KR20190033897A - Clostridium scindens having inhibitory effect against Clostridium difficile - Google Patents

Abstract

The present invention relates to a Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain having an effect of inhibiting the growth of Clostridium difficile. The Clostridium scindens strain is effective for treating CDI and ameliorating symptoms thereof.

Description

Clostridium difficile (Clostridium difficile) having an inhibitory effect on the growth of Clostridium difficile {Clostridium scindens having inhibitory effect against Clostridium difficile}

The present invention relates to a Clostridium strain having a Clostridium difficile growth inhibitory effect or a culture thereof and a pharmaceutical composition for treating Clostridium difficile infection (CDI ) comprising the same.

CDI (Clostridium difficile infection) is a disease caused by the growth of Clostridium difficile and the simultaneous production of antibiotic-associated diarrhea antibiotics-associated diarrhea, AAD).

C. difficile has a relatively high resistance to the treatment of fungicides such as alcohol, and it is difficult to properly manage and remove the spore form which can survive for extreme environments for many years. According to a recent report by the US CDC, the number of CDI patients per year is about 500,000, and the death toll is about 14,000 cases per year. In general, recurrence is frequent after treatment, especially in patients with recurrent episodes of general antibiotic treatment is difficult to see the major effects are classified as a major disease.

The current standard of care for CDI is antibiotic prescription and is known to have a relatively low success rate and a high recurrence rate. Briefly, both metronidazole and vancomycin have a limited success rate in treatment and have a recurrence rate of 20-30%. In the first recurrence, the success rate is 70% and the recurrence rate is reduced to 35% It is inappropriate to use for intractable severe CDIs and recurrent CDIs. Fidaxomicin inhibits C. difficile toxin A, B synthesis and spore formation, and recurrence rate is lower than other antibiotics. However, the price is high, and zone, vomiting fever, dizziness, increase of hepatic enzymes are still problematic. The possibility of Nitazoxanide as a therapeutic agent has also been suggested, but the related research is still insufficient. Recently, a non-antibiotic approach has been proposed for the prevention and treatment of CDIs using fecal microbiota transplantation (FMT), vaccine, and administration of key microbial groups. FMT, which administers donor feces to the patient's intestinal tract, is one of the methods of treatment for intractable or recurrent CDIs. A total of 28 studies involving 317 CDI patients were combined and 92% Respectively. However, despite high treatment success rates and low recurrence rates, FMT therapies have a number of limitations, including procedures that can be discomforting to the public, non-standardized procedures, and pathogen transmission. Studies have shown that C. difficile vaccines, including degenerative toxins A and B, are also effective in patients with recurrent CDI, but commercialization is likely to take considerable time.

Useful microorganisms isolated from intestines can cause intestinal ecosystem changes and interaction with the immune system, and consequently maintain space and resource competition with C. difficile. These studies have shown that treatment with the same level of metronidazole or vancomycin actually results in a significantly lower recurrence rate (Ollech et al., 2016. Best Practice & Research Clinical Gastroenterology). Therefore, the process of securing and mass-producing functional beneficial bacteria that can make a great contribution to the prevention / treatment of CDI is very important as a method to supplement / replace chemical antibiotic therapy in the future.

Intestinal microorganisms protect the intestinal epithelium from pathogens such as C. difficile, and when the intestinal microorganism imbalance is caused by specific causes such as antibiotics (dysbiosis), the pathogens penetrate through the intestinal wall and cause disease cause. Recent studies have shown that IL-25 produced by intestinal microflora enhances intestinal wall tight junctions to prevent CDI (Buonomo et al., 2016. Cell Reports), CDI is an intestinal microorganism (Milani et al., 2016. Scientific Reports). As a result of obtaining the clinical samples of CDI patients and confirming the diversity of intestinal microorganisms, intestinal microbial diversity was found to be significantly lower than that of normal persons. The second bile acid produced by intestinal microorganisms, C. difficile, It has been reported that inhibition of C. difficile growth by inhibiting TCA and DCA, which are essential for the development of atherosclerosis (Winston et al., 2016. Anaerobe.).

It is an object of the present invention to provide Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain which is characterized by having an effect of inhibiting the growth of Clostridium difficile .

Yet another object of the present invention is to provide a pharmaceutical composition for treating Clostridium difficile infection (CDI ) characterized by comprising a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) or a culture thereof will be.

Another object of the present invention is to provide a health functional food for CDI ( Clostridium difficile infection) relieving or improving, which comprises a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) or a culture thereof .

To achieve the above object, the present invention provides Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain having an effect of inhibiting the growth of Clostridium difficile .

The growth inhibitory effect of Clostridium difficile may be bile acid dependent.

The concentration of the bile acid may be 0.2 to 2.0% (wt / vol).

Also, the present invention provides a pharmaceutical composition for treating or preventing CDI ( Clostridium difficile infection) , which comprises a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) or a culture thereof .

Also, the present invention provides a health functional food for mitigating or improving CDI ( Clostridium difficile infection) , which comprises a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) or a culture thereof .

The CDI ( Clostridium difficile infection) may be recurrent CDI.

The CDI ( Clostridium difficile infection) may be caused by an intestinal microorganism imbalance.

The present invention relates to a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) having an effect of inhibiting growth of Clostridium difficile , which is effective for CDI treatment and symptom improvement , Especially in the treatment of recurrent CDI and symptom improvement.

Figure 1 shows the results of the inhibitory effect of Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain (CS) against C. difficile growth.
Fig. 2 is a graph showing the effect of C. difficile infection A schematic diagram of establishing a preventative model.
FIG. 3 is a graph showing the distribution of C. difficile infection In the preventive model, Clostridium scindens SNUG 40402 treatment resulted in weight change.
FIG. 4 is a graph showing the effect of C. difficile infection Inhibitory effect of Clostridium scindens SNUG 40402 on C.difficile proliferation was tested .
FIG. 5 shows the distribution of C. difficile infection In the preventive model, the effect of C. difficile Toxin on Clostridium scindens SNUG 40402 was tested .
FIG. 6 is a graph showing the effect of C. difficile infection The effect of C.difficile infection on the prevention of colon length in experimental models.
Figure 7 shows the effect of C. difficile infection This is the experimental result of inflammation reduction effect in the preventive model.

The present invention provides Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain having an effect of inhibiting the growth of Clostridium difficile (C. difficile ).

Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain was isolated from the feces supplied by healthy adult volunteers. Since the strain is an absolute anaerobic strain, a separation process was carried out under an anaerobic system.

The C. difficile is a Gram-positive bacterium, an anaerobic bacterium, has a relatively large length of 2-17 μm, has a monolayer or a protrusion-like structure, and is a fungus grown in a human field. Clostridium difficile is apt to occur even after the administration of antibiotics such as clindamycin, lincomycin, amoxicillin, cefalothin, cefazoline, etc., After the bacterial gun has been destroyed, it can be colonized by toxic C. difficile .

Clostridium difficile produces a toxin A (intestinal toxin) with a molecular weight of about 310,000 and a toxin B (cytotoxin) with a molecular weight of about 270,000. The toxin A first kills the intestinal epithelial cells and then enters the intestine at the site of toxin B damage and exerts its toxic effects.

Growth inhibitory effects on Clostridium difficile silane (Clostridium difficile) strain can be confirmed by the inhibition assay (Inhibition assay).

The growth inhibitory effect may be due to the baiCD gene of Clostridium scindens strain. The baiCD gene can encode 7 alpha-dehydrogenase. 7 alpha-dehydrogenase gene can convert primary bile acid cholic acid to secondary bile acid deoxycholic acid (DCA), which can inhibit the growth of C. difficile.

In addition, growth inhibition of C. difficile may be due to spore germination inhibition.

The Clostridium scindens SNUG 40402 strain is a deposited strain and deposited on May 29, 2017 in the BRC with the deposit number KCTC 13277 BP.

The growth inhibitory effect of Clostridium difficile may be bile acid dependent.

In the present invention, the concentration of bile acid capable of exhibiting the effect of inhibiting the growth of Clostridium difficile may be 0.2 to 2.0% (wt / vol).

The present invention provides a pharmaceutical composition for treating or preventing CDI ( Clostridium difficile infection) , which comprises a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) or a culture thereof.

CDI may be due to spore forming bacteria, specifically by Clostridium difficile strains, and may also involve diarrhea, high temperature, and abnormal pain.

The CDI may be one of antibiotic-associated diarrhea.

In addition, CDI may be caused by patients receiving broad-spectrum antibiotics (especially clindamycin) that alter the ecological balance of intestinal flora.

CDI may be the result of a patient with normal cholestasis in the liver.

Further, the CDI may be mild CDI, severe CDI, complex CDI or recurrent CDI, and more preferably it may be recurrent CDI. The severity of CDI can be judged by the age, the use of systemic antibiotics, the number of white blood cells, albumin, and serum creatine.

More specifically, mild CDI may have a leukocyte count of less than 15,000 cells / ml, serum creatinine may be less than 1.5 times the premorbid level, severe CDI is more than 15,000 cells / ml, serum creatinine is 1.5 times the premorbid level And complicated CDI may be accompanied by hypotension, shock, and fever, and megacolon, and recurrent CDI may mean recurrence within 8 weeks of successful treatment for CDI.

The pharmaceutical composition according to the present invention can be administered to mammals including human in various routes. The mode of administration may be any conventional manner and may be administered, for example, by oral, skin, intravenous, intramuscular, subcutaneous, and the like routes, preferably orally. The pharmaceutical compositions of the present invention may be formulated into oral preparations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups and aerosols, or parenteral forms such as transdermal preparations, suppositories, Oral formulations, and the like. The pharmaceutical composition of the present invention may be pharmaceutically acceptable and may further contain adjuvants such as physiologically acceptable carriers, excipients and diluents.

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used can be used.

 In embodiments where the pharmaceutical composition of the present invention is applied to humans, the pharmaceutical composition of the present invention may be administered alone, but is generally administered in a pharmacological manner selected in consideration of the mode of administration and standard pharmaceutical practice For example, the composition containing Clostridium cinnamicus strain of the present invention may be administered in the form of tablets containing starch or lactose, in the form of capsules containing the active ingredient alone or in the form of excipients, May be administered orally, buccally or sublingually in the form of an elixir or suspension containing the emulsifying or coloring agent.

The dosage of the pharmaceutical composition containing Clostridium cinnamicum strain of the present invention may vary depending on the age, weight, sex, dosage form, health condition and disease severity of the patient and may be determined at a predetermined time interval It may be administered once to several times per day. For example, the daily dose may be 0.1 to 500 mg / kg, preferably 0.5 to 300 mg / kg, based on the active ingredient content. The above-mentioned dosage is an average case, and the dose may be high or low depending on individual differences. If the daily dose of the mixed-extract-containing composition of the present invention is less than the above-mentioned dosage, no significant effect can be obtained. If the daily dose exceeds the above-mentioned range, it is not only economical but also causes an undesirable side effect It may be within the above range.

Yet another example of the present invention is a health functional food for relieving or improving CDI ( Clostridium difficile infection) characterized by comprising a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) or a culture thereof .

The health functional food may be various beverages, fermented milk, food additives, and the like.

The content of Clostridium cinnamicum strain as an active ingredient contained in the health functional food is not particularly limited depending on the form of the food and the intended use, and may be, for example, 0.01 to 15% by weight of the total food weight ,

The health beverage composition may be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

In the health functional food of the present invention, there is no particular limitation on the liquid ingredient other than the above-mentioned Clostridium cinnamicum strain as an essential ingredient in the indicated ratio in the beverage, and various flavors or natural carbohydrates As an additional component.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, A salt thereof, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like.

In addition, the health functional food of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the health functional food of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples or experimental examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Experimental Example 1 Clostridium scindens  SNUG 40402 (KCTC 13277 BP). difficile Inhibitory effect on growth

Our team concentrated on candidate strains from the feces supplied by more than 40 healthy adult volunteers. Considering that most of the candidate strains are obligate anaerobes, they performed all the processes under the anaerobic system of Gobiap Lab.

In the C. difficile growth inhibition experiment, the inhibitory effect of C. difficile was confirmed by using the supernatant after culturing the isolate for 24 hours. C. difficile inhibitory effect The strains used for the comparative experiments were C. difficile KCTC 5009 strain and C. difficile ATCC43255 strain, and the growth inhibitory effects of the strains were compared. The OD of the C. difficile strain cultivated in the liquid medium for 4 hours was diluted to 0.3 and re-inoculated into the liquid medium at a concentration of 2%. The CD culture solution and the prepared test strain supernatant were mixed at a ratio of 1: 1, For 24 hours, and the OD was measured to evaluate the CD growth rate.

Considering that C. difficile affects the spore germination and growth, the same experiment was carried out by adding the appropriate concentration of bile into the C. difficile culture medium so that the candidate strain could carry out the bile metabolism. Respectively. The concentration of bile acids in the small intestine of the body was 0.2 to 2.0% (wt / vol.) (Kristoffersen et al., 2007. Journal of Bacteriology). The concentration of bile acid was adjusted to 0 ~ 2.0% (wt / vol.) And CDI inhibition effect of candidate strains was confirmed in vitro. Specifically, in order to perform the C. difficile bacterial inhibition assay, the test strains were inoculated into a liquid medium supplemented with bile acids at 0, 0.5, and 1% concentrations, and cultured at 37 ° C. for 24 hours. The OD of the C. difficile strain cultivated in the liquid medium for 4 hours was diluted to 0.3 and re-inoculated into the liquid medium at a concentration of 2%. The CD culture solution and the prepared test strain supernatant were mixed at a ratio of 1: 1, For 24 hours, and the OD was measured to evaluate the CD growth rate.

As a result, it was confirmed that the CD inhibition of CS was increased in a bile acid-dependent manner for two different CD strains (see FIG. 1).

Experimental Example 2 Mouse model experiment to confirm CDI prevention

In the CDI-preventive animal model, CI7BL / 6 mice, which had been intoxicated with intestinal microbial strain for 3 days, were orally administrated with antibiotics for 2 days, and then they were infected with clindamycin intraperitoneal injection and C. difficile , Weight change) and survival were measured daily for 3 weeks (see FIG. 2).

As a result, the Clostridium scindens SNUG 40402 of the present invention showed a significant effect of preventing weight loss compared to the infection control and other Clostridium scindens standard strains, and this phenomenon was continuously observed in the recovery phase (See FIG. 3).

To give the feces after C.difficile infected for 24 hours in a group fed a CS on In vivo was examined for the degree of proliferation of fecal C. difficile. For this purpose, the weight of the obtained feces was measured and suspended in anaerobically prepared phosphate buffered saline (PBS). Subsequently, the diluted suspension was applied to C. difficile selection medium and anaerobically cultured at 37 ° C. for 2 days. . As a result, Clostridium scindens SNUG 40402 significantly inhibited the intestinal C. difficile proliferation as compared to the infected control and other Clostridium scindens standard strains (see FIG. 4).

To confirm the presence of C. difficile related to the primary cause of C. difficile toxin production of the growth inhibitory and CDI symptoms appeared in the group fed the CS on In vivo, the amount of Toxin A / B of the fecal suspension was obtained after 24 hours infection And analyzed using an ELISA kit for quantitation of C. difficile toxin.

As a result, Clostridium scindens SNUG 40402 significantly inhibited the increase of toxin caused by intestinal C. difficile compared to the control and other Clostridium scindens standard strains (see FIG. 5).

On day 3 of the infection with the most severe symptoms of CDI, mice were euthanized to assess the progression of colitis.

Clostridium scindens SNUG 40402 had the effect of inhibiting the reduction of colon length induced by CD infection compared to the infected control (see Figure 6)

In order to investigate the reduction of CDI-induced colonic inflammation, colon tissues of euthanized mice were obtained, RNA changes were blocked using RNA-later and stored at -80 degrees deep freezer until analysis. RNA was extracted from the obtained colon tissues using a total RNA extraction kit, and the expression level of TNF-α, an inflammation inducer, was quantitatively analyzed using real-time quantitative PCR in a library transformed into cDNA using a cDNA synthesis kit.

As a result, in the case of Clostridium scindens SNUG 40402, the expression of TNF-α, which is a major inflammatory index, was significantly decreased, and thus it was confirmed that the effect of the SNUG 40402 on the reduction of intestinal inflammation was suppressed (See Fig. 7)

[Agency]

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC13277BP

Funding date: 2017529

Claims (9)

A strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) characterized by having the effect of inhibiting the growth of Clostridium difficile . The strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) according to claim 1, wherein the effect of inhibiting growth of Clostridium difficile is bile acid dependent. The strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) according to claim 1, wherein the concentration of bile acid is 0.2 to 2.0% (wt / vol). A pharmaceutical composition for treating or preventing CDI ( Clostridium difficile infection) , which comprises Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain or a culture thereof. A health functional food for alleviating or improving CDI ( Clostridium difficile infection) characterized by comprising Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain or culture thereof. The method of claim 4, wherein the CDI (Clostridium difficile infection) is recurrent CDI in that features a pharmaceutical composition for the treatment or prevention (Clostridium difficile infection) of CDI. The method of claim 5, wherein the CDI (Clostridium difficile infection) is recurrent health functional food for mitigating CDI (Clostridium difficile infection) CDI, characterized in that the, or improved. The method of claim 4, wherein the CDI (Clostridium difficile infection) is CDI (Clostridium difficile infection) treatment or prevention a pharmaceutical composition, characterized in that the imbalance caused by the intestinal microflora. The method of claim 5, wherein the CDI (Clostridium difficile infection) are health functional food for (Clostridium difficile infection) CDI, characterized in that the intestinal microflora or improving unbalance mitigation.

Images