Patent Application
20220125858
The present invention relates to an antibacterial composition against drug-resistant bacteria or pro-inflammatory bacteria. The present invention also relates to a pharmaceutical composition or method for treating, ameliorating, or preventing diseases caused by drug-resistant bacteria or pro-inflammatory bacteria.
Various commensal bacteria are resident on mucous membranes such as the digestive tract and the oral cavity, forming a flora as a whole. The resident flora plays a very important role in the physiology and health maintenance of the host. A compositional abnormality of the resident flora is called dysbiosis, and has been gradually revealed to be a cause of various diseases. The advancement of the elucidation of the resident mucosal flora may have a high possibility of developing new disease countermeasures and treatments for various diseases, but the detailed mechanism of such flora has not been sufficiently clarified due to its complexity.
Humans produce and swallow about 1.5 liters of saliva every day. Normally, bacteria contained in saliva (oral bacteria) simply pass through the intestinal tract and do not colonize. However, in some situations, oral bacteria may colonize in the intestinal tract. Particularly in Crohn's disease, liver cirrhosis, and colorectal cancer, it has been reported that colonization of oral bacteria in the intestinal tract was observed even at the early stages of disease onset. In addition, it is known that the colonized oral bacteria affect the pathological conditions of the diseases (NPLs 1 to 6).
Meanwhile, by allowing oral bacteria of patients with Crohn's disease and the like to colonize in the intestinal tract to induce Th1 cells, the present inventors succeeded in isolating, culturing, and identifying the bacteria involved in the onset of the diseases (PTL 1). More specifically, the present inventors found that as a result of oral administration of saliva derived from a patient with Crohn's disease to germ-free mice, interferon gamma (IFN-γ)-producing CD4-positive T-cells (Th1 cells) significantly increased in the large intestine. Then, the present inventors succeeded in isolating and culturing the Kp2H7 strain, which is considered to belong to Klebsiella pneumoniae, from the intestines of the mice in which this increase in Th1 cells was observed. Furthermore, it was also clarified that the bacteria derived from the saliva of patients with Crohn's disease were involved in the development of enteritis by colonizing in the intestinal tract and inducing the proliferation or activation of Th1 cells.
Moreover, the present inventors found that when Kp2H7 strains were orally administered to SPF (specific-pathogen-free) mice, intestinal colonization of these bacterial strains was not observed, unlike the case of the germ-free mice. Furthermore, it was also clarified that by administering an antibiotic to the SPF mice, these bacterial strains might be able to colonize in the intestinal tracts of the mice. Then, from these results, the present inventors considered that the intestinal tract contains intestinal bacteria that inhibit the intestinal colonization of Th1 cell-inducible bacteria such as the Kp2H7 strain, and administration of the antibiotic eliminates the intestinal bacteria from the intestinal tract, making the intestinal colonization of the bacteria possible.
Therefore, the present inventors tried to identify the bacteria that suppress the intestinal colonization of Th1 cell-inducible bacteria among human intestinal bacteria. As a result, the present inventors succeeded in isolating and culturing 68 strains, 37 strains, and 42 strains of intestinal bacterial strains from fecal samples derived from 3 healthy subjects (#K, #F, and #1), respectively, and in determining the sequence of 16S rDNA of each bacterial strain. Furthermore, it was clarified that administration of these bacterial strains suppressed intestinal colonization of Th1 cell-inducible bacteria (PTL 2).
The present invention has an object to find an intestinal bacterium having antibacterial activity against drug-resistant bacteria or pro-inflammatory bacteria, and to provide an antibacterial composition against drug-resistant bacteria or pro-inflammatory bacteria containing the intestinal bacterium as an active ingredient as well as a pharmaceutical composition or method for treating, ameliorating, or preventing diseases caused by drug-resistant bacteria or pro-inflammatory bacteria.
The present inventors have made earnest studies to achieve the above object, and have clarified as a result that the above-mentioned bacteria that suppress the intestinal colonization of Th1 cell-inducible bacteria (68 strains of intestinal bacteria derived from healthy subject #K, 37 strains of intestinal bacteria derived from healthy subject #F, and 42 strains of intestinal bacteria derived from healthy subject #1) can suppress the intestinal colonization of multidrug-resistant bacteria (carbapenem-resistant Enterobacteriaceae, vancomycin-resistant enterococci, Clostridium difficile, Campylobacter jejuni) and pro-inflammatory bacteria (adherent-invasive Escherichia coli).
Furthermore, regarding the ability to suppress bacterial colonization in the intestine, the present inventors have succeeded in selecting 18 strains from the 37 strains of intestinal bacteria derived from healthy subject #F, which are capable of exerting the same level of suppression ability as the 37 strains. Thus, the present invention has been completed.
Specifically, the present invention provides the following.
[1] An antibacterial composition against drug-resistant bacteria or pro-inflammatory bacteria comprising: an intestinal bacterium as an active ingredient.
[2] The antibacterial composition according to [1], wherein the intestinal bacterium is at least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69 to 105 or a base sequence having at least 90% identity to the base sequence.
[3] The antibacterial composition according to [1], wherein the intestinal bacterium is at least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 or a base sequence having at least 90% identity to the base sequence.
[4] The antibacterial composition according to [1], wherein the intestinal bacterium is at least one bacterium specified by any of accession numbers NITE BP-03147 to 03164.
[5] The antibacterial composition according to [1], wherein the intestinal bacterium is at least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 147 or a base sequence having at least 90% identity to the base sequence.
[6] The antibacterial composition according to [1], wherein the intestinal bacterium is at least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 68 or a base sequence having at least 90% identity to the base sequence.
[7] The antibacterial composition according to [1], wherein the intestinal bacterium is at least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 106 to 147 or a base sequence having at least 90% identity to the base sequence.
[8] The antibacterial composition according to any one of [1] to [7], which is a pharmaceutical composition.
[9] The antibacterial composition according to any one of [1] to [7], which is a pharmaceutical composition for treating, ameliorating, or preventing an infectious disease or an inflammatory disease.
According to the present invention, by suppressing the colonization and the like of drug-resistant bacteria or pro-inflammatory bacteria in the intestinal tract, it is possible to suppress the proliferation or activation of these bacteria, and it is possible to treat, ameliorate, or prevent diseases caused by these bacteria.
<Intestinal Bacteria>
In the present invention, the intestinal bacteria contained as an active ingredient of the antibacterial composition have an antibacterial action against drug-resistant bacteria or pro-inflammatory bacteria (hereinafter also referred to as “drug-resistant bacteria and the like”) in the intestinal tract.
In the present invention, the “antibacterial activity” means an activity of suppressing the activity of bacteria, more specifically, an activity of suppressing the proliferation or colonization of bacteria or killing bacteria, and examples thereof include the activity of suppressing the colonization of bacteria in the intestines and the activity of eliminating bacteria from the intestines.
The “Intestinal bacteria” mean bacteria present in the intestinal tract of an animal. Examples of animals in which such bacteria are present include humans and non-human animals (such as mice, rats, monkeys, pigs, cattle, horses, sheep, goats, chickens, ducks, ostriches, domestic ducks, dogs, cats, rabbits, and hamsters), but among these animals, humans are preferable.
In the present invention, the “intestinal bacteria” may be a single strain of bacteria or a mixture of bacterial strains composed of multiple strains of bacteria. Note that when composed of multiple strains of bacteria, it is desirable that at least one of the bacterial strains has antibacterial activity against drug-resistant bacteria and the like. Further, in such a case, the multiple strains of bacteria may even include a bacterial strain that does not have the antibacterial activity, such as a bacterial strain having an action of enhancing the activity of a bacterial strain having the antibacterial activity, a bacterial strain having an action of maintaining the proliferation or colonization of a bacterial strain having antibacterial activity, and a bacterial strain having an action of suppressing the inhibitory activity of a bacterium that inhibits the antibacterial activity.
In the present invention, examples of the “intestinal bacteria” include at least one bacterium having a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 147 or a base sequence having at least 70% identity to the base sequence, at least one bacterium having a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 68 or a base sequence having at least 70% identity to the base sequence, at least one bacterium having a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69 to 105 or a base sequence having at least 70% identity to the base sequence (for example, at least one bacterium having a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 or a base sequence having at least 70% identity to the base sequence), and at least one bacterium having a DNA composed of a base sequence set forth in any of SEQ ID NOs: 106 to 147 or a base sequence having at least 70% identity to the base sequence.
The sequence indicated by each SEQ ID NO is the sequence of 16S rDNA of each bacterium K68, F37, and 143 in the attached document. Tables 1 to 4 below show the correspondence between each bacterium, the SEQ ID NO indicating the sequence of each 16S rDNA, and each bacterial species estimated from the sequence. In addition, K, F, and I represent intestinal bacteria isolated from the feces of three healthy subjects (Japanese) (see PTL 2).
Tables 1 to 4 show the top-hit species names and RefSeq accessions as a result of a BLAST search for the sequences set forth in the SEQ ID NOs on the RefSeq 16s DNA sequence database (as of Jan. 8, 2020). Note that it is generally said % identity>97% can identify species, and >94% can identify genus. Therefore, it should be understood that bacterial strains with a % identity of >94% are bacteria that can be identified at the genus level.
The “at least 70% identity” in the intestinal bacteria of the present invention means that the identity to each base sequence is preferably 80% or more, more preferably 85% or more, further preferably 90% or more (for example, 91% or more, 92% or more, 93% or more, and 94% or more), more preferably 94% or more (for example, 95% or more, 96% or more, 97% or more, and 98% or more), and particularly preferably 99% or more.
In addition, the homology or identity of a sequence (amino acid sequence or nucleotide (base) sequence) can be determined by using the BLAST (Basic Local Alignment Search) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When analyzing the homology or identity between sequences by BLAST, it can be determined by using, for example, the BLAST of the National Center for Biotechnology Information (NCBI) (for example, by using the default, that is, initially set parameters).
In the present invention, the “intestinal bacterium” which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 147 or a base sequence having at least 70% identity to the base sequence is preferably at least 15 bacteria in the intestinal bacterial group, more preferably at least 30 bacteria in the intestinal bacterial group, further preferably at least 75 bacteria in the intestinal bacterial group, more preferably at least 120 bacteria in the intestinal bacterial group, further preferably at least 135 bacteria in the intestinal bacterial group, more preferably at least 140 bacteria in the intestinal bacterial group, further preferably 147 intestinal bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 147 or a base sequence having at least 70% identity to the base sequence, and particularly preferably 147 bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 147.
In the present invention, the “intestinal bacterium” which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 68 or a base sequence having at least 70% identity to the base sequence is preferably at least 7 bacteria in the intestinal bacterial group, more preferably at least 15 bacteria in the intestinal bacterial group, further preferably at least 35 bacteria in the intestinal bacterial group, more preferably at least 60 bacteria in the intestinal bacterial group, further preferably at least 65 bacteria in the intestinal bacterial group, more preferably 68 intestinal bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 68 or a base sequence having at least 70% identity to the base sequence, and particularly preferably 68 bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 68. In addition, the “intestinal bacterium” which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 68 or a base sequence having at least 70% identity to the base sequence desirably has resistance to ampicillin. Also, the present invention uses 46 bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 46 or a base sequence having at least 70% identity to the base sequence.
In the present invention, the “intestinal bacterium” which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69 to 105 or a base sequence having at least 70% identity to the base sequence is preferably at least 4 bacteria in the intestinal bacterial group, more preferably at least 8 bacteria in the intestinal bacterial group, further preferably at least 18 bacteria in the intestinal bacterial group, more preferably at least 29 bacteria in the intestinal bacterial group, further preferably at least 33 bacteria in the intestinal bacterial group, more preferably at least 35 bacteria in the intestinal bacterial group, further preferably 37 intestinal bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69 to 105 or a base sequence having at least 70% identity to the base sequence, and particularly preferably 37 bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69 to 105. In addition, the “intestinal bacterium” which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69 to 105 or a base sequence having at least 70% identity to the base sequence desirably has sensitivity to ampicillin.
In the present invention, the “intestinal bacterium” which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 or a base sequence having at least 70% identity to the base sequence is preferably at least 2 bacteria in the intestinal bacterial group, more preferably at least 5 bacteria in the intestinal bacterial group, further preferably at least 10 bacteria in the intestinal bacterial group, more preferably at least 14 bacteria in the intestinal bacterial group, further preferably at least 15 bacteria in the intestinal bacterial group, more preferably at least 16 bacteria in the intestinal bacterial group, further preferably at least 17 bacteria in the intestinal bacterial group, more preferably 18 intestinal bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 or a base sequence having at least 70% identity to the base sequence, and particularly preferably 18 bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 (bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 69, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 80, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 85, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 86, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 87, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 88, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 89, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 90, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 91, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 92, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 94, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 96, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 98, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 99, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 100, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 101, bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 103, and bacteria which include a DNA composed of a base sequence set forth in SEQ ID NO: 105).
Note that typical examples of 18 bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 are accession bacterial strains shown in Table 5 below. All of the bacterial strains were deposited with the National Institute of Technology and Evaluation (NITE, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Room 122) on Mar. 2, 2020.
Note that as long as the antibacterial action and the like against drug-resistant bacteria or pro-inflammatory bacteria are not impaired, the intestinal bacteria according to the present invention also include bacteria (such as derivative strains and inducible strains) bred from each of these bacteria by mutation treatment, genetic recombination, genome editing, selection of natural mutant strains, and the like.
In the present invention, the “intestinal bacterium” which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 106 to 147 or a base sequence having at least 70% identity to the base sequence is preferably at least 4 bacteria in the intestinal bacterial group, more preferably at least 9 bacteria in the intestinal bacterial group, further preferably at least 22 bacteria in the intestinal bacterial group, more preferably at least 34 bacteria in the intestinal bacterial group, further preferably at least 39 bacteria in the intestinal bacterial group, more preferably at least 41 bacteria in the intestinal bacterial group, further preferably 42 intestinal bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 106 to 147 or a base sequence having at least 70% identity to the base sequence, and particularly preferably 42 bacteria each of which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 106 to 147. In addition, the “intestinal bacterium” which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 106 to 147 or a base sequence having at least 70% identity to the base sequence desirably has sensitivity to ampicillin.
Further, in the present invention, an aspect of the “intestinal bacterium” is an intestinal bacterium that exhibits resistance to at least one compound selected from the group consisting of spectinomycin, and/or sensitivity to at least one compound selected from the group consisting of ampicillin, tylosin, and chloroform. In addition, another aspect includes intestinal bacteria that exhibit resistance to metronidazole and/or sensitivity to at least one compound selected from the group consisting of vancomycin and tylosin.
In addition, as shown in Examples described later, the above-mentioned intestinal bacteria have been isolated by the present inventors and are useful because they exert an antibacterial action against drug-resistant bacteria, pro-inflammatory bacteria, and the like. Therefore, the present invention may also provide the following.
(1) At least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69 to 105 or a base sequence having at least 90% identity to the base sequence.
(2) At least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 or a base sequence having at least 90% identity to the base sequence.
(3) At least one bacterium specified by any of accession numbers NITE BP-03147 to 03164.
(4) At least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 1 to 147 or a base sequence having at least 90% identity to the base sequence.
(5) At least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69 to 105 or a base sequence having at least 90% identity to the base sequence.
(6) At least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 106 to 147 or a base sequence having at least 90% identity to the base sequence.
(7) The bacterium according to any one of (1) to (6), which is a bacterium having an antibacterial action against drug-resistant bacteria, pro-inflammatory bacteria, or bacteria that induce proliferation or activation of Th1 cells in the intestinal tract.
<Antibacterial Composition and Pharmaceutical Composition>
The composition of the present invention may be any as long as it contains the above-mentioned intestinal bacterium, and the bacterium may be a live bacterium or a dead bacterium. In addition, when the compositions can be used in combination, and they are ingested or absorbed in combination as a result (in the case of combined compositions), the above-mentioned intestinal bacteria can also be present separately in two or more compositions.
The composition of the present invention can be in the form of a pharmaceutical composition, a quasi-drug composition, food and drink (including animal feed), or a reagent used for research purposes (such as in vitro or in vivo experiments).
Since the composition of the present invention exhibits antibacterial activity against drug-resistant bacteria and the like, it is preferably used as a pharmaceutical composition, a quasi-drug composition, or food or drink for treating, preventing, or ameliorating diseases caused by the bacterium.
The composition of the present invention can be formulated by a known pharmaceutical method. For example, it can be used in the form of capsules, tablets, pills, liquids, powders, granules, fine granules, film coating preparations, pellets, troches, sublingual preparations, chews, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, coating preparations, ointments, platers, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, and the like, for the purpose of administration by oral, parenteral (for example, intestinal, intramuscular, intravenous, tracheal, intranasal, transdermal, intradermal, subcutaneous, intraocular, vaginal, intraperitoneal, rectal, or inhalation), or multiple combinations of these routes.
In these formulations, they can be appropriately combined with a pharmacologically or food and drink acceptable carrier, specifically, sterilized water, saline, buffer solution, medium, vegetable oil, solvent, base, emulsifier, suspension, surfactant, stabilizer, flavor agent, air freshener, excipient, vehicle, antiseptic, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer agent, coating agent, lubricant, colorant, sweetener, thickener, flavor modifier, solubilizer, or other additives.
In addition, in these formulations, from the viewpoint of e.g., more efficiently exerting antibacterial activity against drug-resistant bacteria and the like in the intestinal tract, particularly in a formulation intended for oral administration, the composition of the present invention may be combined with a composition that enables efficient delivery into the intestinal tract. The composition capable of such delivery into the intestinal tract is not particularly limited, and a known composition can be appropriately employed, and examples thereof include pH-sensitive compositions, compositions that suppress release to the intestinal tract (such as cellulosic polymers, acrylic acid polymers and copolymers, and vinyl acid polymers and copolymers), bioadhesive compositions that specifically adhere to the intestinal mucosa (such as polymers described in U.S. Pat. No. 6,368,586), protease inhibitor-containing compositions, and compositions specifically degraded by intestinal enzymes).
In addition, when the antibacterial composition of the present invention is used as a pharmaceutical composition, it may further contain a known substance used for treating, preventing, or ameliorating diseases caused by drug-resistant bacteria and the like (such as other antibacterial agents, anti-inflammatory agents, and immunosuppressants), or may be used in combination with such a substance.
When the composition of the present invention is used as food and drink, the food and drink may be, for example, a health food, a functional food, a food for specified health use, a food with nutrient function claims, a food with functional claims, a dietary supplement, a food for patients, or animal feed. Specific examples of the food and drink include liquid foods such as fermented beverages, oil-containing products, soups, milk beverages, refreshing beverages, tea beverages, alcoholic beverages, energy drinks, and jelly-like beverages, carbohydrate-containing foods, processed livestock foods, and processed fishery foods; and processed vegetable foods, semi-solid foods, fermented foods, confectionery, retort products, microwave-compatible foods, and the like. It is also possible to list healthy food and drink prepared in the form of powder, granules, tablets, capsules, liquid, paste, or jelly. The food and drink according to the present invention can be produced by a production technique known in the art. In the food and drink, components (such as nutrients) effective for ameliorating or preventing diseases caused by drug-resistant bacteria and the like may be added. In addition, it may be a multifunctional food and drink by combining it with other ingredients or other functional foods that exhibit functions other than the amelioration and the like.
The product (pharmaceutical product, quasi-drug, food and drink, reagent) of the composition of the present invention or the instruction manual thereof may be provided with an indication that it exhibits antibacterial activity against drug-resistant bacteria and the like, or is used for treating, ameliorating, or preventing diseases caused by the drug-resistant bacteria and the like. Further, in relation to the food and drink, the product or the like of the composition of the present invention may be labeled with a health function as a food with health claims (food for specified health use, food with nutrient function claims, food with functional claims) so that the form, target, and the like can be distinguished from general foods. Here, “the product or the instruction manual is labeled” means that a label is attached to the main body, container, packaging, or the like of the product, or a label is attached to the instruction manual, package insert, promotional material, other printed matter, or the like that discloses product information. Further, the composition of the present invention may be in the form of a kit.
Further, as described above, a pharmaceutical composition can be produced by a known formulation technique using the intestinal bacterium and the like of the present invention. Therefore, the present invention also provides the use of the intestinal bacterium and the like of the present invention for producing a pharmaceutical composition for treating, ameliorating, or preventing diseases caused by drug-resistant bacteria and the like.
<Treatment Method and the Like>
The present invention also provides a method for treating, ameliorating, or preventing diseases caused by drug-resistant bacteria and the like in a target, wherein the target is allowed to ingest the above-mentioned antibacterial composition or pharmaceutical composition, or the above-mentioned intestinal bacteria as an active ingredient thereof (hereinafter also collectively referred to as “the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof”).
In the present invention, the “drug-resistant bacterium” means a bacterium that has resistance to antibacterial agents (such as antibiotics) and the drugs are ineffective or difficult to be effective. Further, the drug may be one drug or multiple drugs. That is, the drug-resistant bacteria according to the present invention also include multidrug-resistant bacteria. Such bacteria are not particularly limited, but examples thereof include carbapenem-resistant Enterobacteriaceae (CRE, KPC-2-producing Klebsiella pneumoniae, and the like), vancomycin-resistant enterococci (VRE, bacteria with vancomycin-resistant gene (vanA), and the like), Clostridium difficile, and Campylobacter jejuni. More specific examples include Klebsiella pneumoniae (ATCC BAA-1705), Enterococcus faecium (Orla-Jensen) Schleifer and Kilpper-Balz (ATCC 700221), Clostridioides difficile (Prevot) Lawson et al. (ATCC 43255, bacterial strain designation: VPI 10463), Clostridioides difficile (Prevot) Lawson et al. (ATCC BAA-1382, bacterial strain designation: 630), and Campylobacter jejuni 81-176 (ATCC BAA2151).
The “diseases caused by drug-resistant bacteria” include infectious diseases caused by drug-resistant bacteria. They also include diseases associated with the infection. Examples of such diseases include respiratory infectious diseases such as sepsis, peritonitis, meningitis, gastroenteritis, and pneumonia, urinary tract infectious diseases, surgical site infectious diseases, soft tissue infectious diseases, and medical device-related infectious diseases (such as medical device-related bloodstream infectious diseases).
In the present invention, the “pro-inflammatory bacterium” means a bacterium that induces inflammation in the intestinal tract, and examples thereof include adherent-invasive Escherichia coli (AIEC). More specifically, AIEC LF82 can be mentioned.
The “diseases caused by pro-inflammatory bacteria” include diseases caused or involved in inflammation caused by the bacteria. Examples of such diseases include inflammatory bowel diseases (including chronic inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, and inflammatory bowel disease).
The pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof can be used for animals including humans, but there are no particular restrictions on animals other than humans, and various domestic animals, poultry, pets, laboratory animals, and the like can be targeted.
In addition, the ingestion target of the intestinal bacterium and the like of the present invention includes animals carrying the drug-resistant bacteria and the like, regardless of the onset of diseases caused by the drug-resistant bacteria and the like. From the viewpoint of prevention, an animal that does not possess the bacteria or is suspected of possessing them may be allowed to ingest the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof.
The method for ingesting the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof is not particularly limited, and may be oral administration or parenteral administration (for example, administration into the intestinal tract), and in the case of oral administration, from the viewpoint of further improving the effects of the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof, it is preferable that the ingestion target of the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof has reduced production of gastric acid by ingestion of a proton pump inhibitor (PPI) or the like.
In addition, in the case of ingesting the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof, the amount ingested can be appropriately selected by those skilled in the art according to the target's age, weight, disease symptoms, health conditions, composition type (such as pharmaceutical product, and food and drink), ingestion method, and the like.
Preferable embodiments of the antibacterial composition and the pharmaceutical composition as well as the therapeutic method and the like of the present invention have been described above, but the present invention is not limited to the above embodiments.
As shown in Examples described later, regarding the ability to suppress intestinal colonization possessed by the Th1 cell-inducible bacterium Klebsiella 2H7 strain (Kp2H7), the present inventors have succeeded in selecting strains capable of exerting the same level of suppression ability as the 37 strains of intestinal bacteria derived from healthy subject #F. Therefore, the present invention may also provide the following aspects with respect to antibacterial compositions and pharmaceutical compositions as well as treatment methods and the like.
<1> An antibacterial composition against Th1 cell-inducible bacteria, containing an intestinal bacterium as an active ingredient, wherein the intestinal bacterium is at least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 or a base sequence having at least 90% identity to the base sequence.
<2> The antibacterial composition according to <1>, which is a pharmaceutical composition.
<3> The antibacterial composition according to <1> or <2>, which is a pharmaceutical composition for treating, ameliorating, or preventing diseases caused by Th1 cells.
<4> A method for suppressing proliferation or activation of Th1 cells in a target, a method for suppressing immunity in the target, or a method for treating, ameliorating, or preventing diseases caused by Th1 cells in the target, including allowing the target to ingest the antibacterial composition according to any one of <1> to <3> or at least one bacterium which includes a DNA composed of a base sequence set forth in any of SEQ ID NOs: 69, 80, 85 to 92, 94, 96, 98 to 101, 103, and 105 or a base sequence having at least 90% identity to the base sequence.
The “Th1 cell-inducible bacterium” according to the present invention is a bacterium that normally exists in the human oral cavity, and induces the proliferation or activation of Th1 cells by colonizing in the intestinal tract. It is preferably a bacterium that belongs to Klebsiella, more preferably belonging to Klebsiella pneumoniae or Klebsiella aeromobilis, and induces the proliferation or activation of Th1 cells in the intestinal tract. Further, the “Th1 cell-inducible bacterium” is preferably a bacterium that easily colonizes in an intestinal environment in which the diversity has changed as compared with the healthy state due to the administration of an antibacterial agent. It is also a bacterium that easily colonizes in an intestinal environment in which the diversity has changed as compared with the healthy state due to colitis or the like.
For an example of the “Th1 cell-inducible bacterium,” PTL 1 can be referred to, and typical examples include Kp2H7 strain, Kal1E12 strain, 34E1 strain, BAA-1705 strain, 700603 strain, and 40B3 strain belonging to Klebsiella. Among these, the Kp2H7 strain or the Kal1E12 strain is more preferable, and the Kp2H7 strain is particularly preferable. See Table 6 for details of these bacteria.
In addition, examples of the “Th1 cell-inducible bacterium” of the present invention include a bacterium which contains a DNA composed of a nucleotide sequence having 90% or more (91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more) identity to the nucleotide sequence encoding the 16S rRNA of Kp2H7 strain, Kal1E12 strain, 34E1 strain, BAA-1705 strain, 700603 strain, or 40B3 strain, and also include a bacterium which contains a DNA composed of a nucleotide sequence having 70% or more (preferably 80% or more, more preferably 85% or more, further preferably 90% or more, and more preferably 94% or more (for example, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more)) homology or identity to the nucleotide sequence specific to Kp2H7 strain, Kal1E12 strain, 34E1 strain, BAA-1705 strain, 700603 strain, or 40B3 strain.
In the present invention, the “Th1 cell” is a subgroup of CD4 positive helper T-cells (Th cells) and means a cell that enhances cell-mediated immunity. In addition, the meaning of “activation of Th1 cells” includes the production of Th1 cytokines (such as IFN-γ) by the cells, induction of macrophages by the cytokines, activation of cells such as cytotoxic T-cells (CTL), and enhancement of cell-mediated immunity by the activation. Further, the meaning of “induction of the proliferation or activation of Th1 cells” also includes induction of differentiation from naive T-cells to Th1 cells which leads to proliferation or activation of Th1 cells.
The effect of inducing proliferation or activation of Th1 cells in the intestinal tract can be evaluated by quantitatively detecting Th1 cell-specific markers (such as CD4 and IFN-γ). Such quantitative detection can be performed by a known method, and can be performed by methods for detection using antibodies (immunological methods) such as flow cytometry, imaging cytometry, ELISA method, radioimmunoassay, immunohistochemical staining, immunoprecipitation, immunoblotting, and antibody array analysis.
Regarding whether or not any bacterium or the like has an effect of inducing proliferation or activation of Th1 cells in the intestinal tract, for example, it can be determined that the bacterium or the like has an effect of inducing proliferation or activation of Th1 cells in the intestinal tract when the proportion of IFN-γ+ cells in CD4+TCRβ+ T-cells in the intestinal tract detected by flow cytometry is 10% or more (it is preferable to determine, when the proportion is 25% or more, that the bacterium or the like has an effect of inducing proliferation or activation of Th1 cells in the intestinal tract, and it is more preferable to determine, when the proportion is 30% or more, that the bacterium, substance, or the like has an effect of inducing proliferation or activation of Th1 cells in the intestinal tract).
In the present invention, the “diseases caused by Th1 cells” mean diseases induced by proliferation or activation of Th1 cells, and examples thereof include inflammatory bowel diseases (including chronic inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, and inflammatory bowel disease), type 1 diabetes, rheumatoid arthritis, experimental autoimmune encephalomyelitis (EAE), multiple sclerosis, autoimmune diseases such as systemic lupus erythematosus, and chronic inflammatory diseases. In addition, the “immunity” suppressed in the present invention includes not only mucosal immunity (such as intestinal immunity) but also systemic immunity. Furthermore, not only cell-mediated immunity but also humoral immunity is included.
In addition, when the antibacterial composition of the present invention is used as a pharmaceutical composition, it may further contain known substances (such as anti-inflammatory agents and immunosuppressants) used for the treatment, prevention, or amelioration of diseases caused by Th1 cells, or may be used in combination with such substances.
Note that for other phrases in <1> to <4>, refer as appropriate to the above-mentioned <Antibacterial Composition and Pharmaceutical Composition> and <Treatment Method and the Like>.
<Composition for Examining Diseases Caused by Drug-Resistant Bacteria and the Like>
In the present invention, the presence of intestinal bacteria that can suppress the colonization and the like of drug-resistant bacteria and the like in the intestinal tract has been clarified. Therefore, by detecting the presence of the intestinal bacteria, it becomes possible to examine diseases caused by drug-resistant bacteria and the like.
Therefore, the present invention provides a composition for examining diseases caused by the following drug-resistant bacteria and the like.
A composition for examining diseases caused by drug-resistant bacteria and the like, containing an antibody that specifically recognizes the intestinal bacterium and the like of the present invention.
A composition for examining diseases caused by drug-resistant bacteria and the like, containing a polynucleotide for detecting a nucleotide sequence specific to the drug-resistant bacteria and the like of the present invention.
In the present invention, the “antibody that specifically recognizes the intestinal bacterium and the like of the present invention” may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of antibody (for example, Fab, Fab′, F(ab′)2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc(Fv)2, diabody, multispecific antibody, or a polymer thereof) as long as it can specifically recognize the bacterium. If the antibody of the present invention is a polyclonal antibody, it can be obtained by immunizing a host animal with an antigen (peptide, polynucleotide, sugar chain, lipid, or the like derived from the intestinal bacterium and the like of the present invention) and purifying it from its antiserum by a conventional means (such as salting-out, centrifugation, dialysis, or column chromatography). In addition, the monoclonal antibody can be produced by a hybridoma method or a recombinant DNA method.
Further, as the antibody used for the examination of the present invention, it is possible to use an antibody bound with a labeling substance. By detecting the labeling substance, it is possible to directly measure the amount of antibody bound to the intestinal bacterium and the like of the present invention or a substance derived from the bacterium. The labeling substance is not particularly limited as long as it can bind to an antibody and can be detected by a chemical or optical method, and examples thereof include fluorescent dyes (such as GFP), enzymes (such as HRP), and radioactive substances.
In addition to the antibody component, the examination composition of the present invention may contain additional components acceptable as a composition. Examples of the additional components include carriers, excipients, disintegrants, buffer agents, emulsifiers, suspensions, stabilizers, preservatives, antiseptics, saline, labeling substances, and secondary antibodies. In addition to the above examination compositions, it is possible to combine substrates required for detection of labeling substances, positive controls and negative controls, buffer solutions used for diluting or washing the sample, tubes or plates used for the reaction of the sample with the antibody of the present invention, and the like, or use as an examination kit for diseases caused by drug-resistant bacteria and the like is possible. In addition, when an unlabeled antibody is used as an antibody standard sample, it is possible to combine a labeled substance (such as secondary antibody, protein G, or protein A) that binds to the antibody. Further, the kit for examining diseases caused by drug-resistant bacteria and the like can include an instruction manual for use of the kit.
Further, the examination composition of the present invention can be combined with an apparatus for detecting the antibody of the present invention. Examples of the apparatus include a flow cytometry apparatus and a microplate reader.
In the present invention, the “polynucleotide for detecting a nucleotide sequence specific to the intestinal bacterium and the like of the present invention” is not particularly limited as long as the sequence specific to the bacterium is detected, and examples thereof include polynucleotides according to any of the following (a) and (b), each having a chain length of at least 15 nucleotides.
(a) A polynucleotide that is a pair of primers designed to sandwich the specific nucleotide sequence, and
(b) A polynucleotide that is a primer or probe that hybridizes to a nucleotide sequence containing the specific nucleotide sequence.
The polynucleotide of the present invention has a base sequence complementary to the nucleotide sequence of the intestinal bacterium and the like of the present invention. Here, “complementary” does not have to be perfectly complementary as long as it hybridizes. These polynucleotides usually have 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% homology to the nucleotide sequence.
The “chain length” of the polynucleotide of the present invention, when used as a primer, is usually 15 to 100 nucleotides, preferably 17 to 30 nucleotides, and more preferably 20 to 25 nucleotides. In addition, when used as a probe, it is usually 15 to 1000 nucleotides, and preferably 20 to 100 nucleotides.
The polynucleotide of the present invention may be a DNA or RNA, or may have its nucleotide replaced with an artificial nucleic acid such as LNA (registered trademark, crosslinked nucleic acid), ENA (registered trademark, 2′-O, 4′-C-Ethylene-bridged nucleic acids), GNA (glycerol nucleic acid), TNA (threose nucleic acid), or PNA (peptide nucleic acid) in part or all of it.
Note that the polynucleotide of the present invention can be chemically synthesized using a commercially available nucleotide automatic synthesizer or the like. Further, as the polynucleotide used for the examination of the present invention, it is possible to use a polynucleotide bound with a labeling substance. The labeling substance is not particularly limited as long as it can bind to a polynucleotide and can be detected by a chemical or optical method, and examples thereof include fluorescent dyes (such as DEAC, FITC, R6G, TexRed, and Cy5) as well as dyes such as DAB (chromogen), enzymes, and radioactive substances in addition to fluorescent dyes.
In addition to the above-mentioned polynucleotide, the examination composition of the present invention may contain additional pharmacologically acceptable components. Examples of the additional components include buffer agents, emulsifiers, suspensions, stabilizers, antiseptics, and saline.
In addition to the above examination compositions, it is possible to combine standard samples such as substrates required for detection of labeling substances added to the polynucleotide, positive controls and negative controls, and buffer solutions used for diluting or washing the sample, thereby combining tubes or plates used for the reaction of the sample with the polynucleotide of the present invention, or use as an examination kit for diseases caused by drug-resistant bacteria and the like is possible. Moreover, the kit for examining diseases caused by drug-resistant bacteria and the like can include an instruction manual for use of the kit.
In addition, the examination composition of the present invention can be combined with an apparatus for detecting a nucleotide sequence specific to the intestinal bacterium and the like of the present invention. Examples of the apparatus include a thermal cycler, a sequencer, and a microarray.
Furthermore, the present invention also provides a method for examining diseases caused by drug-resistant bacteria and the like using the above-mentioned antibody, polynucleotide, or examination composition. Specifically, the present invention provides
a method for examining diseases caused by drug-resistant bacteria and the like, including the steps of contacting the antibody, polynucleotide, or examination composition with a sample isolated from a subject, and detecting the presence or absence of an intestinal bacterium and the like of the present invention in the intestinal tract by the contact.
The subject is not particularly limited, and examples thereof include animals such as humans suspected of suffering from a disease caused by drug-resistant bacteria and the like. Further, the sample isolated from the subject is not particularly limited, but a fecal sample of a subject, a culture thereof, or a polypeptide, polynucleotide, sugar chain, lipid or the like extracted therefrom is preferably used in the method of the present invention.
As the method for detecting the presence or absence of an intestinal bacterium and the like of the present invention by contacting the sample with the antibody of the present invention or an examination composition containing the same, examples thereof include methods for detection using antibodies (immunological methods) such as ELISA method, immunoblotting, antibody array analysis, immunohistochemical staining, flow cytometry, imaging cytometry, radioimmunoassay, and immunoprecipitation.
Further, as the method for detecting the presence or absence of an intestinal bacterium and the like of the present invention by contacting the sample with the polynucleotide of the present invention or an examination composition containing the same, it is possible to use PCR (RT-PCR, real-time PCR, and quantitative PCR), DNA microarray analysis, Northern blotting, 16s rRNA sequencing, next-generation sequencing (synthetic sequencing (sequencing-by-synthesis, for example, sequencing by Solexa Genome Analyzer or Hiseq (registered trademark) 2000 manufactured by Illumine), pyrosequencing (for example, sequencing by a sequencer GSLX or FLX manufactured by Roche Diagnostics (454) (so-called 454 sequencing)), and ligase reaction sequencing (for example, sequencing by SoliD (registered trademark) or 5500x1 manufactured by Life Technologies)), bead array method, in situ hybridization, dot blotting, RNase protection assay, mass spectrometry, genomic PCR method, and Southern blotting.
In the present invention, the “examination” of a disease caused by drug-resistant bacteria and the like includes not only the presence or absence of the onset of the disease but also the risk of the onset of the disease, and if the presence of an intestinal bacterium and the like of the present invention is detected in the intestinal tract by the above-mentioned method, it can be determined that a disease caused by drug-resistant bacteria and the like has not developed or the risk of developing the disease is low.
Diagnosis of a disease caused by drug-resistant bacteria and the like in a subject is usually made by a doctor (including a person who has been instructed by a doctor), but the data obtained by the method of the present invention is useful for diagnosis by a doctor. Therefore, the method of the present invention can also be expressed as a method of collecting and presenting data useful for diagnosis by a doctor.
Further, the present invention can also provide a companion diagnostic method using the above-mentioned examination method and a drug thereof. That is, the present invention also provides the following.
A method for determining the effectiveness of the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof in the treatment, amelioration, or prevention of diseases caused by drug-resistant bacteria and the like, including the steps of contacting the antibody, polynucleotide, or examination composition with a sample isolated from a subject, and detecting the presence or absence of the intestinal bacteria and the like by the contact, wherein in the step, when the presence of the bacteria is not detected, it is determined that the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof in the subject is highly effective in treating, ameliorating, or preventing the disease.
A method for treating, ameliorating, or preventing diseases caused by drug-resistant bacteria and the like, including a step of allowing a patient determined by the determination method to have high effectiveness of the pharmaceutical composition and the like of the present invention or the active ingredient and the like thereof to ingest the pharmaceutical composition and the like or the active ingredient and the like thereof.
A composition for treating, ameliorating, or preventing diseases caused by drug-resistant bacteria and the like, containing the intestinal bacterium and the like of the present invention as an active ingredient, wherein the composition is ingested by a subject determined to have high effectiveness by the determination method.
<Method for Screening Intestinal Bacteria Having Antibacterial Activity Against Drug-Resistant Bacteria and the Like in Intestinal Tract>
It has also been clarified for the first time by the present inventors that among intestinal bacteria, there are bacteria that suppress the colonization of drug-resistant bacteria and the like in the intestinal tract. Therefore, the present invention provides a method for screening intestinal bacteria having antibacterial activity against drug-resistant bacteria and the like, including the following steps.
A step of allowing a non-human germ-free animal to ingest drug-resistant bacteria and the like and test intestinal bacteria in the intestinal tract,
a step of detecting the drug-resistant bacteria and the like in the intestinal tract of the non-human germ-free animal, and
a step of determining the test intestinal bacteria as intestinal bacteria having antibacterial activity against the drug-resistant bacteria and the like when the number of bacteria detected in the step is reduced as compared with the case where the test intestinal bacteria are not ingested.
The “drug-resistant bacteria and the like” are as described above. The “non-human germ-free animal” means a non-human animal that is born and grown under germ-free conditions. Examples of non-human animals include, but are not limited to, mice, rats, monkeys, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, domestic ducks, dogs, cats, rabbits, and hamsters. Moreover, in these animals, mice are preferably used.
The test intestinal bacteria to be ingested by non-human germ-free animals may be any bacteria present in the intestines of animals, examples of such animals include humans and non-human animals (such as mice, rats, monkeys, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, domestic ducks, dogs, cats, rabbits, and hamsters). In addition, the test intestinal bacteria to be ingested by non-human germ-free animals may be isolated intestinal bacteria, and examples thereof include a sample containing intestinal bacteria (for example, a fecal sample of the animal or a culture thereof).
Further, the method for “ingesting” the test intestinal bacteria and drug-resistant bacteria and the like to a non-human animal is not particularly limited, and is usually performed by oral administration, but may be parenteral administration (for example, administration into the intestinal tract). In addition, the test intestinal bacteria and drug-resistant bacteria and the like may be ingested at the same time, the drug-resistant bacteria and the like may be ingested by the non-human animal after the test intestinal bacteria are ingested by the animal, or the test intestinal bacteria may be ingested by the non-human animal after the drug-resistant bacteria and the like are ingested by the animal.
The “detection” of drug-resistant bacteria and the like in the intestinal tract can be performed by detecting a nucleotide sequence specific to the drug-resistant bacteria and the like. Examples of the detection method include PCR (RT-PCR, real-time PCR, and quantitative PCR), DNA microarray analysis, Northern blotting, 16s rRNA sequencing, next-generation sequencing (synthetic sequencing (sequencing-by-synthesis, for example, sequencing by Solexa Genome Analyzer or Hiseq (registered trademark) 2000 manufactured by Illumina), pyrosequencing (for example, sequencing by a sequencer GSLX or FLX manufactured by Roche Diagnostics (454) (so-called 454 sequencing)), and ligase reaction sequencing (for example, sequencing by SoliD (registered trademark) or 5500x1 manufactured by Life Technologies)), bead array method, in situ hybridization, dot blotting, RNase protection assay, mass spectrometry, genomic PCR method, and Southern blotting.
Further, the “detection” of drug-resistant bacteria and the like in the intestinal tract can be performed, for example, by detecting an amino acid sequence specific to the drug-resistant bacteria and the like. Examples of the detection method include methods for detection using antibodies (immunological methods) such as ELISA method, immunoblotting, antibody array analysis, immunohistochemical staining, flow cytometry, imaging cytometry, radioimmunoassay, and immunoprecipitation. The timing of detection is not particularly limited, and those skilled in the art can appropriately adjust the timing according to the type and the like of animal to be used.
Note that if the screening method of the present invention fails to select intestinal bacteria having antibacterial activity against drug-resistant bacteria and the like in a single trial, an intestinal sample containing the obtained bacteria is ingested by a new non-human germ-free animal as the next test intestinal bacteria, and the above-mentioned screening is performed multiple times, making it possible to isolate the intestinal bacteria having the antibacterial activity.
As shown in the upper part of
Specifically, the germ-free mice used were C57BL/6N at 4 to 8 weeks of age (CLEA Japan, Inc.), which were kept in a rearing vinyl isolator (germ-free isolator) (manufactured by ICM Co., Ltd.; ICM-1B) under the conditions of free-drinking water and feeding for one week or longer to acclimate them to the environment. The age at the start of the experiment was 8 to 14 weeks of age. The same applies to the other Examples in the present specification.
A bacterial solution of Klebsiella was placed in an LB liquid medium and cultured overnight at 37° C. to adjust the OD value to 1.2 (equivalent to 1*10{circumflex over ( )}9 CFU/mL), and 200 μL/individual (equivalent to 2*10{circumflex over ( )}8 CFU/individual) of bacterial solution was administered into the stomach of mice using a sonde.
For the fecal samples, feces provided by Japanese healthy subject volunteers (#A, #F, #I, #J, and #K) were diluted 5-fold by weight with a glycerol PBS solution (final concentration of glycerol: 20% by volume), which was filtered through a 100 μm diameter filter, and the resultant was stored as a stock solution at −80° C. At the time of fecal administration, the stock solution was diluted 10-fold with PBS in an anaerobic chamber, and administered in an amount of 200 μL/individual into the stomach of each mouse using a sonde.
Mouse fecal samples were dissolved in a solution of glycerol (final concentration 20%) and EDTA (final concentration 10 mM) mixed in PBS at a proportion of 50 mg feces/mL. The fecal lysate was seeded after appropriate dilution in DHL medium containing 50 mg/L ampicillin and mg/L spectinomycin, and after culturing at 37° C. overnight, the number of colonies was counted and the number of CFUs per 1 g of feces was calculated.
In the other Examples in the present specification, bacterial solution of Klebsiella and feces were administered in the same manner, and CFUs were counted in the same manner.
As a result, as shown in the lower part (graph) of
Each of the frozen fecal samples derived from feces of healthy subjects F, K, and I (feces F, feces K, and feces I) prepared in Example 1 was thawed at room temperature, diluted with PBS, and cultured in an agar plate of EG medium, modified GAM agar medium (Nissui Pharmaceutical Co., Ltd.; 05426), REINFORCED CLOSTRIDIAL AGAR (RCM AGAR) (Thermo Fisher Scientific Inc; CM0151), or Schaedler blood medium (manufactured by Wako; 517-45805) under an anaerobic environment of at 37° C. and 10% CO2, and the colonies formed were isolated. From feces F, 37 strains were isolated, 42 strains were isolated from feces I, and 47 strains were isolated from feces K. After that, the feces K was isolated again, and finally 68 strains were isolated from the feces K.
For the isolated bacteria, the gene sequence was analyzed and the bacterial species was estimated by 16S rDNA analysis by the Sanger method. Sequence analysis was performed using 3130 DNA Analyzer manufactured by Thermo Fisher Scientific and a primer set having the following sequences.
Here, R: A or G, Y: C or T, and M: A or C.
Furthermore, the genome sequences of the 37 bacterial strains derived from feces F were determined using a next-generation sequencer. That is, genome sequencing was performed using MiSeq manufactured by Illumina and Sequel manufactured by Pacific Biosciences, and all genome sequences were obtained by hybrid assembly using Unicycler. By extracting the 16S rRNA sequence using RNAmmer for each of these genome sequences, a more accurate sequence was obtained, including both terminal sequences that could not be determined by the 16S rDNA sequence determined by the Sanger method.
Tables 1 to 4 show the results of this analysis. In addition,
As shown in the upper part of
The isolated bacteria were cultured in mGAM liquid medium, EG medium, or CM0149 medium at 37° C. in an anaerobic chamber for 24 to 48 hours, and mixed. The mixed solution was concentrated 5 times, and 200 μL/individual (equivalent to total bacterial amount 1*10{circumflex over ( )}9 CFU/animal) of bacterial solution was administered into the stomach using a sonde. The mixed isolated bacterial strains in the following Examples were also administered in the same manner.
As a result, as shown in the lower part (graph) of
As shown in the upper part of
As shown in the upper part of
As a result, as shown in the lower part of
In addition, the change over time of the abundance ratio of each isolated bacterium in the total bacterial amount was analyzed together with that of Kp2H7.
As shown in
As shown in
As a result, as shown in
From the 37 strains, 18 strains were selected by excluding strains belonging to Bacteroidetes, strains duplicate at the 16S rDNA level, strains disappeared by ampicillin administration, and strains showing behavior unrelated to Klebsiella (see
Then, as shown in the upper part of
As a result, as shown in the lower part (graph) of
The 18 strains shown in
As a result, as shown in the lower part (graph) of
From the above, it was suggested that all of the four groups shown in
Further, in the experiment shown in
As a result, as shown in
As shown in Example 8, in the experiment of removing the bacteria of each group from F18mix, F15mix obtained by removing the 3 strains of other Phyla had the lowest ability to eliminate Klebsiella. Therefore, paying attention to this group, the present inventors prepared groups each obtained by removing any one of these 3 strains (E. coli, Bifidobacterium, and Fusobacterium) from the 18 strains derived from feces F. Then, as shown in the upper part of
As a result, as shown in the lower part (graph) of
In order to explore the mechanism of eliminating Klebsiella of F37mix, the present inventors focused on whether host immunity was involved in this mechanism. Therefore, as shown in the upper part of
As a result, as shown in the lower part (graph) of
Next, the effect of eliminating the isolated bacterial strains on pathogens and drug-resistant bacteria other than the Kp2H7 strain was evaluated. Note that in the analysis, the primers shown in Table 8 were used as primers specific to the bacteria.
First, as shown in the upper part of
Note that a bacterial solution of CRE was placed in an LB liquid medium and cultured overnight at 37° C. to adjust the OD value to 1.2 (equivalent to 1*10{circumflex over ( )}9 CFU/mL), and 200 μL/individual (equivalent to 2*10{circumflex over ( )}8 CFU/individual) of bacterial solution was administered into the stomach of mice using a sonde. Regarding the CFU count for CRE, DHL medium containing 30 mg/L ampicillin and 30 mg/L spectinomycin was used as a selective medium and cultured overnight at 37° C. under aerobic conditions.
In addition, the mice one month after administration of the mixed isolated bacteria were dissected, and the large intestine was fixed with 4% PFA and embedded in paraffin to prepare sliced sections. These sections were stained with hematoxylin solution and eosin solution to observe inflammatory images of the tissues.
As shown in the lower part (graph) of
As shown in the upper part of
Note that a bacterial solution of VRE was placed in an LB liquid medium and cultured overnight at 37° C. to adjust the OD value to 1.2 (equivalent to 1*10{circumflex over ( )}9 CFU/mL), and 200 μL/individual (equivalent to 2*10{circumflex over ( )}8 CFU/individual) of bacterial solution was administered into the stomach of mice using a sonde. Regarding the CFU count for. VRE, VRE medium (Nippon Becton) was used and cultured overnight at 37° C. under aerobic conditions.
As a result, as shown in the lower part (graph) of
As shown in the upper part of
Note that a bacterial solution of AIEC LF82 was placed in an LB liquid medium and cultured overnight at 37° C. to adjust the OD to 1.2 (equivalent to 1*10{circumflex over ( )}9 CFU/mL), and 200 μL/individual (equivalent to 2*10{circumflex over ( )}8 CFU/individual) of bacterial solution was administered into the stomach of mice using a sonde. Regarding the CFU count for AIEC LF82, MacConkey medium containing 1 mg/L cefotaxime was used as a selective medium and cultured overnight at 37° C. under aerobic conditions to calculate the CFU.
As a result, as shown in the lower part (graph) of
As shown in the upper part of
Note that a bacterial solution of Kp-ESBL was placed in an LB liquid medium and cultured overnight at 37° C. to adjust the OD to 1.2 (equivalent to 1*10{circumflex over ( )}9 CFU/mL), and 200 μL/individual (equivalent to 2*10{circumflex over ( )}8 CFU/individual) of bacterial solution was administered into the stomach of mice using a sonde. Regarding the CFU count for Kp-ESBL, DHL medium containing 30 mg/L ampicillin and 30 mg/L spectinomycin was used as a selective medium and cultured overnight at 37° C. under aerobic conditions for calculation.
As a result, as shown in the lower part (graph) of
As shown in the upper part of
The bacterial solution of Campylobacter jejuni was placed in a TS liquid medium, placed in an anaerobic jar, together with a slightly aerobic AnaeroPack; and cultured is, 42° C. fox 48 hours, and the bacterial solution was administered into the stomachs of the mice using a sonde.
For Campylobacter jejuni, CFU and qPCR were used to show the bacterial amount.
CHROMagar Campylobacter was used for CFU counting, which was placed in an anaerobic jar together with a slightly aerobic AnaeroPack and cultured at 42° C. for 48 hours.
The qPCR measurement was performed according to the following procedure.
LightCycler (registered trademark) 48011 (Roche; 05015243001) and Thunderbird (registered trademark) SYBR (registered trademark) qPCR Mix (TOYOBO; QPS-201X5) were used to amplify and quantify with Campylobacter jejuni genome-specific primers and universal bacterial primers, and the calculated DNA concentration ratio was used as the abundance ratio of Campylobacter jejuni.
Note that the primer sets set forth in SEQ ID NOs: 220 and 221 were used as Campylobacter jejuni genome-specific primers for qPCR, and the primer sets set forth in SEQ ID NOs: 222 and 223 were used as universal bacterial primers.
In addition, the bacterial genome was extracted by the following steps.
A PBS solution containing EDTA and glycerol (final concentration of EDTA: 10 mM, and final concentration of glycerol: 20% by volume) was added 5 times by weight to 10 mg of mouse feces, and the mixture was crushed and suspended with vigorous shaking and stirring. Added to 100 μL of sample solution was 800 μL of 10 mM Tris/10 mM EDTA buffer solution (pH 8.0, also referred to as “TE10” hereinafter) having 15 mg of lysozyme (manufactured by Sigma-Aldrich, Lysozyme from chicken egg white; L4919) and 5 μL of RNase (manufactured by Thermo Fisher Scientific, PureLink RNase A (20 mg/mL); 12091-021) dissolved therein, and the mixture was shaken at 37° C. for 1 hour. Subsequently, 2,000 U of Achromopeptidase (registered trademark) (Wako; 015-09951) was added, and the mixture was shaken at 37° C. for 30 minutes for lysis. Then, 50 μL of a 20% SDS TE10 solution and 50 μL of a TE10 solution having Proteinase K (Roche, Proteinase K, recombinant, PCR Grade; 0311585.101) at a final concentration of 20 mg/ml dissolved therein were added, and the mixture was shaken at 55° C. for 60 minutes. Next, DNA was obtained from 400 μL of the solution using Maxwell (registered trademark) RSC Cultured Cells DNA Kit (Promega).
As shown in
As shown in the upper part of
The bacterial solution of C. difficile was subjected to spore formation, adjusted to around 1×10{circumflex over ( )}5 cells, and administered into the stomachs of the mice using a sonde. The spore formation was performed by culturing in Clospore medium for 8 days in an anaerobic chamber at 37° C. and washing the medium with PBS, followed by treatment with sonication, addition of Lysoizyme and trypsin, and treatment at 45° C. for 6 hours and then at 70° C. for 10 minutes.
For C. difficile, qPCR was used to quantify the bacterial amount. As the primers for qPCR, the primer set shown in SEQ ID NOs: 224 and 225 was used.
As a result, as shown in the lower part of
As described above, by suppressing the colonization of drug-resistant bacteria and pro-inflammatory bacteria in the intestinal tract, the present invention makes it possible to treat, ameliorate, or prevent diseases caused by these bacteria. Therefore, the present invention is extremely useful in the development, treatment, amelioration, prevention, and the like of pharmaceutical products relating to infectious diseases caused by drug-resistant bacteria or pro-inflammatory bacteria.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/009423 | 3/5/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62815101 | Mar 2019 | US |
