Method for predicting response to biological therapeutic agents and method for treating various diseases using the same

Abstract

The present invention can provide a personalized treatment method that can maximize the therapeutic effect by predicting the therapeutic efficacy of a biological therapeutic agent based on the distribution of therapeutic bacterial strains or phyla in a patient's intestinal sample and providing a biological agent containing bacteria that are not in a competitive relationship as a personalized treatment option.

Description

[Technical Field]

[0001] The present invention relates to a method for predicting the response of biological therapeutic agents and a method for treating various diseases, including metabolic disorders, using the predicted results. More specifically, the present invention relates to providing personalized medical care by selecting a biological agent containing bacteria that are not competitively excluded as a treatment option based on the distribution of phylogenetic groups of therapeutic bacteria in a patient's sample. [Background technology]

[0002] Obesity is a serious disease with no effective treatment and is continuing to increase worldwide. Obesity is different from other diseases in that it is accompanied by related diseases such as metabolic syndrome, hypertension, diabetes, hyperlipidemia, arteriosclerosis, ischemic heart disease, fatty liver, and cholelithiasis. Metabolic syndrome is a disease characterized by abdominal obesity, impaired glucose tolerance, hypertension, and hemostatic disorders.

[0003] Currently available obesity treatments rely on chemical substances and are broadly divided into appetite suppressant-type obesity treatments and lipolysis inhibitor-type obesity treatments. However, appetite suppressant-type obesity treatments are substances that act on the central nervous system, and therefore have been largely withdrawn due to the fatal problem of inducing serious side effects when taken for a long period of time. Meanwhile, orlistat (Xenical, manufactured by Roche) is the only lipolysis inhibitor-type obesity treatment that has successfully passed clinical trials and entered the market. (R) and Alli (R) ) has been reported to cause diarrhea, fatty stools, and other symptoms, and to cause serious liver damage when taken for a long period of time, leading to a re-examination of the safety of orlistat by the US FDA. As such, most obesity treatments currently on the market have serious side effects, and obesity treatments that utilize gut bacteria are attracting attention as a promising treatment for obesity and related disorders.

[0004] Pharmabiotics is a combination of the words pharmaceutical and probiotics, and is defined as bacteria or metabolic products produced by bacteria that have proven medical benefits for health or disease (Hill, 2010). For pharmacological products to be approved as medicines by regulatory authorities such as the European Food Safety Authority and the US Food and Drug Administration, they must demonstrate sustained and objective physiological and medical effects.

[0005] The efficacy of pharmabiotic products can vary greatly between individuals due to significant inter-individual human microbiome variability, which is mediated by various factors such as the subject's (patient's) age, health status, diet, use of antibiotics, consumption of health functional foods, etc. Therefore, there is an urgent need to develop technology that can select personalized pharmabiotics.

[0006] For example, Akkermansia muciniphila strains, recognized as the next generation microbiome, are attracting attention as candidates for innovative new drugs against obesity, metabolic syndrome, type 2 diabetes, hyperlipidemia, and non-alcoholic fatty liver disease. Despite their importance, the specific mechanisms of these Akkermansia strains are not yet clear. This is because most strain impact assessment studies have focused on the standard strain, Akkermansia muciniphila BAA-835. T This is because the method is being used to detect a single species of Akkermansia. Although there are many different Akkermansia strains in the intestines, only whole genome level identification methods are used to detect them, which has the limitation that accurate research is not possible. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have discovered for the first time that probiotic strains do not exist in the intestine in a complex form where various strains or phylogroups coexist, but rather in a form where a single strain or phylogroup predominates and exhibits a competitive and entrenched relationship with other strains or phylogroups, thereby completing the present invention.

[0008] One object of the present invention is to provide a method for predicting a patient's responsiveness to a biological therapeutic agent by utilizing the competitive advantage, colonization inhibition, and competitive borrowing relationships between enterobacteria in gastrointestinal cells.

[0009] Another object of the present invention is to provide a method for isolating bacterial genes from easily obtainable fecal samples and analyzing them by qPCR to predict a patient's response to biological therapy.

[0010] It is yet another object of the present invention to provide a method for predicting a patient's response to biological therapy by a phylogenetic group-specific discriminator region within the 16S rRNA gene.

[0011] A further object of the present invention is to provide a marker composition for predicting the response of patients with metabolic disorders to biotherapeutics, including pharmacological bacteria.

[0012] Another object of the present invention is to provide a method for treating various diseases, such as metabolic disorders, by providing a biological therapeutic agent as a personalized treatment option that can maximize therapeutic effects by utilizing competitive advantage, colonization inhibition, or competitive borrowing relationships between intestinal bacteria.

[0013] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0014] In one aspect of the present invention to achieve the above-mentioned object, (a) determining the distribution of target bacteria by strain or phylogenetic group through gut microbiota analysis of the patient; (b) identifying whether a competitive relationship exists between the strain or phylogenetic group identified in the previous step as the dominant species in the patient's gut and the target strain or phylogenetic group; (c) if the strain or phylogenetic group identified as the dominant species in the intestine in the previous step is identified as being in a competitive relationship with the target bacterial strain or phylogenetic group, determining that the patient will likely have a low responsiveness to the biological therapeutic agent containing the relevant strain or phylogenetic group.

[0015] The intestinal microbiota analysis may include a step of performing quantitative PCR (qPCR) on DNA extracted from a patient's fecal sample using a primer pair or probe specific to the sodium ion-translocating decarboxylase subunit beta gene of a specific strain or phylogenetic group of bacteria.

[0016] Additionally, gut microbiota analysis may include a step of identifying and characterizing the phylogenetic group of DNA extracted from a patient's fecal sample using a phylogenetic group-specific gene identifier region specific to the 16S rRNA gene of bacteria of a particular strain or phylogenetic group.

[0017] The step of identifying whether or not a competitive relationship exists may include treating a culture supernatant of a strain or phylogenetic group identified as a dominant species in the patient's intestine with a target strain or phylogenetic group and determining whether the growth of the target strain or phylogenetic group is inhibited.

[0018] Another aspect of the present invention is (a) determining the strain or phylogenetic distribution of Akkermansia bacteria to be used as a biological therapeutic agent by analyzing the patient's intestinal microbiota; (b) identifying whether a competitive relationship exists between the Akkermansia phylogenetic group identified as the dominant gut species in the previous step and the target Akkermansia species or phylogenetic group; (c) if it is identified that the Akkermansia phylogenetic group identified as the predominant intestinal species in the previous step and the target Akkermansia bacterium are in a competitive borrowing relationship, determining that the patient will likely have a low responsiveness to a biological therapeutic agent containing the Akkermansia species or phylogenetic group.

[0019] Another aspect of the present invention is (a) determining the distribution of target bacteria by strain or phylogenetic group through a patient's intestinal microbiota analysis; (b) identifying whether a competitive borrowing relationship exists between the strain or phylogenetic group identified as the dominant species in the previous step and the target strain or phylogenetic group; (c) if it is determined that the strain or phylogenetic group identified as the dominant species in the previous step is not in a competitive relationship with the target bacterial strain or phylogenetic group, selecting a biological therapeutic agent containing the strain or phylogenetic group as a treatment option and administering it to the patient. [Effects of the Invention]

[0020] According to the present invention, it is possible to specifically detect each of the four phylogenetic groups AmIa, AmIb, AmII, and AmIV belonging to Akkermansia strains that form the intestinal microbial community. The present invention not only enables accurate and clear detection of Akkermansia strains, the distribution of which may vary depending on various diseases, but also specifically and accurately detects each of the four phylogenetic groups belonging to Akkermansia strains. This makes it possible to analyze the prevalence of specific diseases and the corresponding strains, and based on this, provide personalized diets or drugs containing the target strains.

[0021] No matter how many pharmabiotic bacteria are ingested, if they cannot reach the intestines alive or cannot settle and dominate in the intestines, they will not be able to exert their efficacy at all. However, according to the present invention, it is possible to select and administer pharmabiotics that can successfully settle in the intestines of each patient by analyzing the intestinal flora of the patient, thereby maximizing the therapeutic effect of pharmabiotics.

[0022] According to the present invention, a method for verifying personalized probiotics, prebiotics, foods, health functional foods, and pharmaceuticals based on a patient's intestinal flora is provided, thereby providing an effective analytical method for screening effective strains that can treat personalized metabolic disorders, etc. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows three phylogenetic group identification regions present in the 16S rRNA gene sequence of Akkermansia that can specifically identify the Akkermansia phylogenetic groups (AmI, AmII, and AmIV). [Figure 2a] Figure 2a shows a phylogenetic tree of 92 Akkermansia species based on 16S rRNA gene sequences. [Figure 2b] FIG. 2b is a graph showing the average nucleotide sequence homology based on whole genome comparison of 92 Akkermansia strains. [Figure 3] Figure 3(A) shows the phylogenetic classification of 92 species of Akkermansia based on the sequences of the marker gene sodium ion-translocating decarboxylase subunit beta. Figure 3(B) shows the intra- and inter-phylogroup genetic differences of the marker gene. [Figure 4a]Figure 4a shows a circular phylogram of 22 Akkermansia ASVs (Amplicon sequence variants) identified by 16S rRNA sequences of 92 human-derived Akkermansia genomes and gut microbiota analysis of 890 Korean individuals. [Figure 4b] Figure 4b is a distribution map showing the distribution of Akkermansia by phylogenetic group in the intestines of 890 healthy Korean individuals. [Figure 5a] Figure 5a is a circular phylogenetic tree showing the presence or absence of intestinal Akkermansia and their distribution patterns by phylogenetic group in seven countries, including Korea. [Figure 5b] Figure 5b is a distribution map showing the phylogenetic distribution of intestinal Akkermansia in seven countries, including Korea. [Figure 6] FIG. 6 shows the results of evaluating the analytical performance of Akkermansia phylogenetic group-specific primers (AmIa, AmIb, AmII) according to one embodiment of the present invention. [Figure 7] FIG. 7 shows the changes in fecal levels and maintenance of colonization after single administration of representative strains of each phylogenetic group of Akkermansia to germ-free mice. [Figure 8] Figure 8(A) is a graph showing the competitive relationship between various Akkermansia phylogenetic group strains when co-administered in germ-free mice. This graph shows the colonization of the intestines of mice when co-administered with various Akkermansia phylogenetic group strains (BAA-835T:AmIa, EB-AMDK19:AmIb, EB-AMDK39:AmII). Figure 8(B) is a graph showing the competitive relationship between various Akkermansia phylogenetic group strains when co-administered in germ-free mice. This graph shows the colonization of the intestines of mice when co-administered with two Akkermansia phylogenetic group strains (EB-AMDK19:AmIb, EB-AMDK39:AmII). [Figure 9] Figure 9(A) is a photograph showing the electrophoresis results of PCR using strain-specific and phylogenetic group-specific primers, and Figure 9(B) is a melting curve plot of qPCR using strain-specific and phylogenetic group-specific primers. [Figure 10] FIG. 10 is a graph showing the change in intestinal Akkermansia strains when cross-administered with AmI and AmII strains in germ-free mice. DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0025] When a part in this specification "comprises" a certain element, this does not mean excluding other elements, but rather means further including other elements, unless specifically stated to the contrary.

[0026] As used herein, the terms "patient" and "subject" may be used interchangeably and may refer to a human or non-human animal. These terms include mammals, such as humans, non-human primates, livestock (e.g., cows, pigs, sheep, goats, poultry), pets (e.g., dogs, cats, horses, rabbits), and rodents (e.g., guinea pigs, hamsters, mice).

[0027] As used herein, the terms "treat," "treatment," and the like mean to temporarily or permanently relieve symptoms, eliminate the cause of symptoms, or prevent or slow the onset of symptoms of a disease or condition.

[0028] As used herein, the terms "biotherapeutic agent" and "biopharmaceutical agent" are used interchangeably and refer to drugs effective in preventing, treating, or curing diseases or illnesses, including bacteria (probiotics). In embodiments herein, a "biotherapeutic agent" consists of or includes anaerobic bacteria or strictly anaerobic bacteria.

[0029] As used herein, the term "target strain" refers to therapeutic bacteria (probiotics) used as biological therapeutic agents for a particular subject or patient.

[0030] As used herein, the term "gut microbiota analysis" refers to a test that analyzes the composition and / or distribution of various bacteria present in the intestine by genetic analysis of bacteria or microbiota excreted in feces.

[0031] As used herein, the term "primer" refers to a short nucleic acid sequence with a short, free 3'-hydroxyl group that forms a base pair with a complementary template and serves as a starting point for template replication. A primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer solution and temperature.

[0032] As used herein, the terms "sequence homology," "percent homology," or "percent homology" refer to the degree to which sequences are identical on a nucleotide-by-nucleotide basis over the comparison window.

[0033] As used herein, the term "metabolic disorder" refers to obesity, metabolic syndrome, insulin deficiency or insulin-resistance related disorders, diabetes (e.g., type 2 diabetes), glucose intolerance, dyslipidemia, atherosclerosis, hypertension, cardiac pathology, stroke, nonalcoholic fatty liver disease, hyperglycemic conditions, fatty liver, dyslipidemia, immune system dysfunction associated with overweight and obesity, cardiovascular disease, high cholesterol, elevated triglycerides, asthma, sleep apnea, osteoarthritis, neurodegeneration, gallbladder disease, fragile X syndrome, inflammatory diseases, immune disorders, atherogenic dyslipidemia, and cancer. In other embodiments, the metabolic disorder is a metabolic disorder associated with overweight and / or obesity, i.e., a metabolic disorder associated with or that can be caused by overweight and / or obesity. Examples of metabolic disorders associated with overweight and / or obesity include, but are not limited to, metabolic syndrome, insulin deficiency or insulin-resistance related disorders, diabetes (e.g., type 2 diabetes), glucose intolerance, dyslipidemia, atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholic fatty liver disease, hyperglycemic conditions, fatty liver, dyslipidemia, immune system dysfunction associated with overweight and obesity, cardiovascular disease, high cholesterol, elevated triglycerides, asthma, sleep apnea, osteoarthritis, neurodegeneration, gallbladder disease, fragile X syndrome, inflammatory and immune disorders, atherogenic dyslipidemia, and cancer.

[0034] The term "dominant species" as used herein refers to a bacterial species or phylogenetic group that survives and reproduces more predominantly than other bacterial species or phylogenetic groups in the patient's intestine. A method for analyzing the composition and distribution of various bacteria in the human intestine can be performed through genetic analysis of the indigenous intestinal microbiota excreted in feces.

[0035] One aspect of the present invention is (a) determining the distribution of target bacteria by strain or phylogenetic group through gut microbiota analysis of the patient; (b) identifying whether a competitive relationship exists between the strain or phylogenetic group identified in the previous step as the dominant species in the patient's gut and the target strain or phylogenetic group; (c) if the strain or phylogenetic group identified as the dominant species in the intestine in the previous step is identified as being in a competitive relationship with the target bacterial strain or phylogenetic group, determining that the patient will likely have a low responsiveness to the biological therapeutic agent containing the relevant strain or phylogenetic group.

[0036] As used herein, gut microbiota analysis involves analyzing DNA from a gut sample, which is a fecal sample, and DNA is extracted from the fecal sample prior to genetic analysis. The method for extracting probiotic DNA from a fecal sample is not particularly limited, but an example is the use of a silica membrane column, such as those included in commercially available DNA extraction kits, in combination with high-speed bead-beating extraction, chemical cell lysis, and mechanical disruption as a final purification step.

[0037] The identification of strains or phylogenetic groups of the patient's intestinal bacteria can be performed using any suitable classical method known in the art, typically by bacterial gene quantification, which measures the amount or relative abundance of specific nucleic acid sequences in a sample.

[0038] The gut microbiota analysis can be performed by qPCR on DNA extracted from a patient's fecal sample using primer pairs or probes specific for specific strains or phylogenetic groups of bacteria.

[0039] In a preferred embodiment, quantification of the sodium ion-translocating decarboxylase subunit beta gene of a target bacterium can be performed using one or more oligonucleotide molecules of strain- or phylogenetic group-specific primers in Table 1 below, or sequences having 75% or more sequence identity thereto.

[0040] [Table 1]

[0041] Preferably, oligonucleotide sequences having 75% or greater sequence identity described herein have at least 80%, at least 90%, at least 95%, more preferably 96%, 97%, 98%, 99%, or 100% sequence identity with the relevant sequence (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and / or SEQ ID NO:8, respectively); nucleotide molecules with 100% sequence identity are particularly preferred. Additionally, these oligonucleotide sequences with 75% or greater sequence identity can have the same number of nucleotides.

[0042] For example, the target bacterium Akkermansia may be analyzed by qPCR using an AmIa-specific primer having 75% or more sequence homology to the sequence of SEQ ID NO:1 or SEQ ID NO:2; an AmIb-specific primer having 75% or more sequence homology to the sequence of SEQ ID NO:3 or SEQ ID NO:4; an AmII-specific primer having 75% or more sequence homology to the sequence of SEQ ID NO:5 or SEQ ID NO:6; or an AmIV-specific primer having 75% or more sequence homology to the sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0043] Alternatively, total DNA extracted from human fecal or mucosal or tissue samples can be analyzed by 16S rRNA gene sequencing, such as Sanger, 454 pyrosequencing, MiSeq or HiSeq technology.

[0044] Phylogenetic group differentiation based on the 16S rRNA gene of the target bacteria analyzed by sequencing can be performed using the strain-specific or phylogenetic group-specific gene sequences shown in Figure 1 and Table 2 below. That is, Akkermansia phylogenetic groups can be differentiated by phylogenetic group differentiation region 1; phylogenetic group differentiation region 2; and phylogenetic group differentiation region 3.

[0045] [Table 2]

[0046] Preferably, oligonucleotide sequences having 75% or greater sequence identity to the sequences of the phylogenetic group-specific identifier regions described herein have at least 80%, at least 85%, at least 90%, at least 95%, and more preferably 96%, 97%, 98%, 99%, or 100% sequence identity with the genetic sequence of the relevant phylogenetic group identifier region (e.g., phylogenetic group identifier region 1, phylogenetic group identifier region 2, or phylogenetic group identifier region 3, respectively); nucleoside molecules with 100% sequence identity are particularly preferred. Furthermore, these oligonucleotide sequences with 75% or greater sequence identity share the same number of nucleotides.

[0047] For example, if the target bacterium is Akkermansia, the Akkermansia phylogenetic group-specific identifier region is phylogenetic group identifier region 1 having 75% or more sequence identity to the gene sequence of the AmI-specific identifier region of SEQ ID NO: 9, the gene sequence of the AmII-specific identifier region of SEQ ID NO: 10, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO: 11; a phylogenetic group identifier region 2 having 75% or more sequence identity to the gene sequence of the AmI-specific identifier region of SEQ ID NO: 12, the gene sequence of the AmII-specific identifier region of SEQ ID NO: 13, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO: 14; or The phylogenetic group identifier region 3 may have 75% or more sequence identity to the gene sequence of the AmI-specific identifier region of SEQ ID NO: 15, the gene sequence of the AmII-specific identifier region of SEQ ID NO: 16, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO: 17.

[0048] The step of identifying whether or not a competitive relationship exists can be performed by treating a culture supernatant of an Akkermansia phylogenetic group that has been confirmed to be a dominant species in the intestine of a subject with a target bacterial strain or phylogenetic group and confirming whether growth is inhibited.

[0049] For example, an Akkermansia strain belonging to the same phylogenetic group as the Akkermansia identified as the dominant species in the intestine of a subject is cultured for 24 hours, and then centrifuged to obtain a cell-free culture supernatant. The pH of the culture supernatant is adjusted to neutral, and the target bacterial strain to be administered is added at a ratio of 20% (v / v). Cultivability is confirmed after 24 hours to identify whether a competitive relationship exists.

[0050] In the present invention, the target bacterium is preferably Akkermansia. Akkermansia has phylogenetic groups AmIa, AmIb, AmII, and AmIV. Among the Akkermansia phylogenetic groups, AmIa and AmIb are inhibited by phylogenetic groups II and IV, while phylogenetic group II inhibits AmIa and AmIb but is not inhibited by AmIa and AmIb. phylogenetic groups AmIa and AmIb have a competitive and entrenched relationship. phylogenetic group IV inhibits the growth of AmIa, AmIb, and phylogenetic group II, but is not inhibited by AmIa, AmIb, and phylogenetic group II.

[0051] The method of the present invention can be used to select or treat patients for the treatment of metabolic disorders, but is not necessarily limited to metabolic disorders. The method of the present invention can also be used to maximize the therapeutic effect of various treatments using pharmacobiotics or postbiotics, such as inflammatory diseases, brain diseases, atopic diseases, and cancer. In the present invention, the metabolic disorder patient can be, but is not necessarily limited to, a patient with metabolic syndrome, insulin deficiency, insulin-resistance-related disorders, diabetes, glucose intolerance, dyslipidemia, atherosclerosis, hypertension, preeclampsia, stroke, non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, Crohn's disease, ulcerative colitis, irritable bowel syndrome, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, high cholesterol, elevated triglycerides, asthma, atopy, sleep apnea syndrome, osteoarthritis, neurodegeneration, gallbladder disease, or atherogenic dyslipidemia.

[0052] Another aspect of the present invention is a marker composition for predicting a patient's responsiveness to a biological therapeutic agent containing Akkermansia bacteria, which may include an AmIa-specific primer having 75% or more sequence identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2; an AmIb-specific primer having 75% or more sequence identity to the sequence of SEQ ID NO:3 or SEQ ID NO:4; an AmIIa-specific primer having 75% or more sequence identity to the sequence of SEQ ID NO:5 or SEQ ID NO:6; or an AmIV-specific primer having 75% or more sequence identity to the sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0053] A further aspect of the invention relates to a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, or an oligonucleotide sequence that is 75% or more identical to said sequences. Preferably, the 75% or more identical oligonucleotide sequence has at least 80%, at least 85%, more preferably 90%, at least 95%, more preferably 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and / or SEQ ID NO: 4.

[0054] Another aspect of the present invention is a marker composition for predicting the responsiveness of a patient to a biological therapeutic agent containing Akkermansia bacteria, the marker composition comprising a specific phylogenetic group identifier region capable of identifying the patient's Akkermansia phylogenetic group, the identifier region may include phylogenetic group identifier region 1 having 75% or more sequence identity to the gene sequences of phylogenetic group identifier region 9 to SEQ ID NO: 11, phylogenetic group identifier region 2 having 75% or more sequence identity to the gene sequences of SEQ ID NO: 12 to SEQ ID NO: 14, and phylogenetic group identifier region 3 having 75% or more sequence identity to the gene sequences of SEQ ID NO: 15 to SEQ ID NO: 17. The marker composition may also include a sequence selected from the group consisting of Akkermansia phylogenetic group identifier region 9 to SEQ ID NO: 17, or an oligonucleotide sequence having 75% or more identity to any of the sequences. Preferably, the oligonucleotide sequences that are 75% or more identical have at least 80%, at least 85%, more preferably 90%, at least 95%, more preferably 96%, 97%, 98%, 99%, or 100% sequence identity with phylogenetic group identifier region 1, phylogenetic group identifier region 2, or phylogenetic group identifier region 3.

[0055] Another aspect of the present invention is (a) determining the strain or phylogenetic distribution of Akkermansia bacteria to be used as a biological therapeutic agent by analyzing the patient's intestinal microbiota based on the 16S rRNA gene; (b) identifying whether a competitive relationship exists between the Akkermansia species or phylogenetic group identified as the dominant intestinal species in the previous step and the target Akkermansia species or phylogenetic group; (c) if the Akkermansia species or phylogenetic group identified as the predominant intestinal species in the previous step is identified to be in a competitive and entangled relationship with the target Akkermansia bacterium, determining that the patient is likely to have a low responsiveness to a biological therapeutic agent containing the Akkermansia strain or phylogenetic group. The patient may be a patient with a metabolic disorder.

[0056] Yet another aspect of the present invention is (a) determining the distribution of target bacteria by strain or phylogenetic group through a patient's intestinal microbiota analysis; (b) identifying whether a competitive borrowing relationship exists between the strain or phylogenetic group identified as the dominant species in the previous step and the target strain or phylogenetic group; (c) if it is determined that the strain or phylogenetic group identified as the dominant species in the previous step is not in a competitive relationship with the target bacterial strain or phylogenetic group, selecting a biological therapeutic agent containing the target bacterial strain or phylogenetic group as a treatment option and administering it to the patient.

[0057] The metabolic disorder may be, but is not necessarily limited to, selected from the group consisting of metabolic syndrome; insulin deficiency or insulin-resistance related disorders, diabetes, glucose intolerance, dyslipidemia, atherosclerosis, hypertension, preeclampsia, stroke, non-alcoholic fatty liver disease, hyperglycemic conditions, hepatic steatosis, dyslipidemia, inflammatory diseases including Crohn's disease, ulcerative colitis, irritable bowel syndrome, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, high cholesterol, elevated triglycerides, asthma, atopy, sleep apnea, osteoarthritis, neurodegeneration, gallbladder disease, and atherogenic dyslipidemia.

[0058] The present invention will be described in more detail below with reference to examples. These examples are merely for the purpose of more specifically illustrating the present invention, and the scope of the present invention is not limited by these examples.

[0059] Example Example 1 Example 1.1: Genetic differences between Akkermansia clades We collected 44 Akkermansia isolates from 19 Korean individuals and sequenced their whole genomes using the PacBio method. To investigate the genome diversity of Akkermansia, we downloaded 48 complete Akkermansia genomes from the NCBI Reference Sequence project (RefSeq) database (https: / / www.ncbi.nlm.nih.gov / refseq / ).

[0060] Referring to Table 3, the complete genomes of a total of 92 Akkermansia species showed diverse genome sizes ranging from 2.66 to 3.30 Mbp (average 2.8 Mbp). This indicates that the genome sizes of various Akkermansia species differ significantly by 0.64 Mbp. The genome sizes of almost all Akkermansia species isolated from humans are consistent with those of ATCC BAA-835. T The number of protein-coding genes in the 92 available Akkermansia genomes varied from 2122 to 2782. The 92 Akkermansia genomes also had the same number of rRNA genes, with the rRNAs containing 5S, 16S, and 23S being 3, 3, and 3, respectively, identical to each other.

[0061] [Table 3-1]

[0062] [Table 3-2]

[0063] [Table 3-3]

[0064] [Table 3-4]

[0065] 1.2. Genetic differences between Akkermansia clades 1.2.1. Pyrotyping of 92 Akkermansia genomes of human origin based on phylogenetic classification Phylogenetic classification of the 92 human-derived Akkermansia genomes was performed using two methods: 16S rRNA gene and whole-genome sequence analysis. 16S rRNA genes were isolated from the 92 human-derived Akkermansia genomes. The 16S rRNA genes isolated from the 92 human-derived Akkermansia genomes were aligned using the clustal omega (v1.2.4) program. Phylogenetic classification was performed on the aligned sequences using the neighbor-joining method in the MEGA11 program. Detailed options included the Bootstrap method (1000) and the Kimura 2-parameter model. The derived phylogenetic tree is shown in Figure 2a. For phylogenetic classification based on the 92 human-derived Akkermansia whole genomes, evolutionary distances were estimated using the pyani v0.2.7 program with the -m ANIb setting, and are shown in Figure 2b.

[0066] Figure 2 shows the phylogenetic classification results for the 92 complete Akkermansia genomes of human origin. Figure 2a shows the phylogenetic classification based on the 16S rRNA sequences, while Figure 2b shows the phylogenetic classification based on the whole genome. Based on the 16S rRNA sequences, it can be confirmed that the 92 human-derived Akkermansia species are classified into three major phylogenetic groups (AmI, AmII, and AmIV). Based on the phylogenetic classification based on the whole genome, it can also be confirmed that the 92 human-derived Akkermansia species are classified into three major phylogenetic groups (AmI, AmII, and AmIV). Of the 92 human-derived Akkermansia species, 74 were classified into AmI, 13 into AmII, and 5 into AmIV. Based on the phylogenetic classification based on the whole genome, the AmI phylogenetic group, which contains the most Akkermansia species, was classified into AmIa (22 bases, BAA-835) based on an average base identity (ANI) value of 97%. T It was confirmed that these genes were subdivided into AmIb (52 genes, including the EB-AMDK19 strain) and AmIb (52 genes, including the EB-AMDK19 strain).

[0067] Phylogenetic classification based on 16S rRNA gene sequences divides the genomes of 92 human-derived Akkermansia species into three major phylogenetic groups. Therefore, we investigated whether there are any phylogenetic-specific regions in the 16S rRNA genes that can specifically distinguish between phylogenetic groups. Specifically, we aligned the 16S rRNA genes of 92 human-derived Akkermansia species using the clustal omega (v1.2.4) program and attempted to classify them into phylogenetic groups. We identified phylogenetic-specific regions that were consistent within phylogenetic groups but different between phylogenetic groups (see Figure 1 and Table 2). Furthermore, to confirm the specificity of the phylogenetic-specific regions, we evaluated the genetic differences within and between phylogenetic groups in the phylogenetic-specific regions. The genetic differences were calculated based on the percentage similarity calculated using the blastn-short setting in the blastn program.

[0068] Genetic evaluation within and between phylogenetic groups for phylogenetic group identification region 1 confirmed a genetic difference of 0.270 ± 0.731% within the AmI phylogenetic group. Genetic identity was confirmed within the AmII or AmIV phylogenetic group. Comparison between phylogenetic groups revealed a genetic difference of 5.814 ± 0.000% between the AmI and AmII phylogenetic groups, 8.642 ± 0.000% between the AmI and AmIV phylogenetic groups, and 11.111 ± 0.000% between the AmII and AmIV phylogenetic groups. Genetic evaluation within and between phylogenetic groups for phylogenetic group identification region 2 confirmed genetic identity within the phylogenetic group. In comparisons between phylogenetic groups, the AmI and AmII phylogenetic groups showed a genetic difference of 4.545 ± 0.000%, the AmI and AmIV phylogenetic groups showed a genetic difference of 5.128 ± 0.000%, and the AmII and AmIV phylogenetic groups showed a genetic difference of 2.564 ± 0.000%. In the case of phylogenetic group identification region 3, which is composed of 12 short base sequences, genetic differences could not be confirmed using the blastn program. However, it was confirmed that the sequence was consistent within a phylogenetic group and specifically different between phylogenetic groups.

[0069] 1.2.2. Genetic distance based on comparisons within the same clade or between other clades We attempted to confirm genetic differences within and between Akkermansia clades on a whole-genome or core-genome basis. To calculate the genetic distances within and between clades on a whole-genome basis, we applied the pyani v0.2.7 program with the -m ANIb setting to calculate average nucleotide identity (ANI). The derived percentage similarity values ​​were inversely used to calculate the whole-genome genetic distances within and between clades, as shown in Table 4. To calculate the genetic distances within and between clades on a core-genome basis, we used Roary (v3.11.2), a fast, standalone pan-genome pipeline. Specifically, to evaluate the genetic distances based on the core gene alignment, we performed a Roary analysis on annotated assemblies in GFF3 format generated by Prokka (v1.13.4). The core-genome aligned sequences derived by the Roary analysis were classified by clade, and the percentage similarity within and between clades was calculated using the blastn program. The derived percentage similarity values ​​were then inverted to calculate the core genome genetic distances within and between phylogenetic groups, as shown in Table 4.

[0070] As shown in Table 4 below, it can be seen that the genetic distance within the same phylogenetic group is very low at less than 2%, while the genetic distance with other phylogenetic groups is 12-18%. From the above, it can be seen that Akkermansia shows specific genetic distances depending on the phylogenetic group into which it is classified. In other words, Akkermansia is clearly divided into three major phylogenetic groups.

[0071] [Table 4]

[0072] 1.3. Construction of Akkermansia phylogenetic group-specific primers A search was conducted for marker genes that exist in a single copy across all Akkermansia genomes and show clear differences between phylogenetic groups. A search for orthologous genes based on the 92 human-derived Akkermansia genomes obtained in the previous example was performed using the get_homologues software (https: / / github.com / eead-csic-compbio / get_homologues). Specifically, orthologous genes were searched for using the OrthoMCL algorithm as a clustering criterion for the GenBank file format for the 92 Akkermansia genomes. Among the genes searched, genes that exist in a single copy across all Akkermansia phylogenetic groups (AmIa, AmIb, AmII, and AmIV) were identified. The identified gene was sodium ion-translocating decarboxylase subunit beta. The corresponding gene sequence was obtained from the 92 Akkermansia genomes and then aligned using the clustal omega (v1.2.4) program. Phylogenetic classification was performed on the aligned sequences using the Neighbor-joining method in the MEGA11 program. Detailed options included the Bootstrap method (1000) and the Kimura 2-parameter model, and the resulting phylogenetic diagram is shown in Figure 3(A). Furthermore, to confirm whether the identified genes exhibit specific genetic differences between phylogenetic groups, the degree of similarity (%) within and between phylogenetic groups was calculated using the blastn program, and the results are shown in Figure 3(B).

[0073] As shown in Figure 3(A), phylogenetic classification based on the sodium ion-translocating decarboxylase subunit beta gene obtained through orthologous gene analysis confirmed that the entire genomes of 92 Akkermansia species were classified into phylogenetic groups (AmIa, AmIb, AmII, and AmIV). These results indicate that the sodium ion-translocating decarboxylase subunit beta gene can be used as a marker gene. As shown in Figure 3(B), the marker sodium ion-translocating decarboxylase subunit beta gene showed high similarity in intra-Akkermansia phylogenetic group comparisons but low similarity in inter-Akkermansia phylogenetic group comparisons. These results confirmed that the marker sodium ion-translocating decarboxylase subunit beta gene is suitable for designing phylogenetic group-specific primers.

[0074] To design phylogenetic group-specific primers based on the marker gene, the sodium ion-translocating decarboxylase subunit beta gene, marker gene sequences were obtained from the whole genomes of 92 Akkermansia species and aligned using the clustal omega (v1.2.4) program. Conserved and highly diverse gene regions were identified for each Akkermansia phylogenetic group. Phylogenetic group-specific primers were then created for the highly diverse gene regions using the blastn program, as shown in Table 1.

[0075] Example 2: Confirmation of the distribution pattern of Akkermansia phylogenetic groups in the human intestine Example 2.1: Typing of human intestinal Akkermansia phylogenetic groups based on gut microbiota analysis We attempted to type human intestinal Akkermansia phylogenetic groups using publicly available metagenome data by identifying the Akkermansia phylogenetic group-specific identifier regions in the 16S rRNA gene (see Table 2 and Figure 1). The metagenomic data for 890 Korean individuals used in this study was obtained from the MCBI database (BioProject: PRJEB33905). The sequence data for the 16S rRNA gene was converted into an ASV frequency table. The ASV table was generated using the DADA2 pipeline of the QIIME2 program (version 2019.01). ASVs corresponding to Akkermansia were extracted from the obtained ASV table and aligned with the 16S rRNA gene sequences obtained from the whole genomes of 92 human-derived Akkermansia strains. Based on this, phylogenetic groups were typed for each ASV using the Akkermansia phylogenetic group identifier regions.

[0076] The present inventors have confirmed that Akkermansia phylogenetic groups or phylogenetic groups can be distinguished based on the 16S rRNA V3-V4 region (see Figure 4a). Of the 22 ASVs corresponding to Akkermansia, 13 were identified as AmI, 8 as AmII, and 1 as AmIV (see Figure 4). Based on this, the proportion of Akkermansia present in the intestines of 890 Korean individuals was analyzed based on their presence or absence, and the proportion of Akkermansia present by phylogenetic group, if present. The results are shown in Figure 4b. Figure 4b demonstrates that Akkermansia does not exist in a complex form consisting of multiple phylogenetic groups, but rather in a form dominated by a single phylogenetic group. Furthermore, the coexistence of AmI and AmII phylogenetic groups was extremely rare, occurring in less than 1% of cases. AmIV was also confirmed to be extremely rare.

[0077] 2.2. Confirmation of the distribution of Akkermansia in the intestines of people from various countries Metagenomic data from various countries other than Korea were obtained from the NCBI and MG-RAST databases. Specifically, Chilean metagenomic data was obtained from PRJEB16755, Nigerian metagenomic data from mgp83994, China (Beijing) metagenomic data from PRJNA480547, China (Shanghai) metagenomic data from PRJNA382861, Japanese metagenomic data from PRJDB4360, and Spanish metagenomic data from PRJNA350839. Analysis of the metagenomic data and identification of Akkermansia phylogenetic groups using the analysis were performed as described in Example 2.1, and the results are shown in Figure 5.

[0078] Referring to Figure 5, coexistence of AmI and AmII was observed very rarely. In other words, the unique distribution pattern of Akkermansia, which is a monophyletic dominance pattern based on the characteristic point that the coexistence of AmI and AmII is extremely low, was confirmed to be a characteristic of Akkermansia not only derived from Koreans but also derived from various countries. These results confirmed that the phenomenon of borrowing between Akkermansia lineages AmI and AmII is observed not only in Korea but also in various countries. In other words, it was confirmed that the approach of the present invention can be used not only in Korea but also in various countries.

[0079] 3. Confirmation of specific borrowing patterns among Akkermansia lineages 3.1. Identifying competitive borrowing relationships among Akkermansia lineages We analyzed the effects of cell-free supernatants derived from strains representing each phylogenetic group on each other's growth. To do so, we inoculated EB-AMDK19 (AmI phylogenetic group), EB-AMDK39 (AmII phylogenetic group), and EB-ABDH76 (AmIV phylogenetic group) strains at 0.1% concentration in a culture medium (30 g / L tryptic soy broth (TSB), 2.5 g / L porcine gastrointestinal mucin, 0.1 mg / L cyanocobalamin, and 0.5 g / L L-cysteine ​​hydrochloride) and centrifuged (10,000 rpm, 10 min, 4°C) for 24 hours to separate the supernatant and bacterial pellet. The cell-free supernatant was prepared by filtering the separated supernatant through a 0.2 μm syringe filter. To confirm the effect of the cell-free supernatants collected from each Akkermansia phylogenetic group on different types of Akkermansia phylogenetic groups, the strains from each phylogenetic group were inoculated into the culture medium at 0.1% and treated at 20% (v / v) of the culture medium. The absorbance values ​​after 24 hours were compared with those of the control group to confirm the effect on growth (see Table 5).

[0080] [Table 5]

[0081] Referring to Table 5, the cell-free supernatant derived from EB-AMDK19, a representative strain of the Akkermansia AmI phylogenetic group, did not affect the growth of EB-AMDK39, a representative strain of the Akkermansia AmII phylogenetic group, or EB-ABDH76, a representative strain of Akkermansia AmIV. The cell-free supernatant derived from EB-AMDK39, a representative strain of the Akkermansia AmII phylogenetic group, specifically inhibited the growth of EB-AMDK19 but did not affect the growth of EB-ABDH76. The cell-free supernatant derived from EB-ABDH76, a representative strain of Akkermansia AmIV, specifically inhibited the growth of EB-AMDK19 and EB-AMDK39.

[0082] 3.2. Identification of specific inhibitory patterns among phylogenetic groups To verify whether representative strains from each phylogenetic group could represent the specific inhibitory profiles of each group, we further selected 14 strains from the AmI phylogenetic group and three strains from the AmII phylogenetic group from the whole-genome phylogenetic classification (Fig. 2) and analyzed their inhibitory profiles. Specifically, four strains from the AmII phylogenetic group (EB-AMDK39, EB-AMDK40, EB-AMDK41, and EB-AMDK43) were cultured at 0.1% concentration in a culture medium containing 30 g / L tryptic soy broth (TSB), 2.5 g / L porcine gastrointestinal mucin, 0.1 mg / L cyanocobalamin, and 0.5 g / L L-cysteine ​​hydrochloride. After 24 hours, the supernatant and bacterial pellet were separated by centrifugation at 10,000 rpm for 10 minutes at 4°C. The separated supernatant was filtered through a 0.2 μm syringe filter to prepare a cell-free supernatant. To confirm the effect of cell-free supernatants from the four AmII phylogenetic group strains on the growth of 15 AmI phylogenetic group strains and four AmII phylogenetic group strains, the culture medium was inoculated with 15 AmI phylogenetic group strains and four AmII phylogenetic group strains at 20% (v / v) of the culture medium. The absorbance values ​​after 24 hours were compared with those of the control group to confirm the effect on growth (Table 6).

[0083] [Table 6]

[0084] As shown in Table 6, all cell-free supernatants derived from four AmII phylogenetic group strains specifically inhibited the growth of 15 AmII phylogenetic group strains (2-10% of medium control). Furthermore, no specific inhibitory patterns were observed among AmII phylogenetic group strains (>93% of medium control). This confirmed that representative strains from each phylogenetic group (EB-AMDK19, EB-AMDK39, EB-ABDH76) exhibited specific inhibitory patterns among Akkermansia phylogenetic groups.

[0085] 3.3. Confirmation of colonization in the mouse intestine by single administration of each Akkermansia strain All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). Specifically, germ-free mice (C57BL / 6) were housed and maintained in sterile flexible film isolators (Class Biological Clean Ltd.) at 23°C, 40-60% relative humidity, and a 12-hour light-dark cycle. Six-week-old female germ-free mice (C57BL / 6) housed and maintained in this environment were randomly divided into three groups of four mice per group, as shown in Table 7 below. Fresh fecal samples from the germ-free mice used in the experiments were cultured under aerobic and anaerobic conditions, and microbial contamination was routinely monitored.

[0086] To confirm whether each Akkermansia phylogenetic group can colonize germ-free mice, Akkermansia muciniphila BAA-835 was used as a strain representing Akkermansia phylogenetic group AmIa. T Akkermansia muciniphila EB-AMDK19 was selected as the strain representing AmIb, and Akkermansia muciniphila EB-AMDK39 was selected as the strain representing AmII, and these were used in the experiment. The representative strains of each phylogenetic group mentioned above were each cultured at 1 x 10 viable cells per 150 μL of PBS containing 25% glycerol and 0.05% cysteine. 8 Cryopreservation vials were prepared at CFU concentrations.

[0087] Using the cryopreservation vials prepared by the above method, the representative strains of each Akkermansia phylogenetic group (Akkermansia muciniphila BAA-835 T , Akkermansia muciniphila EB-AMDK19, Akkermansia muciniphila EB-AMDK39) 1 × 10 live bacteria 8 Each experimental group was orally administered 150 μL of CFU once daily for two days (see Table 7). After oral administration, fresh feces were periodically collected from each experimental group and stored frozen at -80°C for use in determining colonization by each Akkermansia phylogenetic group.

[0088] [Table 7]

[0089] To confirm the analytical sensitivity and specificity of the Akkermansia phylogenetic group-specific primers shown in Table 1, DNA fragments containing the DNA sequence of each target marker were constructed.

[0090] [Table 8]

[0091] The DNA fragments were serially diluted (10 3 ~10 9 ) and quantitative PCR was performed using this as template to test analytical sensitivity. Quantitative PCR experiments were performed using a quantitative PCR kit (TOPreal SYBR Green High-ROX PreMIX, Enzynomics) and an ABI Quantstudio 3 Real-Time PCR Instrument, 96-well, 0.2 mL (A28132). Results confirmed the ability of the Akkermansia phylogenetic group-specific primers (AmIa, AmIb, and AmII) to quantify each Akkermansia phylogenetic group in a concentration-dependent manner (see Figure 6).

[0092] Following oral administration of representative strains of each Akkermansia phylogenetic group, DNA was extracted from fresh feces collected periodically from each experimental group using a fecal DNA extraction kit (QIAamp PowerFecal Pro DNA Kit, QIAGEN). Quantitative PCR was performed using the Akkermansia phylogenetic group-specific primers listed in Table 1 and a quantitative PCR kit (TOPreal SYBR Green High-ROX PreMIX, Enzynomics) to assess changes in the levels and colonization patterns of each Akkermansia phylogenetic group in the feces after administration. Specifically, 9 μL of DNA template, 10 μL of PreMIX, and 10 pmol of phylogenetic group-specific primers were used per well, for a total reaction volume of 20 μL. PCR conditions included an initial incubation at 50°C for 4 minutes, followed by 95°C for 10 minutes, followed by 95°C for 30 seconds, followed by 56°C for 30 seconds, repeated 40 times. Then, by substituting the CT values ​​derived by quantitative PCR for each fecal collection time, time, and experimental group into the trend lines for each Akkermansia phylogenetic group-specific primer in Figure 6, changes in the levels of each Akkermansia phylogenetic group in the feces per gram of feces and the retention pattern of colonization were confirmed, as shown in Figure 7.

[0093] As shown in Figure 7 (A:BAA-835 T When Akkermansia phylogenetic group strains were administered alone (A: administered EB-AMDK19, B: administered EB-AMDK39), it was confirmed that all phylogenetic group strains were observed at high levels after administration. Furthermore, it was confirmed that the elevated genome equivalents (log10) / g feces specific to each phylogenetic group-specific primer was maintained, thereby confirming that colonization levels were sustained.

[0094] 3.4. Analysis of colonization patterns in the mouse intestine following simultaneous administration of Akkermansia strains Six-week-old germ-free female mice (C57BL / 6) were randomly divided into two groups, with four mice in each group, as shown in Table 9 below. Representative strains of each Akkermansia phylogenetic group to be used in the experiment were added at a concentration of 1 x 10 viable bacteria per 150 μL of PBS containing 25% glycerol and 0.05% cysteine. 8Cryopreservation vials were prepared at CFU concentrations.

[0095] Using the cryopreservation vials prepared by the above method, a representative strain (BAA-835) of the Akkermansia phylogenetic group was T , EB-AMDK19, EB-AMDK39) live cells 1 × 10 8 150 μL of CFU was orally administered to the experimental group once a day for a total of two days (see Table 9). After oral administration, fresh feces were periodically collected from each experimental group and frozen at -80°C to confirm changes in the intestinal levels of each Akkermansia phylogenetic group.

[0096] Changes in the intestinal levels of each Akkermansia strain due to simultaneous administration of Akkermansia strains were confirmed by performing quantitative PCR on gDNA extracted from fresh feces collected for each experimental group at each time point after simultaneous administration of Akkermansia strains, and the results are shown in Figure 8.

[0097] [Table 9]

[0098] Referring to FIG. 8A, the diverse Akkermansia phylogenetic tree (BAA-835 T When EB-AMDK19:AmIa, EB-AMDK19:AmIb, and EB-AMDK39:AmII were co-administered, the AmIa and AmIb subpopulations, which belong to the AmI phylogenetic group, continuously decreased after administration and reached the detection limit. In contrast, the AmII subpopulation maintained a high level of genome equivalents (log10) / g feces specific to the AmII phylogenetic group primers from administration until the end of the experiment, demonstrating a competitive advantage over the AmI subpopulation in the intestine.

[0099] As shown in Figure 8B, when two Akkermansia phyla, AmIa and AmIb, were administered simultaneously, AmIa, a member of the AmI phyla group, decreased continuously after administration and reached the detection limit, as shown in Figure 8A. In contrast, high levels of AmII phyla were detected in the feces from administration until the end of the experiment. This indicates that AmI and AmII phyla compete for colonization in the host intestinal tract.

[0100] The specificity of the Akkermansia phylogenetic group-specific primers was confirmed by quantitative PCR analysis. After quantitative PCR, phylogenetic group-specific primers were applied to gDNA extracted from time-specific fecal samples collected by co-administration of various Akkermansia phylogenetic groups. The PCR products were then subjected to electrophoresis. Electrophoresis was performed by spotting the PCR products on a 2.0% agarose gel containing NtRON's RedSafe Nucleic Acid Staining Solution (20,000x). The results were interpreted by comparing the bands that appeared after electrophoresis with the amplicon sizes (bp) of the Akkermansia phylogenetic group-specific primers (Figure 9A).

[0101] Furthermore, the specificity of the Akkermansia phylogenetic group-specific primers was confirmed from the melting curve plots derived from the quantitative PCR analysis. After quantitative PCR was completed, phylogenetic group-specific primers were applied to gDNA extracted from time-separated fecal samples obtained by co-administration of various Akkermansia phylogenetic groups. Changes in the melting curve plots were observed. Results were interpreted by comparing the melting curve plots from the first day of administration with those from the end of the experiment (Figure 9B).

[0102] Figure 9A shows the results of electrophoresis of quantitative PCR products using Akkermansia species-specific and phylogenetic group-specific primers. As can be seen from the results in Figure 9, the AmII phylogenetic group remained at a high level from the initial administration to the end of the experiment, confirming that the appearance of a single amplified band using phylogenetic group-specific primers was maintained on electrophoresis. In contrast, the AmIa and AmIb phylogenetic groups belonging to the AmI phylogenetic group rapidly decreased immediately after administration, confirming that the appearance of a single amplified band disappeared on electrophoresis. Furthermore, the same observations were confirmed in the melting curve plots of quantitative PCR using Akkermansia species-specific and phylogenetic group-specific primers in Figure 9B. The AmII phylogenetic group remained dominant from immediately after administration to the end of the experiment, confirming that the melting curve plots at the initial and final stages of the experiment matched and coincided. In contrast, the AmIa and AmIb phylogenetic groups rapidly decreased, confirming that the melting curve plots at the end of the experiment did not match those at the initial stage, indicating a false positive. The above allows confirmation of the specificity of the Akkermansia phylogenetic group-specific primers.

[0103] 3.5. Analysis of colonization patterns in the mouse intestine after cross-administration of Akkermansia lineages In order to confirm the changes in the intestinal Akkermansia strains when other types of Akkermansia strains are orally administered after the intestinal Akkermansia strains have been identified, 6-week-old female germ-free mice (C57BL / 6) were randomly divided into two groups, with four mice per group, as shown in Table 10 below. Representative strains of each Akkermansia strain to be used in the experiment were dissolved in 150 μL of PBS containing 25% glycerol and 0.05% cysteine ​​at 1 x 10 viable bacteria. 8 Cryopreservation vials were prepared at CFU concentrations.

[0104] Using the cryopreservation vials prepared by the above method, 1 × 10 live cells of Akkermansia phylogenetic group strains (EB-AMDK19, EB-AMDK39) were stored. 8150 μL of CFU was orally administered to each experimental group once a day for a total of two days. 17 days after administration, 1 × 10 viable cells of strains from related phylogenetic groups (EB-AMDK19, EB-AMDK39) were administered orally. 8 Each experimental group was orally administered 150 μL of CFU once daily for two days. After oral administration, fresh feces were periodically collected from each experimental group and frozen at -80°C to confirm changes in the intestinal levels of each Akkermansia strain. Changes in the intestinal levels of each Akkermansia strain due to cross-administration of Akkermansia strains were confirmed by quantitative PCR using gDNA extracted from fresh feces collected from each experimental group at each time point after cross-administration of Akkermansia strains. The results are shown in Figure 10.

[0105] [Table 10]

[0106] 10A, EB-AMDK39, which represents the AmII phylogenetic group, was administered first, and colonization of the intestine was confirmed by the genome equivalents (log10) / g feces value specific to the AmII phylogenetic group primers. After identifying the Akkermansia phylogenetic group in the intestine as the AmII phylogenetic group (17 days after AmII phylogenetic group administration), EB-AMDK19, which belongs to the AmIb phylogenetic group, was administered to observe changes in the Akkermansia phylogenetic group in the intestine due to cross-administration. The genome equivalents (log10) / g feces value specific to the AmII phylogenetic group primers was maintained until the end of the experiment, and the genome equivalents (log10) / g feces value specific to the AmIb phylogenetic group primers did not increase, confirming that the predominant Akkermansia species in the intestine remained in the AmII phylogenetic group.

[0107] 10B, EB-AMDK19, which represents the AmIb phylogenetic group, was administered first, and colonization of the intestine was confirmed by the genome equivalents (log10) / g feces value specific to the AmIb phylogenetic group primers. After identifying the Akkermansia phylogenetic group in the intestine as the AmIb phylogenetic group (17 days after AmIb phylogenetic group administration), EB-AMDK39, which belongs to the AmII phylogenetic group, was administered to observe changes in the Akkermansia phylogenetic group in the intestine due to cross-administration. The genome equivalents (log10) / g feces value specific to the AmIb phylogenetic group primers was maintained until the end of the experiment, and the genome equivalents (log10) / g feces value specific to the AmII phylogenetic group primers did not increase, confirming that the predominant Akkermansia species in the intestine remained in the AmIb phylogenetic group. Taken together, these results confirm that when a specific Akkermansia phylogenetic group has colonized the intestine, it is not easy for exogenous Akkermansia bacteria of other phylogenetic groups to colonize the intestine.

[0108] It should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention is defined by the claims below rather than the detailed description, and all modifications and variations derived from the claims should be construed as being included within the scope of the present invention.

Claims

1. (a) determining the distribution of Akkermansia bacteria by strain or phylogroup through gut microbiota analysis of the patient; (b) identifying whether a competitive exclusion relationship exists between the strain or phylogenetic group of Akkermansia bacteria identified in the previous step as the predominant species in the patient's intestine and the target strain or phylogenetic group of Akkermansia bacteria; (c) if it is identified that the strain or phylogenetic group of Akkermansia bacteria confirmed as the dominant species in the intestine in the previous step and the target strain or phylogenetic group of Akkermansia bacteria are in a competitive exclusion relationship, determining that the patient will likely have a low responsiveness to a biological therapeutic agent containing the target strain or phylogenetic group of Akkermansia bacteria.

2. 2. The method of claim 1, wherein the intestinal microbiota analysis comprises performing quantitative PCR (qPCR) on DNA extracted from a fecal sample of the patient using a primer pair or probe specific to a sodium ion-translocating decarboxylase subunit beta gene of a strain or phylogenetic group of bacteria of the genus Akkermansia.

3. The primer is an AmIa-specific primer having 95% or more sequence homology to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; an AmIb-specific primer having 95% or more sequence homology to the sequence of SEQ ID NO: 3 or SEQ ID NO: 4; an AmII-specific primer having 95% or more sequence identity to the sequence of SEQ ID NO:5 or SEQ ID NO:6; or The method for predicting the responsiveness of a patient to a biological therapeutic agent according to claim 2, wherein the AmIV-specific primer has a sequence homology of 95% or more to the sequence of SEQ ID NO:7 or SEQ ID NO:

8.

4. The method of claim 1, wherein the intestinal microbiota analysis comprises a step of identifying a phylogenetic group and confirming distribution of DNA extracted from a fecal sample of the patient using a phylogenetic group-specific gene identification region specific to the 16S rRNA gene of bacteria belonging to a strain or phylogenetic group of Akkermansia bacteria.

5. The phylogenetic group-specific identifier region specific to the 16S rRNA gene of bacteria of the genus Akkermansia is: a phylogenetic group identifier region 1 having 95% or more sequence identity to the gene sequence of the AmI-specific identifier region of SEQ ID NO:9, the gene sequence of the AmII-specific identifier region of SEQ ID NO:10, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO:11; a phylogenetic group identifier region 2 having 95% or more sequence identity to the gene sequence of the AmI-specific identifier region of SEQ ID NO: 12, the gene sequence of the AmII-specific identifier region of SEQ ID NO: 13, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO: 14; or The method for predicting a patient's responsiveness to a biological therapeutic agent described in claim 4, characterized in that the phylogenetic group identifier region 3 has a sequence identity of 95% or more to the gene sequence of the AmI-specific identifier region of SEQ ID NO: 15, the gene sequence of the AmII-specific identifier region of SEQ ID NO: 16, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO:

17.

6. The method for predicting a patient's responsiveness to a biological therapeutic agent described in claim 1, characterized in that the step of identifying whether or not a competitive exclusion relationship exists includes a step of treating a culture supernatant of a strain or phylogenetic group of Akkermansia bacteria confirmed to be a dominant species in the patient's intestine with the target strain or phylogenetic group of Akkermansia bacteria to confirm whether growth is inhibited.

7. A method for predicting a patient's responsiveness to a biological therapeutic agent as described in claim 1, characterized in that the Akkermansia bacteria have phylogenetic groups AmIa, AmIb, AmII, and AmIV, and there is a competitive exclusion relationship between the phylogenetic groups, whereby phylogenetic groups AmIa and AmIb are inhibited by AmII and AmIV but do not inhibit phylogenetic groups AmII and AmIV, and phylogenetic group AmII is inhibited by phylogenetic group AmIV but does not inhibit phylogenetic group AmIV.

8. The method for predicting a patient's responsiveness to a biological therapeutic agent according to claim 1, wherein the patient is a patient suffering from a metabolic disorder, an inflammatory disease, or an atopic disease.

9. 9. The method of claim 8, wherein the metabolic disorder is selected from the group consisting of metabolic syndrome, insulin deficiency, insulin-resistance-related disorders, diabetes, glucose intolerance, dyslipidemia, atherosclerosis, hypertension, preeclampsia, stroke, non-alcoholic fatty liver disease, hyperglycemic conditions, hepatic steatosis, dyslipidemia, Crohn's disease, ulcerative colitis, inflammatory diseases including irritable bowel syndrome, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, high cholesterol, elevated triglycerides, asthma, atopy, sleep apnea syndrome, osteoarthritis, neurodegeneration, gallbladder disease, and atherogenic dyslipidemia.

10. A marker composition for predicting the responsiveness of a metabolic disorder patient to a biotherapeutic containing an Akkermansia bacterium, the marker composition comprising: an AmIa-specific primer having 95% or more sequence homology to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; an AmIb-specific primer having 95% or more sequence homology to the sequence of SEQ ID NO: 3 or SEQ ID NO: 4; an AmII-specific primer having 95% or more sequence identity to the sequence of SEQ ID NO:5 or SEQ ID NO:6; and / or A marker composition for predicting reactivity, comprising an AmIV-specific primer having a sequence homology of 95% or more to the sequence of SEQ ID NO:7 or SEQ ID NO:

8.

11. The marker composition comprising: a phylogenetic group identifier region 1 having 95% or more sequence identity to the gene sequence of the AmI-specific identifier region of SEQ ID NO:9, the gene sequence of the AmII-specific identifier region of SEQ ID NO:10, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO:11; a phylogenetic group identifier region 2 having 95% or more sequence identity to the gene sequence of the AmI-specific identifier region of SEQ ID NO: 12, the gene sequence of the AmII-specific identifier region of SEQ ID NO: 13, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO: 14; and / or The marker composition for predicting reactivity described in claim 10, further comprising a phylogenetic group identifier region 3 having 95% or more sequence homology to the gene sequence of the AmI-specific identifier region of SEQ ID NO: 15, the gene sequence of the AmII-specific identifier region of SEQ ID NO: 16, or the gene sequence of the AmIV-specific identifier region of SEQ ID NO: 17.

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