KR20190033902A - Lactobacillus plantarum having inhibitory effect against Clostridium difficile - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum SNUG 10271 (KCTC 13278 BP) strain having an effect of inhibiting the growth of Clostridium difficile. The Lactobacillus plantarum strain is effective for treating CDI and ameliorating symptoms thereof.

Description

(Lactobacillus plantarum having inhibitory effect against Clostridium difficile) having the effect of inhibiting the growth of Clostridium difficile (Lactobacillus plantarum having inhibitory effect against Clostridium difficile)

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

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 intestines, is one of the methods of treatment for refractory or recurrent CDIs, which combined a total of 28 studies of 317 CDI patients with a recovery rate of 92% It is also showing a high recurrence prevention effect. However, despite the high success rates and low recurrence rates, these FMT therapies have various constraints such as procedures that can be discomforting to the public, non standardized procedures, and pathogen propagation. 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 verifying the diversity of intestinal microorganisms, intestinal microbial diversity of CDI patients was found to be significantly lower than that of normal individuals . Difficile has been reported to inhibit the growth of C. difficile by inhibiting TCA and DCA, which are essential for sporulation (Winston et al., 2016. Anaerobe.).

It is an object of the present invention to provide a strain Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) 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 CDI ( Clostridium difficile infection) characterized by comprising a strain of Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) or a culture thereof will be.

Yet another object of the present invention is to provide a health functional food for alleviating or improving CDI ( Clostridium difficile infection) characterized by comprising a strain of Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) or a culture thereof .

To achieve the above object, the present invention provides a Lactobacillus plantarum strain SNUG 10271 ( KCTC 13278 BP) having an effect of inhibiting the growth of Clostridium difficile .

The effect of inhibiting the growth of Clostridium difficile may be non-specific for bile acids.

Also, the present invention provides a pharmaceutical composition for treating or preventing CDI ( Clostridium difficile infection) , which comprises a strain of Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) or a culture thereof .

The present invention also provides a health functional food for alleviating or improving CDI ( Clostridium difficile infection) characterized by comprising a strain of Lactobacillus plantarum SNUG 10271 ( KCTC 13278 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 Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) having an effect of inhibiting the growth of Clostridium difficile , which is effective for CDI treatment and symptom improvement , Especially in the treatment of recurrent CDI and symptom improvement.

FIG. 1 shows the results of comparative experiments on the effect of inhibiting the growth of Clostridium difficile (C. difficile) .
Figure 2 shows the results of the inhibitory effect of Lactobacillus plantarum strain SNUG 10271 ( KCTC 13278 BP) on the growth of C. difficile.
FIG. 3 shows an experimental procedure for establishing a C. difficile infection prevention model.
FIG. 4 shows the results of experiments for preventing weight loss by Lactobacillus plantarum strain SNUG 10271 ( KCTC 13278 BP) in a C. difficile infection prevention model.
FIG. 5 shows results of an experiment for increasing survival rate by strain Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) in a model for preventing C. difficile infection.
FIG. 6 is a profile analysis result of DCA and LCA, which are bile acids in feces before and after C. difficile infection.
Figure 7 shows the result of microbiomic assay between the LP administration group and the infected control group.
FIG. 8 shows experimental results for correlating microbial species associated with C. difficile infection.
FIG. 9 shows the result of a functional microbiome analysis (microbiome metabolism pathway) experiment between the LP administration group and the infected control group.
FIG. 10 shows the result of analysis of a gene related to a secondary bile acid biosynthetic pathway in the feces microbiome of the LP-treated group and the infected control group.
Figure 11 shows the correlation between the amount of bile acid in the feces and the functional microbiome analysis.
FIG. 12 shows the results of an experiment for the change of the bile acid composition according to the LP culture (in vitro).
Fig. 13 shows the results of experiments on the conversion of TCA and TDCA according to LP culture (in vitro).
14 shows an experimental procedure for establishing a model for preventing the recurrence of C. difficile infection.
FIG. 15 shows the results of experiments on the recovery of body weight due to administration of LP in the prevention of recurrence of C. difficile infection.

The present invention provides a strain Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) having an effect of inhibiting the growth of Clostridium difficile (C. difficile ) .

Lactobacillus plantarum strain SNUG 10271 ( KCTC 13278 BP) 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. C. difficile is apt to occur even after the administration of antibiotics such as Clindamycin, Lincomycin, Amoxicillin, Cefalothin, Cefazoline and the like, Colonization may be caused by toxic C. difficile after destruction of normal intestinal flora due to antibiotics. 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.

The growth inhibitory effect of Clostridium difficile strain can be ascertained through an inhibition assay, but is not limited thereto.

The growth inhibition of C. difficile may be, but is not limited to, spore germination inhibition.

The strain Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) is a deposited strain and deposited on May 29, 2017 in the BRC with the deposit number KCTC 13278 BP.

The effect of inhibiting the growth of Clostridium difficile may be non-specific for bile acids.

The present invention provides a pharmaceutical composition for treating or preventing CDI ( Clostridium difficile infection) , which comprises a strain of Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) or a culture thereof.

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

The CDI may develop in patients whose bile secretion is not normally achieved in the liver.

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.

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 the CDI can be judged by the age, the use of a systemic remedy, 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 dose of the pharmaceutical composition containing Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity, Depending on the judgment of the doctor or pharmacist, it may be administered once or several times a day at intervals of a certain time. 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 method for treating CDI ( Clostridium difficile infection) relieving or improving a health functional food ( Lactobacillus plantarum) strain SNUG 10271 ( KCTC 13278 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 drink 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 critical, 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 Candidate strains for CDI prevention and treatment

Our team concentrated on candidate strains from the feces supplied by more than 40 healthy adult volunteers. In order to compare the efficacy, C. difficile inhibition experiments were carried out by culturing several commercial strains and twelve species of 12 subspecies. Considering that most of the candidate strains are obligate anaerobes, they performed all the processes under the anaerobic system of COBIO Lab. 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.

As a result, Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) strain could be selected as a strain having excellent growth inhibitory ability against C. difficile KCTC 5009 strain and C. difficile ATCC43255 strain. The Lactobacillus plantarum strain SNUG 10271 ( KCTC 13278 BP) was found to be superior to C. difficile strain growth inhibitors in comparison to the other strains Bacteroides caccae , Bacteroides ovatus , Faecalibacterium prausnitzi and Roseburia intestinalis 1).

Experimental Example 2 Lactobacillus plantarum ( Lactobacillus plantarum) SNUG 10271  ( KCTC 13278 BP) (Lp) strain C. difficile  Inhibitory effect on growth

In the C. difficile growth inhibition experiment, each isolate was incubated for 24 hours and the inhibitory effect of C. difficile was confirmed by using the obtained supernatant. Considering that the metabolism of bile affect the spore germination and growth of C. difficile, adding an appropriate concentration of bile in the C. difficile culture medium so that the candidate strains to the bile metabolism and the growth inhibitory effects proceeds the same experiment 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 C. difficile inhibitory effect of candidate strains was confirmed in vitro by adjusting the concentration of bile acid at 0 ~ 2.0% (wt / vol.). Specifically, in order to perform an inhibition assay for C. difficile , the test strains were inoculated into a liquid medium containing bile acids at 0, 0.5, 1%, and 2% concentrations, cultured at 37 ° C. for 24 hours, Respectively. 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 when the concentration of bile acid increases, the CD inhibition of LP increases, but CD can be inhibited even in the absence of bile acid (FIG. 2). In other words, it can be seen that the LP has a bile acid concentration-dependent inhibition effect and an independent CD inhibition at the same time.

Experimental Example 3. Mouse model to identify CDI prevention (preventive model)

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. 3).

 LP had a significant effect of preventing weight loss compared to the control group, and this phenomenon continued in the recovery phase (see FIG. 4).

The survival rate also significantly increased the survival rate of the resultant LP compared with the control (see FIG. 5).

Experimental Example 4. Experimental study on the efficacy of candidate strains

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

After 24 h of C. difficile infection, the concentrations of DCA (deoxycholic acid) and LCA (lithocholic acid) in the feces were measured in the LP group fed in vivo, and the concentrations of DCA and LCA in the feces were significantly increased (See FIG. 6).

In addition, microbiome analysis was performed between the LP-treated group and the infected control group. For this purpose, bacterial DNA was extracted from the feces obtained at day 7 of the animal experiment (24 hours after C. difficile infection) using Qiagen FastStool DNA isolation kit And polymerase chain reaction (PCR) was carried out using a primer 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, it was confirmed that Enterobacteriaceae was significantly increased in the infected control group after 24 hours of C. difficile infection in mice, whereas Clostridium sp., Coprococcus ssp., And Ruminococcaceae, which are known as SCFA producing strains, . For the statistical analysis, the LefSe method was used (see FIG. 7).

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

 Following this analysis, a functional microbiomic analysis was performed between the LP-treated group and the infected control group. For this, OTU and biom files picked using Greengenes DB (gg_13.5.fasta) and Qiime pipeline were analyzed by PICRUSt The metabolic pathways were then converted to functional metadenomas and then the statistical significance of the metabolic pathways was analyzed using LefSe analysis.

Significantly increased pathway of microbiome metabolism, including Vitamin B6 metabolism, amino acid metabolism, Nitrogen metabolism, Glycoxylate and carboxylate metabolism, energy metabolism, and Methane metabolism, was found in the LP - treated group compared to the infected control group. (See FIG. 9).

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

In order to analyze the correlation between the amount of bile acid in the feces and the functional microbiome analysis, five of the bile acids in the feces were directly quantified based on the change of the function of the bile acid-related microorganisms in the feces during LP administration, And microbial metabolism pathways.

Analysis showed that DCA and LCA, which were significantly increased in the LP-treated group, showed a very high correlation with the changed microbial metabolic pathway by LP administration (see FIG. 11).

To investigate the changes of bile acid composition according to Lp culture in vitro, mixed bile acid containing CA, CDCA (chenodeoxycholic acid), LCA, DCA (deoxycholic acid) and UDCA (Ursodeoxycholic acid) wt / vol) Lp was inoculated and cultured. The changes of bile acid composition before and after culture were quantitatively analyzed by HPLC-MS / MS.

As a result, CA was decreased by LP culture, and CDCA and LCA were increased in concentration (see FIG. 12). In addition, when LP was cultured in medium containing TCU (taurocholic acid), TDCA (taurodeoxycholic acid), and TCA / TDCA mixture, deconjugation of TCA and TDCA occurred and CA and DCA were produced (see FIG. 13) ). It was confirmed that there is a BSH (bile salt hydrolase) of LP and the effect is there.

Experimental Example 5 Mouse model to identify recurrence of CDI (relapse model)

Confirmation of recurrence of CDI The animal model was confirmed by oral administration of C. difficile after two days of ingestion of the candidate strain. After 21 days, the antibiotics were reused and the recurrence was confirmed by weight change of C. difficile (see FIG. 14)

As a result, LP had a significant effect of recovering body weight as compared with the control group (see FIG. 15).

[Agency]

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC13278BP

Funding date: 2017529

Claims (8)

A strain Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) having the effect of inhibiting the growth of Clostridium difficile . 2. The Lactobacillus plantarum strain SNUG 10271 ( KCTC 13278 BP) according to claim 1, wherein the effect of inhibiting the growth of Clostridium difficile is bile acid-independent. A pharmaceutical composition for treating or preventing CDI ( Clostridium difficile infection) , which comprises a strain of Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) or a culture thereof. A health functional food for alleviating or improving CDI ( Clostridium difficile infection) characterized by comprising a strain of Lactobacillus plantarum SNUG 10271 ( KCTC 13278 BP) or a culture thereof. 4. The method of claim 3 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 4, wherein the CDI (Clostridium difficile infection) is recurrent health functional food for mitigating CDI (Clostridium difficile infection) CDI, characterized in that the, or improved. 4. The method of claim 3 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 4, wherein the CDI (Clostridium difficile infection) are health functional food for improving or mitigating (Clostridium difficile infection) CDI, characterized in that the imbalance caused by the intestinal microflora.

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