibit diseases or delay the onset of the diseases by administration of the pharmaceutical composition, and "treatment" may refer to all actions in which symptoms of the diseases are improved or beneficially changed by the administration of the pharmaceutical composition.

According to a feature of the present disclosure, the enteric pathogenic bacteria-inducing disease may be at least one selected from the group consisting of food poisoning, typhoid, peritonitis, ulcerative colitis, Crohn's disease, intestinal Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, nervous enteritis syndrome, small intestinal bacterial overgrowth, intestinal acute diarrhea and ischemic enteritis. However, the present disclosure is not limited thereto, and the enteric pathogenic bacteria-inducing diseases may include all symptoms caused by infection of enteric pathogenic bacteria.

According to yet another feature of the present disclosure, the pharmaceutical composition may include the microorganism in an amount of 10⁵ to 10¹² CFU/mL, or the culture medium of the microorganism in an amount of 10⁵ to 10¹² ml/g, based on the total weight of the pharmaceutical composition. Preferably, the pharmaceutical composition may include the microorganism in an amount of 10⁷ to 10¹⁰ CFU/mL, or the culture medium of the microorganism in an amount of 10⁷ to 10¹⁰ ml/g, based on the total weight of the pharmaceutical composition. However, the content of the microorganism or culture medium of the microorganism is not limited thereto.

According to yet another feature of the present disclosure, the enteric pathogenic bacteria secrete the toxicity, and the microorganism may be effective in improving enteric disease by removing the secreted toxicity in the intestine. For example, when a pharmaceutical composition including a Bacteroides vulgatus (Accession number: KCTC18713P) microorganism or a culture medium thereof is administered, the level of enteric toxin by pathogenic bacteria may be reduced by about five times than before administration.

Meanwhile, the pharmaceutical composition according to an embodiment of the present disclosure can be formulated in various forms. For example, when the pharmaceutical composition is prepared in a solid formulation, the pharmaceutical composition may be prepared in the form of tablets, pills, powders, granules, and capsules. Such a solid formulation may further include one or more excipients, for example, starch, calcium carbonate, sucrose, lactose or gelatin. In addition, the composition of the solid formulation may further include lubricants such as magnesium stearate, talc and the like in addition to simple excipients. Furthermore, when the pharmaceutical composition according to one embodiment of the present disclosure is prepared as a liquid formulation, the pharmaceutical composition may be prepared in forms of aqueous solutions, suspensions and pharmaceutically acceptable fats and oils, alcohols or organic solvents, esters, emulsions, syrups or elixirs, suspensions reconstituted from non-foam granules, and foam formulations reconstituted from foam granules. At this time, the composition of the liquid formulation may be administered orally in a liquid dosage formulation in combination with an inert carrier. Suitable inert carriers may be pharmaceutically acceptable inert carriers such as ethanol, glycerol, and water. Furthermore, the composition of the liquid formulation may further include solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners and melt aids. In addition, the composition of the liquid formulation may further include colorants and flavoring agents to increase patient acceptance.

The pharmaceutical composition according to one embodiment of the present disclosure may be administered to a subject by oral or parenteral administration. More specifically, the parenteral administration may include intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration.

Meanwhile, a suitable formulation, dosage, and mode of administration of the pharmaceutical composition may include factors such as the age, weight, gender, the extent of disease symptoms, food ingested, time of administration, route of administration, rate of excretion and response sensitivity of the subject. In addition, the skilled physician or veterinarian may readily determine and prescribe the dosage and mode of administration of the pharmaceutical composition effective for the desired treatment.

Meanwhile, according to a feature of the present disclosure, the pharmaceutical composition may further include at least one metabolite selected from the group consisting of cholic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, isovaleric acid, and trimethylamine.

At this time, the metabolite may enhance the antibacterial activity of the microorganism according to various embodiments of the present disclosure.

According to another feature of the present disclosure, the metabolite may include a metabolite of the microorganism according to various embodiments of the present disclosure but is not limited thereto.

Preferably, the pharmaceutical composition of the present disclosure may include at least one metabolite selected from the group consisting of butyric acid, isobutyric acid, propionic acid, valeric acid, and isovaleric acid.

According to yet another feature of the present disclosure, the metabolite may be present at a concentration of 0.1 to 30 mM with respect to the composition. Preferably, the metabolite may be present at a concentration of 15 to 25 mM with respect to the pharmaceutical composition. More preferably, the metabolite may be present at a concentration of 18 to 22 mM with respect to the pharmaceutical composition. However, the concentration of the metabolite is not limited thereto, and may be set in various ranges according to a type of subject to be administered with the pharmaceutical composition, a desired therapeutic effect, and the like.

According to yet another feature of the present disclosure, the pharmaceutical composition may further include short-chain fatty acids.

In order to solve the problems as described above, the present disclosure provides a food composition for improving enteric infection by enteric pathogenic bacteria, including a Bacteroides vulgatus (Accession number: KCTC18713P) microorganism thereof with antibacterial activity for enteric pathogenic bacteria or a culture medium, as an active ingredient.

At this time, a kind of food to which the food composition of the present disclosure may be added is not particularly limited. Examples of the foods which may be added with the food composition of the present disclosure may include drinks, meat, sausages, bread, biscuit, rice cake, chocolate, candies, snacks, cookies, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcohol drinks, vitamin complex, and the like, and include all general health functional foods.

According to the feature of the present disclosure, the food composition may include the Bacteroides vulgatus (Accession number: KCTC18713P) microorganism in an amount of 10⁵ to 10¹² CFU/mL, or the culture medium of the Bacteroides vulgatus (Accession number: KCTC18713P) microorganism in an amount of 10⁵ to 10¹² ml/g, based on the total weight of the food composition.

According to another feature of the present disclosure, the enteric pathogenic bacteria may secrete the toxicity, and the Bacteroides vulgatus (Accession number: KCTC18713P) microorganism may remove the secreted toxicity in the intestine.

According to yet another feature of the present disclosure, the food composition of the present disclosure may further include at least one metabolite selected from the group consisting of cholic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, isovaleric acid, and trimethylamine.

According to yet another feature of the present disclosure, the metabolite may be present at a concentration of 0.1 to 30 mM with respect to the food composition.

According to yet another feature of the present disclosure, the food composition of the present disclosure may further include short-chain fatty acids (SCFAs).

The present disclosure has an effect of providing a novel microorganism that inhibits enteric pathogenic bacteria.

At this time, the microorganism may induce balance of enteric microbial community or substantially induce the removal of the toxin generated by pathogens causing enteric infection and the inhibition of growth of pathogens causing enteric infection, thereby being used as the pharmaceutical compositions for preventing or treating diseases caused by enteric pathogenic bacteria, and furthermore, the food composition for improving enteric infection by enteric pathogenic bacteria.

Therefore, the present disclosure may contribute to reducing the use of antibiotics and to solving problems caused by the use of antibiotics.

Furthermore, the present disclosure has an effect of providing an effective content of the microorganism in the pharmaceutical composition.

Further, the present disclosure provides a pharmaceutical composition and a food composition including a metabolite that contributes to the enhancement of antibacterial activity for enteric pathogenic bacteria, thereby providing a better therapeutic prognosis than a composition composed of a microorganism alone.

The effects according to the present disclosure are not limited by the contents exemplified above, and more various effects are included in the present specification.

The above and other aspects, features and other advantages of the present disclosure will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 illustrates a process of isolating microorganisms with antibacterial activity for enteric pathogenic bacteria, according to an embodiment of the present disclosure;

FIGS. 2A and 2B illustrate experimental results of analyzing colony formation of Vibrio cholerae after co-culture of microorganisms with antibacterial activity for enteric pathogenic bacteria, according to an embodiment of the present disclosure;

FIG. 2C illustrates experimental results of analyzing a concentration of toxin by Vibrio cholerae after co-culture of microorganisms with antibacterial activity for enteric pathogenic bacteria, according to an embodiment of the present disclosure;

FIG. 2D illustrates results of analyzing proliferation of Vibrio cholerae in the small intestine, large intestine, and feces of a mouse model according to whether microorganisms with antibacterial activity for enteric pathogenic bacteria are taken and whether Vibrio cholerae is infected, according to an embodiment of the present disclosure;

FIG. 3A illustrates a change in enteric microbial community, depending on antibiotic treatment;

FIG. 3B illustrates results of analyzing proliferation of Vibrio cholerae in the small intestine, colon, and feces of a germ-free mouse model according to a state of microorganisms with antibacterial activity for enteric pathogenic bacteria, according to an embodiment of the present disclosure;

FIG. 3C illustrates results of analyzing proliferation of Vibrio cholerae in the small intestine of an infant mouse model according to inoculation of microorganisms with antibacterial activity for enteric pathogenic bacteria, according to an embodiment of the present disclosure;

FIG. 4A illustrates a change in enteric microbial community, depending on antibiotic treatment;

FIG. 4B illustrates a change in metabolites isolated from the cecum depending on antibiotic treatment;

FIG. 4C illustrates a change in growth inhibition of Vibrio cholerae according to the treatment of metabolites, used in various compositions according to an embodiment of the invention;

FIG. 4D illustrates a change in growth stimulation of Vibrio cholerae according to the treatment of metabolites, used in various compositions according to an embodiment of the invention; and

FIG. 4E illustrates a change in infection of Vibrio cholerae according to the presence of microorganisms with antibacterial activity for enteric pathogenic bacteria and metabolites, used in various compositions according to an embodiment of the invention.

Advantages and features of the present disclosure, and methods for accomplishing the same will be more clearly understood from exemplary embodiments described in detail below with reference to the accompanying drawings. However, the present disclosure is not limited to the embodiments set forth below and will be embodied in various different forms. The present embodiments are just for rendering the disclosure of the present disclosure complete and are set forth to provide a complete understanding of the scope of the invention to a person with ordinary skill in the technical field to which the present disclosure pertains, and the present disclosure will only be defined by the scope of the claims.

Hereinafter, a method for isolating microorganisms used in various embodiments of the present disclosure and its inhibitory ability for enteric pathogenic bacteria will be described in detail with reference to Examples 1 and 2.

At this time, as an example of the enteric pathogenic bacteria, Vibrio cholerae is described, but a pathogenic bacteria inhibitory effect of microorganisms used in various embodiments of the present disclosure is not limited thereto. For example, the microorganism according to an embodiment of the present disclosure may have inhibitory ability for at least one enteric pathogenic bacterium selected from the group consisting of Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Salmonella choleraesuis, Salmonella derby, Staphylococcus aureus, enterotoxigenic E. coli, Listeria monocytogenes, Campylobacter jejuni, and Clostridium perfringens. Furthermore, the microorganism may be used as a pharmaceutical composition for the treatment or prevention of various diseases induced by enteric pathogenic bacteria infection.

Example 1: Method for isolating microorganisms used in various embodiments of the present disclosure

FIG. 1 illustrates a process of isolating microorganisms with antibacterial activity for enteric pathogenic bacteria according to an embodiment of the present disclosure.

Referring to FIG. 1, first, feces were collected from three 12 to 15-week-old mice of females of C57BL6/J for isolation of microorganisms with antibacterial activity for enteric pathogenic bacteria. Subsequently, a bacterial suspension was prepared using a pre-substituted BBE (Bacteroides Bile Esculin) broth medium under an anaerobic condition, and then diluted step by step by 10-step dilution. Next, the diluted suspension was spread on the BBE solid medium and incubated in an anaerobic incubator at 37°C for 48 to 72 hours. Next, formed colonies were again spread on a fresh BBE solid medium to confirm single bacteria.

By the above method, Bacteroides vulgatus species of the microorganisms constituting the enteric microbial community was obtained.

At this time, the obtained Bacteroides vulgatus microorganism was Bacteroides vulgatus VIC01 (Accession Number: KCTC18713P), which may be used in various embodiments.

Example 2: First evaluation of antibacterial activity of microorganisms used in various embodiments of the present disclosure

Hereinafter, first evaluation results for the microorganisms used in various embodiments of the present disclosure will be described with reference to FIGS. 2A to 2C.

FIGS. 2A and 2B illustrate experimental results of analyzing colony formation of Vibrio cholerae after co-culture of microorganisms with antibacterial activity for enteric pathogenic bacteria according to an embodiment of the present disclosure. FIG. 2C illustrates experimental results of analyzing a concentration of toxin by Vibrio cholerae after co-culture of microorganisms with antibacterial activity for enteric pathogenic bacteria according to an embodiment of the present disclosure. FIG. 2D illustrates results of analyzing proliferation of Vibrio cholerae in the small intestine, large intestine, and feces of a mouse model according to whether microorganisms with antibacterial activity for enteric pathogenic bacteria are taken and whether Vibrio cholerae is infected, according to an embodiment of the present disclosure.

Referring to FIG. 2A, colonies of Vibrio cholerae were formed after co-culture of microorganisms isolated from mouse feces and Vibrio cholerae under anaerobic conditions for one day by the method described above in Example 1. More specifically, in contrast to a medium cultured only Vibrio cholerae (Only Vc), in a medium co-cultured with the microorganisms isolated from mouse feces and Vibrio cholera (Bv and Vc), colony formation of Vibrio cholerae did not occur.

Such isolated Bacteroides vulgatus microorganisms may have inhibitory activity for enteric pathogenic bacteria, particularly Vibrio cholera. In this case, the Bacteroides vulgatus microorganisms may have activity to inhibit the activity or growth of Vibrio cholerae, and furthermore to kill Vibrio cholerae.

Referring to FIG. 2B, the viable cell count of Vibrio cholerae in a medium cultured only with Vibrio cholerae (Only Vc) was about 8 times higher than the viable cell count of live Vibrio cholerae in a medium co-cultured with the isolated Bacteroides vulgatus microorganism and Vibrio cholera (Bv and Vc). That is, such a result may mean that the isolated Bacteroides vulgatus microorganism inhibits formation of colonies of enteric pathogenic bacteria, particularly Vibrio cholerae.

Meanwhile, considering that the pH of the co-cultured medium was 7.51 and the pH of the medium cultured with only Vibrio cholerae was 6.40, the control of Vibrio cholerae of the Bacteroides vulgatus microorganism may be less associated with a decrease in pH.

Referring to FIG. 2C, through an ELISA experiment using an antibody for detecting CTX A and B subunit, it is illustrated a result of measuring CTX, a Vibrio cholerae toxin in a supernatant of the medium cultured only with Vibrio cholerae (Only Vc) and the medium co-cultured with the isolated Bacteroides vulgatus microorganism and Vibrio cholera (Bv and Vc). More specifically, a level of toxins of CTX in the medium co-cultured with the isolated Bacteroides vulgatus microorganism and Vibrio cholerae (Bv and Vc) was reduced about 5 times or more unlike the medium (Only Vc) cultured only with Vibrio cholerae.

Meanwhile, the level of CTX observed in the co-culture may be a measure of the residual amount of cholerae toxin already produced in a single culture before the initial co-culture.

Referring to FIG. 2D, with respect to a mouse model in which live Bacterioides vulgatus that may be a microorganism having bacterial inhibitory ability used in various embodiments of the present disclosure was ingested at 10⁹ CFU, after 10⁹ CFU of Vibrio cholerae was inoculated, the small intestine, large intestine and feces were isolated and then the number of Vibrio cholerae in each isolated sample was measured.

At this time, a mouse model ingesting PBS instead of live Bacteroides vulgatus was set as a negative control (Con).

More specifically, in the small intestine, large intestine, and feces obtained from a mouse model (Bv) ingesting Bacteroides vulgatus, fewer Vibrio cholerae than the control was shown. These results may mean that the proliferation of the pathogen, Vibrio cholerae was reduced by Bacteroides vulgatus.

According to the results of Examples 1 and 2 above, the Bacteroides vulgatus microorganism isolated from the mouse may be used as the microorganism with antibacterial activity for enteric pathogenic bacteria used in various embodiments of the present disclosure.

Furthermore, the microorganism may be used as a composition for inhibiting infection of enteric pathogenic bacteria, a pharmaceutical composition for treating or preventing diseases caused by the enteric pathogenic bacteria, and a food composition for improving enteric infection by enteric pathogenic bacteria. As a result, the pharmaceutical composition and the food composition according to an embodiment of the present disclosure may have an improved effect for at least one selected from the group consisting of food poisoning, typhoid, peritonitis, ulcerative colitis, Crohn's disease, intestinal Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, nervous enteritis syndrome, small intestinal bacterial overgrowth, intestinal acute diarrhea and ischemic enteritis.

Meanwhile, the pharmaceutical composition may include the microorganism in an amount of 10⁵ to 10¹² CFU/mL, or the culture medium of the microorganism in an amount of 10⁵ to 10¹² ml/g, based on the total weight of the pharmaceutical composition. However, the content of the microorganism or culture medium of the microorganism is not limited thereto.

Example 3: Second evaluation of antibacterial activity of microorganisms used in various embodiments of the present disclosure

Hereinafter, second evaluation results for the microorganisms used in various embodiments of the present disclosure will be described with reference to FIGS. 3A to 3C.

FIG. 3A illustrates a change in enteric microbial community, depending on antibiotic treatment. FIG. 3B illustrates results of analyzing proliferation of Vibrio cholerae in the small intestine, colon, and feces of a germ-free mouse model according to a state of microorganisms with antibacterial activity for enteric pathogenic bacteria according to an embodiment of the present disclosure. FIG. 3C illustrates results of analyzing proliferation of Vibrio cholerae in the small intestine of an infant mouse model according to inoculation of microorganisms with antibacterial activity for enteric pathogenic bacteria according to an embodiment of the present disclosure.

First, referring to FIG. 3A, changes in species-level of the enteric microbial community were shown for a mouse model group inoculated with Vibrio cholerae to an untreated control group (Con-Vc), a mouse model group inoculated with Vibrio cholerae after administration of streptomycin (SM-Vc), a mouse model group inoculated with Vibrio cholerae after administration of bencomycin (VAN-Vc), and a mouse model group inoculated with Vibrio cholerae after administration of clindamycin (CL-Vc), in addition to a mouse model group as an untreated control group (Con), a mouse model group administered with streptomycin (SM), a mouse model group administered with vancomycin (VAN), and a mouse model group administered with clindamycin (CL). At this time, the species-level analysis was performed via 16S rRNA sequencing analysis using PCR.

More specifically, in most groups, an abundance ratio of Bacteroides vulgatus species of Bacteroidetes phylum was higher than that of other species.

On the other hand, when comparing the mouse model group administered with bencomycin (VAN) and the mouse model group inoculated with Vibrio cholerae after the administration of bencomycin (VAN-Vc), a relative abundance ratio of Bacteroides vulgatus after infection with Vibrio cholerae was increased.

That is, this result may mean a change in the microbial colony due to enteric pathogen infection and may mean that the Bacteriodes vulgaris microorganism is associated with an immune response according to enteric Vibrio cholerae infection of a host.

Next, referring to FIG. 3B (a), in the following experiment, a germ-free (GF) mouse model group infected with 5 Х 10⁵ CFU/mL of Vibrio cholerae after ingesting 50 mL of PBS (Negative control, NC), a germ-free mouse model group infected with 5 Х 10⁵ CFU/mL of Vibrio cholerae after ingestion of 2 x 10⁹ CFU/mL of heat-treated Bacteroides vulgatus (Heat-killed B. vulgatus) microorganism, and a germ-free mouse model group infected with 5 Х 10⁵ CFU/mL of Vibrio cholerae after ingestion of 2 x 10⁹ CFU/mL of live Bacteroides vulgatus (Live B. vulgatus) microorganism were analyzed. At this time, the infection with Vibrio cholerae was performed after 24 hours of ingestion of Bacteroides vulgatus microorganism (or PBS), and the analysis was performed after 24 hours of the infection of Vibrio cholerae.

More specifically, referring to FIG. 3B (b), as results of the proliferation analysis of Vibrio cholerae in the small intestine, colon and feces, in the mouse model group ingesting the heat-treated Bacteroides vulgatus (Heat-killed B. vulgatus) microorganism, in all of the small intestine, colon and feces, the number of colonies of Vibrio cholerae formed was shown to be similar to that of the PBS-ingested mouse model group. In contrast, the mouse model group ingesting the live Bacteroides vulgatus (Live B. vulgatus) microorganism had a reduced level of Vibrio cholerae about two times or more than the control group.

That is, this result may mean that the Bacteroides vulgatus microorganism used in various embodiments of the present disclosure inhibits colony formation of Vibrio cholerae.

Further, referring further to FIG. 3C (a), in the following experiment, a 5-day-old infant mouse model group infected with 1 Х 10⁸ CFU/mL of Vibrio cholerae after ingesting 50 mL of PBS (Negative control, NC), and an infant mouse model group infected with 1 Х 10⁸ CFU/mL of Vibrio cholerae after ingesting 1 Х 10⁹ CFU/mL of live Bacteroides vulgatus (Live B. vulgatus) were analyzed. At this time, the infection with Vibrio cholerae was performed after 12 hours of ingestion of Bacteroides vulgatus microorganism (or PBS), and the analysis was performed after 12 hours of the infection of Vibrio cholerae.

More specifically, referring to FIG. 3C (b), as a result of proliferation analysis of Vibrio cholerae in the intestine, in the mouse model group ingesting the live Bacteroides vulgatus (Live B. vulgatus) microorganism, the level of Vibrio cholerae was significantly reduced compared to the control group. Furthermore, referring to the results of analyzing fluid accumulation induced by infection, in the mouse model group ingesting the live Bacteroides vulgatus (Live B. vulgatus) microorganism, a fluid accumulation rate was significantly lower than that of the mouse model group of the control group.

That is, this result may mean that the Bacteroides vulgatus microorganism used in various embodiments of the present disclosure inhibits colony formation of Vibrio cholerae, particularly, enteric colony formation by infection of Vibrio cholerae in an infant mouse model.

According to the results of Example 3 above, the Bacteroides vulgatus microorganism may be used as a microorganism with antibacterial activity for enteric pathogenic bacteria, particularly, Vibrio cholerae.

Meanwhile, the content of Bacteroides vulgatus microorganisms in the pharmaceutical composition or the food composition may be 10⁵ to 10¹² CFU/mL, preferably 10⁷ to 10¹⁰ CFU/mL, and more preferably 10⁸ to 10⁹ CFU/mL, but is not limited thereto.

Example 4: Evaluation for metabolites used in various embodiments of the present disclosure

Hereinafter, evaluation results of antibacterial activity for metabolites further added to compositions according to various embodiments of the present disclosure will be described with reference to FIGS. 4A to 4E.

FIG. 4A illustrates a change in enteric microbial community, depending on antibiotic treatment. FIG. 4B illustrates a change in metabolites isolated from the cecum depending on antibiotic treatment. FIG. 4C illustrates a change in growth inhibition of Vibrio cholerae according to the treatment of metabolites, used in various compositions according to an embodiment of the invention. FIG. 4D illustrates a change in growth stimulation of Vibrio cholerae according to the treatment of metabolites, used in various compositions according to an embodiment of the invention. FIG. 4E illustrates a change in infection of Vibrio cholerae according to microorganisms with antibacterial activity for enteric pathogenic bacteria and metabolites, used in various compositions according to an embodiment of the invention.

First, referring to FIGS. 4A (a) and (b), with respect to each of a specific pathogen free (SPF) mouse model group as an untreated control group and a clindamycin-administered mouse model group, a result of analyzing changes in microbial colony in feces after infection with 5 x 10⁸ CFU of Vibrio cholerae is illustrated.

More specifically, in the administration of clindamycin, a level of Bacteroidetes phylum was relatively reduced. Meanwhile, the level of Proteobacteria phylum was increased. That is, this result may mean that the change in enteric microbial community of mice occurred by antibiotic administration of clindamycin.

Meanwhile, after infection with Vibrio cholerae, in a mouse group administered with clindamycin, as compared with before infection with Vibrio cholerae, the level of the Proteobacteria phylum was reduced, but the Proteobacteria phylum containing 8.81% of Vibrio cholerae was still maintained at a high level.

At this time, the mouse group administered with clindamycin may exhibit antibiotic resistance according to the decrease of the Bacteroidetes phylum including Bacteroides vulgatus microorganism having antibacterial activity for Vibrio cholerae.

Referring further to FIGS. 4B (a) and (b), metabolites having a high abundance ratio were observed in 50 mg of cecal contents obtained from each of a specific pathogen free mouse model group (SPF control mice) as a control group and a clindamycin-administered mouse model group (CL-treated mice). At this time, the metabolites present in the cecal contents may be detected by Capillary Electrophoresis-Time of Flight Mass Spectrometry (CE-TOFMS) analysis.

More specifically, referring to FIG. 4B (a), the specific pathogen free mouse model group of the control having a relatively high abundance ratio of enteric Bacteroidetes phylum had a high abundance ratio for a metabolite such as butyric acid, isobutyric acid, cholic acid, propionic acid, valeric acid, isovaleric acid, trimethylamine, glucose 6-phosphate, fructose 6-phosphate, 2-hydroxyglutaric acid, 2-oxoglutaric acid, N-acetylglutamic acid, 3-(4-hydroxyphenyl) propionic acid, trans-glutaconic acid, and N-acetylaspartic acid.

Meanwhile, among the aforementioned metabolites, cholic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, and isovaleric acid may be short-chain fatty acids (SCFAs) providing an effective barrier against invasion of pathogens and inhibiting activity for pathogen.

At this time, the short-chain fatty acid may be a metabolite produced by Bacteroides vulgatus microorganism used in various embodiments of the present disclosure and may be associated with antibacterial activity for enteric pathogenic bacteria.

Meanwhile, referring to FIG. 4B (b), the clindamycin-administered mouse group having a relatively high abundance ratio of enteric Proteobacteria phylum had a high abundance ratio for the metabolite such as urea, N-acetylgalactosamine/N-acetylmannosamine/N-acetylglucosamine, N-acetylneuraminic acid, creatine, gluconic acid, gluonic acid, glucuronic acid-1/galacturonic acid-1, glucaric acid, gluconolactone, galactosamine, glucosamine, mannosamine, putrescine, N-acetylalanine, and fumaric acid.

Referring to FIG. 4C, among the metabolites found in the mouse group having a relatively high abundance ratio of Proteobacteria phylum, it is illustrated an evaluation result of inhibition of growth of Vibrio cholerae for A. fumaric acid, B. urea, and C. N-acetylgalactosamine (NAG), D. mannosamine, E. putrescine, F. gluconic acid, G. galacturonic acid, H. glucaric acid, and I. gluconolactone. Furthermore, among the metabolites found in the mouse group having a relatively high abundance ratio of Bacteroidetes phylum, it is further illustrated an evaluation result of inhibition of growth of Vibrio cholerae for J. cholic acid, K. isobutyric acid, L. propionic acid, M. valeric acid, and N. isovaleric acid.

In this evaluation, an M9 medium cultured with Vibrio cholerae was treated with 20 nM of the remaining metabolites except for N-acetylgalactosamine, gluconic acid, gluconolactone, mannoseamine, and cholic acid, and the growth of Vibrio cholerae was analyzed by measuring an OD₆₀₀ value. On the other hand, N-acetylgalactosamine, gluconic acid, gluconolactone, mannoseamine and cholic acid were treated at concentrations of 4 nM, 4 nM, 10 nM, 1 nM, and 0.2 nM, respectively, in the M9 medium cultured with Vibrio cholerae, and then the OD₆₀₀ value was measured. At this time, a culture group of Vibrio cholerae without addition of the metabolite was set as a control.

More specifically, in the case of a medium added with A. fumaric acid, B. urea, C. N-acetylgalactosamine, D. mannosamine, E. putrescine, F. gluconic acid, G. galacturonic acid, H. glucaric acid, and I. gluconolactone among the metabolites, the growth of Vibrio cholerae was similar to the control or more active than the control.

Particularly, A. fumaric acid, B. urea, C. N-acetylgalactosamine, E. putrescine, and H. glucaric acid stimulated the growth of Vibrio cholerae.

Furthermore, in the case of all media added with J. cholic acid, K. isobutyric acid, L. propionic acid, M. valeric acid, and N. isovaleric acid among the metabolites, the growth of Vibrio cholerae was inhibited as compared with the control.

In other words, the above results may indicate that in a mouse group with antibiotic resistance, metabolites A to I, which are present at relatively high concentrations, may contribute to the antibiotic resistance of a host by rather stimulating the growth of Vibrio cholerae.

Furthermore, in the mouse group with a relatively high abundance ratio of the Bacteroidetes phylum including Bacteroides vulgatus microorganism, the microorganism of the present disclosure, the metabolites J to N present at high concentrations inhibit the growth of Vibrio cholera. Thus, they can provide antibacterial activity for enteric pathogenic bacteria such as Vibrio cholerae.

At this time, the metabolites of K. isobutyric acid, L. propionic acid, M. valeric acid, and N. isovaleric acid may be short-chain fatty acids produced by the Bacteroides vulgatus microorganism and may provide antibacterial activity to Vibrio cholerae.

Thus, the metabolites of J. cholic acid, K. isobutyric acid, L. propionic acid, M. valeric acid, and N. isovaleric acid may be used to enhance antibacterial activity together with the microorganism with antibacterial activity, in the compositions of various embodiments of the present disclosure.

Referring to FIG. 4D, among the metabolites found in the mouse group having a relatively high abundance ratio of Proteobacteria phylum, it is illustrated an evaluation result for stimulation of the growth of Vibrio cholerae for A. fumaric acid, B. urea, and C. N-acetylgalactosamine, D. mannosamine, E. putrescine, F. gluconic acid, G. galacturonic acid, H. glucaric acid, and I. gluconolactone. Furthermore, among the metabolites found in the mouse group having a relatively high abundance ratio of Bacteroidetes phylum, it is further illustrated an evaluation result for stimulation of the growth of Vibrio cholerae for J. cholic acid, K. isobutyric acid, L. propionic acid, M. valeric acid, and N. isovaleric acid.

In this evaluation, the metabolites were treated in the same manner as the evaluation method for the growth inhibition of cholerae of FIG. 4C and then the growth of Vibrio cholerae was analyzed by measuring an OD₆₀₀ value.

More specifically, in the case of a medium added with A. fumaric acid, B. urea, C. N-acetylgalactosamine, D. mannosamine, E. putrescine, F. gluconic acid, G. galacturonic acid, H. glucaric acid, and I. gluconolactone among the metabolites, the growth of Vibrio cholerae was more activated than a control without adding the metabolites.

Particularly, A. fumaric acid, C. N-acetylgalactosamine, F. gluconic acid, and I. gluconolactone stimulated the growth of Vibrio cholerae.

Furthermore, in the case of all media added with J. cholic acid, K. isobutyric acid, L. propionic acid, M. valeric acid, and N. isovaleric acid among the metabolites, the growth of Vibrio cholerae was similar to the control.

In other words, the above results may indicate that in a mouse group with antibiotic resistance, the metabolites A to I, which are present at relatively high concentrations, may contribute to the antibiotic resistance of a host by rather stimulating the growth of Vibrio cholerae.

Furthermore, in the mouse group with a relatively high abundance ratio of the Bacteroidetes phylum including Bacteroides vulgatus microorganism, the microorganism of the present disclosure, the metabolites J to N present at high concentrations inhibit the growth of Vibrio cholera. Thus, they can provide antibacterial activity for enteric pathogenic bacteria such as Vibrio cholerae.

Referring to FIG. 4E, when the relative abundance ratio of Bacteroidetes phylum including the Bacteroides vulgatus microorganism used in various embodiments of the present disclosure is high in the intestine of the host, short-chain fatty acids (SCFAs), including propionic acid, valeric acid and butyric acid produced by Bacteroides vulgatus (B. vulgatus) microorganism may form barriers to the intestinal wall. Thus, the short-chain fatty acids may inhibit colony formation for enteric pathogenic bacteria such as V. cholerae, and as a result, provide antibacterial activity for Vibrio cholerae.

Meanwhile, in the case of a host having a relatively high abundance ratio of Proteobacterial phylum containing E. coli in the intestine (e.g., clindamycin-administered mouse model group), metabolites of N-acetyl amino sugars such as N-acetylgalactosamine, N-acetylglucosamine and N-acetylneuramine may be produced in the intestine. At this time, the N-acetyl amino sugar as described above may be used as a nutrient source of Vibrio cholerae. Thus, the N-acetyl amino sugars may stimulate colony formation for enteric pathogenic bacteria, such as V. cholerae, and as a result, may cause antibiotic resistance.

According to the results of Example 4 above, the Bacteroides vulgatus microorganism may be used as a microorganism with antibacterial activity for enteric pathogenic bacteria, particularly, Vibrio cholerae. Furthermore, the metabolites of cholic acid, butyric acid, isobutyric acid, propionic acid, valeric acid and isovaleric acid which inhibit the colony formation of Vibrio cholerae and have antibacterial activity for the bacteria may be provided as the pharmaceutical composition and the food composition according to various embodiments of the present disclosure together with the Bacteroides vulgatus microorganism.

As a result, the pharmaceutical composition and the food composition according to an embodiment of the present disclosure may have an improved effect for at least one selected from the group consisting of food poisoning, typhoid, peritonitis, ulcerative colitis, Crohn's disease, intestinal Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, nervous enteritis syndrome, small intestinal bacterial overgrowth, intestinal acute diarrhea and ischemic enteritis.

Meanwhile, in various embodiments, the metabolites in the pharmaceutical composition and the food composition may be present at a concentration of 0.1 to 30 mM with respect to the composition but is not limited thereto.

Although the exemplary embodiments of the present disclosure have been described in detail with reference to the accompanying drawings, the present disclosure is not limited thereto and may be embodied in many different forms without departing from the technical concept of the present disclosure. Accordingly, the various embodiments disclosed in the present disclosure are not intended to limit the technical spirit but describe the present disclosure and the technical spirit of the present disclosure is not limited by the following embodiments. Therefore, it should be understood that the above-described exemplary embodiments are illustrative in all aspects and do not limit the present disclosure. The protective scope of the present disclosure should be construed based on the appended claims, and all the technical spirits in the equivalent scope thereof should be construed as falling within the scope of the present disclosure.

[National R & D Projects Supporting This Invention 1]Task identification number:918003-4, Name of government department:Ministry of Agriculture, Food and Rural Affairs, Research Management Specialized Organization:Korea Institute Of Planning and Evaluation For Technology in Food, Agriculture and Forestry, Name of Research Project:Korea Institute Of Planning and Evaluation For Technology in Food, Agriculture and Forestry-Post Genome Multi Ministries Project, Project name:Development of probiotic strains that inhibit infection and relieve intestinal inflammation, Contribution rate:1/2, Managing department:Yonsei University Industry-Academic Cooperation Foundation, Research Period:2018.04.25 ~ 2021.12.31

[National R & D Projects Supporting This Invention 2]Task identification number:2017M3A9F3041233, Name of government department:Ministry of Science and ICT, Research Management Specialized Organization:National Research Foundation of Kor, Name of Research Project:Bio Medical Technology Development Project, Project name:Probiotic Gene Function Study with New Gene Screening System, Contribution rate:1/2, Managing department:Yonsei University, Research Period:2017.05.01. ~ 2021.12.31

Claims

A microorganism of Bacteroides vulgatus (Accession number: KCTC18713P) with antibacterial activity for enteric pathogenic bacteria.
The microorganism of claim 1, wherein the enteric pathogenic bacteria are at least one selected from the group consisting of Vibrio cholerae, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Salmonella choleraesuis, Salmonella derby, Staphylococcus aureus, enterotoxigenic E. coli, Listeria monocytogenes, Campylobacter jejuni, Shigella flexneri, Salmonella enterica, Clostridium difficile, vancomycin-resistant Enterococcus and Clostridium perfringens.
The microorganism of claim 1, wherein the microorganism has a base sequence having at least 80% homology with a base sequence represented by SEQ ID NO:1.
The microorganism of claim 1, wherein the microorganism produces short-chain fatty acids (SCFAs).
A pharmaceutical composition for preventing or treating enteric pathogenic bacteria-inducing diseases, the pharmaceutical composition comprising a Bacteroides vulgatus (Accession number: KCTC18713P) microorganism with antibacterial activity for the enteric pathogenic bacteria or a culture medium thereof, as an active ingredient.
The pharmaceutical composition of claim 5, wherein the enteric pathogenic bacteria-inducing disease is at least one selected from the group consisting of food poisoning, typhoid, peritonitis, ulcerative colitis, Crohn's disease, intestinal Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, nervous enteritis syndrome, small intestinal bacterial overgrowth, intestinal acute diarrhea and ischemic enteritis.
The pharmaceutical composition of claim 5, wherein the enteric pathogenic bacteria are at least one selected from the group consisting of Vibrio cholerae, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Salmonella choleraesuis, Salmonella derby, Staphylococcus aureus, enterotoxigenic E. coli, Listeria monocytogenes, Campylobacter jejuni, Shigella flexneri, Salmonella enterica, Clostridium difficile, vancomycin-resistant Enterococcus and Clostridium perfringens.
The pharmaceutical composition of claim 5, wherein the microorganism has a base sequence having at least 80% homology with a base sequence represented by SEQ ID NO:1.
The pharmaceutical composition of claim 5, wherein the pharmaceutical composition includes the microorganism in an amount of 10⁵ to 10¹² CFU/mL, or the culture medium of the microorganism in an amount of 10⁵ to 10¹² ml/g, based on the total weight of the pharmaceutical composition.
The pharmaceutical composition of claim 5, wherein the enteric pathogenic bacteria secrete the toxicity, and the microorganism removes the secreted toxicity in the intestine.
The pharmaceutical composition of claim 5, further comprising:

at least one metabolite selected from the group consisting of cholic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, isovaleric acid, and trimethylamine.
The pharmaceutical composition of claim 11, wherein the metabolite is present at a concentration of 0.1 to 30 mM with respect to the composition.
The pharmaceutical composition of claim 5, further comprising:

short-chain fatty acids (SCFAs).
A food composition for improving enteric infection by enteric pathogenic bacteria, the food composition comprising a Bacteroides vulgatus (Accession number: KCTC18713P) microorganism with antibacterial activity for the enteric pathogenic bacteria or a culture medium thereof, as an active ingredient.
The food composition of claim 14, wherein the food composition includes the microorganism in an amount of 10⁵ to 10¹² CFU/mL, or the culture medium of the microorganism in an amount of 10⁵ to 10¹² ml/g, based on the total weight of the food composition.
The food composition of claim 14, wherein the enteric pathogenic bacteria secrete the toxicity, and the microorganism removes the secreted toxicity in the intestine.
The food composition of claim 14, further comprising:

at least one metabolite selected from the group consisting of cholic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, isovaleric acid, and trimethylamine.
The food composition of claim 17, wherein the metabolite is present at a concentration of 0.1 to 30 mM with respect to the food composition.
The food composition of claim 14, further comprising:

short-chain fatty acids (SCFAs).