Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor

Definitions

• the present invention relates to an antibacterial composition containing a gluconic acid consuming bacterium as an active ingredient, and more specifically, to an antibacterial composition against a second bacterium that consumes gluconic acid, containing a first bacterium that consumes gluconic acid as an active ingredient.
• the present invention also relates to a method for screening bacteria having antibacterial activity against bacteria that consume gluconic acid.
• a variety of resident bacteria are present in the mucous membranes of the digestive tract, oral cavity, etc., and together they form the flora.
• the resident bacterial flora plays a very important role in maintaining the physiology and health of the host. Abnormalities in the composition of the resident bacterial flora are called dysbiosis, and it is gradually becoming clear that they are the cause of various diseases. Further elucidation of the resident mucosal bacterial flora is highly likely to lead to the development of new disease countermeasures and treatments for various diseases, but due to its complexity, the detailed mechanisms have not yet been fully elucidated.
• the present inventors have succeeded in isolating, culturing, and identifying bacteria that are involved in the onset of the disease by colonizing the intestine (intestinal tract) and inducing Th1 cells from bacteria (oral bacteria) contained in the saliva of patients with Crohn's disease and the like (Patent Document 1). More specifically, the present inventors have found that oral administration of saliva from a Crohn's disease patient to germ-free mice resulted in a significant increase in IFN- ⁇ -producing CD4-positive T cells (Th1 cells) in the large intestine.
• the inventors hypothesized the existence of human intestinal bacteria that suppress the colonization of Th1 cell-inducing bacteria in the intestine, and attempted to identify them.
• they isolated and cultured 37 strains of intestinal bacteria from a fecal sample derived from a healthy individual (subject number: #F), and succeeded in determining the 16S rDNA sequence of each strain.
• bacteria that suppress the colonization of the aforementioned Th1 cell-inducing bacteria in the intestine (37 strains of intestinal bacteria derived from healthy individual #F, 42 strains of intestinal bacteria derived from healthy individual #I, 47 strains of intestinal bacteria derived from healthy individual #K, etc.) can also suppress the colonization of multidrug-resistant bacteria and inflammation-inducing bacteria in the intestine.
• the inventors selected 31 strains of intestinal bacteria (F31mix) by removing overlapping bacteria from 37 strains of intestinal bacteria derived from healthy subject #F, and further succeeded in selecting 18 strains (F18mix) that can exhibit the same level of bacterial colonization inhibition ability (Patent Document 3).
• the present invention was made in consideration of the problems with the prior art, and aims to clarify the mechanism by which inflammation-inducing bacteria, drug-resistant bacteria, and the like can be inhibited from colonizing the intestine, and to provide an antibacterial composition against such bacteria.
• the inventors first used a transposon to create a mutant library (Kp2H7_tp) of the Kp2H7 strain.
• the library was then administered to germ-free mice, followed by administration of F18mix or 13 strains (F31-18mix) consisting of F31mix and F18mix only, and analysis was performed over time to determine which genes were mutated in the Kp2H7 strains contained in the stool and the proportion of these mutant strains present.
• gntR is a gene related to the metabolism of gluconic acid. As shown in Figure 5A, Klebsiella, E. coli, etc. have an Enter-Dodoroff pathway (ED pathway) in addition to the normal glycolysis (EMP pathway) as a metabolic pathway for sugars, and gluconic acid enters this ED pathway directly and is metabolized to pyruvic acid in three steps.
• ED pathway Enter-Dodoroff pathway
• EMP pathway normal glycolysis
• gntR is known to act repressively against gntU, gntK, edd, and eda, which are genes that metabolize gluconic acid via the ED pathway.
• the inventors discovered that the Kp2H7 strain and the 10 types of bacteria in F31mix (including the 8 types of bacteria in F18mix) are both bacteria that consume gluconic acid, and that the 10 types of bacteria inhibit the colonization of the Kp2H7 strain and the like in the intestine by competing with each other for the consumption of gluconic acid.
• 9 types of bacteria in the 42 strains of intestinal bacteria derived from healthy individual #I and 13 types of bacteria in the 47 strains of intestinal bacteria derived from healthy individual #K are bacteria that consume gluconic acid.
• the present invention provides the following aspects:
• An antibacterial composition against a second bacterium that consumes gluconic acid comprising a first bacterium that consumes gluconic acid as an active ingredient.
• composition described in [1], wherein the first bacterium is a bacterium that contains a gene encoding a gluconate dehydrogenase and a gene encoding a gluconate transporter in the same gene cluster.
• a method for screening bacteria having antibacterial activity against bacteria that consume gluconic acid comprising the steps of detecting the ability of a test bacterium to consume gluconic acid, and determining that the test bacterium has the antibacterial activity if the test bacterium is found to have the ability in the step.
• a method for screening bacteria having antibacterial activity against bacteria that consume gluconic acid comprising the steps of: 1.
• a method comprising: detecting at least one gene selected from the group consisting of a gene encoding gluconate kinase and a gene encoding gluconate dehydrogenase, and a gene encoding a gluconate transporter in a test bacterium; and determining that the test bacterium has the antibacterial activity if the genes detected in the step are found to be included in the same gene cluster.
• a method for screening bacteria having antibacterial activity against bacteria that consume gluconic acid comprising the steps of: 1.
• a method comprising: detecting a gene encoding gluconate dehydrogenase and a gene encoding a gluconate transporter in a test bacterium; and determining that the test bacterium has the antibacterial activity if the genes detected in the step are found to be included in the same gene cluster.
• a bacterium comprising at least one gene selected from the group consisting of a gene encoding a gluconate kinase and a gene encoding a gluconate dehydrogenase, and a gene encoding a gluconate transporter in the same gene cluster.
• gluconic acid-consuming bacteria such as the 32 species (10 species derived from healthy individual #F, 9 species derived from healthy individual #I, and 13 species derived from healthy individual #K) to compete with the consumption of gluconic acid by the second bacteria or the like.
• first bacteria such as the 32 species (10 species derived from healthy individual #F, 9 species derived from healthy individual #I, and 13 species derived from healthy individual #K) to compete with the consumption of gluconic acid by the second bacteria or the like.
• first bacteria such as the 32 species (10 species derived from healthy individual #F, 9 species derived from healthy individual #I, and 13 species derived from healthy individual #K)
• first bacteria such as the 32 species (10 species derived from healthy individual #F, 9 species derived from healthy individual #I, and 13 species derived from healthy individual #K)
• FIG. 1 is a graph showing the time course of changes in the bacterial load of Kp2H7 strain in feces in CFU when germ-free mice were administered Klebsiella (Kp2H7 strain) and, one week later, administered 37 strains (F37mix) or 18 strains (F18mix) of enterobacteria isolated from fecal samples of healthy subject #F.
• FIG. 1 shows an outline of the construction of a Kp2H7 strain mutant library using a transposon.
• FIG. 1 is a graph showing the time-dependent change in the bacterial load of the Kp2H7 strain in stool in terms of CFU when the Kp2H7 strain mutant library (Kp2H7_tp) was administered to germ-free mice, and then one week later, F18mix or 13 strains (F31-18mix) obtained by excluding F18mix from F31mix were administered.
• This is a graph showing the percentage of genes mutated in Kp-2H7 analyzed (Tn-seq) in feces collected on days 0, 4, 10, and 28 in the test process shown in Figure 3. In the figure, genes with a mutation rate of 1% or less at all collection points are shown in black.
• the white bar graph shows the gntR gene.
• FIG. 1 shows an overview of the normal glycolysis (EMP pathway) and the Enter-Dodoroff (ED) pathway found in Klebsiella and the like.
• FIG. 1 shows an overview of the changes in progression of the EMP and ED pathways when there is a mutation in the gntR gene.
• 1 is a graph showing the results of measuring the concentration of gluconic acid in feces when F18mix or F31-18mix was administered to germ-free mice (GF).
• GF germ-free mice
• 1 is a graph showing the time course of gluconic acid concentration in feces when germ-free mice were administered with the Kp2H7 strain and then with F18mix one week later.
• This graph shows the amount of Klebsiella bacteria (CFU) in the stool and the ratio of the amount of the mutant strain to the wild-type strain in the stool (Competition index ( ⁇ gntR/WT)) when equal amounts of a mixture of the Kp2H7 wild-type strain and a gntR-deficient strain were administered to germ-free mice, and then F18mix or F31-18mix was administered two days later.
• CFU Klebsiella bacteria
• This graph shows the amount of Klebsiella bacteria (CFU) in the stool and the ratio of the amount of the mutant strain to the wild-type strain in the stool (Competition index ( ⁇ gntK/WT)) when equal amounts of a Kp2H7 wild-type strain and a gntK-deficient strain were mixed and administered to germ-free mice, and then F18mix or F31-18mix was administered two days later.
• 1 is a graph showing the amount of Klebsiella in the feces of germ-free mice colonized with Kp2H7 and F18mix, which were fed diets containing 0%, 2.5%, or 10% gluconic acid (GA) one week after the feeding.
• 1 is a graph showing the results of analyzing the expression of the gntK gene in the Kp2H7 strain when the Kp2H7 strain was administered to germ-free mice and then, two days later, F18mix or F31-18mix was administered.
• 1 is a graph showing the results of measuring the gluconic acid consumption concentration by LC-MS for 31 strains of enterobacteria isolated from stool samples of healthy subject #F. The graph shows the average values of the measured concentrations obtained from three independent tests.
• 1 is a graph showing the results of measuring the gluconic acid consumption concentration of pathogenic microorganisms using LC-MS.
• FIG. 13 is a graph showing the time course of changes in the bacterial load of the Kp2H7 strain in feces in CFU when germ-free mice were administered the Kp2H7 strain and then administered F18mix, F8mix, or F10mix one week later.
• F8mix represents the 8 strains shown in FIG. 13 that were found to have gluconic acid consumption ability in F18mix.
• F10mix represents the 10 strains excluding F8mix from F18mix.
• FIG. 16B is a graph showing the time-dependent change in the bacterial count of the Kp2H7 strain in the stool (in CFU) when germ-free mice were administered the Kp2H7 strain, then administered F18mix one week later, and then fed food containing 0%, 2.5%, or 10% gluconic acid (GA) from the 21st day.
• 16B is a graph showing the time course of the bacterial load of the Kp2H7 strain in stool on Day 28 in the test shown in FIG. 16A, expressed as CFU.
• FIG. 16B is a graph showing the time-dependent changes in the bacterial load (relative load) of the Kp2H7 strain and F18mix in stool in terms of CFU in the test shown in FIG. 16A.
• 16B is a graph showing the time-dependent changes in the bacterial load (absolute load) of the Kp2H7 strain and F18mix in stool in terms of CFU in the test shown in FIG. 16A.
• This is a graph showing the results of RNA-seq analysis of bacteria in feces from germ-free mice administered Kp-2H7 alone or Kp-2H7 and F18mix two days after collection of fecal samples.
• pathways composed of genes whose expression was increased in the Kp-2H7 and F18mix group compared to the Kp-2H7 alone group in the case of "Kp + F18-mix > Kp only" are shown.
• GF on CL-2 shows the results for the group that ate a nutritious diet containing gluconic acid
• GF on AIN93G shows the results for the group that ate a diet that did not contain gluconic acid.
• Each bacterial strain isolated from healthy individuals #F, #K, and #I was cultured in a medium containing 300 ⁇ M gluconic acid, and the gluconic acid concentration in the culture supernatant was measured.
• This figure shows an overview of the gene cluster related to the classical pathway (gluconate kinase) in each strain.
• Each bacterial strain isolated from healthy individuals #F, #K, and #I was cultured in a medium containing 300 ⁇ M gluconic acid, and the gluconic acid concentration in the culture supernatant was measured.
• This figure shows an overview of the gene cluster related to the alternative pathway (gluconate dehydrogenase) in each strain.
• Each bacterial strain isolated from healthy individuals #F, #K, and #I was cultured in a medium containing 300 ⁇ M gluconic acid, and the gluconic acid concentration in the culture supernatant was measured.
• This figure shows an overview of the gene cluster related to gluconate transporter in each bacterial strain.
• This figure shows the results of culturing each bacterial strain isolated from healthy individuals #F, #K, and #I, which were not found to possess genes involved in gluconic acid metabolism, in a medium containing 300 ⁇ M gluconic acid, and measuring the gluconic acid concentration in the culture supernatant.
• 1 shows an overview of the gluconate metabolic pathways (classical or alternative pathways) involving gluconate kinase or gluconate dehydrogenase.
• 1 is a graph showing the results of analyzing the time-dependent change in fecal concentration of the Kp2H7 strain in germ-free mice, in which 18 strains (F18mix) or 13 strains (F13mix) of enterobacteria isolated from fecal samples of healthy individual #F, or no strain, were administered to germ-free mice, and then 2 weeks later, the Kp2H7 strain was administered to the mice (4 mice per administration group).
• the inventors have previously found that 37 types of enterobacteria strains (37 types of bacteria shown in Table 1 below) isolated and cultured from fecal samples from healthy individual #F can suppress the colonization of inflammation-inducing bacteria such as the Kp2H7 strain in the intestine.
• the inventors also selected 31 strains of enterobacteria (F31mix) by removing overlapping bacteria from the 37 strains of enterobacteria, and further succeeded in selecting 18 strains (F18mix) that can exert the same level of intestinal colonization suppression ability.
• the 37 species of bacteria listed in Table 1 correspond to f01 to f37 listed in Patent Documents 2 and 3. Their 16S rDNA sequences are shown in SEQ ID NOs: 1 to 37, respectively.
• “F31mix” and “F18mix” are bacteria selected in Patent Document 3, and bacteria belonging to these are indicated with black circles in Table 1.
• their identification, deposit number, and date of deposit are shown in Table 1. All of these deposited bacterial strains have been deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (NITE, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818).
• the inventors have now clarified that the Kp2H7 strain and the 10 types of bacteria in F31mix (including the 8 types of bacteria in F18mix) are both bacteria that consume gluconic acid, and that by competing with each other in the consumption of gluconic acid, the 10 types of bacteria suppress the colonization of the Kp2H7 strain and the like in the intestine.
• the 10 types of bacteria are indicated with a + (plus) in Table 1. Meanwhile, a - (minus) indicates that the bacteria do not consume gluconic acid. Also, "nd" indicates that the bacteria have not been evaluated for their ability to consume gluconic acid.
• the present inventors have also found that 42 types of enterobacteria strains (42 types of bacteria excluding I14 in Table 2 below) isolated and cultured from fecal samples derived from healthy individual #I can suppress colonization of inflammation-inducing bacteria such as the Kp2H7 strain in the intestine.
• I01 to I13 and I15 to I43 in Table 2 correspond to I01 to I13 and I14 to I42 in Patent Documents 2 and 3, respectively.
• sequences of their 16S rDNA are shown in SEQ ID NOs: 168 to 209, respectively.
• enterobacteria strains 47 types of bacteria shown in Table 3 below
• 47 types of enterobacteria strains isolated and cultured from fecal samples from healthy individual #K can suppress the colonization of inflammation-inducing bacteria such as the Kp2H7 strain in the intestine.
• K01 to K44 and K46 to K47 in Table 3 correspond to K01 to K44 and K45 to K46 in Patent Documents 2 and 3, respectively.
• the sequences of their 16S rDNA are shown in SEQ ID NOs: 210 to 255, respectively.
• the bacteria that were found to consume gluconic acid are indicated with a + (plus) in Tables 2 and 3.
• a - (minus) indicates that the bacteria do not consume gluconic acid.
• nd indicates that the bacteria have not been evaluated for their ability to consume gluconic acid.
• the present invention relates to a method for suppressing colonization of inflammation-inducing bacteria or drug-resistant bacteria (second bacteria) in the intestine by using gluconic acid-consuming bacteria (first bacteria) such as the above 32 species (10 species derived from healthy individual #F, 9 species derived from healthy individual #I, and 13 species derived from healthy individual #K) to compete with the gluconic acid consumption of the bacteria. That is, the present invention provides an antibacterial composition against the second bacteria that consume gluconic acid, which contains the first bacteria that consume gluconic acid as an active ingredient.
• first bacteria such as the above 32 species (10 species derived from healthy individual #F, 9 species derived from healthy individual #I, and 13 species derived from healthy individual #K)
• antibacterial means the inhibition of bacterial activity, more specifically, the inhibition of bacterial proliferation, growth or colonization, or the death of bacteria, and examples of this include the inhibition of bacterial colonization in the intestines and the elimination of bacteria from the intestines.
• gluconic acid refers to a carboxylic acid produced by oxidizing the carbon atom at position 1 of glucose, also known as (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoic acid. Furthermore, the gluconic acid of the present invention includes not only this carboxylic acid, but also gluconolactone (glucono- ⁇ -lactone) in an equilibrium state in an aqueous solution.
• Constant of gluconic acid means at least that the bacterium takes in gluconic acid from the surrounding environment, and may also include converting the taken-in gluconic acid into other compounds (catabolism or assimilation, metabolism or utilization, etc.).
• the surrounding environment There are no particular limitations on the surrounding environment, but examples include the intestine of the host and culture medium.
• the term "bacteria that consume gluconic acid” refers to bacteria that have the ability to consume gluconic acid. Whether or not a bacterium has the ability to consume gluconic acid can be determined by, for example, culturing the bacterium in a medium containing gluconic acid, as shown in the examples below, if the amount of gluconic acid remaining in the medium is significantly reduced compared to before the culture, the bacterium can be evaluated as having the ability to consume gluconic acid.
• a culture solution of a bacterium in stationary phase is added to a 300 ⁇ M gluconic acid-containing medium at a volume ratio of 100:1, and the bacterium is cultured at 37° C. under anaerobic conditions for 48 hours.
• concentration of gluconic acid remaining in the culture supernatant is 100 ⁇ M or less (preferably 70 ⁇ M or less, more preferably 50 ⁇ M or less, even more preferably 30 ⁇ M or less, more preferably 25 ⁇ M or less, even more preferably 20 ⁇ M or less, and more preferably 15 ⁇ M or less)
• the bacterium can be evaluated as having the ability to consume gluconic acid.
• the bacteria can be ingested by an animal, and the amount of gluconic acid in the animal's feces can be detected. If the amount is significantly lower than that in the animal that has not been ingested the bacteria, the bacteria can be evaluated as having the ability to consume gluconic acid.
• the animals There are no particular limitations on the animals, and examples include humans, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, etc.
• the "first bacterium that consumes gluconic acid” is not particularly limited as long as it is a bacterium that has the ability to consume gluconic acid and can be established in the intestine, but is an intestinal bacterium that preferably produces a residual gluconic acid concentration in the above-mentioned method of 30 ⁇ M or less, more preferably 25 ⁇ M or less, even more preferably 20 ⁇ M or less, more preferably 15 ⁇ M or less, even more preferably 10 ⁇ M or less, more preferably 5 ⁇ M or less, and even more preferably 2 ⁇ M or less.
• bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 10 bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 17, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 18, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 20, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 21, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 22, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 23, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 29, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO: 32, bacteria having at least 90% identity to the sequence described in SEQ ID NO: 37, Bacteria having a DNA that shows at least 90% identity to the sequence set forth in SEQ ID NO: 168, bacteria having a DNA that shows at least 90% identity to the sequence set forth in SEQ ID NO: 168, bacteria having
• bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 10 bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 17, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 18, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 21, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 22, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 29, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 32, a bacterium having a DNA that exhibits at least 90% identity to the sequence set forth in SEQ ID NO: 37; a bacterium having a DNA that exhibits at least 90% identity to the sequence set forth in SEQ ID NO: 168; a bacterium having a DNA that exhibits at least 90% identity to the sequence set forth in SEQ ID NO: 169; a bacterium having a DNA that exhibit
• the first bacterium may be at least one bacterium (preferably 2 or more (e.g., 3 or 4), more preferably 5 or more (e.g., 6 or 7), and particularly preferably 8) selected from the group consisting of bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 17, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 18, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 20, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 21, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 22, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 23, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 32, and bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 37.
• the first bacterium is preferably at least one bacterium selected from the group consisting of bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 18, bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 21, bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 22, and bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 37, more preferably two or more bacteria, even more preferably three or more bacteria, and particularly preferably four bacteria.
• a bacterium that contains at least one gene selected from the group consisting of a gene encoding gluconate kinase and a gene encoding gluconate dehydrogenase, and a gene encoding a gluconate transporter in the same gene cluster can have the ability to consume gluconate.
• glucose kinase gluconokinase
• gntK a phosphotransferase (EC 2.7.1.12) that catalyzes the following reaction (see FIG. 19E).
• gluconate kinases include proteins that contain an amino acid sequence that shows homology to at least one of the amino acid sequences in SEQ ID NOs: 76 to 85 (see Tables 9 to 12 below).
• glucose dehydrogenase means a dehydrogenase (EC 4.2.1.39) that catalyzes the following reaction (see FIG. 21).
• Gluconic acid ⁇ 2-keto-3-deoxygluconic acid (KDG) + H 2 O.
• gluconate dehydrogenase examples include proteins that contain an amino acid sequence that shows homology to at least one of the amino acid sequences in SEQ ID NOs: 86 to 117 (see Tables 9 to 11 below).
• gluconate transporter refers to a protein that transports sugar acid molecules such as gluconic acid or sugar-keto acid.
• gluconate transporters include proteins that contain an amino acid sequence that shows homology to at least one of the amino acid sequences in SEQ ID NOs: 118 to 167 (see Tables 9 to 12 below).
• “showing homology” means showing a coverage of 60% or more and a match rate of 60% or more with respect to the amino acid sequence described in each of the above SEQ ID NOs.
• coverage query cover, qcovhsp
• match rate id, pident
• 60% or more means that the coverage and match rate are each independently preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even 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, 99% or more, 100%).
• a “gene cluster” refers to a group of genes that are encoded on the same chromosome and are closely spaced within a range of several thousand to tens of thousands of base pairs on the genome sequence.
• the "several thousand to tens of thousands of base pairs” specifically refers to within 10,000 base pairs (10,000 bp), within 9,000 base pairs (9,000 bp), or within 8,000 base pairs (8,000 bp).
• the gene cluster of the present invention may take the form of an operon.
• examples of bacteria that contain a gene encoding gluconate kinase and a gene encoding a gluconate transporter in the same gene cluster include bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 37, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 248, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 168, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 169, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 199, and bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 203 (see “Gluconate kinase + Transporter [gene cluster (+)]" in Figure 19A).
• examples of bacteria that contain a gene encoding a gluconate kinase and a gene encoding a gluconate transporter in the same gene cluster include the 403 species of bacteria shown in Tables 17 to 40 below, and among these, the 63 species of bacteria with a value of "1" in the "gut_microbes" item in Tables 17 to 40 are preferred.
• examples of bacteria containing a gene encoding gluconate dehydrogenase and a gene encoding a gluconate transporter in the same gene cluster include bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 17, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 19, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 188, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 189, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 213, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 243, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 21, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 32, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO: 212, bacteria having DNA that is at least 90% identical to the sequence described in SEQ ID NO
• examples of bacteria that contain a gene encoding gluconate dehydrogenase and a gene encoding a gluconate transporter in the same gene cluster include the 70 species of bacteria shown in Tables 13 to 16 below, and among these, the 10 species of bacteria with a value of "1" in the "gut_microbes" field in Tables 13 to 16 are preferred.
• the first bacterium is used to suppress colonization of the second bacterium described below in the intestine. Moreover, as shown in the examples described below, the suppression can be enhanced by using the first bacterium in combination with a specific bacterium that does not consume gluconic acid. Therefore, in the present invention, in addition to the first bacterium, a bacterium that does not consume gluconic acid (for example, a bacterium having an effect of enhancing the gluconic acid consumption ability of the first bacterium, or a bacterium having an effect of maintaining the growth or colonization of the first bacterium) may be used in combination.
• a bacterium that does not consume gluconic acid for example, a bacterium having an effect of enhancing the gluconic acid consumption ability of the first bacterium, or a bacterium having an effect of maintaining the growth or colonization of the first bacterium
• Bacteria that do not consume gluconic acid refers to bacteria that do not have the ability to consume gluconic acid. For example, as shown in the examples below, if the bacteria are cultured in a medium containing gluconic acid and the amount of gluconic acid remaining in the medium has not significantly decreased before culturing, the bacteria can be evaluated as not having the ability to consume gluconic acid. More specifically, examples of such bacteria include enterobacteria that have a residual gluconic acid concentration of 250 ⁇ M or more (preferably 270 ⁇ M or more, more preferably 280 ⁇ M or more, and even more preferably 290 ⁇ M or more) in the above method.
• non-gluconic acid consuming bacteria to be used in combination with the first bacterium examples include bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:1, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:2, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:3, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:4, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:5, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:7, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:9, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:11, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO:12, bacteria having a DNA that is at least 90% identical to the sequence set forth in SEQ ID NO
• non-gluconic acid consuming bacteria include, for example, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO:1, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO:12, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO:19, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO:24, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO:26, bacteria having DNA exhibiting at least 90% identity to the sequence described in SEQ ID NO:28, A, bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 30, bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 31, bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 33, and bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 35.
• At least one bacterium (preferably two or more (e.g., three or four), more preferably five or more (e.g., six, seven, eight or nine), and particularly preferably ten bacteria) is selected from the group consisting of bacteria having A, bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 30, bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 31, bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 33, and bacteria having DNA that shows at least 90% identity to the sequence described in SEQ ID NO: 35.
• the non-gluconic acid consuming bacterium is at least one bacterium selected from the group consisting of bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 1, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 19, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 24, and bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 35, more preferably two or more bacteria, even more preferably three or more bacteria, and particularly preferably four bacteria.
• the first bacterium to be used in combination is at least one bacterium selected from the group consisting of bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 18, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 21, bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 22, and bacteria having DNA showing at least 90% identity to the sequence described in SEQ ID NO: 37.
• the bacteria according to the present invention may be one strain of bacteria, or a mixture of bacterial strains composed of multiple strains of bacteria.
• the bacteria according to the present invention include not only bacteria that have 16SrDNA containing a specific sequence (for example, bacteria identified by accession number NITE BP-03147 that have 16SrDNA containing the sequence set forth in SEQ ID NO: 1), but also bacteria that have DNA that shows at least 90% identity to the specific sequence.
• sequence identity means that the identity for each sequence is 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more), preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more), and more preferably 99% or more (particularly preferably 100%).
• sequence identity can be determined, for example, using the BLAST (Basic Local Alignment Search) program (Altschul et al. J. Mol. Biol., 215:403-410, 1990). The program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl.
• BLAST from the National Center for Biological Information (NCBI) can be used (for example, using default, i.e., initial setting parameters).
• NCBI National Center for Biological Information
• the bacteria of the present invention may also be bacteria bred by mutation treatment, genetic recombination, selection of natural mutants, etc.
• mutant strain means that it includes a specific bacterium (strain) mutated by a method well known to those skilled in the art to the extent that the properties are not changed, a strain bred by selection of natural mutants, etc., or a strain that a person skilled in the art can confirm to be equivalent thereto.
• the bacteria of the present invention are not limited to the strains deposited or registered in a specified institution as described above (hereinafter also referred to as "deposited strains"), but also include strains that are substantially equivalent thereto (also referred to as "derived strains” or "derived strains").
• strains substantially equivalent to the deposited strain means strains that belong to at least the same species as the deposited strain.
• a strain substantially equivalent to the deposited strain may be, for example, a derived strain with the deposited strain as a parent strain.
• Derivative strains include strains bred from the deposited strain and strains that have arisen naturally from the deposited strain.
• the "second bacterium that consumes gluconic acid” is not particularly limited as long as it is a bacterium that has the ability to consume gluconic acid and can be established in the intestine, but it is preferable that the second bacterium has a lower ability to consume gluconic acid than the first bacterium, from the viewpoint that the establishment of the second bacterium in the intestine is suppressed by competing with the first bacterium in gluconic acid consumption. More specifically, the second bacterium is an intestinal bacterium that has a residual gluconic acid concentration in the above method of preferably 70 ⁇ M or less, more preferably 50 ⁇ M or less, and even more preferably 30 ⁇ M or less.
• Such second bacteria include, for example, bacteria belonging to the Enterobacteriaceae family, more specifically, bacteria belonging to Klebsiella, Escherichia, Proteus, Salmonella, and Pseudomonas.
• bacteria belonging to the Enterobacteriaceae family include bacteria belonging to the Staphylococcus, Bacillus, Peptostreptococcus, Megasphaera, and Enterococcus.
• Klebsiella pneumoniae Klebsiella pneumoniae (Kp2H7 strain, 34E1 strain, BAA-1705 strain, 700603 strain, 40B3 strain, etc.) and Klebsiella aerogenes (Ka11E12 strain, etc.).
• Klebsiella pneumoniae Kp2H7 strain, 34E1 strain, BAA-1705 strain, 700603 strain, 40B3 strain, etc.
• Klebsiella aerogenes Ka11E12 strain, etc.
• Bacteria belonging to the Escherichia genus include, for example, Escherichia coli, such as adherent-invasive Escherichia coli (AIEC) (E. coli LF82, etc.) and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli ATCC BAA2777, etc.).
• Bacteria belonging to the Proteus genus include, for example, Proteus mirabilis (P. mirabilis JCM1669T, etc.) and Proteus vulgaris (P. vulgaris JCM20013, etc.).
• Bacteria belonging to the Salmonella genus include, for example, Salmonella enteritidis (S. enterica subsp.
• bacteria belonging to the genus Pseudomonas include P. aeruginosa (P. aeruginosa ATCC10145, etc.).
• bacteria belonging to the genus Staphylococcus include Staphylococcus aureus (S. aureus JCM16555, etc.), Staphylococcus mutans, and Staphylococcus epidermidis.
• bacteria belonging to the genus Bacillus include Bacillus cereus (B. cereus JCM2152, etc.).
• bacteria belonging to the genus Peptostreptococcus include Peptostreptococcus prevotii.
• bacteria belonging to the genus Megasphaera include Megasphaera elsdenii.
• bacteria belonging to the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium.
• the second bacterium may be a bacterium that causes a disease by colonizing the intestine.
• diseases caused by the second bacterium include sepsis, peritonitis, meningitis, enteritis, gastroenteritis (infectious gastroenteritis, etc.), respiratory infections (pneumonia, etc.), urinary tract infections, surgical site infections, soft tissue infections, medical device-related infections (medical device-related bloodstream infections, etc.), inflammatory bowel disease (chronic inflammatory bowel disease such as Crohn's disease and ulcerative colitis), autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, experimental immune-mediated encephalitis (EAE), multiple sclerosis, and systemic lupus erythematosus, chronic inflammatory diseases, opportunistic infections caused by methicillin-resistant Staphylococcus aureus infection (MRSA), food poisoning (Bacillus cereus infection, etc.), and toxic shock syndrome (TSS).
• MRSA methicillin
• the composition of the present invention is an antibacterial composition against a second bacterium that consumes gluconic acid, containing a first bacterium that consumes gluconic acid as an active ingredient.
• the bacterium according to the present invention (the first bacterium described above, and further the non-gluconic acid consuming bacterium described above) contained in such a composition may be not only a live bacterium but also a culture thereof.
• the culture may be one containing the bacterium (a culture medium (culture liquid, solid medium, etc.) containing the grown bacterium), and the form thereof is not particularly limited and may be either liquid or solid, and may also be in a dried form (e.g., a culture dried product, etc.) as long as it can be restored and resumed growth in the intestine of the host after administration.
• a dried product can be prepared by drying a suspension in which the bacterial cells are dispersed in a solvent (water, etc.).
• the suspension may be dried by appropriately adding a drying protection agent described later.
• the drying method is not particularly limited, and examples thereof include a freeze-drying method, a spray-drying method, and a heat-drying method, with the freeze-drying method being preferred.
• composition of the present invention may be in the form of a pharmaceutical composition (medicinal products, quasi-drugs, etc.), a food composition (food and beverages, animal feed, etc.), or a reagent for use in cell experiments, model animal experiments, etc.
• a pharmaceutical composition medicinal products, quasi-drugs, etc.
• a food composition food and beverages, animal feed, etc.
• composition of the present invention when used as a pharmaceutical composition, it can be formulated by known pharmaceutical methods.
• it can be used orally or parenterally as capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, troches, sublinguals, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc., but oral ingestion is preferred from the viewpoint of non-invasive and easy ingestion.
• carriers acceptable pharmacologically or as food and beverages specifically, physiological saline, sterilized water, culture medium (modified GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.), enriched Clostridium medium, BHI medium, BL medium, LB medium, EG medium, etc.), drying protection agents (sugars or sugar alcohols such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, etc.; amino acids such as arginine, histidine, etc.; propyl alcohols such as propyl alcohols, propyl glycerides ...
• polyols such as poly(ethylene glycol), glycerol, poly(ethylene glycol), and poly(propylene glycol), vegetable oils, solvents, excipients, bases, emulsifiers, suspending agents, surfactants, stabilizers, flavorings, fragrances, vehicles, preservatives, binders, diluents, isotonicity agents, soothing agents, bulking agents, disintegrants, buffers (phosphates, citrates, acetates, etc.), coating agents, lubricants, colorants, sweeteners, thickening agents, flavorings, dissolution aids, or other additives.
• polyols such as poly(ethylene glycol), glycerol, poly(ethylene glycol), and poly(propylene glycol), vegetable oils, solvents, excipients, bases, emulsifiers, suspending agents, surfactants, stabilizers, flavorings, fragrances, vehicles, preservatives, binders, diluents, is
• the bacteria of the present invention may be combined with a composition that enables efficient delivery to the intestine, particularly in preparations intended for oral administration.
• a composition that enables delivery to the intestine there are no particular limitations on the composition that enables delivery to the intestine, and known compositions can be appropriately adopted, such as pH-sensitive compositions, compositions that suppress release to the intestine (cellulose-based polymers, acrylic acid polymers and copolymers, vinyl acid polymers and copolymers, etc.), bioadhesive compositions that specifically adhere to the intestinal mucosa (e.g., polymers described in U.S. Patent No. 6,368,586), compositions containing protease inhibitors, and compositions that are specifically decomposed by intestinal enzymes.
• the composition of the present invention can be used to treat or prevent the disease.
• Treatment includes not only complete recovery from the disease, but also alleviating or ameliorating the symptoms of the disease, inhibiting its progression, and inhibiting its recurrence.
• prevention includes inhibiting or delaying the onset of the disease, or inhibiting its recurrence.
• composition of the present invention when used as a food composition, it may be, for example, a health food, functional food, food for specified health uses, food with nutrient functions, food with functional claims, nutritional supplements, food for the sick, or animal feed.
• Functional foods are generally classified into four categories based on their mechanism of action: probiotics, biogenics, prebiotics, and synbiotics, but in the present invention, it may take the form of a probiotic.
• composition of the present invention can also be ingested as various foods and beverages.
• foods and beverages include liquid foods such as functional drinks, energy drinks, jelly-like drinks, soft drinks, milk drinks, tea drinks, alcoholic drinks, and soups; fermented foods and beverages such as yogurt and drinkable yogurt; oil-containing products such as edible oils, dressings, mayonnaise, and margarine; carbohydrate-containing foods such as rice, noodles, and bread; processed livestock foods such as ham and sausage; processed seafood foods such as kamaboko, dried fish, and salted fish; processed vegetable foods such as pickles; semi-solid foods such as jelly; fermented foods such as miso; various confectioneries such as Western confectioneries, Japanese confectioneries, candies, chewing gum, gummy candies, frozen desserts, and frozen desserts; retort products such as curry, thickened sauce, and Chinese soup; instant foods and microwave-compatible foods such as instant soup and instant miso soup.
• health foods and beverages prepared in powder, granules, tablets, capsules
• the food and drink compositions proposed in this invention can be provided and sold as food and drink with a label indicating their health uses.
• labeling includes all acts of informing consumers of said uses, and may include expressions that directly recognize said uses or that can recall or infer said uses, which may be attached to the composition itself, or to the container, packaging material or attached document that contains the composition.
• labeling may include information related to the composition of the present invention, such as flyers, pamphlets, pop art, catalogs, posters, books, storage media such as DVDs, or advertisements on electronic bulletin boards or the Internet, which display and advertise the effectiveness of the composition of the present invention.
• composition of the present invention may contain one or more components that are effective in the antibacterial action against the second bacterium (for example, a component that is effective in the proliferation of the bacterium of the present invention in the intestine). It may also be combined with known components, drugs, or foods for treating or preventing the above-mentioned disease. Furthermore, it may be combined with other components, drugs, or foods that exhibit functions other than the above-mentioned antibacterial action, treatment, and prevention.
• the composition of the present invention can also be in the form of a composition that is ingested in combination with food containing a low concentration of gluconic acid.
• low concentration means, for example, that the concentration of gluconic acid in the food is preferably 2.5% by weight or less, more preferably 2% by weight or less, even more preferably 1.5% by weight or less, even more preferably 1% by weight or less, even more preferably 0.5% by weight or less, and particularly preferably 0% by weight.
• Foods that contain low concentrations of gluconic acid include, for example, bananas, blueberries, strawberries, grapefruit, carrots, eggplants, potatoes, pumpkins, gluten-free bread (rice flour bread, bran flour bread, soy flour bread, etc.), rice, oats, fermented foods such as hard cheese, tofu, and sugar.
• "taking in combination” includes taking the bacteria of the present invention and a food containing a low concentration of gluconic acid at the same time or at different times, and by the same route or different routes.
• it is preferable to take the bacteria of the present invention after taking the food from the viewpoint that the influence of gastric acid is reduced and the administered bacteria is more likely to be established in the intestine.
• the number of times that the bacteria of the present invention and the food are taken may be the same or different. Therefore, the composition of the present invention may be an embodiment in which the bacteria of the present invention and the food are contained in one composition, or an embodiment in which the bacteria of the present invention and the food are contained in separate compositions.
• the present invention also provides a method for inhibiting a second bacterium that consumes gluconic acid in the intestine of a subject, comprising the step of administering or ingesting to the subject a bacterium according to the present invention (the first bacterium described above, and also the non-gluconic acid consuming bacterium described above), or a composition containing said bacterium (the composition of the present invention described above).
• inhibitor means inhibition of bacterial activity, more specifically inhibition of bacterial proliferation, growth or colonization, or the death of bacteria, and examples of this include inhibition of bacterial colonization in the intestines and elimination of bacteria from the intestines.
• the "subject" in the present invention is an animal, including a human.
• animals other than humans there is no particular limitation on animals other than humans, and various livestock, poultry, pets, laboratory animals, etc. can be the subject.
• Specific examples include, but are not limited to, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, etc.
• subjects in the present invention include, for example, a person whose intestines have the second bacterium colonized, a person who is likely to have the second bacterium colonized in the intestines, a person who is suffering from the above-mentioned disease, a person who is likely to have the above-mentioned disease, or a person who is likely to suffer from the above-mentioned disease.
• Non-therapeutic use is a concept that does not include medical procedures, i.e., treatment of the human body through therapy. Examples include health promotion.
• the dosage or intake amount is appropriately selected depending on the age, weight, health condition, type of composition (pharmaceutical, food or beverage, etc.), form of active ingredient (e.g. live bacteria, culture), etc. of the subject.
• the content or amount of the bacteria (viable bacteria) according to the present invention is not particularly limited, but is preferably 1x10 to the power of 9 to 1x10 to the power of 12, more preferably 1x10 to the power of 9 to 1x10 to the power of 11 in the composition.
• the units are CFU/g or CFU/mL, or cells/g or cells/mL.
• CFU stands for colony forming unit.
• the daily dosage of the microorganism (live bacteria) of the present invention is not particularly limited, but is preferably 1x109 to 1x1013 per day, and may be 1x109 to 1x1012, or even 1x109 to 1x1011.
• the unit is CFU/day or cells/day.
• the amount of culture can be, for example, equivalent to the amount of bacterial cells.
• the dosage of the bacteria of the present invention or the composition of the present invention described above is as described above, but it may be administered or ingested once or multiple times (e.g., two or three times) per day.
• the period of administration or ingestion may be discontinued depending on the condition of the subject, but it may also be administered or ingested continuously without discontinuing. Note that “continuous” may mean every day or at intervals, but in terms of effectiveness, it is preferable to administer or ingest the bacteria of the present invention or the composition of the present invention described above every day.
• the present invention provides a method for screening bacteria having antibacterial activity against bacteria that consume gluconic acid, comprising: a step of detecting the ability of a test bacterium to consume gluconic acid; and a step of determining that the test bacterium has the antibacterial activity if the test bacterium is found to have the consumption ability in the step.
• the present invention also provides a method for detecting, in a test bacterium, at least one gene selected from the group consisting of a gene encoding gluconate kinase and a gene encoding gluconate dehydrogenase, and a gene encoding a gluconate transporter; and, when it is found that the genes detected in the above step are included in the same gene cluster, determining that the test bacterium has the antibacterial activity.
• a method for screening for bacteria having antibacterial activity against bacteria that consume gluconic acid is also provided.
• the "bacteria that consume gluconic acid” in this screening method is not particularly limited, but may be the second bacterium described above.
• the "test bacteria” used in this screening method is also not particularly limited, but may be, for example, bacteria present in the intestines of animals. Such animals include humans and non-human animals (mice, rats, monkeys, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, etc.).
• the test bacteria may be isolated bacteria, but may also be a sample containing the bacteria (for example, a fecal sample from the animal, or a culture thereof).
• Detection of gluconic acid consumption ability can be performed, for example, as described above, by culturing the bacterium in a medium containing gluconic acid and detecting the amount of gluconic acid remaining in the medium. If the amount of gluconic acid is significantly reduced compared to that before culturing, the bacterium is determined to have gluconic acid consumption ability.
• the bacterium is cultured for 48 hours using the method shown in the Examples below, and the concentration of gluconic acid remaining in the culture supernatant is 100 ⁇ M or less (preferably 70 ⁇ M or less, more preferably 50 ⁇ M or less, even more preferably 30 ⁇ M or less, more preferably 20 ⁇ M or less, even more preferably 10 ⁇ M or less, more preferably 3 ⁇ M or less), the bacterium can be determined to have gluconic acid consumption ability.
• an animal can be made to ingest bacteria, and the amount of gluconic acid in the animal's feces can be detected. If the amount is significantly lower than when the animal is not ingested, it can be determined that the bacterium has the ability to consume gluconic acid.
• the animals There are no particular limitations on the animals, and examples include humans, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, etc.
• the method for detecting the amount of gluconic acid is not particularly limited, and an example of the method is a detection method using a liquid chromatograph mass spectrometer (LC-MS/MS), as shown in the examples below.
• LC-MS/MS liquid chromatograph mass spectrometer
• the "gluconate kinase,” “gluconate dehydrogenase,” and “gluconate transporter,” as well as the “gene cluster” in this screening method, are as described above. Whether or not at least two types of genes according to the present invention are contained in the same gene cluster can be determined, for example, by using the next generation sequencing (NGS) method, as shown in the examples described later.
• NGS next generation sequencing
• next-generation sequencing methods include synthetic sequencing (sequencing-by-synthesis, for example, sequencing using Illumina's Solexa genome analyzer, Hiseq, or Miseq), pyrosequencing (for example, sequencing using Roche Diagnostics (454)'s GSLX or FLX sequencer (so-called 454 sequencing)), ligase reaction sequencing (for example, sequencing using Life Technologies' SoliD or 5500xl), and ion semiconductor sequencing (for example, Thermo Fisher Scientific's Ion Torrent technology).
• synthetic sequencing for example, sequencing using Illumina's Solexa genome analyzer, Hiseq, or Miseq
• pyrosequencing for example, sequencing using Roche Diagnostics (454)'s GSLX or FLX sequencer (so-called 454 sequencing)
• ligase reaction sequencing for example, sequencing using Life Technologies' SoliD or 5500xl
• ion semiconductor sequencing for example, Thermo Fisher Scientific's Ion Torrent technology.
• mice Germ-free mice were purchased from CLEA Japan, Inc. or Sankyo Lab, aged 4 to 8 weeks. They were kept in a vinyl breeding isolator (germ-free isolator) (ICM Corporation; ICM-1B), and 8 to 14 week old mice were used in the following experiments.
• GF Germ-free mice
• ICM Corporation ICM-1B
• Kp2H7 strain Klebsiella pneumoniae 2H7 strain
• Patent Documents 1 to 3 For the isolated bacteria (bacteria belonging to F31-18mix or F18mix) administered to the germ-free mice, please refer to Patent Documents 2 and 3 and Table 3 below. Note that F31-18mix refers to the 13 strains excluding F18mix from F31mix.
• the bacterial solution of the Kp2H7 strain was placed in LB liquid medium, cultured overnight at 37°C, and adjusted to an OD of 1.2 (equivalent to 1*10 9 CFU/mL). Then, 200 ⁇ L/mouse (equivalent to 2*10 8 CFU/mouse) of the bacterial solution was administered into the stomach of a mouse using a sonde.
• 200 ⁇ L/mouse (equivalent to 1*10 8 CFU/mouse) of the bacterial solution was administered into the stomach of a mouse using a sonde.
• the isolated bacteria were cultured in mGAM liquid medium, EG medium, or CM0149 medium in an anaerobic chamber at 37°C for 24 to 48 hours.
• the resulting culture solutions of the isolated bacteria were mixed in equal amounts.
• the mixtures were then concentrated 5 times to prepare F18mix, F31-18mix, F8mix (described below), and F10mix (F18-8mix) (described below).
• 200 ⁇ L/animal (equivalent to a total bacterial amount of 1*10 9 CFU/animal) of the bacterial solution was administered into the stomach using a sonde.
• mice stool samples were dissolved in a solution of glycerol (final concentration 20%) and EDTA (final concentration 10 mM) mixed in PBS at a ratio of 50 mg stool/mL.
• the stool solution was diluted to an appropriate concentration on DHL medium containing 50 mg/L ampicillin and 50 mg/L spectinomycin, and then plated. After overnight incubation at 37°C, the number of colonies was counted and the number of CFU per gram of stool was calculated.
• CL-2 (Nippon CLEA) was usually used as mouse feed.
• the feed was changed to AIN93G (Oriental Yeast) from Day 21 onwards.
• AIN93G was given to the mice without gluconic acid (0%) or with gluconic acid added to a concentration of 2.5% by weight (80 ⁇ mol/g) or 10% by weight (320 ⁇ mol/g).
• the gluconic acid content of CL-2 used for normal breeding was 5 ⁇ mol/g. In all experiments, the mice were allowed to feed ad libitum.
• mice were administered Kp2H7 and F18mix, and then fed a diet containing an adjusted concentration of gluconic acid, and the changes in the bacterial load of each bacterium in the mice were examined. Specifically, feces from each mouse were collected once a week on Day 21 and Day 25 after the diet change, and bacterial genomes were extracted from the feces.
• the primers specific to each bacterium are as follows:
• Kp2H7 gene-deficient strain was prepared using the Quick and Easy E. coli Gene Deletion Kit (Gene Bridges, Heidelberg) according to the kit's protocol.
• the pRED/ET plasmid was introduced into the Kp2H7 strain by electroporation, and the strain was cultured at 30°C using LB agar medium containing 30 mg/L tetracycline to select the gene-introduced strain.
• the selected pRED/ET-introduced strain was placed in LB liquid medium and cultured at 30°C overnight, and then 1 mL of the culture was placed in 40 mL of LB liquid medium and cultured at 30°C until the OD reached 0.2.
• L-arabinose was then added to 0.3%, and the culture was changed to 37°C and cultured for another hour, after which the bacterial liquid was collected and competent cells were prepared.
• a DNA linear chain with the desired recombination sequence added to both ends of the FRT-PGK-gb2-neo-FRT cassette was prepared by PCR according to the protocol of the kit. The resulting DNA was then introduced into the competent cells by electroporation, and the cells were cultured on LB agar medium containing 90 mg/L kanamycin to select gene-deficient strains.
• gluconic acid concentration 1 was measured as follows. In order to examine the gluconic acid consuming ability of the strain, 10 ⁇ L of the bacterial solution was added to 1 mL of 300 ⁇ M gluconic acid-containing modified GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) and cultured at 37° C. under anaerobic conditions (in an anaerobic chamber) for 48 hours. The resulting culture solution was centrifuged, and the gluconic acid concentration in the supernatant was measured using LC-MS/MS.
• the bacterial solution used was a bacterial solution obtained by placing a bacterial strain picked from a single colony in a medium, culturing it overnight (pre-cultivating) under anaerobic conditions at 37° C., and reaching the stationary phase.
• a strain with a significantly decreased gluconic acid concentration (100 ⁇ M or less) was determined to be a strain capable of consuming gluconic acid.
• Example 2 the concentration of gluconic acid in feces was measured by dissolving mouse feces in 20 mg/mL MQ and centrifuging the solution, and then measuring the concentration of gluconic acid in the supernatant using LC-MS/MS. Liquid chromatography was performed using an ExionLC AD, and mass spectrometry was performed using an SCIEX TQ6500+. The measurement conditions were as follows:
• the glycerol stock of the transposon-inserted Kp strain thus prepared was administered to germ-free C57BL/6 mice at 200 ⁇ L per mouse. Seven days later, the mice were divided into three groups, either administered the isolated bacteria mix (F18 mix or F31-18 mix) or not administered anything. The day the isolated bacteria was administered was designated Day 0, and feces were collected from the mice on Days 0, 4, 10, and 28, suspended in 50 mg/mL PBS, and then cultured on LB agar medium containing 90 mg/L kanamycin. After overnight culture at 37°C, all colonies were collected and DNA was extracted.
• the extracted genomic DNA was fragmented into pieces of approximately 300 bases in length, and polyC bases were added to the 3' ends of the DNA fragments.
• the sequence near the transposon was amplified using a biotin-added primer containing a sequence complementary to the transposon sequence and a primer containing polyG bases.
• the target fragment was purified and amplified with streptavidin beads, and sequenced with an Illumina HiSeq2500 to obtain the sequence near the transposon.
• the first 24 bp of the obtained read sequences were trimmed to remove PCR primer complementary sequences and mosaic sequences.
• Illumina sequence adapter trimming and filtering based on sequence quality were performed using Trimmomatic-v0.39 [A. M. Bolger, M. Lohse, and B. Usadel, 'Trimmomatic: a flexible trimmer for Illumina sequence data', Bioinformatics, vol. 30, no. 15, pp. 2114-2120, Aug. 2014, doi:10.1093/BIOINFORMATICS/BTU170.
• the read sequences correspond to the mutation positions of the transposon-inserted Kp strains, and by assuming that each Kp mutant strain has one transposon mutation in its genome, a comparative analysis was performed by regarding TPM as the mutation rate of each gene in the sample.
• RNA was isolated from stool samples using NucleoSpin RNA (MACHEREY-NAGEL). Libraries for RNA sequencing were prepared using TruSeq Stranded mRNA Library Prep (Illumina Inc.) and sequenced in 150-bp paired-end mode using HiSeq X (Illumina Inc.). In order to analyze the transcriptome profile of Kp-2H7 with and without F18-mix (same as F18mix in Patent Document 3), the genome sequence of Kp-2H7 and the genome sequence of F18-mix were concatenated to create a reference genome (hereinafter referred to as the concatenated reference genome).
• Paired-end reads were sequenced using the 'ILLUMINACLIP:2:30:10 LEADING:3 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:30' options of Trimmomatic version 0.39 and the '-q20-p80' options of FASTX-Toolkit version 0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Unpaired reads were excluded from further analysis.
• gluconic acid concentration 2 was measured as follows. To evaluate the bacterial gluconic acid utilization in vitro, the isolated strain was cultured in mGAM medium or RCM medium containing 300 ⁇ M gluconic acid under anaerobic conditions at 37 ° C. for 48 hours. The supernatant of each culture medium was collected, and the gluconic acid concentration was measured using an ExionLC AD and a SCIEX Triple Quad 6500 + LC-MS / MS system (both manufactured by Sciex).
• each fecal sample was suspended in water (50 mg / mL), and the carbon concentration in the culture supernatant was measured by LC-MS / MS.
• chromatographic separation was performed using an Intrada Organic Acid column 150 ⁇ 2 mm (manufactured by Imtakt), the column temperature was 40 ° C., and the injection volume per time was 2 ⁇ L.
• the flow rate was 0.2 mL/min.
• the detailed MS conditions are as follows: curtain gas, 30 psi; collision gas, 9 psi; ion spray voltage, -4500 V; temperature, 400°C; ion source gas 1, 50 psi; ion source gas 2, 80 psi.
• Illumina MiSeq and PacBio Sequel platforms were used for bacterial whole genome sequencing.
• Illumina MiSeq libraries were prepared with a target insert size of 550 bp using the TruSeq DNA PCR-free library prep kit (Illumina). All the obtained Illumina MiSeq sequence sequences were trimmed and filtered using FASTX-toolkit (version 0.0.13).
• PacBio Sequel libraries were prepared using SMRTbell template prep kit 1.0. Genome assembly was performed using the hybrid assembler Unicycler using both types of sequence data. Genome classification was performed using the classify_wf of GTDB-tk version 2.3.0 and the GTDB database R214.
• NCBI taxonomy of the FastANI reference genome associated with the genome of each strain was searched using NCBI-genome-download version 0.3.3 (DOI: 10.5281/zenodo.8192432) and the NCBI classification database unknownlineage.dmp (downloaded on 2023/1514). Genes were predicted using the '--kingdom Bacteria --rnamer' option of Prokka version 1.14.0 and rnamer version 1.2.
• mice Germ-free mice, 4-8 week-old sterilized C57BL/6N mice, were purchased from CLEA Japan, Inc. or Sankyo Lab. They were kept in a vinyl breeding isolator (germ-free isolator, ICM-1B), and 8-14 week-old mice were used in the following experiments.
• CL-2 (CLEA Japan) was used as mouse feed.
• the isolated bacteria were cultured in mGAM liquid medium, EG medium, or CM0149 medium in an anaerobic chamber at 37°C for 24 to 48 hours.
• the resulting culture solutions of the isolated bacteria were mixed in equal amounts.
• the mixtures were then concentrated 5 times to prepare F18mix and F13mix (F31-18mix).
• 200 ⁇ L/mouse (equivalent to a total bacterial amount of 1*10 9 CFU/mouse) of the bacterial solution was administered into the stomach of the mouse using a sonde.
• the bacterial solution of the Kp2H7 strain was placed in LB liquid medium, cultured overnight at 37°C, and adjusted to an OD of 1.2 (equivalent to 1* 109 CFU/mL), and then diluted 100,000 times. 200 ⁇ L/mouse (equivalent to 2* 103 CFU/mouse) of the bacterial solution was administered into the stomach of each mouse using a sonde.
• mice stool samples were dissolved in a solution of glycerol (final concentration 20%) and EDTA (final concentration 10 mM) mixed in PBS at a ratio of 50 mg stool/mL.
• the stool solution was diluted to an appropriate concentration on DHL medium containing 50 mg/L ampicillin and 50 mg/L spectinomycin, and then plated. After overnight incubation at 37°C, the number of colonies was counted and the number of CFU per gram of stool was calculated.
• Example 1 Analysis of the mechanism of bacterial inhibition in the intestine by F18mix using transposon-inserted Kp2H7 strain
• the present inventors have previously revealed that the Kp2H7 strain is thought to belong to Klebsiella pneumoniae as a bacterium that colonizes the intestinal tract and is involved in the onset of enteritis by inducing the proliferation or activation of Th1 cells (Patent Document 1).
• Patent Document 1 the present inventors have assumed the presence of bacteria that suppress the colonization of such Th1 cell-inducing bacteria in human intestinal bacteria, and have attempted to identify them.
• transposome a complex of a transposon and a transposase
• the transposon contained a kanamycin resistance gene, and since the introduced transposon becomes a kanamycin-resistant strain when incorporated into the chromosome of the Kp2H7 strain, we selected mutant strains in which the transposon had been inserted on a kanamycin-containing medium.
• the heterologous library (Kp2H7_tp) was administered to germ-free mice, followed by administration of F18mix or F31-18mix. Feces were then collected on days 0, 4, 10, and 28, and the genes in the Kp2H7 strains contained in the feces were analyzed for mutations and the proportion of mutant strains present (Tn-seq). The proportion of mutated genes was shown for each mouse at each time point ( Figure 4). As a result, strains with mutations in gntR were predominant in the F31-18mix-administered group and the group administered only Kp2H7_tp (Kp only), while a gradual decrease in gntR mutant strains was observed in the group administered F18mix.
• gntR is a gene related to the metabolism of gluconic acid. As shown in Figure 5A, Klebsiella, E. coli, etc. have an Enter-Dodoroff pathway (ED pathway) in addition to the normal glycolysis (EMP pathway) as a metabolic pathway for sugars, and gluconic acid enters directly into this ED pathway and is metabolized to pyruvate in three steps.
• ED pathway Enter-Dodoroff pathway
• EMP pathway normal glycolysis
• gntR is known to act repressively against gntU, gntK, edd, and eda, which are genes that metabolize gluconic acid via the ED pathway.
• Example 2 Analysis of gluconic acid consumption ability of F18mix and Kp2H7 strains Based on the above speculation, the concentration of gluconic acid in feces was actually measured.
• F18mix was administered to germ-free mice, the gluconic acid in feces was significantly reduced compared to F31-18mix or germ-free mice, as shown in Figure 6.
• Kp2H7 strain was administered to germ-free mice and then F18mix was administered, the gluconic acid in feces was reduced as shown in Figure 7.
• the same amount of bacteria of the Kp2H7 strain (wild strain) and the gntR-deficient strain ( ⁇ gntR) were mixed and administered to germ-free mice, and two days later, F18mix or F31-18mix was administered, and the amount of bacteria of the Kp2H7 strain in the stool of those mice was measured.
• the amount of bacteria of the gntR-deficient strain decreased more quickly than the wild strain by administration of F18mix.
• the amount of bacteria of the Kp2H7 strain in the stool did not change significantly in the F31-18mix-administered group or the group administered only the Kp2H7 strain (Kp only) was analyzed.
• the Kp2H7 strain (wild strain) and the gntK-deficient strain ( ⁇ gntK) were mixed in the same amount and administered to germ-free mice. Two days later, F18mix or F31-18mix was administered, and the amount of Kp2H7 strain in the feces of the mice was measured. As a result, as shown in the graph on the left side of Figure 10, the gntK-deficient strain in the feces was significantly reduced in the F31-18mix group and the group administered only the Kp2H7 strain (Kp only).
• mice with the Kp2H7 strain and F18mix colonized in the intestines were prepared in the same manner as above.
• the diet of these mice was changed to CL-2 (gluconic acid content: 5 ⁇ mol/g) from the administration of F18mix until Day 20, and from Day 21 onwards to diet containing 0%, 2.5% (80 ⁇ mol/g) or 10% (320 ⁇ mol/g).
• CL-2 gluconic acid content: 5 ⁇ mol/g
• the bacterial load of the Kp2H7 strain in the feces was significantly increased in the group administered diet containing 10% gluconic acid.
• F18mix or F31-18mix was administered to mice in which only the Kp2H7 strain had been established, and the expression of the gntK gene in the Kp2H7 strain was analyzed two days later. As a result, as shown in Figure 12, the expression of the gene was low in the F18mix-administered group.
• Example 3 Identification of gluconic acid consuming bacteria in F18mix
• various isolated strains were cultured in modified GAM bouillon containing 300 ⁇ M gluconic acid. Then, after 48 hours, the culture supernatant was collected and the gluconic acid concentration was measured, and the strains in which a decrease in gluconic acid concentration was observed were determined to be strains capable of consuming gluconic acid. As a result, as shown in FIG. 13, it was revealed that 8 strains in F18mix were strains capable of consuming gluconic acid. Furthermore, it was revealed that 2 strains capable of consuming gluconic acid were included in the 13 strains of F31-18mix.
• strains f17 (18.4 ⁇ M), f18 (1.7 ⁇ M), f20 (16.5 ⁇ M), f21 (4.2 ⁇ M), f22 (9.0 ⁇ M), f23 (15.4 ⁇ M), f32 (15.0 ⁇ M) and f37 (9.2 ⁇ M), and two strains, f10 (14.2 ⁇ M) and f29 (8.2 ⁇ M), were found to have gluconic acid consumption ability.
• the concentration in parentheses indicates the residual gluconic acid concentration (average of three measured values) after 48 hours of culture in each strain.
• the residual gluconic acid concentration (average of three measured values) in 21 strains other than these 10 strains was all more than 270 ⁇ M.
• Example 4 Analysis of gluconic acid consumption ability in pathogenic microorganisms
• pathogenic microorganisms other than Klebsiella can consume gluconic acid.
• bacteria belonging to the Enterobacteriaceae family including Klebsiella (as well as Escherichia coli, Salmonella enteritidis, Proteus vulgaris, Proteus mirabilis, and Pseudomonas aeruginosa) consumed gluconic acid.
• Staphylococcus aureus and Bacillus cereus can also consume gluconic acid.
• Campylobacter Campylobacter upsaliensis, Campylobacter jejuni
• Streptococcus Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus sanguinis
• Enterococcus faecium Enterococcus faecium
• Clostridium Clostridium difficile, Clostridium perfringens
• Example 5 Effect of Combined Use of Gluconate-Consuming Bacteria and Non-Gluconating Bacteria in F18mix
• the F18 strain (F18mix) that consumes gluconic acid in the F18 strain
• the 10 strain (F18-8mix (F10mix)) that does not consume gluconic acid in the F18mix were administered.
• the amount of Kp2H7 strain bacteria in the feces decreased in the order of the F18mix administration group, the F8mix administration group, and the F10mix administration group.
• F8mix when F18mix was divided into strains that consume gluconic acid (F8mix) and those that do not (F10mix), F8mix showed a higher inhibitory ability against the Kp2H7 strain than F10mix. This is thought to be because F8mix and Kp2H7 strains compete for and consume gluconic acid in the intestine, resulting in greater inhibition of the growth of Klebsiella and other bacteria than F10mix.
• F18mix when comparing F18mix and F8mix, F18mix reduced the amount of Kp2H7 strain bacteria more than F18mix. This suggests that using a more specific strain rather than just using a strain that consumes gluconic acid can result in interactions and other effects that result in a greater inhibitory effect on Klebsiella.
• Example 6 Analysis of the ability of F18mix to suppress bacteria in the intestine depending on the concentration of gluconic acid Since most of the gluconic acid in the intestine is delivered by food, we observed the relationship between the food and the amount of Klebsiella bacteria in mice administered F18mix.
• mice After the Kp2H7 strain was established in germ-free mice, they were administered F18mix and fed CL-2 until day 21. From that point on, the mice were fed AIN-93G with different gluconic acid contents. As a result, as shown in Figures 16A and 16B, when mouse feed containing 0% or 2.5% gluconic acid by weight was used, a decrease in the amount of bacteria of the Kp2H7 strain was observed. On the other hand, when mouse feed containing 10% gluconic acid by weight was used, an increase in the amount of bacteria of the Kp2H7 strain was observed.
• Example 7 Analysis of the proliferation of F18mix in the intestine
• Bacterial genomes were extracted from the obtained feces, and the amount of each bacterium was quantified. The amount of bacteria was confirmed in both relative and absolute amounts.
• Figures 16C and 16D a rapid decrease in the amount of bacteria of the Kp2H7 strain was observed from the day of administration of F18mix to the 21st day.
• a tendency was observed in which the amount of bacteria of non-gluconic acid consuming bacteria f19 and f24 increased significantly. This suggests that even bacteria other than gluconic acid consuming bacteria may have some Klebsiella inhibitory effect.
• Example 8 Gene Expression Analysis in the Intestine Germ-free mice were administered either Kp-2H7 alone or Kp-2H7 + F18-mix. Fecal samples were collected from the mice 2 days after administration of F18-mix, and RNA-seq analysis of the bacteria in the feces was performed. Regarding the expression level of Klebsiella-derived RNA, KEGG pathway analysis was performed on genes with significant differences between groups (Benjamini-Hochberg corrected p-value ⁇ 0.001). A pathway consisting of 10 or more genes whose expression was increased or decreased in the Kp-2H7 + F18-mix group compared to the Kp-2H7 alone group) is shown in Figure 17A. In addition, the expression profile of genes involved in sugar metabolism is shown in Figure 17B.
• Example 9 Analysis of gluconic acid concentration in the intestine
• Germ-free mice were fed CL-2 (manufactured by CLEA Japan, Inc.) as a nutritious feed containing gluconic acid, or AIN93G (manufactured by Oriental Yeast Co., Ltd.) as a feed not containing gluconic acid.
• the gluconic acid concentration in the feces of these germ-free mice was then measured by LC-MS.
• Figure 18A the amount of gluconic acid in the feces varies depending on the diet, but since gluconic acid was detected even when it was not contained in the feed, it is believed that gluconic acid in the feces is supplied from the feed (food) and the host.
• Kp-2H7 was also administered to germ-free mice fed CL-2, and on the 21st day the diet was changed from CL-2 to AIN93G. Fecal CF for Kp-2H7 was shown as median ⁇ IQR. As a result, as shown in Figure 18B, the amount of Kp-2H7 bacteria in the feces decreased after the change to AIN93G. Therefore, it is thought that the change in the concentration of gluconic acid derived from the diet affected the change in the amount of Kp-2H7 bacteria.
• Example 10 Gene cluster analysis in gluconic acid consuming bacteria
• genes that are thought to be involved in gluconic acid metabolism and that can form gene clusters are shown in Figures 19A to 19D.
• strains capable of utilizing gluconate have gluconate kinase and gluconate transporter, or gluconate dehydrogenase and gluconate transporter, as a gene cluster, and also have the KDGK and Eda genes required for subsequent metabolism in nearby or separate gene clusters.
• gluconate dehydrogenase (gad), gluconate kinase (gntK), and transporter protein sequences identified in Figures 19A to 19D were collected and used to create a gluconate metabolism-related gene database (DB).
• DB gluconate metabolism-related gene database
• bacteria in which the enzyme (gad or gntK) and the transporter are in close proximity were searched for under the following conditions:
• the enzyme and transporter are encoded in the same DNA.
• the distance between the enzyme and transporter is less than 10,000 bp. No more than two genes are encoded between the enzyme and transporter.
• 70 species (gad) and 403 species (gnt) were identified as bacteria that satisfy all three of these conditions and have the enzyme and transporter genes in close proximity (the 70 species (gad) are shown in Tables 13 to 16, and the 403 species (gnt) are shown in Tables 17 to 40.
• Tables 13 and 14 Tables 15 and 16, Tables 17 and 18, Tables 19 and 20, Tables 21 and 22, Tables 23 and 24, Tables 25 and 26, Table 27).
• Tables 29 and 30, Tables 31 and 32, Tables 33 and 34, Tables 35 and 36, Tables 37 and 38, and Tables 39 and 40 correspond to each other, and the former tables (lower numbers) show information on the bacteria and gluconate dehydrogenase (gad) or gluconate kinase (gntK), and the latter tables show information on the gluconate transporters corresponding to the bacteria listed in the former tables and the "gut_microbes" described below.
• bacteria that can compete with disease-causing bacteria such as Kp-2H7 for gluconic acid consumption and thereby suppress the colonization of the bacteria in the intestine are preferably bacteria with a gluconic acid amount of 100 ⁇ M or less in the central bar graphs in Figures 19A to 19D and at least one of the above 70 species (gad) and 403 species (gnt), more preferably bacteria with a gluconic acid amount of 100 ⁇ M or less in the central bar graphs in Figures 19A to 19D and at least one of the above 10 species (gad) and 68 species (gnt), and particularly preferably bacteria with a gluconic acid amount of 100 ⁇ M or less in the central bar graphs in Figures 19A to 19D.
• Example 12 Inhibitory effect of F18mix or F13mix on colonization of the intestine by Kp2H7 strain
• Germ-free mice were divided into three groups (four mice per group): a group administered 18 strains of enterobacteria isolated from fecal samples of healthy individual #F (F18-mix), a group administered 13 strains (F13-mix), and a group administered nothing (Kp only).
• F18-mix a group administered 18 strains of enterobacteria isolated from fecal samples of healthy individual #F
• F13-mix group administered 13 strains
• Kp only group administered nothing
• the first two groups were administered F18mix or F13mix
• the three groups were administered the Kp2H7 strain, and the amount of bacteria in the stool Kp2H7 strain was analyzed over time. The results are shown in FIG. 20.
• the present invention by suppressing the colonization of gluconate-consuming bacteria, such as drug-resistant bacteria and inflammation-inducing bacteria, in the intestinal tract, it is possible to treat or prevent diseases caused by these bacteria. Therefore, the present invention is extremely useful in the development of medicines for the treatment, improvement, prevention, etc. of infectious diseases caused by the above-mentioned bacteria.

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