Antibacterial composition, bacterium inhibition method, and treatment or prevention method of gastroenteritis

Abstract

To provide a new function and use of Weizmannia coagulans.SOLUTION: A composition includes a culture supernatant of a bacterial strain belonging to Weizmannia coagulans and has an activity of inhibiting growth of Clostridioides difficile.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to an antibacterial composition, a method for inhibiting bacteria, and a method for treating or preventing gastroenteritis. More specifically, the present disclosure relates to an antibacterial composition that contains a culture supernatant of a bacterial strain belonging to Weizmannia coagulans and inhibits the growth of Clostridioides difficile. [Background technology]

[0002] Weizmania coagulans SANK70258 (synonym: Bacillus coagulans SANK70258) is a spore-forming lactic acid bacterium with high lactic acid production ability that was isolated from green malt in 1949. A food preparation containing Weizmania coagulans SANK70258 (trade name: Lacris) has been on the market for over 50 years since 1966.

[0003] Bacterial diarrhea in livestock and poultry induces a decrease in the growth rate and mortality. To prevent bacterial diarrhea and increase productivity, antibiotics have been added to feed. In recent years, considering the emergence of antibiotic-resistant bacteria and problems such as antibiotic residues in livestock products, the use of probiotics has been proposed as an alternative method to antibiotic use.

[0004] Patent Document 1 discloses a therapeutic composition for the treatment or prevention of gastrointestinal infections caused by pathogenic bacteria, the composition comprising a therapeutically effective concentration of one or more species or strains of lactic acid-producing bacteria in a pharma- ceutically acceptable carrier suitable for administration to the gastrointestinal tract of a vertebrate, wherein the lactic acid-producing bacteria retain the ability to reduce both the colonization rate of the pathogenic bacteria and potential physiologically deleterious effects resulting from the colonization. (See Claim 1). In this composition, the lactic acid-producing bacteria may be Bacillus coagulans or the like (See Claim 2), and the pathogenic bacteria may be Clostridium perfringens or the like. However, the antibacterial activity of the composition against Clostridioides difficile is unclear. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special table number 2002-523372 Summary of the Invention [Problem to be solved by the invention]

[0006] The main objective of this disclosure is to provide new functions and uses of Weizmania coagulans. [Means for solving the problem]

[0007] In order to solve the above problems, this disclosure provides the following [1]-

[15] . [1] A composition comprising a culture supernatant of a bacterial strain belonging to Weizmannia coagulans, and having activity to inhibit the growth of Clostridioides difficile. [2] The bacterial strain has a circular bacteriocin-related gene group including the acnB, acnA, acnC, acdD, and acnE genes on its chromosome in this order from the 5' to the 3' end; The composition of [1], wherein the distance between the start codon of the acnA gene and the start codon of the acnC gene is within 300 bps. [3] The composition of [1] or [2], wherein the bacterial strain is one or more selected from the group consisting of SANK70258 strain, BC01 strain, and BC99 strain. [4] Any of the compositions [1]-[3] further having activity to inhibit the growth of Clostridium bacteria and / or Bacillus bacteria. [5] The composition according to [4], wherein the Clostridium bacterium is one or more selected from the group consisting of Clostridium perfringens, Clostridium ramosum, Clostridium botulinum, Clostridium tetani, and Clostridium sordellii. [6] The composition according to [4], wherein the Bacillus bacterium is Bacillus cereus or Bacillus anthracis. [7] Any of the compositions [1]-[6] which is a food, beverage, supplement, feed or medicine.

[0008] [8] A method for inhibiting the growth of Clostridioides difficile in the gastrointestinal tract of an animal other than a human, comprising: The composition includes a culture supernatant of a bacterial strain belonging to Weizmania coagulans, and has an activity of inhibiting the growth of Clostridioides difficile. method. [9] A method for treating or preventing gastroenteritis caused by a pathogenic bacterium, including Clostridioides difficile, in an animal other than a human, comprising: Orally administering to the animal a therapeutically effective amount of a composition comprising a culture supernatant of a bacterial strain belonging to Weizmania coagulans and having activity of inhibiting the proliferation of Clostridioides difficile. method.

[10] The bacterial strain has a circular bacteriocin-related gene cluster comprising the acnB, acnA, acnC, acdD and acnE genes on its chromosome in this order from the 5' to the 3' end; The method according to [8] or [9], wherein the distance between the start codon of the acnA gene and the start codon of the acnC gene is within 300 bps.

[11] Any of the methods according to [8] to

[10] , wherein the bacterial strain is one or more selected from the group consisting of SANK70258 strain, BC01 strain and BC99 strain.

[12] Any of the methods [8]-

[11] , wherein the composition further has activity of inhibiting the growth of Clostridium bacteria and / or Bacillus bacteria.

[13] The method according to

[12] , wherein the Clostridium bacterium is one or more selected from the group consisting of Clostridium perfringens, Clostridium ramosum, Clostridium botulinum, Clostridium tetani, and Clostridium sordellii.

[14] The method according to

[12] , wherein the Bacillus bacterium is Bacillus cereus or Bacillus anthracis.

[15] Any of the methods [9]-

[14] , wherein the composition is a food, beverage, supplement, feed or medicine. Effect of the Invention

[0009] The present disclosure provides new functions and uses of Weizmania coagulans. [Brief description of the drawings]

[0010] [Figure 1] The results of evaluating the growth inhibitory activity of culture supernatants (test solutions) of SANK70258, BC01, BC99, MTCC5856, GBI-30, 6086, CP3425, and NBRC12583 strains against Clostridioides difficile are shown below. The test solutions were used either without dilution or after dilution with phosphate buffer 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold. Vancomycin was used as a positive control. [Diagram 2] The results of evaluating the growth inhibitory activity against Clostridium perfringens of culture supernatants (test solutions) of SANK70258, BC01, BC99, MTCC5856, GBI-30, 6086, CP3425, and NBRC12583 strains are shown below. The test solutions were used either without dilution or after dilution with phosphate buffer 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold. Vancomycin was used as a positive control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Preferred embodiments for carrying out the present disclosure will be described below. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and the scope of the present disclosure should not be interpreted as being narrow.

[0012] The composition disclosed herein comprises a culture supernatant of a bacterial strain belonging to Weizmania coagulans, and has activity of inhibiting the growth of Clostridioides difficile. "Growth-inhibiting activity" (growth-inhibiting activity) is intended to include killing bacteria in addition to stopping the growth of bacteria and slowing down the rate of growth. The growth-inhibiting activity can be evaluated by a conventionally known method using the increase or decrease in the number of bacteria during a predetermined culture period as an index. For example, antibacterial tests using the MIC method (see Example 1) and the paper disk method are known. In these evaluation methods, when the number of bacteria after a predetermined period of culture in the presence of the composition according to the present disclosure is suppressed to 90% or less, 80% or less, preferably 70% or less, 60% or less, more preferably 50% or less, 40% or less, even more preferably 30% or 20% or less, and particularly preferably 10% or less, 5% or less of the number of bacteria in the absence of the composition according to the present disclosure, the growth-inhibiting activity of the composition according to the present disclosure is demonstrated.

[0013] Examples of bacterial strains belonging to Weizmania coagulans include SANK70258 strain, BC01 strain, BC99 strain, MTCC5856 strain, GBI-30, 6086 strain, CP3425 strain, NBRC12583 strain, P-22 strain, lilac-01 strain, SIM-7 DSM14043 strain, C101 strain, GBI-1 strain, GBI-20 strain, and GBI-40 strain, etc. Mutants derived from these strains may also be used as long as they have the effects of the present disclosure.

[0014] Examples of bacterial strains belonging to Weizmania coagulans include strains that have a circular bacteriocin-related gene group comprising the acnB, acnA, acnC, acdD and acnE genes in this order from the 5' to the 3' side on their chromosome, and in which the distance between the start codon in the coding region of the acnA gene and the start codon in the coding region of the acnC gene is within 300 bps. Cyclic bacteriocins are bacteriocins with a cyclic structure in which the N-terminus and C-terminus are peptide-bonded. The acn gene cluster is a group of genes involved in the production of cyclic bacteriocins, and includes the acnA, acnB, acnC, acnD, acnE, and acnF genes ("Amylocyclicin, a Novel Circular Bacteriocin Produced by Bacillus amyloliquefaciens FZB42", Romy Scholz et al, Journal of Bacteriology, 2014, Vol. 196, No. 10, p. 1842-1852). acnA encodes the precursor of cyclic bacteriocins, and acnC is thought to function in maturation of cyclic bacteriocins. In addition, acnD and acnE are thought to function in membrane transport by ABC transporters. In the present disclosure, it has been suggested that the growth inhibitory activity of a bacterial strain belonging to Weizmania coagulans against Clostridioides difficile and Clostridium perfringens is related to the close distance between acnA and acnC on the genome of the cell line. A strain in which the distance between the start codons of the acnA gene and the acnC gene is within 300 bps, preferably within 270 bps, may exhibit higher growth inhibitory activity against Clostridioides difficile and Clostridium perfringens. Specific examples of bacterial strains in which the distance between the start codons of the acnA gene and the acnC gene is within 300 bps include the SANK70258 strain, the BC01 strain, and the BC99 strain.

[0015] As a bacterial strain belonging to Weizmania coagulans, the SANK70258 strain is preferred in terms of stability of supply and ease of acquisition.

[0016] The bacterial strain belonging to Weizmania coagulans can be cultured by a conventionally known method. Commercially available cell lines can be used for the culture (for example, Mitsubishi Chemical Corporation's "Lacris-S", "Lacris-15", and "Feed-use Lacris-10", as well as products from Kerry Inc., SABINSA, Aterio Bio, UNIQUEBIOTECH, Asahi Biocycle, etc.). The culture supernatant of the cell line can also be prepared by a conventionally known method.

[0017] In addition to Clostridioides difficile, the compositions disclosed herein may also exhibit growth inhibitory activity against Clostridium bacteria, Bacillus bacteria, Listeria, and Vibrio.

[0018] Examples of Clostridium bacteria include Clostridium perfringens, Clostridium ramosum, Clostridium botulinum, Clostridium tetani, and Clostridium sordellii. Examples of the Bacillus bacteria include Bacillus cereus and Bacillus anthracis. Examples of Listeria bacteria include Listeria monocytogenes. Examples of Vibrio bacteria include Vibrio parahaemolyticus.

[0019] The composition disclosed herein may contain a culture supernatant (active ingredient) of a bacterial strain belonging to Weizmania coagulans, and its specific form may be a food, beverage, supplement, feed, or medicine. "Food" includes health foods, functional foods, health foods (special health foods, nutrient functional foods, functional food, etc.), dietary supplements, and nutritional supplements. The form of the food can be selected appropriately, such as solid, liquid, or paste. "Beverages" includes soft drinks, dairy drinks and alcoholic drinks. The "supplement" may be in any form, such as tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. In addition, in order to protect the supplement from gastric acid and act in the intestines, it may be in the form of an enteric coating having different solubility at different pH levels. "Feed" is obtained by mixing the active ingredient with fresh grass or hay, green forage crops (such as green corn), grains (such as corn, milo, barley, oats, rice, foxtail millet, barnyard millet, millet, sorghum, etc.), grain by-products (such as rice bran and bran), root vegetables, straw, oilseed meals (peanut meal, cottonseed meal, sunflower meal, rapeseed meal, sesame meal, linseed meal, etc.), etc. A "medicine" can be obtained by formulating an active ingredient with a known pharmacologically acceptable carrier, excipient, or other additive.

[0020] The compositions disclosed herein may be suitably used to inhibit the growth of Clostridioides difficile in the gastrointestinal tract of animals other than humans, or to treat or prevent gastroenteritis caused by pathogenic bacteria, including Clostridioides difficile, in animals other than humans.

[0021] By orally administering to an animal a therapeutically effective amount of the composition according to the present disclosure, for example as feed or medicine, the proliferation of Clostridioides difficile and the like in the gastrointestinal tract can be suppressed, and gastroenteritis caused by pathogenic bacteria including Clostridioides difficile can be treated or prevented.

[0022] Gastroenteritis can be caused, inter alia, by Clostridioides difficile. Clostridioides difficile gastroenteritis can present with a wide range of symptoms, from loose stools to bloody diarrhea, abdominal pain, and fever. Treatment of Clostridioides difficile gastroenteritis with the compositions of the present disclosure can include alleviation or amelioration of any one or more of these symptoms.

[0023] The target animals may be, but are not limited to, birds such as chickens, ducks, and wild ducks, and mammals such as pigs, cows, horses, sheep, goats, dogs, and cats. The therapeutically effective amount may vary depending on the target animal, and may be appropriately adjusted and optimized within the scope of conventional industrial animal breeding techniques.

[0024] When the composition disclosed herein is used to inhibit the growth of Clostridioides difficile and the like in the gastrointestinal tract or to treat or prevent gastroenteritis, the composition disclosed herein may contain an antibiotic or antifungal agent or may be used in combination with these. EXAMPLES

[0025] Example 1: Preparation of Weizmannia coagulans extracellular products Culture supernatants of bacterial strains belonging to W. coagulans (SANK70258, BC01, BC99, MTCC5856, GBI-30, 6086, CP3425, and NBRC12583) were prepared as test solutions.

[0026] Each cell line was plated on agar medium containing BCP and cultured overnight at 50°C in a standard incubator. The plate was scraped with saline, the collected material was diluted with saline to OD600 = 10, and 50 μL was applied to a BCP-added agar plate medium and cultured at 50°C for 7 hours. The cells were scraped again with saline, and the collected material was diluted with saline to an OD600 of 1. 50 μL of the diluted material was applied to a BCP-added agar plate and cultured at 50° C. for 16 hours. The plate was scraped with 1 mL of phosphate buffer (100 mM NaH2PO4, 100 mM Na2HPO4), the collected material was centrifuged at 12,000 g for 10 minutes, and the supernatant was collected. The supernatant was diluted with phosphate buffer to OD600=50, and the bacteria were removed through a 0.2 μm filter to obtain a test solution.

[0027] [Example 2: Antibacterial test using MIC method] The test solution prepared in Example 1 was evaluated for its growth inhibitory activity against C. difficile and C. perfringens.

[0028] C. difficile and C. perfringens were inoculated into standard liquid media, cultured with shaking at 37°C, and the culture medium was harvested when it reached the logarithmic growth phase. The culture medium was diluted to OD600 = 0.0001 and inoculated into liquid media dispensed in 100 μL portions into each well of a 96-well microplate. 8 μL of the test solution prepared in Example 1 was added to each well, and the growth of C. difficile and C. perfringens was evaluated based on absorbance while culturing for 24 hours at 35° C. The test solution was used for the test either without dilution or after diluting 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold with phosphate buffer. As a positive control to determine the presence or absence of growth inhibitory activity, 2 μL of vancomycin in two-fold serial dilutions (0.00625 to 0.8 mg / mL) was used.

[0029] The results are shown in Figures 1 and 2. Vancomycin completely inhibited the growth of C. difficile up to 32-fold dilution (approximately 0.5 μg / mL) (see Figure 1). The SANK70258, BC01, and BC99 strains showed growth inhibitory activity up to 4-fold or 8-fold dilution. On the other hand, the MTCC5856 strain showed growth inhibitory activity only in the original solution (undiluted), and the GBI-30, 6086, CP3425, and NBRC12583 strains showed no growth inhibitory activity at all. Against C. perfringens, the SANK70258, BC01 and BC99 strains showed growth inhibitory activity at 2-fold dilution, whereas the MTCC5856, GBI-30, 6086, CP3425 and NBRC12583 strains showed no growth inhibitory activity at all (see FIG. 2). These results demonstrated that among the bacterial strains belonging to W. coagulans, the SANK70258, BC01 and BC99 strains exhibited superior antibacterial activity against C. difficile and C. perfringens.

[0030] [Example 3: Analysis of the genome structure of W. coagulans] We analyzed the structures of the acn gene clusters in the genomes of the SANK70258, BC01, BC99, MTCC5856, GBI-30, 6086, CP3425, and NBRC12583 strains.

[0031] The genome was extracted from the precipitate from the centrifugation step in Example 1, and a whole genome library was prepared. The sequence was then read using a nanopore sequencer, and the full-length genome sequence was obtained by de novo assembly. The structure of the acn gene cluster is shown in Table 1. In the table, the "intergenic distance" for each gene indicates the distance (bp) between the start codon in the coding region of the gene and the start codon in the coding region of the next gene (the gene located on the 3' side) of the gene.

[0032] [Table 1]

[0033] The CP3425 and NBRC12583 strains did not contain any genes of the acn gene cluster. In the SANK70258, BC01, and BC99 strains, which showed significant growth inhibitory activity against C. difficile and C. perfringens, the distance between acnA, the precursor of the cyclic bacteriocin, and acnC, which is responsible for its maturation, was 264 bases. On the other hand, in the MTCC5856 and GBI-30, 6086 strains, which showed inferior growth inhibitory activity, the distance between acnA and acnC was 21,347 bases. These results suggest that the close distance between acnA and acnC is related to the significant growth-inhibitory activity of the SANK70258, BC01, and BC99 strains. This close distance may have altered the translation efficiency into proteins and the interaction of the translation products to increase the expression level of cyclic bacteriocins.

Claims

1. A composition comprising a culture supernatant of a bacterial strain belonging to Weizmannia coagulans, and having activity of inhibiting the proliferation of Clostridioides difficile.

2. the bacterial strain has a circular bacteriocin-related gene group comprising the acnB, acnA, acnC, acdD and acnE genes on its chromosome in this order from the 5' to the 3' side; The composition according to claim 1 , wherein the distance between the start codon of the acnA gene and the start codon of the acnC gene is within 300 bps.

3. The composition according to claim 2, wherein the bacterial strain is any one or more selected from the group consisting of SANK70258 strain, BC01 strain and BC99 strain.

4. The composition according to claim 3, further having activity of inhibiting the growth of bacteria of the genus Clostridium and / or Bacillus.

5. The composition according to claim 4, wherein the Clostridium bacterium is at least one selected from the group consisting of Clostridium perfringens, Clostridium ramosum, Clostridium botulinum, Clostridium tetani, and Clostridium sordellii.

6. The composition according to claim 4, wherein the Bacillus bacterium is Bacillus cereus or Bacillus anthracis.

7. The composition according to any one of claims 1 to 6, which is a food, a beverage, a supplement, an animal feed or a medicine.

8. 1. A method for inhibiting the growth of Clostridioides difficile in the gastrointestinal tract of an animal other than a human, comprising: The method includes a step of orally administering to the animal a composition comprising a culture supernatant of a bacterial strain belonging to Weizmania coagulans and having activity of suppressing the growth of Clostridioides difficile. method.

9. 1. A method for treating or preventing gastroenteritis caused by pathogenic bacteria, including Clostridioides difficile, in an animal other than a human, comprising: The method includes orally administering to the animal a therapeutically effective amount of a composition comprising a culture supernatant of a bacterial strain belonging to Weizmania coagulans and having activity of suppressing the proliferation of Clostridioides difficile. method.

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