Use of novel microbiome as gastric cancer diagnostic and prognostic marker

Abstract

It has been identified in the present invention that, in addition to Helicobacter felis, strains causing cell hyperplasia, metaplasia, dysplasia, and the like in gastric tissues. Therefore, a gastric cancer mouse model in which gastric lesions are induced in mice can be provided using a gastric microbial transplantation (GMT) technology, and various gastrointestinal diseases can be diagnosed and the prognosis thereof can be predicted by measuring the concentrations of the strains.

Description

Novel microbiome applications as diagnostic and prognostic markers for gastric cancer

[0001] The present invention relates to innovative new drugs and personalized healthcare, and more particularly to the use of a novel microbiome as a diagnostic and prognostic marker for gastric cancer.

[0002]

[0003] In April 2023, the suppository 'rbx2660' (FDA approved on November 30, 2022) and the oral medication 'SER-109' were the first microbial therapeutics commercialized for the treatment of recurrent C. difficile enteritis (FDA approved on April 26, 2023). This marks the first microbiome-based drug treatment and presents limitless potential for microbial therapeutics utilizing the microbiome. However, the currently developed microbiome therapeutics are limited by their unclear mechanism of action, as they are a mixture of dozens of microorganisms. Meanwhile, there are 105 domestic microbiome LBP programs, rapidly growing through preclinical, phase 1, and 2 clinical trials, primarily targeting inflammatory bowel disease (IBD), asthma, psoriasis, and metabolic diseases.

[0004] Epidemiologically, gastric metaplasia is a typical precursor lesion of gastric cancer, and extensive research is being conducted as a target for treatment development and gastric cancer prevention. While anti-Helicobacter pylori eradication therapy can halt intestinal metaplasia, studies demonstrating its normalization (regression) are lacking. In this regard, dysbiosis has been identified as a key driver of gastrointestinal diseases, including cancer. Therefore, there is an urgent need to develop standardized and purified microbiome therapeutics, either in the form of a single species or a microbial community with a proven mechanism of action.

[0005] Meanwhile, Grand View Research predicted that the microbiome therapeutics market will grow from the current $114 million (approximately KRW 150 billion) to over $1.06 billion (KRW 1.43 trillion) by 2030, and the Ministry of Food and Drug Safety and international regulatory agencies are developing and revising guidelines for the development of clear microbiome therapeutics.

[0006]

[0007] One object of the present invention is to provide a diagnostic method, composition, kit and device for diagnosing or predicting the prognosis of a gastrointestinal disease using a microbiome containing multiple strains and a preparation for measuring the level of the microbiome.

[0008] Another object of the present invention is to provide a method for providing information for diagnosing or predicting the prognosis of a gastrointestinal disease using a microbiome containing multiple strains and a preparation for measuring the level of the microbiome.

[0009]

[0010] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0011]

[0012] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0013]

[0014] In one embodiment of the present invention, a microbiome is provided comprising two or more strains selected from the group consisting of Streptococcus mitis, Neisseria perflava, Haemophilus parahaemolyticus, Gemella haemolysans, and Haemophilus parainfluenzae.

[0015] The above strain may be isolated. As used herein, the term "isolated" means something that is artificially isolated and available rather than existing in nature.

[0016] The term "culture medium" in this specification may be used interchangeably with "culture supernatant", "conditioned medium" or "conditioned medium", and may mean the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the strain of Streptococcus mitis, Neisseria perflava, Haemophilus parahaemolyticus, Gemella haemolysans or Haemophilus parainfluenzae can grow and survive in vitro. In addition, the culture medium may mean a culture medium obtained by removing the cells from a cell culture medium obtained by culturing the strain. Meanwhile, the liquid from which the bacterial cells have been removed from the culture medium is also called a "supernatant", and can be obtained by leaving the culture medium still for a certain period of time and taking only the liquid in the upper layer excluding the portion that has settled to the lower layer, removing the bacterial cells through filtration, or centrifuging the culture medium to remove the sediment at the lower layer and taking only the liquid at the upper layer. The "bacterial cells" refers to the strain of the present invention itself, and includes the strain itself that has been separated and selected from a sample, etc., or the strain that has been isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and taking the portion that has settled to the lower layer, or by leaving the culture medium still for a certain period of time and then removing the upper liquid because the bacterial cells have settled to the lower layer of the culture medium due to gravity.

[0017] In the present invention, the above-mentioned “Streptococcus mitis” is a mesophilic alpha-hemolytic Streptococcus species that mainly inhabits the oral cavity, is a cocci (spherical), gram-positive, catalase-negative, facultative anaerobe. In the past, it was classified as Streptococcus mitior, and Streptococcus mitis is known to cause various diseases including infective endocarditis.

[0018] In the present invention, the “Neisseria perflava” belongs to the Neisseriaceae family and is known as an aerobic, mesophilic, gram-negative human pathogen. In addition, the “Neisseria perflava” is a cancer-specific human intestinal bacterium, and is an intestinal bacterium that selectively targets colon cancer and rectal cancer.

[0019] In the present invention, the "Hemophilus parahaemolyticus" is a species of anaerobic gram-negative coccus belonging to the genus Hemophilus, identified by Dr. Margaret Pittman in 1953. This species is known to be pathogenic not only to pigs but also to humans. Furthermore, H. parahaemolyticus has been found in the intestines of asymptomatic individuals.

[0020] In the present invention, the “Gemella haemolysans” is a species of Gemella, which was first reported as Neisseria hemolysans in 1938. In 1960, it was reclassified as a new genus when it was found that the strain was sufficiently distinct from Neisseria to require a new genus. Gemella haemolysans is a facultative anaerobe and reacts negatively to both oxidase and catalase tests. They are fermentative and produce a mixture of acetic acid and lactic acid or an equimolar mixture of acetic acid and CO2. For example, Gemella haemolysans ferments glucose to form a mixture of acetic acid and lactic acid in the absence of oxygen, whereas it forms acetic acid and CO2 in the presence of oxygen.

[0021] Gemella bacteria are primarily found in the mucous membranes of humans and other animals, particularly the oral cavity and upper digestive tract. Gemella haemolysins has been implicated in pulmonary exacerbations in patients with cystic fibrosis. As of 2000, 15 cases of human endocarditis have been reported, primarily in men with underlying valve disease and / or poor dentition or orthodontic treatment. Most cases have been treated with a combination of penicillin and gentamicin, with good results.

[0022] In the present invention, the above-mentioned "Haemophilus parainfluenzae" is an opportunistic pathogen associated with endocarditis, bronchitis, otitis media, conjunctivitis, pneumonia, abscesses, and genital infections. Furthermore, it is a Gram-negative, facultative anaerobic coccus that can cause acute bronchitis, exacerbation of chronic obstructive pulmonary disease, and meningitis. The above-mentioned Haemophilus parainfluenzae is also a Gram-negative, facultative anaerobic coccobacillus belonging to the HACEK group, which causes approximately 3% of cases of infective endocarditis.

[0023] In the present invention, the "microbial community" or "microbiome" refers to "the sum total of all microorganisms existing in a specific environment." Diversity includes alpha diversity, beta diversity, and gamma diversity. Alpha diversity determines the distribution of various microorganisms present in a single sample. Beta diversity indicates whether the diversity of samples is similar. Gamma diversity refers to the total diversity of all communities within a geographic region.

[0024] The microbiome provided in the present invention includes two or more strains selected from the group consisting of Streptococcus mitis, Neisseria perflava, Haemophilus parahaemolyticus, Gemella haemolysans, and Haemophilus parainfluenzae, and in other embodiments, it may be a microbiome including three or more, four or more, or all five of the above strains.

[0025] In a comparative example of the present invention, when only a single strain was inoculated, no gastric mucosal damage or inflammation occurred. However, when a microbiome comprising a combination of the above strains was inoculated, gastric mucosal damage and inflammation occurred. This suggests that gastric mucosal damage and inflammation occur due to interactions between strains, and if two or more strains are included and interactions occur between strains, gastric mucosal damage and inflammation may occur.

[0026] In another embodiment of the present invention, the Streptococcus mitis may be deposited under the accession number NCTC 12261, the Neisseria perflava may be deposited under the accession number DSM 18009, the Haemophilus parahaemolyticus may be deposited under the accession number CCUG 3716, the Haemophilus parainfluenzae may be deposited under the accession number ATCC 10379, and the Gemella haemolysans may be deposited under the accession number ATCC 33392.

[0027] However, since the present invention is not a pharmaceutical composition or treatment method for treating gastrointestinal diseases, it is not limited to the above-mentioned accession numbers, and a person skilled in the art will clearly understand that gastrointestinal diseases can be diagnosed and prognosis of gastrointestinal diseases can be predicted using the above-mentioned strains, even if they are not necessarily the strains of the above-mentioned accession numbers.

[0028] In another embodiment of the present invention, the concentration of strains in the microbiome is 1 x 10 4 1 x 10 10 cells / mL, providing a microbiome. In another embodiment, 1 x 10 5 1 x 10 9 cells / mL, 5 x 10 5 5 x 10 9 cells / mL, 1 x 10 5 1 x 10 9 cells / mL, 5 x 10 6 5 x 10 8 cells / mL or 1 x 10 6 1 x 10 8 It may be, but is not limited to, cells / mL.

[0029] In the present invention, the strains were cultured under various culture conditions. However, culture is not limited to the culture conditions specified in the examples. Any method capable of culturing the five strains described above using methods widely known in the relevant technical field is not particularly limited. Furthermore, while all of the microbiomes described above were cultured on solid media and under microaerobic conditions, any method capable of culturing the five strains described above is not particularly limited.

[0030]

[0031] In one embodiment of the present invention, a composition for diagnosing gastrointestinal diseases is provided, which comprises as an active ingredient a preparation for measuring the level of the microbiome.

[0032] The agent for measuring the level of the above microbiome refers to an agent capable of measuring the concentration of the above five strains, and is not particularly limited as long as it can measure the concentration of the strains. In addition, methods for measuring the concentration of the strains using the above sample include a method of isolating the strains from the sample using saline solution or PBS and then measuring the concentration.

[0033] In another embodiment of the present invention, a diagnostic composition is provided, wherein the gastric disease is at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer.

[0034] In the present invention, the stomach is a key digestive organ located just below the esophagus. When a person swallows food, it passes through the esophagus and reaches the stomach. It is the most elastic organ in the human body, expanding to hold approximately 1.5 liters of food in an adult human. Furthermore, the stomach primarily digests proteins. The gastric glands in the stomach wall secrete hydrochloric acid, a highly acidic substance with a pH of approximately 2, and pepsinogen, an inactive form of the digestive enzyme pepsin, which breaks down proteins into peptones. Furthermore, the strong acidity of the stomach also prevents ingested food from decaying within the body. Because this organ provides an extremely hostile environment for bacteria and viruses, most bacteria and viruses that may be mixed in food are killed here. Because the strong acidity of the stomach denatures proteins, the gastric glands secrete a mucus called mucin to protect the stomach wall from being dissolved by the stomach acid itself.

[0035] Therefore, if a microbiome can damage the tissues above, which have a highly elastic and highly acidic environment of about pH 2, where bacteria cannot survive, a person skilled in the art would easily predict that it would also cause tissue damage in other tissues (e.g., intestinal organs such as the duodenum, small intestine, and large intestine, and other organs such as the lungs and liver) that are more favorable for bacteria survival than the harsh environment above.

[0036] In the present invention, "tissue" refers to a mass of cells of the same type within an organ in biology. Tissues are an important unit for studying physiology. Because cells are so small, molecular biology studies typically study humans and higher animals as tissue units. These tissues include animal tissues, such as connective tissue, muscle tissue, nervous tissue, and epithelial tissue, as well as plant tissues and permanent tissues.

[0037] Gastric cancer is classified as a cancer with a poor prognosis, second only to lung cancer, in Korea. The molecular pathophysiology of gastric cancer remains unknown. It is understood to be influenced by four major risk factors: genetic factors, Helicobacter pylori infection, environmental factors, and the microbiome. Chronic gastric mucosal infection leads to atrophic gastritis and intestinal metaplasia over time, which are important precancerous lesions. Currently, H. pylori eradication therapy is considered the most important treatment for gastric cancer prevention. However, the factors that can normalize the precancerous lesions, atrophic gastritis and intestinal metaplasia, remain unknown. Therefore, inducing normalization of intestinal metaplasia is the most effective treatment and prevention method for gastric cancer.

[0038] Meanwhile, Helicobacter pylori (H. pylori) colonization in the gastric mucosa causes acute and chronic inflammation, and bacterial virulence factors and host genetic factors determine the severity of inflammation and pathological sequelae. H+, K+-adenosine triphosphatase (H+, K+-ATPase) of parietal cells is the final step in gastric acid secretion and is affected by H. pylori. In the acute infection stage, H. pylori suppresses the activity of the promoter of the alpha subunit of H+, K+-ATPase, and several cytokines such as interleukin-1β (IL-1β; encoded by IL-1B) and tumor necrosis factor-α released during this process also suppress acid secretion. When H. pylori infection progresses to a chronic state, atrophy in the gastric corpus accelerates, gastric acid secretion decreases, and loss of parietal cells occurs.

[0039] In addition, representative diseases caused by Helicobacter pylori infection include gastritis (atrophic gastritis, chronic gastritis, chronic atrophic gastritis, superficial gastritis), and intestinal metaplasia including metaplasia is known as a representative disease lesion caused by Helicobacter pylori infection.

[0040]

[0041] A composition according to one aspect of the present invention may be capable of predicting or diagnosing at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer. It may mean predicting or diagnosing whether the subject of the above “at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer” is likely to develop at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer, whether the subject is relatively likely to develop at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer, what the causative factor of at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer is, or whether at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer has already developed. In addition, in one aspect of the present invention, “diagnosis of at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer” means confirming the presence or characteristics of a pathological condition in a subject, and for the purpose of one aspect of the present invention, diagnosis may mean confirming whether at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer has occurred. The composition, kit, or method according to one aspect of the present invention can be used to delay or prevent the onset of at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer, and gastric cancer in any specific patient at a high risk of developing the disease through special and appropriate management.In addition, the composition, kit or method according to one aspect of the present invention can be clinically used to determine treatment by early diagnosis of at least one selected from the group consisting of intestinal metaplasia, gastritis, atrophic gastritis, gastric spasm, gastric ulcer and gastric cancer and selecting the most appropriate treatment method.

[0042] In another embodiment of the present invention, a diagnostic composition is provided, wherein the gastric disease is caused by cell hyperplasia, metaplasia, or dysplasia.

[0043] A composition according to one aspect of the present invention may be for predicting or diagnosing gastric cancer or gastric dysplasia. The “prediction of gastric cancer or gastric dysplasia” may mean predicting or diagnosing whether a subject is likely to develop gastric cancer or gastric dysplasia, whether the subject is relatively likely to develop gastric cancer or gastric dysplasia, what the causative factor of gastric cancer or gastric dysplasia is, or whether gastric cancer or gastric dysplasia has already developed. Furthermore, in one aspect of the present invention, “diagnosing gastric cancer or gastric dysplasia” means confirming the presence or characteristics of a pathological condition in a subject, and for the purposes of one aspect of the present invention, diagnosis may mean confirming whether gastric cancer or gastric dysplasia has developed. A composition, kit, or method according to one aspect of the present invention may be used to delay or prevent the onset of gastric cancer or gastric dysplasia in any specific patient who is at a high risk of developing gastric cancer or gastric dysplasia through special and appropriate management. Additionally, the composition, kit or method according to one aspect of the present invention can be clinically used to determine treatment by diagnosing gastric cancer or gastric dysplasia at an early stage and selecting the most appropriate treatment method.

[0044] In another embodiment of the present invention, a diagnostic composition is provided, wherein the diagnosis predicts prognosis.

[0045] In addition, in the present invention, the "diagnosis" means confirming the existence or characteristics of a pathological condition, and in the present invention, the "prognosis" means an act of predicting in advance the course of a disease and the outcome of death or survival. More specifically, the diagnosis of cancer means confirming whether a current patient is suffering from cancer, and the prognosis of cancer means an act of predicting in advance the course of a disease and the outcome of death or survival of a patient suffering from cancer, and more specifically, it means predicting or anticipating the recurrence-free survival period and the overall survival period.

[0046]

[0047] In one embodiment of the present invention, a gastrointestinal disease diagnostic kit comprising the above gastrointestinal disease diagnostic composition as an active ingredient is provided.

[0048] In the present invention, the "kit" refers to a tool that can evaluate the expression level of a biomarker, strain, or microbiome by labeling it with a detectable label using a probe or antibody that specifically binds to the biomarker, strain, or microbiome. In addition to direct labeling of a detectable substance related to the probe or antibody through reaction with a substrate, it also includes indirect labeling in which a chromogenic label is conjugated through reactivity with another directly labeled reagent. The kit may include a chromogenic substrate solution, a washing solution, and other solutions that will react with the label for chromogenic reaction, and may be manufactured including the reagent components used. In the present invention, the kit may be a kit that includes the essential elements required for performing RT-PCR, and may include a test tube, a reaction buffer, deoxynucleotides (dNTPs), Taq polymerase, reverse transcriptase, DNase, RNase inhibitor, sterile water, etc. in addition to each primer pair specific for the marker gene. In addition, the kit may be a kit for detecting a gene for cancer diagnosis that includes the essential elements required for performing a DNA chip. The DNA chip kit comprises a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. The kit of the present invention is not limited thereto, as long as it is known in the art.

[0049] In the present invention, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0050] The kit of the present invention may further comprise one or more other component compositions, solutions, or devices suitable for the analysis method. For example, the kit of the present invention may further comprise essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit comprises a pair of primers specific for a gene encoding a marker protein. The primers are nucleotides having a sequence specific for the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also comprise a primer specific for the nucleic acid sequence of a control gene. In addition, the reverse transcription polymerase reaction kit may comprise a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0051] Additionally, the diagnostic kit for gastrointestinal diseases of the present invention may include essential elements necessary for performing a DNA chip assay. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or fragment thereof are attached, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. The substrate may also include cDNA or oligonucleotides corresponding to a control gene or fragment thereof.

[0052] Additionally, the diagnostic kit for gastrointestinal diseases of the present invention may include the essential components necessary for performing an ELISA. The ELISA kit includes an antibody specific for the protein. The antibody has high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. The ELISA kit may also include an antibody specific for a control protein. In addition, the ELISA kit may include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, enzymes (e.g., conjugated to the antibody), and their substrates or other substances capable of binding to the antibody.

[0053] In the present invention, a fixative for the antigen-antibody binding reaction may be a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, a glass slide glass, etc., but is not limited thereto.

[0054] In addition, in the present invention, the label of the secondary antibody is preferably a conventional chromogen that undergoes a color development reaction, and labels such as fluorescein and dyes such as HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, FITC (poly L-lysine-fluorescein isothiocyanate), and RITC (rhodamine-B-isothiocyanate) can be used, but are not limited thereto.

[0055] In addition, in the present invention, it is preferable to use a chromogenic substrate for inducing color development according to a marker that undergoes a color development reaction, and TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. can be used. At this time, it is more preferable that the chromogenic substrate is provided in a state dissolved in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a marker of a secondary antibody conjugate to generate a chromogenic precipitate, and the presence or absence of the marker proteins is detected by visually confirming the degree of deposition of this chromogenic precipitate.

[0056] In the present invention, the washing solution preferably contains a phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 is more preferred. After the antigen-antibody binding reaction, the washing solution reacts the secondary antibody with the antigen-antibody complex, and then an appropriate amount is added to the fixative and washed 3 to 6 times. The reaction stopping solution can preferably be a sulfuric acid solution (H2SO4).

[0057]

[0058] In one embodiment of the present invention, a method for providing information for diagnosing a gastrointestinal disease is provided, including a step of determining that a gastrointestinal disease has developed or is likely to develop, when two or more strains selected from the group consisting of Streptococcus mitis, Neisseria perflava, Haemophilus parahaemolyticus, Gemella haemolysans, and Haemophilus parainfluenzae are present in a biological sample isolated from a target individual at a higher concentration than that of a normal control group.

[0059] In the present invention, the "target subject" refers to a subject that has developed or is likely to develop a gastrointestinal disease, and may be a mammal including a human, and may be selected from the group consisting of, for example, a human, a rat, a mouse, a guinea pig, a hamster, a rabbit, a monkey, a dog, a cat, a cow, a horse, a pig, a sheep, and a goat, and is preferably a human, but is not limited thereto.

[0060] When the expression level of the marker according to the present invention is measured from a biological sample isolated from the subject of interest in the present invention, it is possible to determine whether or not a disease has developed or the possibility of developing the disease very quickly and easily.

[0061] In the present invention, the term "control group" may be, but is not limited to, the expression level of a biomarker protein or a gene encoding the protein specific to the strain or microbiome in a healthy normal control group, the average or median value of the expression level of the marker protein or the gene encoding it in a biological sample derived from a patient with a gastrointestinal disease, or the average or median value of the expression level of the marker protein or the gene encoding it in a biological sample derived from a patient with a gastrointestinal disease other than a gastrointestinal disease.

[0062] In the above method of the present invention, predicting that the gastrointestinal disease has developed or is likely to develop includes not only predicting the possibility of the development, growth, progression or metastasis of the gastrointestinal disease, but also predicting that the disease developed or suspected to have developed in the target individual is a gastrointestinal disease by distinguishing it from other diseases.

[0063] In the present invention, the composition is intended to be applied to a biological sample isolated from a target subject, and the biological sample includes, but is not limited to, a solid tissue sample, a tissue culture, a liquid tissue sample, a cell, or a cell fragment. Also, non-limiting examples of biological samples include whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint fluid. It may include, but is not limited to, one or more selected from the group consisting of joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid.

[0064] According to another embodiment of the present invention, a method for measuring the concentration of a strain present in the biological sample provides an information providing method using absorbance measurement, turbidity measurement, plate counting method or direct microscopic counting method.

[0065] In addition, methods for measuring the concentration of strains are widely known in the relevant technical field, and are not particularly limited to methods for measuring the concentration of strains or microbiomes.

[0066]

[0067] In one embodiment of the present invention, a gastrointestinal disease diagnosis device is provided, comprising: a measuring unit for measuring the concentration of two or more strains selected from the group consisting of Streptococcus mitis, Neisseria perflava, Haemophilus parahaemolyticus, Gemella haemolysans, and Haemophilus parainfluenzae in a biological sample isolated from a target object; and (b) a judgment unit for determining that a gastrointestinal disease has developed or is likely to develop when the concentration of the strain measured by the measuring unit is higher than that of a normal control group; and (c) a detection unit for outputting the result determined by the judgment unit.

[0068] According to one aspect of the present invention, the composition, diagnostic kit or diagnostic device may be capable of predicting or diagnosing a gastrointestinal disease negative if the concentration of the strain or microbiome decreases by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more or 20% or more. In addition, the composition, diagnostic kit or diagnostic device may be capable of predicting or diagnosing a gastrointestinal disease positive if the concentration of the strain or microbiome increases by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more or 20% or more.

[0069]

[0070] In one embodiment of the present invention, an animal model induced with a gastrointestinal disease is provided, which is prepared by administering the microbiome.

[0071] The animal model of the present invention refers to an animal model of a mammal other than a human, and the mammal other than a human may be selected from the group consisting of mice, rats, monkeys, guinea pigs, dogs, and rabbits, and in one embodiment may be a mouse or a rat, and in another embodiment may be a mouse, but is not limited thereto.

[0072] Therefore, in one embodiment of the present invention, the mammal may be a mammal of 5 to 20 weeks of age, in another embodiment, 5 to 15 weeks of age, in another embodiment, 7 to 13 weeks of age, and in yet another embodiment, 7 to 11 weeks of age.

[0073] The above animal model is an animal model in which a gastrointestinal disease is induced by the strain or microbiome provided in the present invention, and the method of administering the strain or microbiome is not particularly limited other than oral administration.

[0074] In addition, using the gastric microbiota transplantation (GMT) technique, it was confirmed that inflammation, metaplasia, and precancerous lesions were observed at a high rate in the gastric mucosa of the transplanted mice after transplantation into germ-free mice. This is the world's first experimental animal (mouse) model in which a human-like gastric disease was induced in mice beyond species limitations by human gastric microbiota.

[0075]

[0076] The present invention identifies strains that cause hyperplasia, metaplasia, dysplasia, etc. in gastric tissue in addition to Helicobacter felis, and thereby provides a gastric cancer mouse model that induces gastric lesions in mice using gastric microbiota transplantation (GMT) technology, and by measuring the concentration of strains, various gastric diseases can be diagnosed and their prognosis can be predicted.

[0077]

[0078] Figure 1 is a mouse experiment method of the microbiome provided in the present invention.

[0079] Figure 2a shows the gastric tissue and inflammatory response of the negative control group, as confirmed by H&E staining. No gastric tissue or inflammatory response was observed in the negative control group.

[0080] Figure 2b shows the results of H&E staining of gastric tissue from mice inoculated with the microbiome provided by the present invention. Gastric tissue damage and inflammatory responses were observed in all mice inoculated with the microbiome.

[0081] Figure 2c shows the immunohistochemical staining results of gastric tissue from mice inoculated with the microbiome provided by the present invention. In all mice inoculated with the microbiome, an increase in macrophages, an indicator of inflammatory response, hyperplasia of chief cells, an indicator of gastric tissue damage, loss of parietal cells, increased cell proliferation, and increased expression of metaplasia markers were observed.

[0082] Figure 3a shows the H&E staining results when administered with a single strain of Gemella haemolysans. Unlike when administered with a microbiome, no gastric tissue damage or inflammatory response was observed.

[0083] Figure 3b shows the H&E staining results when a single strain of Streptococcus mitis was administered. As a result, unlike when the microbiome was administered, no damage to the gastric tissue or inflammatory response could be observed.

[0084]

[0085] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0086]

[0087] Example

[0088]

[0089] [Example 1] Production of a microbiome cocktail

[0090] To prepare a microbiome cocktail, strains were first identified by pure culture and 16S sequence on solid media. Cells were then harvested from the solid media, resuspended in phosphate-buffered saline (PBS), and washed once with PBS. The cell number was then measured using a QUANTOM Tx Microbial cell counter (Logos biosystems) in PBS. The final microbiome cocktail contained 1 x 10 of each of the five strains. 7 A PBS solution containing 20% ​​glycerol was prepared to contain 10 cells / ㎖. The specific combination of microbiome cocktails and culture conditions are shown in Table 1 below.

[0091] Microbiome strain combination and culture conditions Type of strains to be inoculated Culture medium Culture condition Concentration Injection method Injection medium Streptococcus mitis NCTC 12261 Trypticase soy agar Microaerobic 48-72 h 1 x 10 7 cells / mLTotal 5*10 7 cells / mL 200 μL Oral injection Mixed, 20% glycerol in PBS solution Neisseria perflava DSM 18009 Trypticase soy agar 5% horse blood Microaerobic 48-72 h 1 x 10 7 cells / mLHaemophilus parahaemolyticusCCUG 3716Haemophilus agarMicroaerobic48-72h1 x 10 7 cells / mLGemella haemolysansATCC 10379Trypticase soy agar 5% horse bloodMicroaerobic48-72h1 x 10 7 cells / mLHaemophilus parainfluenzaeATCC 33392Haemophilus agarMicroaerobic48-72h1 x 10 7 cells / mL

[0092] [Example 2] Administration of microbiome cocktail to mice

[0093] The microbiome cocktail prepared in Example 1 was administered to mice after being stored in a deep freezer. Specifically, as shown in Fig. 1, a total of 14 8-week-old germ-free mice were prepared. Two 8-week-old germ-free mice were used as a negative control group, and as a comparative example, five 8-week-old germ-free mice were administered Streptococcus mitis, a single strain instead of a cocktail, three times on days D+0, D+2, and D+4, and five 8-week-old germ-free mice were administered Gemella haemolysans three times on days D+0, D+2, and D+4. The remaining five mice were administered the microbiome cocktail prepared in Example 1 three times on days D+0, D+2, and D+4, and 8 weeks after the first administration, when all mice were 16 weeks old, gastric tissue damage and inflammatory responses were observed.

[0094]

[0095] [Example 3] Confirmation of Tissue Damage and Inflammatory Response of the Cocktail

[0096] The gastric tissue damage and inflammatory response of mice administered the microbiome cocktail prepared in Example 1 were compared with the negative control group.

[0097] Specifically, the gastric tissue damage and inflammatory response of the negative control group and mice administered the microbiome cocktail were compared through H&E staining and immunochemical staining. As shown in Fig. 2(a), no gastric tissue damage or inflammatory response was observed in the negative control group, but as shown in Figs. 2(b) to 2(c), in the case of the mice administered the microbiome cocktail prepared in Example 1, gastric tissue damage and inflammatory response were observed in all mice, and an increase in macrophages, which is an indicator of inflammatory response, hyperplasia of chief cells, which is an indicator of gastric tissue damage, loss of parietal cells, increased cell proliferation, and increased expression of metaplasia indicators were observed through immunochemical staining.

[0098] In addition, using the gastric microbiota transplantation (GMT) technique, it was confirmed that inflammation, metaplasia, and precancerous lesions were observed at a high rate in the gastric mucosa of the transplanted mice after transplantation into germ-free mice. This is the world's first experimental animal (mouse) model in which a human-like gastric disease was induced in mice beyond species limitations by human gastric microbiota.

[0099]

[0100] [Comparative Example] Confirmation of tissue damage and inflammatory response in a single strain

[0101] In comparison with the above Example 3 in which a microbiome cocktail was administered, a comparative experiment was conducted to determine whether gastric tissue damage and inflammatory responses occurred even in the case of a single strain.

[0102] Specifically, when only the Gemella haemolysans strain was administered, as shown in Fig. 3(a), no gastric tissue damage or inflammatory response could be observed, and even when only the Streptococcus mitis strain was administered, as shown in Fig. 3(b), no gastric tissue damage or inflammatory response could be observed.

[0103]

[0104] [conclusion]

[0105] In this way, when a microbiome cocktail rather than a single strain was administered, gastric tissue damage and gastric tissue inflammatory response were confirmed. In addition to Helicobacter felis, strains that cause hyperplasia, metaplasia, and dysplasia in gastric tissue were confirmed. Therefore, when the microbiome cocktail combination (combination of strains) in Table 1 is found in the stomach, it can be expected that intestinal metaplasia, gastric inflammation, and gastric cancer have occurred, or that the probability of occurrence of the above indications is high, or that the prognosis of the indications is poor.

[0106]

[0107] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0108]

Claims

1. A microbial community comprising two or more strains selected from the group consisting of Streptococcus mitis, Neisseria perflava, Haemophilus parahaemolyticus, Gemella haemolysans, and Haemophilus parainfluenzae.

2. In paragraph 1, The concentration of strains in the above microbial community is 1 x 10 6 1 x 10 8 cells / mL, microbial community.

3. A composition for diagnosing gastrointestinal diseases, comprising as an active ingredient a preparation for measuring the level of a microbial community of any one of claims 1 to 2.

4. In paragraph 3, A diagnostic composition, wherein the above gastric disease is at least one selected from the group consisting of intestinal metpalsia, gastritis, gastric spasm, gastric ulcer, and gastric cancer.

5. In paragraph 4, A diagnostic composition wherein the above gastric disease is caused by cell hyperplasia, metaplasia, or dysplasia.

6. In paragraph 4, A diagnostic composition wherein the above gastritis is at least one selected from the group consisting of atrophic gastritis, chronic gastritis, chronic atrophic gastritis, and superficial gastritis.

7. In paragraph 4, A diagnostic composition wherein the above intestinal metaplasia is metaplasia.

8. In paragraph 4, A diagnostic composition wherein the above diagnosis predicts the prognosis.

9. A composition for diagnosing tissue damage, comprising as an active ingredient a preparation for measuring the level of a microbial community of any one of claims 1 to 2.

10. In paragraph 9, A diagnostic composition, wherein the tissue is at least one selected from the group consisting of connective tissue, muscle tissue, nerve tissue, and epithelial tissue.

11. A gastrointestinal disease diagnostic kit comprising the gastrointestinal disease diagnostic composition of Article 3 as an active ingredient.

12. In paragraph 11, The above kit is a diagnostic kit, which is an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

13. In paragraph 11, The above diagnosis is a diagnostic kit that predicts the prognosis.

14. In a biological sample isolated from the target organism, A method for providing information for diagnosing a gastrointestinal disease, comprising: a step of determining that a gastrointestinal disease has developed or is likely to develop when two or more strains selected from the group consisting of Streptococcus mitis, Neisseria perflava, Haemophilus parahaemolyticus, Gemella haemolysans, and Haemophilus parainfluenzae are present in the biological sample at a higher concentration than that of a normal control; 15. In paragraph 14, A method of providing information, wherein the above gastrointestinal disease is at least one selected from the group consisting of intestinal metpalsia, gastritis, gastric spasm, gastric ulcer, and gastric cancer.

16. In paragraph 15, A method for providing information, wherein the above gastritis is at least one selected from the group consisting of atrophic gastritis, chronic gastritis, chronic atrophic gastritis, and superficial gastritis.

17. In paragraph 15, The above intestinal metaplasia is metaplasia, a method of providing information.

18. In paragraph 14, A method for measuring the concentration of strains present in a biological sample is as follows: Information providing method by absorbance measurement, turbidity measurement, plate counting method, real-time polymerase chain reaction (real-time PCR) or direct microscopic counting method.

19. In paragraph 14, A method of providing information, wherein the above diagnosis is for predicting prognosis. 20.(a) A measuring unit for measuring the concentration of two or more strains selected from the group consisting of Streptococcus mitis, Neisseria perflava, Haemophilus parahaemolyticus, Gemella haemolysans, and Haemophilus parainfluenzae in a biological sample isolated from a target object; and (b) a judgment unit that determines that a gastrointestinal disease has developed or is likely to develop if the concentration of the strain measured in the above measurement unit is higher than that of the normal control group; and (c) A gastrointestinal disease diagnosis device, comprising a detection unit that outputs the result determined by the above judgment unit.

21. In paragraph 20, A diagnostic device wherein the above gastrointestinal disease is at least one selected from the group consisting of intestinal metpalsia, gastritis, gastric spasm, gastric ulcer, and gastric cancer.

22. In paragraph 21, A diagnostic device wherein the above gastritis is at least one selected from the group consisting of atrophic gastritis, chronic gastritis, chronic atrophic gastritis, and superficial gastritis.

23. In paragraph 21, The above intestinal metaplasia is a diagnostic device for metaplasia.

24. In paragraph 20, The above diagnosis is a diagnostic device that predicts the prognosis.

25. An animal model in which a gastrointestinal disease is induced, prepared by administering the microbial community of clauses 1 and 2.

26. A method for diagnosing a gastrointestinal disease, comprising as an active ingredient a preparation for measuring the level of a microbial community of any one of claims 1 to 2.

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