KR20230009525A - Lactobacillus plantarum having inhibitory effect against Clostridium difficile - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain having a growth inhibitory effect on Clostridium difficile. The Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain is effective in treating CDI and alleviating symptoms thereof, and is particularly effective in treating recurrent CDI and alleviating symptoms thereof.

Description

Lactobacillus strain having inhibitory effect against Clostridium difficile growth {Lactobacillus plantarum having inhibitory effect against Clostridium difficile}

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

Clostridium difficile infection (CDI) is an antibiotic-associated diarrheal disease ( antibiotics-associated diarrhea (AAD).

C. difficile shows relatively high resistance to disinfectant treatment such as alcohol, and it is difficult to properly manage and remove it because it can build a spore form that can survive in extreme environments for more than several years. According to a recent report by the US CDC, the number of CDI patients per year is around 500,000, and it can be said that it is a serious disease with 14,000 deaths per year. In general, recurrence is frequent even after treatment, and in the case of recurrent patients, it is classified as a major disease because it is difficult to see great efficacy even with general antibiotic treatment.

The standard treatment currently used for CDI treatment is antibiotic prescription, which is known to have a relatively low treatment success rate and high recurrence rate. Briefly, both Metronidazole and vancomycin have limited treatment success rates in the first treatment and show a recurrence rate of 20 to 30%. The treatment success rate is 70% for the first relapse and decreases to 35% for further relapses. It is not suitable for use in refractory severe CDIs and recurrent CDIs. In addition, Fidaxomicin inhibits C. difficle toxin A and B synthesis and sporulation, and the recurrence rate is also lower than other antibiotics. However, it is expensive and causes nausea, vomiting, fever, dizziness, and increased liver enzyme levels. The possibility of nitazoxanide as a treatment has also been suggested, but related studies are still lacking. Recently, non-antibiotic approaches utilizing fecal microbiota transplantation (FMT), vaccines, and administration of core microbes have been proposed for the prevention and treatment of CDIs. FMT, in which stool from a healthy donor is administered to the patient's intestinal tract, is one of the methods of treating refractory or recurrent CDIs. As a result of 28 studies on 317 patients with CDI, the recovery rate was 92%. It also shows a high relapse prevention effect. However, despite the high treatment success rate and low recurrence rate, these FMT treatments have several limitations, such as a procedure process that may cause discomfort to the general public, non-standard treatment, and pathogen propagation. C. difficile vaccines, including toxoids A and B, are also being studied to show efficacy in patients with recurrent CDI, but commercialization is expected to take considerable time.

Useful microorganisms isolated from the intestine can cause changes in the intestinal ecosystem and interactions with the immune system, and as a result maintain space and resource competition with C. difficile. It has been reported that when these are treated, they actually show the same level of therapeutic effect as metronidazole or vancomycin, and the recurrence rate is also significantly lowered (Ollech et al., 2016. Best Practice & Research Clinical Gastroenterology). Therefore, the process of securing and mass-producing functional beneficial strains that can make a significant contribution to the prevention/treatment of CDI can be considered to have very important significance as a method that can supplement/replace chemical antibiotic therapy in the future.

Intestinal microorganisms play a role in protecting the intestinal barrier (intestinal epithelium) from pathogens such as C. difficile , and when an imbalance in intestinal microorganisms is caused by a specific cause (dysbiosis), such as antibiotics, pathogens penetrate the intestinal wall and cause disease. cause. According to a recent study, IL-25 produced by intestinal microorganisms increased the tight junction of the intestinal wall and showed the effect of preventing CDI (Buonomo et al., 2016. Cell Reports). It has been reported that it has a high correlation with the degree of diversity of (Milani et al., 2016. Scientific Reports). As a result of confirming the diversity of intestinal microorganisms by obtaining clinical samples from CDI patients, it was confirmed that the diversity of intestinal microorganisms in CDI patients was significantly lower than that of normal subjects, and the secondary bile acids produced by beneficial strains in the intestinal microflora were C. It has been reported that difficile inhibits the growth of C. difficile by inhibiting TCA and DCA, which are essential for sporulation (Winston et al., 2016. Anaerobe.).

An object of the present invention is to provide a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain characterized by having a Clostridium difficile growth inhibitory effect.

Another object of the present invention is Lactobacillus plantarum KBL396 (KCTC 13278 BP) to provide a pharmaceutical composition for treating Clostridium difficile infection (CDI) characterized in that it comprises a strain or a culture thereof.

Another object of the present invention is Lactobacillus plantarum ( Lactobacillus plantarum ) KBL396 (KCTC 13278 BP) strain or CDI ( Clostridium difficile infection) characterized in that it comprises a culture thereof to provide health functional food for alleviation or improvement is to do

In order to achieve the above object, the present invention provides a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain characterized by having a Clostridium difficile growth inhibitory effect.

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

In addition, the present invention provides a pharmaceutical composition for the treatment or prevention of CDI ( Clostridium difficile infection) comprising a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture thereof.

In addition, the present invention provides a health functional food for mitigating or improving CDI ( Clostridium difficile infection) , characterized in that it comprises a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture thereof.

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

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

The present invention relates to a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain characterized by having a Clostridium difficile growth inhibitory effect, and is effective in treating CDI and improving symptoms, In particular, it is very effective in treating recurrent CDI and improving symptoms.

1 is a result of a comparative experiment on the growth inhibitory effect of Clostridium difficile (C.difficile) .
Figure 2 is a result of the inhibitory effect experiment on the growth of C. difficile of Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain (LP).
Figure 3 relates to the experimental process for establishing a C. difficile infection prevention model.
Figure 4 is a result of the weight loss prevention effect test by the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain in the C.difficile infection prevention model.
Figure 5 is a result of the survival rate increase experiment by the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain in the C. difficile infection prevention model.
6 is a profile analysis result for DCA and LCA, which are bile acids, in feces before and after C. difficile infection.
7 is a microbiome analysis test result between the LP administration group and the infection control group.
8 is an experimental result for understanding the correlation of microbial species associated with C. difficile infection.
9 is a functional microbiome analysis (microbiome metabolic pathway analysis) test results between the LP administration group and the infection control group.
10 is a result of analysis of genes related to the secondary bile acid biosynthesis pathway in the fecal microbiome of the LP-administered group and the infection control group.
11 is a correlation test result between the amount of bile acids in feces and functional microbiome analysis.
12 is an experiment result of changes in bile acid composition according to LP culture (in vitro).
13 is an experimental result of the conversion effect of TCA and TDCA according to LP culture (in vitro).
Figure 14 relates to the experimental process for establishing a C. difficile infection prevention model.
Figure 15 is the result of the weight recovery effect experiment due to LP administration in the C. difficile infection recurrence prevention model.

The present invention provides a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain characterized by having a Clostridium difficile (C.difficile) growth inhibitory effect.

Lactobacillus plantarum strain KBL396 (KCTC 13278 BP) was isolated from feces provided by healthy adult volunteers. Since the strain is an obligate anaerobic strain, the separation process was performed under an anaerobic system.

The Clostridium difficile ( C. difficile ) is a gram-positive, anaerobic bacterium, relatively large at 2-17 μm in length, has a structure such as a flagellum or projection, and is a bacterium that resides in the human intestine. Clostridium difficile is likely to occur even after administration of antibiotics such as Clindamycin, Lincomycin , Amoxicillin, Cefalothin, and Cefazoline, and the above Colonization by virulent C. difficile may occur after destruction of the normal intestinal flora by antibiotics. Clostridium difficile produces toxin A (enterotoxin) with a molecular weight of about 310,000 and toxin B (cytotoxin) with a molecular weight of about 270,000. Toxin A first damages intestinal epithelial cells, and then invades the intestinal membrane at the site of toxin B damage and exerts its toxic effect.

Growth inhibitory effect on Clostridium difficile strains can be confirmed through an inhibition assay, but is not limited thereto.

Growth inhibition of C. difficile may be due to inhibition of spore germination, but is not limited thereto.

The Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain is a deposited strain and was deposited with the Center for Biological Resources on May 29, 2017, and the deposit number is KCTC 13278 BP.

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

The present invention provides a pharmaceutical composition for the treatment or prevention of CDI ( Clostridium difficile infection) comprising a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture thereof.

CDI may be caused by spore-forming bacteria, specifically Clostridium difficile strains. CDI can also be accompanied by diarrhea, high temperature, and unusual pain.

The CDI may occur in patients in whom bile secretion is not normally performed in the liver.

The CDI may be a type of antibiotic-associated diarrhea.

CDI may also have developed in patients receiving broad-spectrum antibiotics (particularly clindamycin) that alter the ecological balance of the intestinal flora.

In addition, CDI may be mild CDI, severe CDI, complex CDI or recurrent CDI, more preferably recurrent CDI. The severity of the CDI can be determined through age, use of systemic antibiotics, white blood cell count, albumin, and serum creatine.

More specifically, mild CDI may have a white blood cell count of less than 15,000 cell/ml and serum creatine less than 1.5-fold premorbid level, and severe CDI may have a white blood cell count of 15,000 cell/ml or more and serum creatine 1.5-fold premorbid level. Complex CDI can involve hypotension, shock, ileus, megacolon, and recurrent CDI can mean recurrence within 8 weeks of successful treatment for CDI.

The pharmaceutical composition according to the present invention can be administered to mammals including humans by various routes. The administration method may be any method commonly used, and may be administered by, for example, oral, dermal, intravenous, intramuscular, subcutaneous, etc. routes, preferably orally. The pharmaceutical composition of the present invention can be prepared in the form of oral formulations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, aerosols, etc., or parenteral formulations in the form of transdermal preparations, suppositories and sterile injection solutions, respectively, according to conventional methods. It may be formulated into a spherical formulation or the like and used. The pharmaceutical composition of the present invention may further contain adjuvants such as pharmaceutically suitable and physiologically acceptable carriers, excipients and diluents.

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used.

In the specific embodiment of applying the pharmaceutical composition of the present invention to humans, the pharmaceutical composition of the present invention can be administered alone, but in general, the pharmaceutical composition selected in consideration of the administration method and standard pharmaceutical practice It may be administered by mixing with a carrier. For example, the composition containing the strain of Clostridium syndens of the present invention may be administered in the form of a tablet containing starch or lactose, or alone or in the form of a capsule containing an excipient, or in the form of a flavoring agent. It may be administered orally, buccally, or sublingually in the form of elixirs or suspensions containing chemicals to color or color.

The dosage of the pharmaceutical composition containing the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity, and the doctor Or, according to the judgment of the pharmacist, it may be administered in divided doses once or several times a day at regular time intervals. For example, the daily dosage may be 0.1 to 500 mg/kg, preferably 0.5 to 300 mg/kg, based on the content of the active ingredient. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences. If the daily dose of the mixed extract-containing composition of the present invention is less than the above dose, a significant effect cannot be obtained, and if it exceeds the dose, it is not only uneconomical but also out of the range of the usual dose, so there is a concern that undesirable side effects may occur. Since it can occur, it is good to set it as the said range.

Another example of the present invention is a health functional food for mitigating or improving CDI ( Clostridium difficile infection) comprising a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture thereof to provide.

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

The content of the Clostridium syndens strain as an active ingredient contained in the health functional food is appropriately not particularly limited depending on the type of food, desired use, etc., for example, 0.01 to 15% by weight of the total food weight. ,

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

Among the health functional foods of the present invention, there is no particular limitation on the liquid component except that the beverage contains the Clostridium syndens strain as an essential component in the indicated ratio, and as in conventional beverages, various flavors or natural carbohydrates, etc. may be included as an additional component.

Examples of the aforementioned natural carbohydrates include monosaccharides, such as disaccharides such as glucose and fructose, such as maltose and sucrose, and polysaccharides such as dextrins and cyclodextrins. Sugar and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The proportion of the natural carbohydrates 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 is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and It may contain salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like.

In addition, the health functional food of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not so critical, but is generally selected from 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 by the following examples or experimental examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

Experimental example 1. Securing candidate strains for CDI prevention and treatment

The research team intensively isolated candidate strains from feces provided by more than 40 healthy adult volunteers. To compare efficacy, C. difficile inhibition experiments were conducted by culturing several commercial strains and a total of 5 strains of 12 strains. In particular, considering that most of the candidate strains are obligate anaerobes, all processes were performed under the anaerobic system of Gobiolab Co., Ltd. The strains used for the C. difficile inhibitory efficacy comparison experiment were the C. difficile KCTC 5009 strain and the C. difficile ATCC43255 strain, and the growth inhibitory effects of the strains were compared. After diluting the OD of the C. difficile strain cultured in a liquid medium for 4 hours to 0.3, re-inoculating the liquid medium at a concentration of 2%, mixing the CD culture medium and the prepared supernatant of the test strain at a ratio of 1: 1 and heating it to 37 ° C. After culturing for 24 hours, the CD growth rate was evaluated by measuring the OD.

As a result, the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain was selected as a strain having excellent growth inhibitory activity against the C. difficile KCTC 5009 strain and the C. difficile ATCC43255 strain. The Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain was found to have superior ability to inhibit the growth of C. difficile strains compared to other strains such as Bacteroides caccae strain, Bacteroides ovatus strain, Faecalibacterium prausnitzi , and Roseburia intestinalis strain (FIG. 1 Reference).

Experimental example 2. Lactobacillus plantarum ( Lactobacillus plantarum) KBL396 (KCTC 13278 BP) (Lp) strain C. difficile Inhibitory effect on growth

In the C. difficile growth inhibition experiment, the inhibitory ability of C. difficile was confirmed using the supernatant obtained after culturing each isolate for 24 hours. Considering that the metabolism of bile affects the spore germination and growth of C. difficile, the same experiment was conducted by adding an appropriate concentration of bile to the C. difficile culture medium so that the candidate strain could metabolize the bile, and the growth inhibitory effect was determined. Observed. The concentration of bile acids in the small intestine of the organism 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.) to confirm the inhibitory effect of the candidate strain on C. difficile in an in vitro environment. Specifically, to perform the C. difficile probiotic inhibition assay, the test strain was inoculated into a liquid medium containing bile acids at concentrations of 0, 0.5, 1%, and 2%, incubated at 37 ° C for 24 hours, and then the supernatant was used. did After diluting the OD of the C. difficile strain cultured in a liquid medium for 4 hours to 0.3, re-inoculating the liquid medium at a concentration of 2%, mixing the CD culture medium and the prepared supernatant of the test strain at a ratio of 1: 1 and heating it to 37 ° C. After culturing for 24 hours, the CD growth rate was evaluated by measuring the OD.

As a result, it was confirmed that although the CD inhibitory ability of LP increased as the bile acid concentration increased, it could inhibit CD even in the absence of bile acid (Fig. 2). That is, it can be seen that LP has both bile acid concentration-dependent inhibitory effect and independent CD inhibitory activity.

Experimental example 3. Mouse Model to Confirm CDI Prevention (Prevention Model)

The CDI prevention animal model is a C57BL/6 mouse in which intestinal microbial strains have been disturbed by drinking antibiotics for 3 days. After oral administration of the candidate strain for 2 days, intraperitoneal administration of clindamycin and infection of C. difficile , the CDI symptoms of the candidate strain ( weight change) and survival were measured daily for 3 weeks (see Fig. 3).

In the case of LP, it showed an effect of preventing weight loss significantly compared to the control group, and this phenomenon was continuously shown even in the recovery phase (see FIG. 4).

In addition, the survival rate also showed an effect of significantly increasing the survival rate in the case of the resultant LP compared to the control group (see FIG. 5).

Experimental example 4. Mechanism study experiments on the efficacy of candidate strains

To study the mechanism of efficacy of the candidate strain, profile analysis of representative bile acids (DCA and LCA) in feces before and after C. difficile infection was performed using LC-MS/MS.

24 hours after C. difficile infection in the group that ate LP in vivo, the concentrations of DCA (deoxycholic acid) and LCA (lithocholic acid) in feces were measured, and it was confirmed that the concentrations of DCA and LCA in feces were significantly increased. (See Fig. 6).

In addition, a microbiome analysis of significant changes between the LP-treated group and the infection control group was performed. For this purpose, bacterial DNA was extracted from feces obtained on the 7th day of the animal experiment (24 hours after infection with C. and polymerized chain reaction (PCR) was performed using primers corresponding to the V4-V5 region of the bacterial 16S rRNA gene. The amplified 16S rRNA gene profile was subjected to NGS sequencing using Illumina Miseq, followed by OTU analysis and taxon analysis using Qiime 1.8 pipeline.

As a result, 24 hours after C. difficile infection in mice, it was confirmed that Enterobacteriaceae were significantly increased in the infected control group, whereas Clostridium sp., Coprococcus ssp., and Ruminococcaceae, known as SCFA-producing strains, were significantly increased in the LP-administered group. . The LefSe method was used for the above statistical analysis (see FIG. 7).

In addition, to identify the microbial species associated with C. difficile infection, 24 hours after infection, microbial species significantly associated with the abundance of C. difficile were analyzed through fecal microbiome analysis. Spearman correlation was used for the correlation analysis. As a result of the analysis, it was confirmed that C. difficile and Clostridium sp., Coprococcus ssp., and Ruminococcaceae showed significant negative correlations (see FIG. 8).

Following the above analysis, functional microbiome analysis was performed that significantly changed between the LP-treated group and the infected control group. Conversion to a functional metagenome was followed by analyzing the statistical significance of altered metabolic pathways using LefSe analysis.

Compared to the infected control group, microbiome metabolic pathways, including vitamin B6 metabolism, amino acid metabolism, nitrogen metabolism, glycoxylate and carboxylate metabolism, energy metabolism, and methane metabolism, were significantly increased in the LP-administered group. (See Fig. 9).

As a result of analyzing changes in the secondary bile acid biosynthesis pathway in the fecal microbiome of the LP-administered group and the infected control group, it was found that genes related to secondary bile acid biosynthesis were increased in the LP-administered group compared to the infected control group (see FIG. 10).

In order to analyze the correlation between the amount of bile acids in feces and the functional microbiome analysis, based on the changes in the function of bile acids-related microorganisms in feces during LP administration, five of the bile acids in feces were directly quantified, and the concentration of bile acids and various Correlation between microbial metabolic pathways was analyzed.

As a result of the analysis, it was confirmed that DCA and LCA, which were significantly increased in the LP administration group, showed a very high correlation with the microbial metabolic pathway changed by LP administration (see FIG. 11).

In vitro, the change in bile acid composition according to Lp culture was examined. wt/vol), Lp was inoculated and cultured in the MRS culture medium, and the change in bile acid composition before and after culture was quantitatively analyzed using HPLC-MS/MS.

As a result, by LP culture, CA decreased, and the concentrations of CDCA and LCA increased (see FIG. 12). In addition, when LP was cultured in a medium containing TCA (taurocholic acid), TDCA (taurodeoxycholic acid), and TCA/TDCA mixture) in MRS, it was confirmed that deconjugation of TCA and TDCA occurred to produce CA and DCA (see FIG. 13). ). It was confirmed that this was due to the existence of bile salt hydrolase (BSH) of LP.

Experimental example 5. Mouse model for confirming CDI recurrence (relapse model)

The animal model for confirming CDI recurrence was confirmed by oral administration of C. difficile 2 days after ingestion of the candidate strain. After 21 days, antibiotic drinking water was taken again, and recurrence was confirmed through weight change of C. difficile (see FIG. 14).

As a result, in the case of LP, it showed a significant weight recovery effect compared to the control group (see FIG. 15).

[Entrusted institution]

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

Accession number: KCTC13278BP

Entrusted date: 2017529

Claims (9)

Lactobacillus plantarum KBL396 having accession number KCTC 13278 BP strain. According to claim 1, wherein the strain Clostridium difficile lei ( Clostridium difficile ) Will have a growth inhibitory effect, the strain. The strain according to claim 2, wherein the Clostridium difficile growth inhibitory effect is bile acid independent. A pharmaceutical composition for treatment or prevention of CDI ( Clostridium difficile infection) comprising the strain according to any one of claims 1 to 3 or its culture. The composition of claim 4, wherein the CDI ( Clostridium difficile infection) is a recurrent CDI. The composition according to claim 4, wherein the CDI ( Clostridium difficile infection) is caused by an imbalance of intestinal microorganisms. A health functional food for relieving or improving CDI ( Clostridium difficile infection ) comprising the strain according to any one of claims 1 to 3 or a culture thereof. The food according to claim 7, wherein the Clostridium difficile infection (CDI) is a recurrent CDI. The food according to claim 7, wherein the Clostridium difficile infection (CDI) is caused by an imbalance of microorganisms in the intestine.

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