Bacteriophages, pharmaceuticals, foods, beverages, supplements, and feeds containing them.

Abstract

To provide a technique useful for the treatment or prevention of diseases such as infection and obesity associated with Clostridium bacteria.SOLUTION: A bacteriophage (A) comprises a genome containing the nucleotide sequence of SEQ ID NO: 1 or (B) comprises a genome containing a nucleotide sequence having at least 85% sequential identity to the nucleotide sequence of SEQ ID NO: 1 and exhibits antibacterial activity on Clostridium bacteria.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This disclosure relates to bacteriophages, and pharmaceuticals, foods, beverages, supplements, and feeds containing them. More specifically, it relates to bacteriophages that exhibit antibacterial activity against Clostridium bacteria. [Background technology]

[0002] Due to the emergence of drug-resistant bacteria and the difficulty in developing new antibiotics, there is growing interest in technologies that use bacteriophages (natural bacterial viruses) to treat or prevent bacterial diseases. For example, Patent Document 1 proposes a technology that uses a bacteriophage that lyses Propionibacterium acnes to treat or prevent acne.

[0003] Since Clostridium bacteria cause infectious diseases and food poisoning, and have also been implicated in obesity (see Non-Patent Document 1), active ingredients that inhibit their growth are expected to be useful in the treatment or prevention of infectious diseases and obesity. Furthermore, it is known that abnormal changes in the gut microbiota (intestinal microbiome) are one of the factors contributing to obesity, and attempts have been made to improve obesity and other related conditions by using bacteriophages that have an infection-targeting property against obesity-inducing bacteria in the gut to bring about positive changes in the gut microbiome (see Non-Patent Literature 2). Non-patent document 2 describes 19 bacteriophages that are infectious against eight bacterial species, including Enterobacter cloacae, but does not describe any bacteriophages that are infectious against Clostridium bacteria, and the genome sequences of these 19 bacteriophages differ significantly from those of the bacteriophages described herein. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2009-501018 [Patent Document 2] International Publication No. 2019 / 226950 [Non-patent literature]

[0005] [Non-Patent Document 1] "Clostridium ramosum Promotes High-Fat Diet-Induced Obesity in Gnotobiotic Mouse Models", Anni Woting et. al., September / October 2014 Volume 5 Issue 5 e01530-14 [Overview of the project] [Problems that the invention aims to solve]

[0006] The primary purpose of this disclosure is to provide technologies useful for the treatment or prevention of diseases such as infectious diseases and obesity involving Clostridium bacteria. [Means for solving the problem]

[0007] To address the above issues, this disclosure provides the following [1]-

[32] . [1] (A) Having a genome containing the nucleotide sequence of Sequence ID No. 1, or, (B) Having a genome containing a nucleotide sequence that has 85% or more sequence identity with the nucleotide sequence of Sequence ID No. 1, and exhibiting antibacterial activity against Clostridium bacteria, Bacteriophage. [2] The bacteriophage of [1], wherein the Clostridium bacterium is Clostridium ramosum, Clostridium botulinum, Clostridium tetani, Clostridium difficile, Clostridium perfringens, or Clostridium sordellii. [3] The bacteriophage of Clostridium, which is Clostridium ramosum, [2]. [4] A bacteriophage identified by accession numbers NITE P-3713, NITE P-3714, or NITE P-3715, one of the bacteriophages listed in [1]-[3]. [5] A bacteriophage of any of [1]-[4] wherein the antibacterial activity is lytic activity or growth inhibitory activity. A pharmaceutical product containing the bacteriophages [6] [1]-[5] as active ingredients. [7] A medicine used for one or more purposes selected from the group consisting of the treatment of obesity, the prevention of obesity, the treatment of infections, the prevention of infections, and the improvement of the gut microbiota. [6] [8] A medicine formulated for oral administration, [7]. Foods, beverages, supplements, or feeds containing the bacteriophages [9] [1]-[5].

[10] Foods, beverages, supplements or feeds used for one or more purposes selected from the group consisting of treating obesity, preventing obesity, treating infections, preventing infections, and improving the gut microbiota. [9]

[0008]

[11] Bacteriophages identified by accession numbers NITE P-3713, NITE P-3714, and NITE P-3715.

[0009]

[12] (A) Having a genome containing the nucleotide sequence of Sequence ID No. 1, or (B) Having a genome containing a nucleotide sequence that has 85% or more sequence identity with the nucleotide sequence of Sequence ID No. 1, and exhibiting antibacterial activity against Clostridium bacteria, An anti-Clostridium bactericide containing bacteriophages.

[13] The anti-Clostridium agent according to

[12] , wherein the Clostridium bacteria are Clostridium ramosum, Clostridium botulinum, Clostridium tetani, Clostridium difficile, Clostridium perfringens, or Clostridium sordellii.

[14] The anti-Clostridium bactericide of

[13] , wherein the Clostridium bacterium is Clostridium ramosum.

[15] The bacteriophage is identified under accession numbers NITE P-3713, NITE P-3714, and NITE P-3715, and is one of any of the anti-Clostridium bacteria agents in

[12] -

[14] .

[16] An anti-Clostridium agent having lytic activity or growth inhibitory activity, one of the

[12] -

[15] antibacterial agents of the genus Clostridium.

[0010]

[17] (A) Having a genome containing the nucleotide sequence of Sequence ID No. 1, or (B) Having a genome containing a nucleotide sequence that has 85% or more sequence identity with the nucleotide sequence of Sequence ID No. 1, and exhibiting antibacterial activity against Clostridium bacteria, Use of bacteriophages for the treatment or prevention of obesity and / or infections, or for the improvement of the gut microbiota.

[18] Use according to

[17] , wherein the Clostridium bacterium is Clostridium ramosum, Clostridium botulinum, Clostridium tetani, Clostridium difficile, Clostridium perfringens, or Clostridium sordellii.

[19] Use according to

[18] , wherein the Clostridium bacterium is Clostridium ramosum.

[20] Use according to any one of

[17] -

[19] , wherein the bacteriophage is identified by accession numbers NITE P-3713, NITE P-3714, and NITE P-3715.

[21] Use according to any one of

[17] -

[20] , wherein the antibacterial activity is lytic activity or growth inhibitory activity.

[0011]

[22] (A) having a genome comprising the nucleotide sequence of SEQ ID NO: 1, or (B) having a genome comprising a nucleotide sequence having 85% or more sequence identity to the nucleotide sequence of SEQ ID NO: 1 and exhibiting antibacterial activity against Clostridium bacteria, Use of a bacteriophage for the production of a medicament, food, beverage, supplement or feed for use in the treatment or prevention of obesity and / or infectious diseases or improvement of the intestinal flora.

[23] The use of Clostridium bacteria such as Clostridium ramosum, Clostridium botulinum, Clostridium tetani, Clostridium difficile, Clostridium perfringens, or Clostridium sordellii,

[22] .

[24] Use of Clostridium ramosum,

[23] the bacterium of the genus Clostridium.

[25] Use of any of

[22] -

[24] , wherein the bacteriophage is identified by accession numbers NITE P-3713, NITE P-3714, or NITE P-3715.

[26] Use of any of

[22] -

[25] wherein the antimicrobial activity is lytic activity or growth inhibitory activity.

[0012]

[27] (A) Having a genome containing the nucleotide sequence of Sequence ID No. 1, (B) Having a genome containing a nucleotide sequence that has 85% or more sequence identity with the nucleotide sequence of Sequence ID No. 1, and exhibiting antibacterial activity against Clostridium bacteria, A method for treating or preventing obesity and / or infection, or for improving the gut microbiota, including a procedure for orally administering an effective dose of a bacteriophage.

[28] The method of

[27] wherein the Clostridium bacterium is Clostridium ramosum, Clostridium botulinum, Clostridium tetani, Clostridium difficile, Clostridium perfringens, or Clostridium sordellii.

[29] The method of

[28] wherein the Clostridium bacterium is Clostridium ramosum.

[30] The bacteriophage is identified by accession numbers NITE P-3713, NITE P-3714, and NITE P-3715, in any of the methods described in

[27] -

[29] .

[31] Any method of

[27] -

[30] wherein the antimicrobial activity is lytic activity or growth inhibitory activity.

[0013]

[32] (A) Having a genome containing the nucleotide sequence of Sequence ID No. 1, (B) Having a genome containing a nucleotide sequence that has 85% or more sequence identity with the nucleotide sequence of Sequence ID No. 1, and exhibiting antibacterial activity against Clostridium bacteria, A substance isolated from bacteriophages that exhibits antibacterial activity against Clostridium bacteria. [Effects of the Invention]

[0014] This disclosure provides a technology useful for treating or preventing diseases such as infectious diseases and obesity, in which Clostridium bacteria are involved. [Brief explanation of the drawing]

[0015] [Figure 1] This is a transmission electron microscope image of the bacteriophage (CR-V-MCC002). [Figure 2]This shows the change in the number of C. ramosum in the feces of mice orally administered bacteriophages. [Figure 3] The results of quantitative detection of bacteriophages in the feces of mice orally administered bacteriophages are shown. [Modes for carrying out the invention]

[0016] The following describes preferred forms for implementing this disclosure. The embodiments described below are merely examples of typical embodiments of this disclosure and should not be interpreted as narrowing the scope of this disclosure.

[0017] The bacteriophage relating to this disclosure is technically characterized by having (A) a genome containing the nucleotide sequence of SEQ ID NO: 1, or (B) a genome containing a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence of SEQ ID NO: 1, and exhibiting antibacterial activity against Clostridium bacteria. The bacteriophage relating to this disclosure exhibits highly specific lytic activity against Clostridium bacteria. Specifically, the bacteriophage relating to this disclosure exhibits lytic activity against Clostridium bacteria, while not exhibiting lytic activity against at least Pseudomonas, Escherichia, and Staphylococcus bacteria. Furthermore, the bacteriophage relating to this disclosure, when ingested orally, survives and multiplies in the gastrointestinal tract environment and exhibits growth inhibitory effects against Clostridium bacteria. Therefore, the bacteriophages relating to this disclosure can be used as anti-Clostridium agents and may also be useful in treating or preventing diseases associated with Clostridium bacteria or in improving the gut microbiota which contains Clostridium bacteria.

[0018] The antibacterial activity of bacteriophages can be evaluated and confirmed according to conventionally known methods, such as the plaque assay method. Bacteria (e.g., Gram-positive bacteria (e.g., Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus), Gram-negative bacteria (e.g., Acinetobacter baumannii, Pseudomonas aeruginosa), or bacteria not classified as either Gram-positive or Gram-negative) are cultured on an agar plate, and the culture is brought into contact with the bacteriophage. If a decrease in plaque size or a decrease in the total number of plaques is observed in the plate that has been contacted with the bacteriophage compared to the plate that has not been contacted with the bacteriophage, it can be determined that the bacteriophage has antibacterial activity.

[0019] (A) A bacteriophage having a genome containing the nucleotide sequence of Sequence ID No. 1 is deposited in Japan under accession number NITE P-3713. This bacteriophage strain is referred to in this disclosure as "CR-V-MCCO02". CR-V-MCCO02 has been demonstrated to have specific antimicrobial activity against Clostridium bacteria.

[0020] Domestic deposits are made to the Patent Microbial Depositary Center (NPMD), Biotechnology Center, National Institute of Technology and Evaluation (NITE) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture), which is a depositary institution pursuant to the provisions of Articles 27-2 and 27-3 of the Enforcement Regulations of the Patent Act and an international depositary authority pursuant to the Budapest Convention on the International Recognition of the Deposit of Microorganisms.

[0021] (B) A bacteriophage having a genome containing a nucleotide sequence that has 85% or more sequence identity with the nucleotide sequence of Sequence ID No. 1, and exhibiting antibacterial activity against Clostridium bacteria, may have specific antibacterial activity against Clostridium bacteria, similar to CR-V-MCCO02. The sequence identity of the nucleotide sequence of SEQ ID NO: 1 is preferably 86% or more, 87% or more, 88% or more, and 89% or more; more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more; and even more preferably 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more. Examples of such bacteriophage strains include those identified under accession numbers NITE P-3714 and NITE P-3715. These bacteriophage strains are referred to in this disclosure as "CR-V-MCCO04" and "CR-V-MCCO05". The genome sequences of CR-V-MCCO04 (SEQ ID NO: 2) and CR-V-MCCO05 (SEQ ID NO: 3) have sequence identity of 85.62% and 85.33% to the genome sequence of CR-V-MCCO02 (SEQ ID NO: 1), respectively.

[0022] Here, "identity" of sequences, in the case of nucleotide sequences, is expressed as a percentage obtained by aligning the two sequences to be compared so that as many bases as possible match, and then dividing the number of matching bases by the total number of bases. During the alignment process, gaps are inserted into one or both of the two sequences being compared as needed. Such sequence alignment can be performed using well-known programs such as BLAST, FASTA, and CLUSTALW. When gaps are inserted, the total number of bases is calculated by counting each gap as one base. If the total number of bases counted in this way differs between the two sequences being compared, the identity (%) is calculated by dividing the number of matching bases by the total number of bases in the longer sequence. The same applies to the identity of amino acid sequences.

[0023] Clostridium bacteria, while not particularly limited, may be Clostridium ramosum associated with obesity, or Clostridium botulinum, Clostridium tetani, Clostridium difficile, Clostridium perfringens, or Clostridium sordellii associated with infections. In particular, Clostridium ramosum is a relevant example.

[0024] Infections associated with Clostridium bacteria include, but are not limited to, gastroenteritis, tetanus, infections of the female reproductive organs (toxic shock syndrome), bacteremia, and sepsis, and may also include food poisoning due to toxins.

[0025] The bacteriophage relating to this disclosure exhibits highly specific lytic activity against Clostridium bacteria, survives and multiplies in the gastrointestinal environment after oral ingestion, and exhibits growth inhibitory effects against Clostridium bacteria. Therefore, it may be useful as an active ingredient (effective ingredient or contributing ingredient) in pharmaceuticals, foods, beverages, supplements, or feeds for the treatment or prevention of obesity and / or infections associated with Clostridium bacteria, or for improving the intestinal microbiota that includes Clostridium bacteria.

[0026] Here, the treatment or prevention of obesity and / or infection means therapeutic measures and preventive or protective measures for obesity and / or infection, and broadly means eliminating, reducing, decreasing the severity of, slowing the progression of, or delaying or preventing the symptoms or underlying causes (bacterial infection) associated with obesity or infection. Furthermore, preventive or protective measures against obesity may include controlling weight gain. Improving the gut microbiota means bringing about changes in the gut microbiota that have a positive impact on health. Specifically, in a gut microbiota that includes Clostridium bacteria as a component, this means reducing the proportion of Clostridium bacteria, increasing the proportion of bacteria other than Clostridium (especially bacteria that have a positive impact on health), or increasing the variety of bacterial species other than Clostridium.

[0027] Pharmaceuticals, foods, beverages, supplements, and feeds may be administered to subjects including humans or to non-human subjects. Oral administration may be particularly preferred for the administration of pharmaceuticals, foods, beverages, supplements, and feeds.

[0028] The animals included in this study are not particularly limited to humans, but examples include mammals such as cows, pigs, horses, sheep, goats, and rabbits, and birds such as chickens, ducks, and geese.

[0029] A pharmaceutical product may further contain, in addition to the active ingredient, pharmaceutically acceptable carriers, excipients, or stabilizers. The pharmaceutical product may be formulated for oral administration as, if necessary, sugar-coated tablets, capsules, elixirs, microcapsules, granules, etc. Furthermore, the pharmaceutical product may be in enteric-coated form (tablets, capsules, etc.).

[0030] Foods include health foods, functional foods, health functional foods (foods for specified health uses, nutrient function foods, foods with functional claims, etc.), health supplements, and nutritional supplements. The form of the food can be selected as appropriate, such as solid, liquid, or paste.

[0031] Beverages include soft drinks, dairy drinks, and alcoholic beverages.

[0032] Supplements may take any form, including tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. They may also be enteric-coated to protect them from stomach acid and allow them to act in the intestines, exhibiting different solubility at different pH levels.

[0033] Feed can be obtained by mixing active ingredients with fresh grass or hay, green forage crops (such as green corn), grains (corn, milo, barley, oats, rice, millet, barnyard millet, foxtail millet, sorghum, etc.), grain by-products (such as rice bran and wheat bran), root vegetables, straw, oilseed meal (such as peanut meal, cottonseed meal, sunflower meal, rapeseed meal, sesame meal, and flaxseed meal).

[0034] Pharmaceuticals, foods, beverages, supplements, and feeds may contain an effective amount of the bacteriophage of this disclosure. The term "effective amount" means an amount of therapeutic agent sufficient to improve one or more symptoms of a disease or disorder, particularly obesity and / or infection. The effective dose of bacteriophages can be appropriately optimized for each application, including pharmaceuticals, foods, beverages, supplements, and feeds. For example, when administering bacteriophages in drinking water and liquid supplemental feeds for birds, the dose may be 3.4 × 10⁶ per day. 6 Plaque formation unit (PFU) or more, preferably 6.1 × 10⁻¹⁴ 6 It is greater than pfu, 5.6 × 10 7 PFU or less, preferably 2.9 × 10⁻¹⁴ 7 PFU or lower may be adopted (EFSA Journal 2021;19(5):6534).

[0035] The amount of bacteriophage in pharmaceuticals, foods, beverages, supplements, and animal feed can be appropriately set according to the effective amount described above. For example, when added to cheese, 1 × 10 7 pfu / g or more 1×10 9 pfu / g or less, preferably 1 × 10⁻⁶ 8 pfu / g or more*9×10 8 A concentration of less than pfu / g may be adopted (GRAS Notice No. 198: https: / / www.cfsanappsexternal.fda.gov / scripts / fdcc / ?set=GRASNotices&id=198&sort=GRN_No&order=DESC&startrow=1&type=basic&search=bacteriophage). Pharmaceuticals, foods, beverages, supplements, and animal feeds may contain at least one of the bacteriophages related to this disclosure, and may contain two or more. If two or more bacteriophages are included, the above-mentioned blending amount shall be the total amount of the two or more bacteriophages.

[0036] The determination of the preventive / therapeutic effects or dosage / administration of pharmaceuticals, foods, beverages, supplements, and feeds can be based on the amount of Clostridium bacteria and / or the amount of the bacteriophage relating to this disclosure in feces. The bacteriophage relating to this disclosure amplifies in the intestinal tract and exhibits antibacterial activity against Clostridium bacteria, resulting in an increase in the amount of bacteriophage and a decrease in the amount of Clostridium bacteria excreted in feces (see Example 3). Therefore, the amount of Clostridium bacteria and / or the amount of the bacteriophage relating to this disclosure in feces can be used as information to determine the preventive / therapeutic effects and to determine the dosage / administration.

[0037] Specifically, a decrease in Clostridium bacteria in feces and / or an increase in bacteriophages as disclosed herein indicate a preventive / therapeutic effect of the medicine, etc. If the degree of decrease in Clostridium bacteria in feces and / or the degree of increase in bacteriophages as disclosed herein exceeds the degree corresponding to the desired effect, the administration regimen may be modified, such as by reducing the dosage of the medicine, food, beverage, supplement and feed, or by extending the application interval. On the other hand, an increase in Clostridium bacteria in feces and / or a decrease in bacteriophages as disclosed herein indicates insufficient preventive / therapeutic effects of the drug, etc. In this case, the administration regimen can be modified, such as increasing the dosage of the drug, food, beverage, supplement, and feed, or shortening the application interval, so that the degree of decrease in Clostridium bacteria in feces and / or the degree of increase in bacteriophages as disclosed herein reaches a level equivalent to the desired effect.

[0038] This disclosure also provides methods for determining the preventive / therapeutic effects or dosage / usage of such pharmaceuticals, foods, beverages, supplements, and feeds. It also provides methods for providing the amount of Clostridium bacteria and / or the amount of bacteriophages related to this disclosure in feces as information to assist in determining the preventive / therapeutic effects or dosage / usage of pharmaceuticals, foods, beverages, supplements, and feeds. The quantification of Clostridium bacteria and / or bacteriophages related to this disclosure in feces can be performed by conventionally known methods, such as next-generation sequencing analysis or quantitative nucleic acid amplification (QPCR). [Examples]

[0039] [Example 1: Isolation of C. ramosum strain-specific bacteriophage] 1. Culture of Clostridium ramosum strain We obtained C. ramosum strains (JCM1298, ATCC25582, see "Bacteriostatic Effect of Orally Administered Bovine Lactoferrin on Proliferation of Clostridium Species in the Gut of Mice Fed Bovine Milk", SUSUMU TERAGUCHI et. al., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1995, pp. 501-506), test strains for host specificity analysis, and strains necessary for mouse animal experiments from the RIKEN Joint Microbial Materials Research Laboratory (JCM), the National Biotechnology Research Center (NBRC) of the National Institute of Technology and Evaluation (ATCC), and the American Type Culture Collection (ATCC). In addition, medium A was prepared for culturing the C. ramosum strain (8.34 g of modified GAM broth medium (05433, "Nissui"), 1.9 g of glucose, and 0.54 g of sodium acetate trihydrate, which was filled up to 200 ml with sterile water and then autoclaved).

[0040] After restoring the freeze-dried powder of the C. ramosum strain by adding a small amount of medium A, the bacterial cells were inoculated into 14 ml culture tubes containing 10 ml of medium A, and incubated statically at 37°C for 2-3 days under anaerobic conditions. After incubation, glycerol stocks were prepared and stored frozen at -80°C. To confirm the degree of growth, a single scoop of the glycerol stock of strain JCM1298 was taken using a 1,000 μl pipette tip and inoculated into a 14 ml culture tube containing 10 ml of medium A. Multiple such tubes were prepared and placed in a plastic container that could seal the culture tubes together with an aneropack (Mitsubishi Gas Chemical Co., Ltd.), and anaerobic culture was performed overnight at 37°C. During the culture, the turbidity of the culture medium was measured over time to confirm the changes over time. As a result, the OD600 nm reached approximately 1 after about 24 hours of culture, so it was decided to use culture medium cultured for about 12-16 hours when using culture medium in the growth phase.

[0041] 2. Lawn formation of C. ramosum strain using the Top Agar method Plaque assays using the double-plate method are commonly used to grow and isolate phages. We investigated whether it was possible to grow the target strain across the entire upper layer (known as a lawn) by pouring a top agar containing the C. ramosum strain onto a lower agar plate of medium A. First, the mixture was poured onto the lower layer agar of medium A onto multiple plates and allowed to solidify. Next, 200 μl of the pre-cultured C. ramosum strain was added to an empty 14 ml culture tube, and then 3 ml of medium A (at approximately 40°C) containing 0.7% agar (Bacto Agar, BD, #214019) was added. After slowly pipetting 5-6 times to avoid creating bubbles, the mixture was poured onto the medium A agar plates and allowed to solidify. When these plates were incubated under anaerobic conditions in an aneropack at 37°C for two days, lawns formed across the entire surface of the plates.

[0042] 3. Phage sampling and isolation Since C. ramosum is a bacterium that inhabits the intestines, it was thought that the phages that infect C. ramosum are likely to be present in irrigation canals and sewage systems into which domestic wastewater flows. Therefore, samples were taken from irrigation canals and sewage systems at 20 locations in Okinawa Prefecture.

[0043] After transferring the sampled water to two 50 ml centrifuge tubes, centrifugation (3,739 × g, 10 min, 25°C) was performed to precipitate any contaminants in the sample. 80 ml of the supernatant was collected and added to a medium vial containing 20 ml of 5x concentration 1 / 2 medium A (containing 0.475 g glucose, 0.135 g sodium acetate, and 2.09 g modified GAM medium in 20 ml), which had been previously autoclaved. To each vial, 1 ml of pre-culture of the C. ramosum strain was added, and the samples were incubated statically at 37°C under anaerobic conditions using an aneropack for 2 days.

[0044] The culture supernatant was transferred to a 1.5 ml microtube and centrifuged (17,800 × g, 10 min, 25°C) to precipitate the bacterial cells. The supernatant was then collected and sterilized by filtering through a 0.2 μm syringe filter. 1 ml of the collected filtrate was transferred to a 14 ml culture tube, to which 200 μl of C. ramosum pre-culture solution was added. 3 ml of 0.7% agar-containing medium A was then added, mixed without creating bubbles, and poured onto medium A agar plates for solidification. This was incubated under anaerobic conditions at 37°C for 2 days. As a result, plaque formation was observed on the top agar in three samples.

[0045] 4. Isolation of phages using the top agar method The observed plaques were collected, diluted, and purified by allowing plaque formation again, thereby obtaining plaques derived from a single phage. Using a 200 μl yellow tip with the tip cut off, single plaques were scooped out along with the top agar and resuspended in 200 μl of modified SM buffer (final concentrations: NaCl 100 mM, CaCl 210 mM, MgSO4 10 mM). These were then sequentially diluted 10-fold with the modified SM buffer. 10 μl of each of these dilutions was spotted onto agar medium A, which had been overlaid with a top agar containing the host strain's culture medium. The mixture was then incubated under anaerobic conditions at 37°C for 1 day to form single plaques. This procedure was repeated twice to purify the phages. The three purified strains were labeled CR-V-MCCO02, CR-V-MCCO04, and CR-V-MCCO05 (accessions: NITE P-03713, NITE P-03714, NITE P-03715).

[0046] 5. Analysis of host specificity of bacteriophages The host specificity of the three isolated phage strains was investigated using a plaque assay. C. ramosum JCM1298 (corresponding to ATCC25582) was used as the C. ramosum strain, and Pseudomonas aeruginosa PAO1, Pseudomonas putida ATCC12633, Escherichia coli DH10B, Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH5α, and Staphylococcus aureus ATCC BAA-2313 were used as test strains with different physiological and biochemical properties.

[0047] Table 1 shows the presence or absence of plaque formation and the degree of plaque formation. The degree of plaque formation was determined by the following formula: 20% or more was marked "+++", 20-10% was marked "++", and less than 10% was marked "+". It was found that the three isolated phage strains specifically infected and lysed the C. ramosum strain. Plaque formation degree = [(Background signal intensity when imaging the plate - Plaque signal intensity) / Background signal intensity] × 100

[0048]

Table 1

[0049] [Example 2: Genome analysis of C. ramosum - specific bacteriophage] 1. Amplification of phage by liquid culture To obtain sufficient amounts of CR-V-MCCO02, CR-V-MCCO04, and CR-V-MCCO05, phage amplification in liquid medium was performed. 50 ml of medium A was prepared in a 250 ml medium bottle, covered with aluminum foil instead of a cap, and autoclaved (115 °C, 15 min). 500 μl of the preculture solution of C. ramosum JCM1298 strain (about 1×10 8 c.f.u.) and phage 1×10 8 p.f.u. (M.O.I. = 1) or 1×10 7 p.f.u. (M.O.I. = 0.1) were added, and static culture was performed at 37 °C for 1 day under anaerobic conditions using an AnaeroPack. The culture solution was centrifuged (3,739×g, 10 min, 25 °C), the supernatant was collected, and then a part of it was transferred to a 1.5 ml tube and centrifuged again (17,800×g, 10 min, 25 °C), followed by filter sterilization using a 0.2 μm filter, and the phage solution was collected in a 5 ml polypropylene tube. A part of the recovered phage solution was sampled, serially diluted 10-fold with modified SM buffer, and 10 μl of each dilution was spotted on a medium A agar medium overlaid with top agar containing C. ramosum JCM1298 strain, and cultured at 37 °C for 1 day under anaerobic conditions using an AnaeroPack. The number of plaques that appeared on the plate after culture was counted, and the titer was determined from the dilution ratio. As a result, phage solutions showing a titer of 1×10 8 p.f.u. / ml or more in the liquid medium could be prepared for all three phage strains, whether M.O.I. = 1 or M.O.I. = 0.1.

[0050] 2. Concentration of phage solution Dispense 40 mL of culture medium from a medium vial into a 50 mL Falcon tube and centrifuge at 5000 × g for 10 minutes at room temperature. Filter the supernatant through a 0.22 μm filter, dispense 20 mL of the solution into 50 mL Falcon tubes, add 30 mL of PEG6000-NaCl solution (final concentration PEG6000 20 w / v%, NaCl 2.5 M) to each tube, mix well, and cool on ice for at least 2 hours. Centrifuge at 8000 × g at 4°C for 30 minutes and remove the supernatant. Resuspend the precipitate in 8 mL of modified SM buffer to obtain the phage concentrate.

[0051] 3. Preparation of phage genome DNA To 8 ml of phage concentrate, 12 ml of PEG6000-NaCl solution was added and thoroughly mixed, then cooled on ice for at least 2 hours. Centrifuge was performed at 8000 × g at 4°C for 30 minutes, and the supernatant was removed. The precipitate was resuspended in 356 μl of sterile water, and then 40 μl of 10 × DNase I buffer, 2 μl of DNase I, and 2 μl of RNase A were added. The mixture was reacted at 37°C for at least 2 hours to degrade the host bacterial DNA and RNA. 60 μl TE buffer, 20 μl 0.5 M EDTA (pH 8.0), 10 μl 10% SDS, and 10 μl Proteinase K solution were added, and the mixture was reacted at 56°C for more than 2 hours, with mixing approximately every 15 minutes using a vortex mixer. 500 μl of phenol-chloroform solution was added and thoroughly mixed to form an emulsion. The mixture was then centrifuged (17,800 × g, 10 min, 25°C), and the supernatant was collected while taking care not to aspirate the intermediate layer. This procedure was repeated until the intermediate layer was no longer visible (approximately 3-4 times). Then, 500 μl of chloroform was added to the collected aqueous layer and thoroughly mixed. The mixture was then centrifuged (17,800 × g, 10 min, 25°C), and the supernatant was collected. To the collected solution, 1 / 10 volume of 3 M sodium acetate solution (pH 5.2) was added and mixed, and then 2-2.5 times the volume of 99% ethanol was added. The filamentous DNA aggregates were collected in a separate tube, and 2-2.5 times the volume of 99% ethanol was added again. The mixture was then left at -30°C for at least 30 minutes. Genomic DNA was precipitated by centrifugation (17,800×g, 10 min, 4°C), and the supernatant was removed. 500 μl of 70% ethanol was added, and centrifugation (17,800×g, 5 min, 25°C) was performed. The supernatant was then removed and the mixture was dried. After redissolving the DNA with 50 μl of TE buffer, a portion of each sample was diluted 10-fold with TE buffer, and the DNA concentration was determined using a micro-spectrophotometer (Table 2). The extracted DNA was treated with the restriction enzyme HhaI and subjected to electrophoresis together with untreated DNA. The treated DNA showed multiple bands, indicating that the phage genome is double-stranded DNA.

[0052] [Table 2]

[0053] 4. Determination of phage genome sequencing Long-read and short-read sequencing data were obtained for the genomic DNA of CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005 using a next-generation sequencer. Hybrid assembly was performed using this data to determine the genomic DNA sequences. Long-read sequencing data were obtained using Nanopore's PromethION, and short-read data were obtained using MGI Tech's DNBSEQ. Short-read data were cleaned using cutadapt, and long-read data were cleaned using porechop and nanofilt, after which assembly was performed using Unicycler. After assembly, base error correction was performed using Medaka and Pilon to obtain a single circularized contig sequence. The sequence lengths for the genomic DNA of CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005 were 105,401 bp (SEQ ID NO: 1), 102,464 bp (SEQ ID NO: 2), and 103,211 bp (SEQ ID NO: 3), respectively. Based on gene prediction analysis performed on the obtained genome sequence using Prokka v1.14.5, it was estimated that approximately 140 genes were contained in the genome.

[0054] 5. Comparison of Phage Genome DNA Sequences The sequence similarity of genomic DNA between CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005 was compared. Using CR-V-MCC002 as a baseline, the sequence similarity between CR-V-MCC004 and CR-V-MCC005 was calculated to be 85.62% and 85.33%, respectively.

[0055] The genomic DNA sequences of CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005 were searched using BLAST on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) against the Nucleotide Collection (nr / nt) database. The results are shown in Table 3-5.

[0056] Search results for the genomic DNA sequence (Sequence ID 1) of CR-V-MCC002 (extracting the top 5 results in terms of query coverage) [Table 3]

[0057] Search results for the genomic DNA sequence (SEQ ID NO: 2) of CR-V-MCC004 (extracting the top 5 results in terms of query coverage) [Table 4]

[0058] Search results for the genomic DNA sequence (SEQ ID NO: 3) of CR-V-MCC005 (extracting the top 5 results in terms of query coverage) [Table 5]

[0059] For all three genome sequences of CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005, the only search result with sufficient query coverage was the sequence registered with accession number BK026068.1 (MAG TPA_asm: Siphoviridae sp. isolate ct4bT3, partial genome, sequence length 100535 bp). The genome sequences of the 19 bacteriophages disclosed in Patent Document 2 were not found. According to a document registered in relation to the sequence with accession number BK026068.1 (Proc Natl Acad Sci USA, 2021, Jun 8;118(23)), the sequence is registered as a MAG (Metagenome-Assembled Genome), and it is presumed that the phage containing this sequence has not been isolated.

[0060] 6. Observation using a transmission electron microscope Since phage classification cannot be determined solely from genome sequencing, the phage (CR-V-MCC002) was imaged using negative staining with a transmission electron microscope (TEM) to confirm its morphology. The phage morphology (Figure 1) was identical to that of a lambda phage, suggesting it belongs to the Siphoviridae family.

[0061] [Example 3: Intestinal Survival Ability Test] Bacteriophages (or phages) are seen as a potential alternative to antibiotics for treating certain bacterial infections and disorders of the gut microbiota. For phages to be effective therapeutic agents for gut microbiota disorders, it is necessary to demonstrate their ability to survive in the gastrointestinal environment. Therefore, we administered a phage solution orally to mice, collected fecal samples, and measured the bacterial flora and phage levels in the feces to verify whether phages could act in the gastrointestinal tract.

[0062] 1. Creation of a simulated gut microbiota cocktail

[0063] To verify the specific inhibitory effect of oral administration of phages on the growth of C. ramosum bacteria in the mouse gut, a bacterial cocktail containing C. ramosum bacteria was colonized in the gut of germ-free mice. For the preparation of the bacterial cocktail, the following eight types were purchased and used, based on the pseudo-intestinal bacterial cocktail described in the literature (mBio 2014 Sep 30;5(5):e01530-14, Microorganisms, 2019 Dec 3;7(12):641). Clostridium ramosum ATCC 25582 Clostridium butyricum NBRC 13949 Blautia producta ATCC 27340 Bifidobacterium longum NBRC 114370 Anaerostipes caccae NBRC 114412 Escherichia coli NBRC 3301 Bacteroides thetaiotaomicron ATCC 29148 Lactiplantibacillus plantarum NBRC 3070

[0064] For each bacterium, cultivation and subculturing were repeated until the OD reached 1.0 or higher, and a 10 mL bacterial suspension with an OD of 1.0 was prepared by diluting it with culture medium as appropriate. The bacterial mixture was centrifuged, and the bacterial pellet was suspended in 400 μl of 8% glycerose / PBS. 150 μl of the bacterial mixture for each bacterial species was mixed to make 1.2 ml, and 150 μl of 8% glycerose / PBS was added to prepare a total of 1.5 ml of mixed stock solution.

[0065] 2. Administration of phages to mice Sixteen germ-free mice (6 weeks old) of the mouse strain C57BL / 6NCr were purchased and divided into three groups (see Table 6). In a vinyl isolator, 1000 μl of mixed stock solution was orally administered for two weeks to colonize the intestinal flora. Group 1 (Vehicle) received 1000 μl of phosphate buffer (pH 8.0) orally on day 15. Groups 2 and 3 (Phage administration) received phage solution orally as a single dose on day 15 or consecutively from days 15 to 18. The phage solution contained 4.8 × 10⁶ of CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005, respectively. 6 PFU, 3.0 × 10 6 PFU, 2.4 × 10 6 The mixture, which was mixed with pfu, was used after replacing the solvent with phosphate buffer (pH 8.0).

[0066] [Table 6]

[0067] 3. Results of comparison between groups of mouse intestinal bacteria Stool samples were collected on days 14, 15, and 18, nucleic acids were extracted from the feces, and the microbial flora was analyzed using a next-generation sequencer. Next-generation sequencing was performed using an Illumina iseq100, targeting the V4 region of the 16S rRNA sequence to obtain a fastq file. Analysis was performed using QIIME2 (https: / / qiime2.org / ) t2), ver. 2020.11. Using DADA2, 5' and 3' terminal bases were removed, quality filtering was performed, and chimeric sequences were removed to obtain representative Amplicon Sequence Variant (ASV) sequences. The bacterial species and genus of the representative sequences were identified using the Naive Bayes classifier in QIIME2. After identification, the relative amounts of bacterial species and genus were obtained for each sample.

[0068] Figure 2 shows the results of comparing the number of C. ramosum bacteria between the groups. Suppression of C. ramosum growth was confirmed in groups 2 and 3 (phage administration). Quantitative detection of C. ramosum in feces suggests the possibility of evaluating the antibacterial effect of the bacteriophage described herein against Clostridium bacteria in the intestinal tract.

[0069] 4. Detection of phages in mouse fecal samples Specific primers for the genomic DNA of CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005 were designed as follows, and absolute quantification was performed by QPCR. A calibration curve was created using double-stranded DNA of the same size as the amplified region as a control sample, and quantification was performed using the calibration curve.

[0070] Primer for CR-V-MCC002 strain P_V002S2_F:5'-TGATGACAGGGGAAAGCCATAC-3'(Sequence ID 4) P_V002S2_R:5'-TCGCTAGGGGTAGCATACAATG-3'(Sequence ID 5) Primer for CR-V-MCC004 strain P_V004S3_F:5'-AAGAATTCCATAAGCACCAC-3'(Sequence ID 6) P_V004S3_R:5'-GTGAAGTAAATAGTGCTAGGAG-3'(Sequence ID 7) Primer for CR-V-MCC005 strain P_V005S1_F:5'-CGCAAACACACAATCATCCG-3'(Sequence ID 8) P_V005S1_R:5'-AAATCACAAGGTGGGGCAAC-3'(Sequence ID 9)

[0071] The results are shown in Figure 3. All three CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005 were detected in the fecal samples, with CR-V-MCC002 being detected in particularly high concentrations. These results support the finding that CR-V-MCC002, CR-V-MCC004, and CR-V-MCC005 are amplified in the intestine and exhibit specific antibacterial activity against Clostridium ramosum. Quantitative detection of the bacteriophages described herein in feces suggests the potential for evaluating the antibacterial effect of bacteriophages against Clostridium bacteria in the intestinal tract. [Sequence Listing Free Text]

[0072] Sequence ID 1: Genomic DNA sequence of CR-V-MCC002 Sequence ID 2: Genomic DNA sequence of CR-V-MCC004 Sequence ID 3: Genomic DNA sequence of CR-V-MCC005 Sequence ID 4: Base sequence of primer P_V002S2_F Sequence ID 5: Base sequence of primer P_V002S2_R Sequence ID 6: Base sequence of primer P_V004S3_F Sequence ID 7: Base sequence of primer P_V004S3_R Sequence ID 8: Base sequence of primer P_V005S1_F Sequence ID 9: Base sequence of primer P_V005S1_R

Claims

1. (A) Having a genome containing the nucleotide sequence of SEQ ID NO: 1, 2, or 3, (B) Having a genome containing a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 1, 2, or 3, and exhibiting antibacterial activity against Clostridium bacteria, Bacteriophage.

2. The bacteriophage according to claim 1, wherein the Clostridium bacterium is Clostridium ramosum, Clostridium botulinum, Clostridium tetani, Clostridium difficile, Clostridium perfringens, or Clostridium sordellii.

3. The bacteriophage according to claim 2, wherein the Clostridium bacterium is Clostridium ramosum.

4. The bacteriophage according to claim 1, wherein the antibacterial activity is lytic activity or growth inhibitory activity.

5. A pharmaceutical product containing the bacteriophage described in claim 1 as an active ingredient.

6. The pharmaceutical product according to claim 5, used for one or more purposes selected from the group consisting of treatment of obesity, prevention of obesity, treatment of infectious diseases, prevention of infectious diseases, and improvement of the intestinal microbiota.

7. The pharmaceutical product according to claim 6, formulated for oral administration.

8. A food, beverage, supplement, or feed containing the bacteriophage described in claim 1.

9. The food, beverage, supplement, or feed according to claim 8, used for one or more purposes selected from the group consisting of treatment of obesity, prevention of obesity, treatment of infectious diseases, prevention of infectious diseases, and improvement of the gut microbiota.

10. A bacteriophage identified by accession number NITE P-3713.

11. A bacteriophage identified by accession number NITE P-3714.

12. A bacteriophage identified by accession number NITE P-3715.

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