Composition containing bacterial strains for improving metabolic health

Abstract

The present invention provides a composition comprising further microorganisms identified based on their spontaneous occurrence in the digestive tract of metabolically healthy individuals, selected based on their ability to maintain and optimize metabolic health and prevent disease. [Solution] A composition is provided comprising a biologically pure strain of the genus Ackermansia, wherein the whole-genome mean nucleotide identity between the Ackermansia species and (i) Ackermansia muciniphila and / or (ii) Ackermansia glycaniphila is less than approximately 95%. Also provided is a composition comprising one or more biologically pure strains of bacteria, and a method for producing and using this composition to treat and / or prevent one or more obesity-related disorders in subjects requiring it.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 004,617, filed on 3 April 2020, which disclosure is incorporated herein by reference in its entirety.

[0002] Provided herein are, in particular, bacterial compositions useful for improving metabolic health in subjects, as well as methods for producing and using such bacterial compositions. [Background technology]

[0003] The human digestive tract contains a complex and diverse ecosystem of microorganisms. Intestinal bacteria are not only symbiotic organisms but also undergo symbiotic co-evolution with their host. The interaction between the gut microbiota and the host is complex. Beneficial gut bacteria have numerous important functions, directly or indirectly influencing various physiological functions of the host, such as providing nutrients, preventing infections caused by intestinal pathogens, and regulating normal immune responses. It is established that imbalances in the composition of the microbiota can lead to various disease states in the host. Therefore, to maintain and improve the health of the host, it is necessary to modify the gut microbiota to achieve, restore, and maintain a favorable balance of the ecosystem and the activity of microorganisms present in the digestive tract.

[0004] First-generation probiotics are viable microorganisms primarily derived from the Lactobacillus and Bifidobacterium genera, often originating from their use as trace components of the digestive tract or as starter cultures for dairy products. Traditionally, first-generation probiotics have primarily targeted gut and immune health. Some, such as B. lactis B420, have been shown to exhibit beneficial activity regarding metabolic health (e.g., reduction of body fat and some improvement in blood glucose and insulin levels). However, current first-generation probiotics do not appear to offer an optimal solution for glucose and insulin metabolism, and therefore do not appear to offer an optimal solution as a potential treatment or preventative measure for type 2 diabetes, prediabetes, or metabolic syndrome. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, what is needed are further microorganisms, identified based on their spontaneous generation in the digestive tract of metabolically healthy individuals, and selected based on their ability to maintain and optimize metabolic health and prevent disease. [Means for solving the problem]

[0006] The subject matter disclosed herein addresses these needs and also provides further advantages.

[0007] Provided herein are, in particular, compositions comprising one or more biologically pure strains of bacteria, and methods for producing and using such compositions to treat and / or prevent one or more obesity-related disorders (e.g., but not limited to obesity, metabolic syndrome, diabetes mellitus, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, dyslipidemia, non-alcoholic fatty liver disease, fatty liver, leptin resistance, decreased resistin levels, and / or cardiovascular diseases) in subjects requiring such treatment.

[0008] Accordingly, in some embodiments, provided herein are (a) a biologically pure strain of Eubacterium eligens; (b) a biologically pure strain of Intestinimonas massiliensis; (c) a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of Prevotella copri deposited in the German Collection of Microorganisms and Cell Cultures (DSM) under no. DSM 33457; and / or (d) a biologically pure strain of an Akkermansia sp., wherein the Akkermansia sp. is (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphila The composition comprises at least one or more biologically pure strains that are not glycaniphilia. In some embodiments, the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila or (ii) Akkermansia glycaniphilia is less than approximately 95%. In some embodiments of any of the embodiments disclosed herein, the composition includes (a) a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of E. eligens deposited with the DSM under no. DSM 33458; and / or (b) a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of I. massiliensis deposited with the DSM under no. DSM 33460.In some embodiments of the embodiments disclosed herein, the composition is (a) a viable strain of E. eligens deposited with DSM under the code DSM 33458 or having all the identifying characteristics of E. eligens deposited with DSM under the code DSM 33458; (b) a viable strain of I. massiliensis deposited with DSM under the code DSM 33460 or having all the identifying characteristics of I. massiliensis deposited with DSM under the code DSM 33460; (c) a viable strain of P. copri deposited with DSM under the code DSM 33457 or having all the identifying characteristics of P. copri deposited with DSM under the code DSM 33457; and / or (d) a viable strain of DSM under the code DSM The composition comprises (A) a single viable strain of an Akkermansia species deposited with DSM 33459, or a viable strain possessing all the identifying characteristics of an Akkermansia species deposited with DSM 33459, either (A) alone or / or (b) in combination with a culture supernatant derived from one or more of these strains. In some embodiments, the composition comprises (b) a biologically pure strain of Intestinimonas massiliensis; and (d) a biologically pure strain of an Akkermansia species, wherein the Akkermansia species is not (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia. In some embodiments, the composition includes (b) a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of I. massiliensis deposited in the DSM under the nomination DSM 33460.In some embodiments of the embodiments disclosed herein, the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila or (ii) Akkermansia glycaniphilia is less than approximately 95%. In some embodiments of the embodiments disclosed herein, the composition comprises (b) a viable strain of I. massiliensis deposited with the DSM under the no. DSM 33460 or having all the identifying characteristics of the I. massiliensis strain deposited with the DSM under the no. DSM 33460; and (d) a viable strain of an Akkermansia sp. deposited with the DSM under the no. DSM 33459 or having all the identifying characteristics of an Akkermansia sp. deposited with the DSM under the no. DSM 33459. In some embodiments of the embodiments disclosed herein, the composition is formulated for oral administration. In some embodiments of the embodiments disclosed herein, the composition is lyophilized or freeze-dried. In some embodiments of the embodiments disclosed herein, the composition is encapsulated or coated. In some of the embodiments disclosed herein, the composition is a food, a food ingredient, a dietary supplement, or a pharmaceutical product. In some of the embodiments disclosed herein, the composition is at least about 1 × 10. 4 CFU / g composition ~ at least about 1 × 10 12The bacteria in the CFU / g composition are present in the composition. In some embodiments of any of the embodiments disclosed herein, the composition is a probiotic. In some embodiments of any of the embodiments disclosed herein, the composition is pasteurized or heat-treated. In some embodiments of any of the embodiments disclosed herein, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient.

[0009] In a further embodiment, provided herein are (a) a biologically pure strain of Eubacterium eligens; (b) a biologically pure strain of Intestinimonas massiliensis; (c) a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of Prevotella copri deposited in the German Collection of Microorganisms and Cell Cultures (DSM) under no. DSM 33457; and / or (d) a biologically pure strain of an Akkermansia sp., wherein the Akkermansia sp. is (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphila The composition comprises isolated bacterial extracellular vesicles (EVs) derived from at least one or more biologically pure strains that are not glycaniphilia. In some embodiments, the composition further comprises one or more bacteria derived from (a), (b), (c), and / or (d). In some embodiments of any of the embodiments disclosed herein, the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia is less than about 95%.In some embodiments of the embodiments disclosed herein, the composition comprises (a) an EV derived from a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of E. eligens deposited with the DSM under no. DSM 33458; and / or (b) an EV derived from a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of I. massiliensis deposited with the DSM under no. DSM 33460. In some embodiments of the embodiments disclosed herein, the composition is derived from (a) an E. eligens strain deposited with DSM under number DSM 33458 or a surviving strain having all the identifying characteristics of the E. eligens strain deposited with DSM under number DSM 33458; (b) an E. massiliensis strain deposited with DSM under number DSM 33460 or a surviving strain having all the identifying characteristics of the I. massiliensis strain deposited with DSM under number DSM 33460; (c) an E. copri strain deposited with DSM under number DSM 33457 or a surviving strain having all the identifying characteristics of the P. copri strain deposited with DSM under number DSM 33457; and / or (d) an E. EVs derived from an Akkermansia sp. deposited with DSM under number 33459, or from a living strain possessing all the identifying characteristics of an Akkermansia sp. deposited with DSM under number DSM 33459, either (A) alone or (B) in combination with a culture supernatant derived from one or more of these strains.In some embodiments of the embodiments disclosed herein, the composition includes (b) EVs derived from a biologically pure strain of Intestinimonas massiliensis; and (d) EVs derived from a biologically pure strain of an Akkermansia sp., wherein the Akkermansia sp. is not (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia. In some embodiments, the composition includes (b) EVs derived from a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of I. massiliensis deposited with the DSM under number DSM 33460. In some embodiments of the embodiments disclosed herein, the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila or (ii) Akkermansia glycaniphilia is less than approximately 95%. In some embodiments of the embodiments disclosed herein, the composition comprises (b) EVs derived from a living strain of I. massiliensis deposited with the DSM under the no. DSM 33460 or having all the identifying characteristics of the I. massiliensis strain deposited with the DSM under the no. DSM 33460; and (d) EVs derived from a living strain of an Akkermansia species deposited with the DSM under the no. DSM 33459 or having all the identifying characteristics of the Akkermansia species deposited with the DSM under the no. DSM 33459. In some embodiments of the embodiments disclosed herein, the composition is formulated for oral administration.In some embodiments of any of the embodiments disclosed herein, this composition is lyophilized or freeze dried. In some embodiments of any of the embodiments disclosed herein, this composition is encapsulated or coated. In some embodiments of any of the embodiments disclosed herein, this composition is a food, a food ingredient, a dietary supplement or a pharmaceutical. In some embodiments of any of the embodiments disclosed herein, at least about 1×10. 4 CFU / g composition ~ at least about 1×10 12 CFU / g bacteria of the composition are present in this composition. In some embodiments of any of the embodiments disclosed herein, this composition is a probiotic. In some embodiments of any of the embodiments disclosed herein, this composition is pasteurized or heat treated. In some embodiments of any of the embodiments disclosed herein, this composition part is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient.

[0010] In other aspects, provided herein is a tablet, a sustained release capsule, a sustained release granule, a powder, a sachet or a gummy comprising any of the compositions (e.g., probiotic compositions) disclosed herein.

[0011] In a further aspect, provided herein is a kit comprising (a) (i) any of the compositions (e.g., probiotic compositions) disclosed herein; or (ii) a tablet, a sustained release capsule, a sustained release granule, a powder, a sachet or a gummy disclosed herein, and b) instructions regarding administration to a subject.

[0012] In yet another embodiment, the foregoing provides a method for treating and / or preventing one or more obesity-related disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the compositions disclosed herein or any of the tablets, sustained-release capsules, sustained-release granules, powders, sachets, or gummies disclosed herein. In some embodiments, the obesity-related disorder is one or more disorders selected from the group consisting of obesity, metabolic syndrome, diabetes mellitus, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, dyslipidemia, non-alcoholic fatty liver disease, fatty liver, leptin resistance, low resistin levels, and / or cardiovascular disorders.

[0013] In additional embodiments, provided herein are methods for producing a composition, comprising combining a biologically pure strain of Intestinimonas massiliensis with a biologically pure strain of an Akkermansia sp., wherein the Akkermansia sp. is not (i) Akkermansia muciniphila or (ii) Akkermansia glycaniphilia. In some embodiments, the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila or (ii) Akkermansia glycaniphilia is less than about 95%. In some embodiments of the embodiments disclosed herein, I. massiliensis includes a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of I. massiliensis deposited in the DSM under the nomination DSM 33460. In some embodiments of the embodiments disclosed herein, I. massiliensis includes the I. massiliensis strain deposited with the DSM under number DSM 33460 or a living strain having all the identifying characteristics of the I. massiliensis strain deposited with the DSM under number DSM 33460; and Akkermansia sp. includes the Akkermansia sp. deposited with the DSM under number DSM 33459 or a living strain having all the identifying characteristics of the Akkermansia sp. deposited with the DSM under number DSM 33459.In some embodiments of any of the embodiments disclosed herein, the method further comprises freeze drying or lyophilizing the composition.

[0014] In other aspects, provided herein is a composition for use in preventing and / or treating one or more obesity-related disorders in a subject that requires it, the composition being for the subject, any of the compositions disclosed herein (e.g., a probiotic composition) or a composition comprising any of the tablets, sustained release capsules, sustained release granules, powders, sachets or gummies disclosed herein. In some embodiments, the obesity-related disorder is one or more disorders selected from the group consisting of obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, dyslipidemia, non-alcoholic fatty liver disease, fatty liver, leptin resistance, decreased resistin levels and / or cardiovascular disease. In other aspects, provided herein is a method for providing a source for producing agmatine in the intestine for the treatment and / or prevention of diabetes, inflammation, oxidative stress, traumatic and neurodegenerative diseases, opioid addiction, mood disorders, cognitive disorders and cancer, the method comprising administering to the subject any of the compositions disclosed herein (e.g., a probiotic composition) or any of the tablets, sustained release capsules, sustained release granules, powders, sachets or gummies disclosed herein.

[0015] Each of the aspects and embodiments described herein can be used in combination, unless explicitly or expressly excluded in relation to that embodiment or aspect.

[0016] Throughout this specification, various patents, patent applications and other types of publications (e.g., magazine articles, electronic data service entities, etc.) are referenced. The disclosures of all patents, patent applications and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.

Brief Description of the Drawings

[0017] [Figure 1A] This study demonstrates the association between differentially abundant fecal 16S rRNA operational taxonomic units (OTUs) measured in lean, healthy individuals compared to obese individuals with prediabetes, and corresponding clinical metabolic markers using Spearman's correlation coefficient analysis. [Figure 1B] This study presents Intestinimonas, Prevotella, Eubacterium, and Akkermansia sp., identified from clinical studies comparing lean, healthy individuals with obese, prediabetic individuals (high BMI, insulin, and glucose levels), along with taxonomically defined OTUs, demonstrating a clear relationship with metabolic health.

[0018] [Figure 2] This shows a phylogenetic tree consisting of strain AF3360009 and strains of the class Verrucomicrobiae included in Ouwerkerk et al., 2016. This tree was reconstructed using the neighbor-joining method with 1000 bootstraps. The numbers represent bootstrap values. The bars in the legend indicate 5% sequence differences. Chlamydia trachomatis was used as the outgroup.

[0019] [Figure 3] The image shows a scanning electron microscope image of strain AF3360009. Compared to YCFA (right), when grown with YCFA and mucin (left), this strain is oval or elongated and has a filamentous structure.

[0020] [Figure 4]The graphs show the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia species, and Intestinimonas massiliensis + Akkermansia species on insulin levels in the DIO mouse model.

[0021] [Figure 5] The graphs show the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia species, and Intestinimonas massiliensis + Akkermansia species on leptin levels in the DIO mouse model.

[0022] [Figure 6A] The graphs show the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia species, and Intestinimonas massiliensis + Akkermansia species on glucose tolerance in the DIO mouse model. [Figure 6B]The graphs show the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia species, and Intestinimonas massiliensis + Akkermansia species on glucose tolerance in the DIO mouse model.

[0023] [Figure 7] The graphs show the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia species, and Intestinimonas massiliensis + Akkermansia species on cholesterol levels in the DIO mouse model.

[0024] [Figure 8] The graph shows the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia species, and Intestinimonas massiliensis + Akkermansia species on resistin levels in the DIO mouse model.

[0025] [Figure 9]This shows a dendrogram of Akkermansia gANI comparing the publicly available genomes of A. muciniphila, A. glycaniphilia, and strain AF3360009.

[0026] [Figure 10] This study demonstrates the effects of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​body weight in the DIO model.

[0027] [Figure 11] This study demonstrates the effects of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​body fat mass.

[0028] [Figure 12] This study shows the effects of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​liver weight.

[0029] [Figure 13] This study demonstrates the effects of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​insulin levels in a DIO model.

[0030] [Figure 14] This shows the measurement of insulin resistance using HOMA-IR.

[0031] [Figure 15]This study demonstrates the effects of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​leptin levels in the DIO model.

[0032] [Figure 16] This study demonstrates the effects of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​plasminogen activator inhibitor 1 (PAI1) levels in the DIO model.

[0033] [Figure 17] This study demonstrates the effects of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​resistin levels in the DIO model.

[0034] [Figure 18] This shows the production of SCFA by species of the genus Akkermansia and I. massiliensis.

[0035] [Figure 19] This shows a sample comparison of agmatine relative peak area using CE-TOFMS.

[0036] [Figure 20] This shows the removal of extracellular ATP by strains of the genus Akkermansia. [Modes for carrying out the invention]

[0037] Numerous studies have shown that probiotic bacteria (e.g., bacteria of the Lactobacillus and Bifidobacterium genera) support the growth of beneficial gut bacterial colonies, and it appears that certain beneficial probiotic strains may even alter the host's metabolic pathways for the better. Microorganisms produce bioactive substances that influence carbohydrate and lipid metabolism and regulate inflammatory processes in both the gut and systemically. Therefore, there is growing interest in identifying nutritional supplements and probiotic foods that are effective in controlling obesity and obesity-related disorders.

[0038] The inventors of this application have surprisingly discovered that microorganisms other than the commonly used probiotics Lactobacillus and Bifidobacterium can successfully alter intestinal metabolism and alleviate obesity-related conditions. These beneficial microorganisms were found to be both enriched in the digestive systems of healthy individuals with normal body weight and deficient in individuals suffering from one or more obesity-related disorders. Supplementation of the diets of mice modeling human obesity with one or more of these beneficial microorganisms resulted in substantial improvements in one or more metrics associated with obesity-related negative conditions.

[0039] I. Definition As used herein, “microorganism” or “microbe” refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0040] As used herein, “probiotics” means a composition consumed by an animal (i.e., consumed as animal feed or as an ingredient in animal feed) that contains viable (i.e., living) microorganisms (i.e., viable and reproductive microorganisms) that, when administered in appropriate amounts, provide a health benefit to the subject (see Hill et al. 2014 Nature Revs Gastro&Hep 11, 506-514, which is incorporated herein by reference in its entirety). Probiotics may contain one or more of the microbial strains described herein (e.g., any of 1, 2, 3, or 4). Probiotics are distinguished from bacterial compositions that have been killed, for example, by pasteurization or heat treatment. In certain embodiments of the methods disclosed herein, administration of non-viable bacterial compositions is also intended for the treatment of one or more metabolic disorders.

[0041] As used herein, a bacterial "strain" refers to a bacterium that remains genetically unchanged when grown or multiplied. This also includes the diversity of the same bacterium.

[0042] "At least one strain" means a single strain, but also means a mixture of strains containing at least two strains of a microorganism. "A mixture of at least two strains" means a mixture of two, three, four, five, six, or more strains. In some embodiments of the mixture of strains, the proportions can vary from 1% to 99%. If the mixture contains more than two strains, these strains may be present in substantially equal proportions or in different proportions in the mixture.

[0043] For the purposes of this disclosure, “biologically pure strain” means a strain that does not contain other bacterial strains in an amount sufficient to prevent replication of that strain or to be detectable by conventional bacteriological techniques. “Isolated,” when used in reference to the organisms and cultures described herein, includes not only biologically pure strains but also any culture of an organism that is grown or maintained in a manner different from that found in nature. In some embodiments, this strain is a mutant, variant, or derivative of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, and / or Akkermansia sp. strains, where Akkermansia sp. is a mutant, variant, or derivative of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, and / or Akkermansia sp. This Akkermansia sp. is not A. muciniphila or A. glycaniphilia, which also provide benefits equivalent to those provided by sp.In some embodiments, this strain is a strain of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, and / or an Akkermansia sp. strain, which is an Akkermansia sp. strain that is not A. muciniphila or A. glycaniphilia, but possesses all the identifying characteristics of an Akkermansia sp. strain. Furthermore, individual strains (Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, and / or Akkermansia sp., where Akkermansia sp. is not A. muciniphila or A. glycaniphilia) or any combination thereof may also yield one or more of the benefits described herein. It will also be evident that the addition of other microbial strains, carriers, additives, enzymes, etc., may yield one or more benefits or improvements to one or more metabolic states in the subject, without constituting substantially different bacterial strains.

[0044] The term "16S rRNA" or "16S ribosomal RNA" refers to the rRNA that constitutes the small subunit of the ribosome in prokaryotes. In bacteria, this sequence can be used to identify and characterize operational taxa.

[0045] The terms “sequence identity” or “sequence similarity,” as used herein, mean that two polynucleotide sequences (i.e., a candidate sequence and a reference sequence) are identical (i.e., 100% sequence identity) or similar (i.e., similar on a nucleotide-to-nucleotide basis) over the entire length of the candidate sequence. When the candidate sequence and the reference sequence are optimally aligned, the candidate sequence may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions). Optimal alignment of sequences to determine sequence identity can be performed using any publicly available local alignment algorithm known in the art (e.g., ALIGN or Megalign (DNASTAR)) or by scrutiny.

[0046] The terms “sequence identity (%)” or “sequence similarity (%)” as used herein in relation to a reference sequence are defined as the percentage of nucleotide residues in a candidate sequence that are identical to residues in a reference polynucleotide sequence after the sequence has been optimally aligned by inserting gaps as necessary to maximize sequence identity %.

[0047] As used herein, the terms “subject” or “patient” mean a mammal (e.g., human). In some embodiments, the subject suffers from a relevant disease, disorder, or condition, which includes, but is not limited to, one or more metabolic disorders, e.g., obesity, metabolic syndrome, diabetes mellitus, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, lipid metabolism disorders, non-alcoholic fatty liver disease, fatty liver, leptin resistance, low resistin levels, and / or cardiovascular disease. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a person who has one or more characteristic features of susceptibility to or risk of the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who has been and / or is being diagnosed and / or treated.

[0048] As used herein, “prevent,” “prevent,” “prevent,” and their grammatical variations mean a method of partially or completely delaying or preventing the onset or recurrence of one or more of the disorders or conditions (e.g., one or more metabolic disorders, e.g., obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, dyslipidemia, non-alcoholic fatty liver disease, fatty liver, leptin resistance, low resistin levels, and / or cardiovascular diseases) and / or their associated symptoms; prohibiting a subject from acquiring or reacquiring the disorder or condition; or reducing the risk of a subject acquiring or reacquiring one or more of the disorders or conditions or their associated symptoms.

[0049] As used herein, the term “reduce” with respect to a particular feature, characteristic, feature, biological process, or phenomenon means a decrease in that particular feature, characteristic, feature, biological process, or phenomenon. This feature, characteristic, feature, biological process, or phenomenon may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%.

[0050] As used herein, “administer” or “to administer” means introducing one or more compositions containing one or more microbial strains to a target, for example, by feeding or oral ingestion. This composition containing one or more microbial strains may also be administered in one or more doses.

[0051] As used herein, “effective amount” means an amount of a composition comprising one or more microbial strains for improving one or more metrics in a subject. Improvement of one or more metrics in a subject (e.g., treatment and / or prevention of any of the following, but not limited to, obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, dyslipidemia, non-alcoholic fatty liver disease, fatty liver, leptin resistance, decreased resistin levels and / or cardiovascular disease) may be measured as described herein or by other methods known in the art.

[0052] In this specification, certain ranges are indicated by a number preceded by the term “approximately.” The term “approximately” is used herein to provide literal support for the exact number preceded by the term, as well as for numbers that are close to or approximate the number preceded by the term. In determining whether a number is close to or approximate to a specifically enumerated number, an unenumerated close or approximate number may be a number that provides a substantial equivalent to the specifically enumerated number in the context in which the number is expressed. For example, with respect to a number, the term “approximately” refers to a range of -10% to +10% of that number, unless the term is otherwise explicitly defined in the context.

[0053] As used herein, the singular forms "a," "an," and "that" include multiple references unless otherwise specified by the context.

[0054] Furthermore, it should be noted that claims may be written in a manner that excludes any optional elements. Therefore, this wording is intended to serve as a prior basis for using exclusive terms such as "only," "unique," and "likely" in connection with the enumeration of elements in the claims or the use of "negative" limitations.

[0055] It should also be noted that, as used herein, the term "essentially consisting of" refers to a composition in which the component following the term is present in the presence of other known components in a total amount less than 30% by weight of the total composition and which neither contributes to nor interferes with the action or activity of the component.

[0056] Furthermore, it should be noted that the term "contains," as used herein, means including, but not limited to, the component following the term "contains." While the component following the term "contains" is required or essential, a composition containing this component may further contain other non-essential or optional components.

[0057] It should also be noted that, as used herein, the term "consisting of" means including and being limited to the component following the term "consisting of." Therefore, the component following the term "consisting of" is required or essential, and no other components are present in this composition.

[0058] Throughout this specification, all numerical upper limits are intended to include all lower numerical limits as if such lower numerical limits were explicitly stated herein. Throughout this specification, all numerical lower limits will include all higher numerical limits as if such higher numerical limits were explicitly stated herein. Throughout this specification, all numerical ranges will include all narrower numerical ranges that fall within such wider numerical ranges as if all such narrower numerical ranges were explicitly stated herein.

[0059] Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains.

[0060] Other definitions of terms may appear throughout this specification.

[0061] II. Composition A. Stock The beneficial microorganism-containing compositions disclosed herein may be used as dietary supplements, food additives, and therapeutic agents for administration to subjects during periods of physiological stress (such as disease or metabolic conditions), or as part of a daily nutritional regimen for disease prevention and promotion of healthy gut metabolism. Probiotics is another term that may be used for these compositions containing viable microorganisms. The term “viable microorganisms” means microorganisms that are metabolically active or capable of differentiating. In some embodiments, the beneficial microorganism-containing compositions disclosed herein include viable probiotic products and / or, in certain embodiments, compositions containing non-viable bacteria (e.g., heat-treated or pasteurized compositions).

[0062] The strains provided herein include biologically pure strains of Eubacterium eligens, biologically pure strains of Intestinimonas massiliensis, biologically pure strains of Prevotella copri, and biologically pure strains of Akkermansia sp., wherein the Akkermansia sp. is not Akkermansia muciniphila or Akkermansia glycaniphilia.

[0063] The E. eligens strain, I. massiliensis strain, P. copri strain, and Akkermansia sp. were deposited with the German Collection of Microorganisms and Cell Cultures GmbH (DSM), Inhoffenstraße 7B, 38124 Braunschweig, Germany on March 4, 2020, and were assigned accession numbers DSM 33458, DSM 33460, DSM 33457, and DSM 33459, respectively. This deposit was made in accordance with the provisions of the Budapest Convention on the International Recognition of the Deposit of Microorganisms in Patent Proceedings. One or more strains provided herein may be used as probiotics in a non-limiting embodiment.

[0064] Examples of the microorganism-containing composition (e.g., probiotic composition) include a composition containing one or more strains of Eubacterium eligens (e.g., any of about 1, 2, 3, 4, 5, 6, 7, or 8 strains or more; e.g., E. eligens strain DSM 33458). E. eligens is a Gram-positive bacterium of the Eubacteriaceae family characterized by its rigid cell wall. Further examples of beneficial microorganism-containing compositions include those comprising one or more strains of E. eligens and one or more strains of I. massiliensis, P. copri and / or Akkermansia sp. (e.g., about 1, 2, 3, 4, 5, 6, 7 or 8 species or more).

[0065] Examples of microorganism-containing compositions (e.g., probiotic compositions) include compositions containing one or more strains of Intestinimonas massiliensis (e.g., about 1, 2, 3, 4, 5, 6, 7, or 8 species or more strains; e.g., I. massiliensis strain DSM 33460). I. massiliensis is a non-motile, Gram-negative rod-shaped bacterium with an average diameter of 0.5 μm and a length of 1.8 μm, lacking spore-forming activity (Durand et al., 2017, New Microbes New Infect., 15:1-2). Further examples of this beneficial microorganism-containing composition include compositions comprising one or more strains of I. massiliensis and one or more strains of E. eligens, P. copri and / or Akkermansia sp. (e.g., any of about 1, 2, 3, 4, 5, 6, 7 or 8 species or more). In some embodiments, this beneficial microorganism-containing composition comprises both I. massiliensis and Akkermansia sp. (e.g., Akkermansia sp. that is not A. muciniphila or A. glycaniphilia, e.g., Akkermansia strain DSM 33459).In addition, when cultured or administered together, one or more I. massiliensis strains (e.g., I. massiliensis strain DSM 33460) and one or more Akkermansia sp. species (e.g., Akkermansia strain DSM 33459) exhibit one or more physiological or metabolic characteristics that are lacking in individually cultured I. massiliensis strains (e.g., I. massiliensis strain DSM 33460) and Akkermansia sp. species (e.g., Akkermansia strain DSM 33459). These characteristics may include, but are not limited to, the following: changes in the amount and / or type of organic acids produced, changes in the metabolic profile, and / or changes in the composition of the culture medium in which the bacteria are cultured together.

[0066] Examples of this beneficial microorganism-containing composition (e.g., probiotic composition) include a composition containing one or more strains of Prevotella copri (e.g., about 1, 2, 3, 4, 5, 6, 7, or 8 strains or more, e.g., P. copri strain DSM 33457). P. copri is a Gram-negative bacterium commonly found in the intestines. Further examples of this beneficial microorganism-containing composition include a composition containing one or more strains of P. copri and one or more strains of I. massiliensis, E. eligens, and / or Akkermansia sp. (e.g., about 1, 2, 3, 4, 5, 6, 7, or 8 strains or more).

[0067] Examples of microorganism-containing compositions (e.g., probiotic compositions) include compositions containing one or more strains of the genus Akkermansia (e.g., about 1, 2, 3, 4, 5, 6, 7, or 8 species or more strains), wherein the Akkermansia species is not A. muciniphila or A. glycaniphilia, but rather a strain (e.g., Akkermansia strain DSM 33459). Until 2016, this genus contained only one known species (i.e., A. muciniphila). In that year, Akkermansia glycanphila, a type of intestinal mucin-degrading bacterium, was first isolated from the feces of a reticulated python (Ouworkerk, et al., 2016, International Journal of Systematic and Evolutionary Microbiology. 66(11):4614-4620). As will be explained in more detail below, although not bound by theory, the inventors believe they have identified a new species of the genus Akkermansia based on whole-genome mean nucleotide identity (gANI) between isolated Akkermansia species (Akkermansia sp.) and A. muciniphila and A. glycanphila, which are below a 95% identical species boundary cutoff (Goris, et al., 2007, Int J Syst Evol Microbiol, 57, 81-91). In some embodiments, the Akkermansia sp. species (e.g., Akkermansia strain DSM 33459) of the microorganism-containing compositions disclosed herein has a gANI of less than 95% compared to the genome of A. muciniphila, for example, about 94%, 93%, 92%, 91%, 90%, 89%, or 88% (e.g., 87.58%).In another embodiment, the Akkermansia sp. (e.g., Akkermansia strain DSM 33459) of the microorganism-containing composition disclosed herein has a gANI of less than 95% compared to the genome of A. glycanphila, and is, for example, about 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, or 71% (e.g., 70.17%). Further examples of beneficial microorganism-containing compositions include compositions comprising one or more strains of the genus Akkermansia and one or more strains of I. massiliensis, E. eligens, and / or P. copri (for example, about 1, 2, 3, 4, 5, 6, 7, or 8 species or more).

[0068] The microorganism-containing compositions (e.g., probiotic compositions) disclosed herein may comprise one or more strains of E. eligens having a 16S ribosomal RNA sequence exhibiting at least about 97.0% sequence similarity (e.g., any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence containing SEQ ID NO: 1. This beneficial microorganism-containing composition (e.g., probiotic compositions) may comprise one or more strains of I. massiliensis having a 16S ribosomal RNA sequence exhibiting at least about 97.0% sequence similarity (e.g., any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence containing SEQ ID NO: 2. This beneficial microorganism-containing composition (e.g., probiotic composition) may contain one or more strains of P. copri having a 16S ribosomal RNA sequence exhibiting at least about 97.0% sequence similarity (e.g., any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence containing Sequence ID No. 3.

[0069] The microorganism-containing compositions disclosed herein (e.g., probiotic compositions) include one or more strains of Eubacterium eligens (e.g., E. eligens strain DSM 33458), one or more strains of Intestinimonas massiliensis (e.g., I. massiliensis strain DSM 33460), one or more strains of the genus Akkermansia (Akkermansia sp.), where Akkermansia sp. is not A. muciniphila or A. glycaniphilia (e.g., Akkermansia strain DSM 33459), and / or one or more strains of Prevotella copri. The composition may include strains of P. copri (e.g., P. copri strain DSM 33457) (i.e., the composition may include actual bacteria (either viable or inviolable) derived from these strains) and / or one or more culture supernatants derived from cultures of these strains (individually or in co-culture).

[0070] B. Formulations Generally, the microorganism-containing compositions disclosed herein (e.g., probiotic compositions) contain bacteria, such as one or more bacterial strains. In some embodiments of the present invention, the compositions are formulated in freeze-dried or lyophilized form. For example, the microorganism-containing compositions may constitute granules or gelatin capsules (e.g., rigid gelatin capsules) containing the bacterial strains disclosed herein.

[0071] In some embodiments, the microorganism-containing compositions disclosed herein include lyophilized bacteria. Lyophilization of bacteria is a well-established procedure in the art. Alternatively, the microorganism-containing composition may include a culture of living, active bacteria.

[0072] In some embodiments, any of the microbial-containing compositions disclosed herein are encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from disintegration before delivery at the target site, for example, by chemical or physical stimuli (e.g., physical disintegration caused by changes in pressure, enzyme activity, or pH). Any suitable encapsulation method may be used. Exemplary encapsulation techniques include encapsulation in porous matrices, adhesion or adsorption on solid carrier surfaces, self-aggregation with flocculants or crosslinkers, and mechanical storage in porous membranes or microcapsules.

[0073] The microorganism-containing compositions disclosed herein may be administered orally, and these compositions may be in the form of tablets, capsules, or powders. Other components (e.g., vitamin C or minerals) may be included as oxygen scavengers and prebiotic substrates to improve in vivo delivery and / or partial or overall establishment and survival. Alternatively, the microorganism-containing compositions disclosed herein (e.g., probiotic compositions) may be administered orally as a food or nutritional product (e.g., a milk or whey-based fermented dairy product) or as a pharmaceutical product.

[0074] The microorganism-containing compositions disclosed herein may be formulated as probiotics. Alternatively, the microorganism-containing compositions disclosed herein may be formulated as non-viable bacterial compositions (e.g., pasteurized or heat-treated bacterial compositions).

[0075] The microorganism-containing compositions disclosed herein contain a therapeutically effective amount of the bacterial strain disclosed herein. A therapeutically effective amount of the bacterial strain is sufficient to produce a beneficial effect on a patient. A therapeutically effective amount of the bacterial strain may be sufficient to result in delivery to the target intestine and / or partial or complete colonization of this intestine.

[0076] For example, a suitable daily dose of this bacterium for adults is approximately 1 × 10⁻⁶. 3 ~Approx. 1×10 11It can be a colony forming unit (CFU), for example, about 1×10 7 ~ about 1×10 10 CFU; in another example, about 1×10 6 ~ about 1×10 10 It can be a GPU; in another example, about 1×10 7 ~ about 1×10 11 CFU; in another example, about 1×10 8 ~ about 1×10 10 CFU; in another example, about 1×10 8 ~ about 1×10 11 CFU can be. In certain embodiments, the dose of the bacterium is at least 10 9 cells per day, for example, at least 10 10 cells, at least 10 11 [[ID=ed=26]]cells or at least 10 12 cells.

[0077] In certain embodiments, the microbial composition contains the bacterial strain in an amount of about 1×10 6 ~ about 1×10 11 CFU / g, for example, about 1×10 8 ~ about 1×10 10 CFU / g. The dose can be, for example, 1 g, 3 g, 5 g and 10 g.

[0078] In certain embodiments, the amount of the bacterial strain is about 1×10 3 ~ about 1×10 11 colony forming units per gram with respect to the weight of the composition.

[0079] In certain embodiments, any of the microbial compositions disclosed herein are administered at a dose of 500 mg to 1000 mg, 600 mg to 900 mg, 700 mg to 800 mg, 500 mg to 750 mg or 7,50 mg to 1000 mg. In certain embodiments, the lyophilized bacteria in any of the microbial compositions disclosed herein are administered at a dose of 500 mg to 100 mg, 600 mg to 900 mg, 700 mg to 800 mg, 500 mg to 750 mg or 750 mg to 1000 mg.

[0080] Typically, probiotics are optionally combined with at least one suitable prebiotic compound. Prebiotic compounds are usually indigestible carbohydrates (e.g., oligosaccharides, polysaccharides, or sugar alcohols) that are not broken down or absorbed in the upper digestive tract. Known prebiotics include commercially available products such as inulin and transgalactooligosaccharides.

[0081] In certain embodiments, the probiotic compositions disclosed herein are formulated to contain a prebiotic compound in an amount of about 1 to about 30% by weight (e.g., about 5 to 20% by weight) with respect to the total weight of the composition. The carbohydrate may be selected from the group consisting of: fructooligosaccharides (i.e., FOS), short-chain fructooligosaccharides, insulin, isomaltoligosaccharides, pectin, xylooligosaccharides (i.e., XOS), chitosan oligosaccharides (i.e., COS), human milk oligosaccharides, beta-glucan, modified gum arabic and indigestible starch, polydextrose, D-tagatose, acacia fiber, carob, oat and citrus fiber. In one embodiment, the prebiotic is a short-chain fructooligosaccharide (for simplicity, referred to herein below as FOSs-cc), which is an indigestible carbohydrate generally obtained by the conversion of beet sugar and containing a saccharose molecule to which three glucose molecules are linked. In some embodiments, any of the prebiotics disclosed herein may be formulated together with additional probiotics derived from the genera Lactobacillus and Bifidobacterium (e.g., B. lactis B420).

[0082] The microorganism-containing compositions disclosed herein may further comprise pharmaceutically acceptable excipients or carriers. Carriers or diluents acceptable for therapeutic use are known in the pharmaceutical field. Examples of suitable carriers include, but are not limited to, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include, but are not limited to, ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent may be made in consideration of the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise, or in addition to, any suitable binder, lubricant, suspending agent, coating agent (e.g., a gastric-resistant enteric coating agent that does not dissolve or decompose until it reaches the small or large intestine) or solubilizer as carriers, excipients, or diluents. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, colorants, and even flavorings may be provided in this pharmaceutical composition. Examples of preservatives include, but are not limited to, esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0083] The microorganism-containing compositions disclosed herein can be formulated as food products. For example, the food product, such as a nutritional supplement, may provide nutritional benefits in addition to the therapeutic effects of the present invention. Similarly, the composition of the present invention can be formulated into a food product to enhance its palatability or to make it more appealing to consume by making it resemble a general food product rather than a pharmaceutical composition. In certain embodiments, the microorganism-containing composition is formulated as a milk-based product. The term “milk-based product” as used herein means any liquid or semi-solid milk-based or whey-based product with varying lipid content. Milk-based products may include, for example,: cow’s milk, goat’s milk, sheep’s milk, skim milk, whole milk, milk or processed products reconstituted from powdered milk and whey without any processing, such as yogurt, curd, fermented milk, fermented whole milk, buttermilk and other fermented milk products. Another important group includes milk beverages, such as whey beverages, fermented milk, condensed milk, infant or baby milk; flavored milk, ice cream; and confectionery and other milk-containing foods.

[0084] In certain embodiments, the microbial compositions disclosed herein comprise a single bacterial strain or species and no other bacterial strain or species. Such compositions may contain only trace or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be cultures in which other biological species are substantially absent. In certain embodiments, the compositions of the present invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 bacterial strains or species. In certain embodiments, the composition comprises 1 to 10 (e.g., 1 to 5) bacterial strains or species.

[0085] Microbial-containing compositions for use in accordance with the methods disclosed herein may or may not require marketing authorization.

[0086] In some cases, the lyophilized bacterial strain is reconstituted before administration. In some cases, this reconstitution is performed using the diluents described herein.

[0087] The microorganism-containing compositions disclosed herein may include pharmaceutically acceptable excipients, diluents, or carriers.

[0088] In certain embodiments, provided herein are pharmaceutical compositions comprising a bacterial strain disclosed herein and pharmaceutically acceptable excipients, carriers, or diluents, wherein the bacterial strain is present in an amount sufficient to treat a disease when administered to a subject in need, the disorder being selected from the group consisting of obesity, metabolic syndrome, diabetes mellitus, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, lipid metabolism disorders, non-alcoholic fatty liver disease, fatty liver, leptin resistance, decreased resistin levels, and / or cardiovascular diseases.

[0089] In certain embodiments, the present invention provides a pharmaceutical composition comprising a carrier selected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol.

[0090] In certain embodiments, the present invention provides a pharmaceutical composition comprising a diluent selected from the group consisting of ethanol, glycerol, and water.

[0091] In certain embodiments, the present invention provides a pharmaceutical composition comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethylcellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride.

[0092] In certain embodiments, the present invention provides a pharmaceutical composition comprising at least one of a preservative, an antioxidant, and a stabilizer.

[0093] In certain embodiments, the present invention provides a pharmaceutical composition comprising a preservative selected from the group consisting of sodium benzoate, sorbin chain, and p-hydroxybenzoic acid esters.

[0094] In a particular embodiment, the present invention provides a pharmaceutical composition wherein the bacterial strain is freeze-dried.

[0095] In a particular embodiment, the pharmaceutical composition is stored in a sealed container at about 4°C or about 25°C, and when the container is placed in an atmosphere of 50% relative humidity, at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the bacterial strain, as measured by colony-forming units, remain after a period of at least about 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years, or 3 years.

[0096] The bacterial strains disclosed herein can be cultured using standard microbiological techniques, such as those described in the Examples section or those known in the Art.

[0097] In additional embodiments, one or more of the bacterial strains disclosed herein may be formulated as a composition (e.g., a pharmaceutical composition) comprising bacterial extracellular vesicles (EVs). As used herein, the terms “extracellular vesicles” or “EVs” refer to a bacterial composition comprising bacterial lipids and bacterial proteins and / or bacterial nucleic acids and / or carbohydrate moieties contained in nanoparticles. The EVs may comprise one, two, three, four, five, ten, or more than ten different lipid species. The EVs may comprise one, two, three, four, five, ten, or more than ten different protein species. The EVs may comprise one, two, three, four, five, ten, or more than ten different nucleic acid species. The EVs may comprise one, two, three, four, five, ten, or more than ten different carbohydrate species. As used herein, the terms “purified EV composition” or “EV composition” refer to a preparation containing EVs isolated from at least one related substance found in the raw materials (e.g., isolated from at least one other bacterial component), or a preparation containing EVs isolated from any substance related to these EVs in any process used to produce such preparation. The term also refers to compositions that are significantly enriched or concentrated. In some embodiments, the EVs are concentrated to 2x, 3x, 4x, 5x, 10x, 100x, 1000x, 10,000x or more than 10,000x.

[0098] The EVs described herein can be prepared using any method known in the art. In some embodiments, EVs are prepared without an EV purification step. For example, in some embodiments, bacteria containing the EVs described herein are killed using a method that preserves the intact bacterial EVs, and the resulting bacterial component (e.g., EVs) is used in the methods and compositions described herein. In some embodiments, these bacteria are killed using antibiotics (e.g., antibiotics described herein). In some embodiments, these bacteria are killed using UV irradiation. In some embodiments, the EVs described herein are purified from one or more other bacterial components. Methods for purifying EVs from bacteria are known in the art. In some embodiments, EVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9):e0134353 (2015) (each of these is incorporated herein by reference in whole). In some embodiments, the bacteria are cultured to a high optical density and then centrifuged to pellet the bacteria (e.g., 10,000 × g over 30 minutes at 4°C). In some embodiments, the culture supernatant is then filtered to remove intact bacterial cells (e.g., through a 0.22 μm filter). In some embodiments, the filtered supernatant is centrifuged to pellet the bacterial extracellular matrix (EVs) (e.g., 100,000–150,000 × g over 1–3 hours at 4°C). In some embodiments, the resulting EV pellets are resuspended (e.g., in PBS), the resuspended EVs are applied to a sucrose gradient (e.g., a discontinuous sucrose gradient of 30–60%), and then centrifuged (e.g., 200,000 × g over 20 hours at 4°C) to further purify the EVs. The EV bands can be collected, washed (e.g., in PBS), and centrifuged to pellet the EVs (e.g., 150,000 × g over 3 hours at 4°C). The purified extracellular viable (EV) can be stored at, for example, -80°C until use. In some embodiments, the EV is further purified by treatment with DNase and / or proteinase K.

[0099] For example, in some embodiments, the bacterial culture disclosed herein may be pelletized by centrifuging at 11,000 × g for 20 to 40 minutes at 4°C. The culture supernatant may be passed through a 0.22 μm filter to remove intact bacterial cells. The filtered supernatant may then be concentrated using methods including, but not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, in the case of ammonium sulfate precipitation, 1.5 to 3 M ammonium sulfate may be slowly added to the filtered supernatant while stirring at 4°C. The precipitate can be incubated at 4°C for 8–48 hours, and then centrifuged at 11,000 × g for 20–40 minutes at 4°C. The resulting pellet contains bacterial extracellular molecules and other residues.

[0100] Using ultracentrifugation, the filtered supernatant may be centrifuged at 100,000 to 200,000 × g for 1 to 16 hours at 4°C. The pellet from this centrifugation contains bacterial extracellular molecules and other residues. In some embodiments, the supernatant may be filtered using filtration techniques (e.g., using an Amicon Ultra spin filter or by tangential flow filtration) to retain species with molecular weights greater than 50 or 100 kDa.

[0101] Alternatively, by connecting the bioreactor to an alternating tangential flow (ATF) system (e.g., Repligen's XCell ATF), EVs can be obtained from the bacterial culture continuously during growth or at a selected point during growth. This ATF system allows intact cells (greater than 0.22 μm) to be retained in the bioreactor, while smaller components (e.g., EVs, free proteins) can be recovered by filtering. For example, the system may be configured so that the filtrate smaller than 0.22 μm then passes through a second 100 kDa filter, thereby allowing the recovery of species such as EVs between 0.22 μm and 100 kDa, and allowing species smaller than 100 kDa to be pumped back into the bioreactor. Alternatively, the system may be configured to allow the replenishment and / or modification of the culture medium in the bioreactor during culture growth. The EVs recovered in this manner can be further purified and / or concentrated with respect to the filtered supernatant by ultracentrifugation or filtration as described above.

[0102] EV obtained by the methods provided herein may be further purified by size-based column chromatography, affinity chromatography, and gradient ultracentrifugation using methods such as, but are not limited to, the use of a sucrose gradient or an Optiprep gradient. Briefly, if the filtered supernatant is concentrated using ammonium sulfate precipitation or ultracentrifugation, the pellet is resuspended in 60% sucrose, 30 mM Tris, pH 8.0 using the sucrose gradient method. If the filtered supernatant is concentrated using filtration, the concentrate is buffered to 60% sucrose, 30 mM Tris, pH 8.0 using an Amicon Ultra column. The sample is applied to a discontinuous sucrose gradient of 35–60% and centrifuged at 200,000 × g for 3–24 hours at 4°C. Briefly, if the filtered supernatant is concentrated using ammonium sulfate precipitation or ultracentrifugation, the pellet is resuspended in 35% Optiprep in PBS using the Optiprep gradient method. In some embodiments, if filtration is used and the filtered supernatant is concentrated, the concentrate is diluted with 60% Optipre to a final concentration of 35% Optipre. The sample is applied to a discontinuous sucrose gradient of 35–60% and centrifuged at 200,000 × g for 3–24 hours at 4°C.

[0103] In some embodiments, to confirm the sterility and isolation of the EV preparation, the EV is serially diluted on agar medium used for the normal culture of the bacteria to be tested and incubated under normal conditions. The non-sterile preparation is passed through a 0.22 μm filter to remove intact cells. To further increase purity, the isolated EV may be treated with DNase or proteinase K.

[0104] III. Method A. Methods for treating or preventing diseases Further provided herein are methods for treating and / or preventing one or more obesity-related disorders, including obesity, metabolic syndrome, diabetes mellitus, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, dyslipidemia, non-alcoholic fatty liver disease, fatty liver, leptin resistance, decreased resistin levels, and / or cardiovascular disease, in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of any of the microorganism-containing and / or EV-containing compositions disclosed herein.

[0105] The Body Mass Index (BMI), calculated as weight in kilograms divided by the square of height in meters, is the most commonly accepted measure of being overweight and / or obese. In adults, a BMI of 25 or higher is considered overweight, obesity is defined as a BMI of 30 or higher, a BMI of 35 or higher is considered a serious comorbidity, and a BMI of 40 or higher is considered morbid obesity. For the purposes of this invention, “obesity” means a BMI of 30 or higher.

[0106] One of the five overweight individuals had metabolic syndrome. Metabolic syndrome is one of the fastest-growing obesity-related health problems in the United States and is characterized by a group of health issues including obesity, high blood pressure, abnormal lipid levels, and high blood sugar. According to the Centers for Disease Control and Prevention (CDC), metabolic syndrome affects nearly a quarter (22%) of the U.S. population, or an estimated 47 million people. Metabolic syndrome can increase the risk of patients developing more serious health problems such as diabetes, heart disease, and stroke.

[0107] Overweight and obese individuals have a higher incidence of heart disease and are therefore more likely to suffer from heart attacks, congestive heart failure, sudden cardiac death, angina, and cardiac abnormalities compared to those who maintain a healthy body mass index. Obesity negatively impacts blood lipid levels, which are elevated in obese individuals. This often increases the risk of heart disease because it leads to elevated triglyceride levels and decreased high-density lipoprotein (also known as HDL). Individuals with excessive body fat not only have lower levels of HDL cholesterol in their blood, but also higher levels of triglycerides and low-density lipoprotein (LDL, also known as "bad cholesterol"). This combination creates optimal conditions for the development of atherosclerotic heart disease.

[0108] Being overweight or obese increases the risk of developing high blood pressure. High blood pressure significantly increases the risk of heart attack, stroke, and kidney failure. In fact, blood pressure increases with weight gain. Even a 10-pound weight loss can lower blood pressure, and weight loss is most effective for overweight individuals and those who already have high blood pressure.

[0109] Obesity is associated with the development of diabetes. More than 80 percent of people with type 2 diabetes, the most common form of diabetes, are obese or overweight. Type 2 diabetes develops when the pancreas's production of insulin is impaired, or when the body's tissues and organs become insulin-resistant. Obesity increases the risk of developing diabetes because, as insulin's ability to control blood glucose is reduced, the body begins to overproduce insulin to regulate blood glucose levels. Over time, the body can no longer maintain blood glucose levels within a normal range. Eventually, it becomes impossible to achieve a healthy blood glucose balance, and type 2 diabetes develops. Furthermore, obesity complicates the management and treatment of type 2 diabetes by increasing insulin resistance and impaired glucose tolerance, and this increase reduces the effectiveness of drug treatments for the disease. In many cases, weight loss to a normal range normalizes blood glucose and restores insulin sensitivity.

[0110] Childhood obesity is a major public health problem, especially in Western countries. Children aged 2 to 18 are considered obese if their BMI is above the 95th percentile. Despite policies aimed at reducing prevalence, childhood obesity has more than doubled in children and more than tripled in adolescents over the past 30 years. As in adults, childhood obesity leads to hypertension, dyslipidemia (abnormal lipid metabolism), chronic inflammation, increased blood clotting tendencies, endothelial dysfunction, and hyperinsulinemia. This clustering of risk factors for cardiovascular disease has been observed in children as young as 5 years of age.

[0111] The methods disclosed herein concern the prevention, inhibition, and treatment of obesity-related disorders. “Obesity-related disorders” as used herein include, but are not limited to, obesity, undesirable weight gain, and binge eating disorders (e.g., binge eating, bulimia, compulsive eating, or lack of appetite control, each of which may optionally lead to undesirable weight gain or obesity), metabolic syndrome, diabetes mellitus, insulin deficiency-related disorders, insulin resistance-related disorders, impaired glucose tolerance, dyslipidemia, non-alcoholic fatty liver disease, fatty liver, leptin resistance, low resistin levels, and / or cardiovascular disease. “Obese” and “obese” as used herein refer to Class I obesity, Class II obesity, Class III obesity, and pre-obese (e.g., “overweight”) as defined by the World Health Organization.

[0112] A reduction in body fat is expected to bring about a variety of primary and / or secondary benefits in subjects (e.g., subjects diagnosed with obesity-related complications such as obesity-related disorders), including, for example: increased insulin responsiveness or reduced glucose tolerance (e.g., in subjects diagnosed with type II diabetes); suppression of blood pressure elevation; suppression of cholesterol levels, and / or LDL, and / or VLDL elevation; and suppression (or reduced risk or progression) of cardiovascular diseases (e.g., ischemic heart disease, arterial vascular disease, angina pectoris, myocardial infarction, and / or stroke), migraines, congestive heart failure, deep vein thrombosis, pulmonary embolism, gallstones, gastroesophageal reflux disease, obstructive sleep apnea, obesity hypoventilation syndrome, asthma, gout, decreased mobility, back pain, erectile dysfunction, urinary incontinence, liver injury (e.g., fatty liver disease, cirrhosis, alcoholic cirrhosis, endotoxin-mediated liver injury), chronic renal failure, leptin resistance, and elevated resistin levels.

[0113] In another embodiment, the Disclosure relates to a method for reducing obesity by administering an effective amount of any of the microorganism-containing and / or EV-containing compositions (e.g., probiotic compositions) disclosed herein to a subject. In some embodiments, the subject's obesity is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​between these percentages) compared to an obese subject that has not been administered one or more of the microorganism-containing and / or EV-containing compositions disclosed herein. Obesity reduction can be measured by any known method in this field, such as a decrease in BMI.

[0114] In another embodiment, the Disclosure relates to a method for reducing one or more of metabolic syndrome, diabetes (e.g., type 2 diabetes), insulin resistance, and / or impaired glucose tolerance by administering an effective amount of any of the microorganism-containing and / or EV-containing compositions (e.g., probiotic compositions) disclosed herein to a subject. In some embodiments, the percentage of one or more of metabolic syndrome, diabetes (e.g., type 2 diabetes), insulin resistance, and / or impaired glucose tolerance is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​between these percentages) compared to a subject diagnosed with one or more of these conditions who has not been administered any of the microorganism-containing and / or EV-containing compositions disclosed herein. A reduction in one or more of metabolic syndrome, diabetes (e.g., type 2 diabetes), insulin resistance, and / or impaired glucose tolerance can be determined by any means known in the art, such as blood glucose measurement and A1C determination.

[0115] In another embodiment, the Disclosure relates to a method for treating one or more liver disorders (e.g., lipid metabolism disorders, non-alcoholic fatty liver disease, and / or fatty liver) by administering an effective amount of any of the microorganism-containing and / or EV-containing compositions disclosed herein (e.g., probiotic compositions) to a subject. In some embodiments, the incidence of liver disorders is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​between these percentages) compared to a subject with liver disorders who has not been administered any of the microorganism-containing and / or EV-containing compositions disclosed herein. The reduction of one or more liver disorders may be determined by any means known in the Art.

[0116] In another embodiment, the Disclosure relates to a method for treating leptin resistance and / or resistin level reduction by administering an effective amount of any of the microorganism-containing and / or EV-containing compositions (e.g., probiotic compositions) disclosed herein to a subject. In some embodiments, leptin resistance is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110% (including all values ​​between these percentages) compared to a subject having leptin resistance and / or resistin level reduction that has not been administered one or more of the microorganism-containing and / or EV-containing compositions disclosed herein. The reduction in leptin resistance and / or resistin level can be determined by any means known in the Art.

[0117] In another embodiment, the Disclosure relates to a method for treating one or more cardiovascular disease-related disorders (e.g., ischemic heart disease, arterial vascular disease, angina pectoris, myocardial infarction, and / or stroke) by administering an effective amount of any of the microorganism-containing and / or EV-containing compositions disclosed herein (e.g., probiotic compositions) to a subject. In some embodiments, the incidence of one or more cardiovascular disease-related disorders is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​between these percentages) compared to a subject having one or more cardiovascular disease-related disorders who has not been administered any of the microorganism-containing and / or EV-containing compositions disclosed herein. The reduction of one or more disorders associated with cardiovascular disease can be determined by any means known in the art.

[0118] In yet another embodiment, any of the microorganism-containing and / or EV-containing compositions (e.g., probiotic compositions) disclosed herein to be administered to a subject comprises one or more strains of E. eligens having a 16S ribosomal RNA sequence exhibiting at least about 97.0% sequence similarity (e.g., any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%) to the 16S ribosomal RNA sequence comprising SEQ ID NO: 1. This beneficial microorganism-containing and / or EV-containing composition (e.g., probiotic composition) may comprise one or more strains of I. massiliensis having a 16S ribosomal RNA sequence exhibiting at least about 97.0% sequence similarity (e.g., any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%) to the 16S ribosomal RNA sequence comprising SEQ ID NO: 2. This beneficial microorganism-containing and / or EV-containing composition (e.g., probiotic composition) may contain one or more strains of P. copri having a 16S ribosomal RNA sequence exhibiting at least about 97.0% sequence similarity (e.g., any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence containing SEQ ID NO: 3.

[0119] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 species) of E. eligens, I. massiliensis, P. copri, and Akkermansia sp. are arranged in a manner of at least approximately 1 × 10 4 CFU / target / day ~ at least approximately 1 x 10 12 The target is administered a CFU / target / day ratio, for example, approximately 1 × 10 4 CFU / target / day, 1 x 10 5 CFU / target / day, 1 x 10 6 CFU / target / day, 1 x 10 7 CFU / target / day, 1 x 10 8 CFU / target / day, 1 x 10 9CFU / target / day, 1 x 10 10 CFU / target / day, 1 x 10 11 CFU / target / day or 1 x 10 12 The target is administered at a rate of CFU / target / day (including all values ​​between these amounts).

[0120] B. Method for preparing a microbial composition Similarly, provided herein are methods for preparing microbial-containing and / or EV-containing compositions (e.g., probiotic compositions), comprising combining a biologically pure strain of Intestinimonas massiliensis with a biologically pure strain of an Akkermansia sp., wherein the Akkermansia sp. is not A. muciniphila or A. glycaniphilia. It is a species of the genus Akkermansia (Akkermansia sp.) and has a whole-genome mean nucleotide identity (gANI) that is at least 95% different from other known species of the genus Akkermansia (Akkermansia sp.). I. massiliensis may contain a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity (e.g., any of approximately 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence of I. massiliensis deposited in the DSM under the number DSM 33460.

[0121] In addition, the method for preparing this composition may further include lyophilizing or freeze-drying the microbial composition. The method may further include the additional step of packaging the feed additive composition for storage or transport.

[0122] C. Administration Preferably, the microorganism-containing and / or EV-containing compositions disclosed herein are administered into the gastrointestinal tract to enable delivery of the bacterial strains of the present invention to the intestines and / or partial or complete colonization. Generally, the compositions of the present invention are administered orally, but may be administered rectally, intranasally, or via the buccal or sublingual route.

[0123] In certain embodiments, the microorganism-containing and / or EV-containing compositions disclosed herein may be administered as a foam, spray, or gel.

[0124] In certain embodiments, the microorganism-containing and / or EV-containing compositions of the present invention disclosed herein may be administered as suppositories such as anal suppositories, for example, in the form of cocoa butter, synthetic hard fats (e.g., suppocire, witepsol), glycerogelatin, polyethylene glycol, or soap glycerin compositions.

[0125] In certain embodiments, the microorganism-containing and / or EV-containing compositions disclosed herein are administered into the gastrointestinal tract via a tube (e.g., a nasogastric tube, an oral gastric tube, a gastric tube, a jejunostomy tube (J tube), a percutaneous endoscopic gastrostomy (PEG)) or a port (e.g., a chest wall port that allows access to a tired stomach, the jejunum, and other suitable access ports).

[0126] The microorganism-containing and / or EV-containing compositions disclosed herein may be administered as a single dose or sequentially as part of a treatment regimen. In certain embodiments, the compositions of the present invention are administered daily.

[0127] In certain embodiments of the present invention, treatment with the microorganism-containing and / or EV-containing compositions disclosed herein, according to the methods disclosed herein, is accompanied by an evaluation of the target intestinal microbiota. If delivery and / or partial or complete establishment of the strain of the present invention is not achieved, and as a result no efficacy is observed, the treatment may be repeated; if delivery and / or partial or complete establishment is successful and efficacy is observed, the treatment may be discontinued. In certain embodiments, the compositions of the present invention may be administered to pregnant animals (e.g., mammals such as humans) to prevent the development of a condition in the utero and / or postnatally in the offspring.

[0128] The composition of the present invention may be administered to patients diagnosed with a disease or condition mediated by histone deacetylase activity, or to patients identified as being at risk of a disease or condition mediated by histone deacetylase activity. This composition may also be administered as a prophylactic measure to prevent the development of a disease or condition mediated by histone deacetylase activity in healthy patients.

[0129] Microbiota-containing and / or EV-containing compositions disclosed herein may be administered to subjects identified as having an abnormal intestinal microbiota. For example, such a patient may have reduced or absent colonization of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, and / or Akkermansia sp. species, excluding A. muciniphila or A. glycaniphilia.

[0130] The microorganism-containing and / or EV-containing compositions disclosed herein may be administered as foods such as nutritional supplements.

[0131] Generally, the microorganism-containing and / or EV-containing compositions disclosed herein are for human treatment, but may be used to treat animals, including monogastric animals (e.g., poultry, pigs, cats, dogs, horses, or rabbits) or polygastric animals (e.g., ruminants). The compositions of the present invention may be useful in enhancing the growth and capabilities of animals. When administered to animals, forced oral administration may be used.

[0132] IV. Kit Further provided herein are kits comprising one or more microbial strains disclosed herein and / or EVs derived from one or more of these microbial strains. This kit, along with appropriate storage, maintenance, and use instructions, is for administering to subjects for the treatment or prevention of one or more obesity-related disorders, and includes Akkermansia sp. species that are not A. muciniphila or A. glycaniphilia (e.g., Akkermansia strain DSM 33459), E. eligens strain (e.g., E. eligens strain DSM 33458), I. massiliensis strain (e.g., I. massiliensis strain DSM 33460), and / or P. copri strain (e.g., P. copri strain DSM 33460). The kit may include one or more strains (e.g., any one of 1, 2, 3, or 4) of the microbial strains provided herein, such as 33457, and / or EVs derived from one or more of these microbial strains. In one embodiment, the kit may include Akkermansia strain DSM 33459 and I. massiliensis strain DSM 33460.

[0133] The present invention can be further understood by referring to the following embodiments, which are provided for illustrative purposes only and are not intended to limit the invention. [Examples] [Examples]

[0134] Isolation of the strain Isolation of Intestinimonas massiliensis: Clinical fecal simples with a higher population of Intestinimonas massiliensis, determined by 16S crowd analysis of all clinical fecal samples, were used in the enrichment protocol, and all procedures were performed under anaerobic conditions. Samples were diluted 1:100 with Basal Bicarbonate Buffered Medium, and a total of 25 ml of seeded medium was sealed in 50 ml glass vials. The vials were incubated at 37°C under anaerobic conditions. Approximately 5 ml of culture was dispensed daily into four tubes (two for DNA extraction and two for strain isolation). Samples were collected on days 4, 5, 6, 7, 8, and 11 after the start of this enrichment. DANs were extracted from samples each day, and 16S crowd sequencing was performed to determine the bacterial population. The sample from day 11 was determined to have the highest proportion of Intestinimonas massiliensis; therefore, one of the strain isolation samples from day 11 was used, diluted, and plated onto agar plates in Basal Bicarbonate Buffer. Basal Bicarbonate Buffered Media: 0.53 g / L sodium phosphate, dibasic; 0.41 g / L potassium phosphate, monobasic; 0.3 g / L ammonium chloride; 0.11 g / L calcium chloride; 0.1 g / L magnesium sulfate heptahydrate; 0.3 g / L sodium chloride; 4 g / L sodium bicarbonate; 0.48 g / L sodium sulfide hydrate; 5 × Wolfe's trace minerals; 1 × standard vitamin solution; 80 mM lactate; 80 mM acetate.

[0135] Isolation of Prevotella copri: The Prevotella copri strain was isolated by directly seeding diluted feces onto BHIB agar plates and incubating anaerobically for 24 hours. BHIB: Brainheart infusion agar (commercial product, BD 221843) supplemented with 10% sheep blood. BHIS was used for growth in liquid broth. BHIS: Brainheart infusion supplemented with yeast extract, vitamin K1, and hemin.

[0136] Isolation of Akkermansia species: Akkermansia strains were isolated from clinical fecal samples by directly inoculating diluted feces onto YCFA medium containing 10 g / L mucin.

[0137] Isolation of Eubacterium eligens: The E. eligens strain was isolated by directly seeding diluted feces onto a BHIB agar plate and incubating anaerobically for 24 hours. BHIB: Brain Heart Infusion agar (commercial product, BD 221843) supplemented with 10% sheep blood. BHIS was used for growth in liquid broth. BHIS: Brain Heart Infusion supplemented with yeast extract, vitamin K1, and hemin.

[0138] Genome sequencing: The genome sequences of all strains were obtained using the same method. The strains were grown on either BHIB or YCFA agar plates. The growths were removed from the agar plates using a large loop, taking care not to remove the agar. Based on the amount of growth, the number of wells to be processed for DNA extraction was determined. For one well, the growths were resuspended in 750 μl of PowerMag Bead Solution, the first solution used in the DNA extraction kit. If the growth was sufficient to spread to multiple wells, additional amounts were used. The cells were resuspended and dispensed into the desired number of wells. The DNA extraction protocol was then performed using the kit instructions. For elution, the amount of elution buffer used per well was slightly reduced to obtain higher DNA concentrations. After completion of DNA extraction, similar wells were grouped together. DNA concentrations were determined using the Invitrogen Quant-It PicoGreen DSDNA Assay Kit.

[0139] Candidates were isolated from fecal samples of healthy donors and identified by whole-genome sequencing. Ranking was performed using statistical analysis (e.g., correlation values ​​(insulin, BMI, glucose, DXATotFAT), prevalence, and the number of lean samples containing this candidate). Candidates were selected based on the availability of correlation analysis and isolation. Four of these candidates were selected for further study (Figures 1A and 1B). [Examples]

[0140] Identification of a new species of the genus Akkermansia Unless otherwise specified, all operations were performed in an anaerobic chamber using a mixed gas of N2 / CO2 / H2 (85 / 10 / 5%).

[0141] The AF33600009 strain was isolated during a second isolation round following a general isolation round, and the target was one of the top candidates. The clinical samples selected for this isolation round were shown to have higher abundances of the top candidates based on 16S crowd analysis already analyzed. This strain was isolated from fecal sample F015V3. The isolation method used in this round was selection against YCFA medium containing mucin at 10 g / L.

[0142] Aliquotes used in the isolation round were pre-prepared from fecal matter from sample F015V3, mixed with glycerol to a final glycerol concentration of 25%. These aliquots were stored at -80°C until needed. One aliquot was removed from the freezer and placed in an anaerobic chamber, where it was thawed at room temperature for approximately 10 minutes. Unless otherwise specified, all operations were performed in an anaerobic chamber. The measurement portion was removed and sequentially diluted with mucin-free YCFA broth. In an omnitray, 100 μl of the aliquot was placed on YCFA + mucin agar. -4 , 10 -5 and 10 -6 The cells were sown in a diluted solution. Bacterial cells were spread using approximately 12 sterile glass beads, and a uniform diluted aliquot was spread onto the agar surface. The plates were incubated at 37°C for approximately 72 hours in an anaerobic box equipped with a small bag to create an anaerobic environment.

[0143] Deoxygenated growth medium (YCFA + 10 g / L mucin) was dispensed into 1 ml deep-well plates at a rate of 350 μl per well. Single colonies were selected and seeded one per well onto the plate containing the pre-dispensed medium. The plate was covered with a breathable cover to allow gas exchange. The plate was incubated at 37°C for approximately 216 hours. The cultures were gently mixed using a 96-well head Integra pipette. Aliquots of the cultures taken for 16S PCR analysis and the remaining cultures were mixed with sterile, deoxygenated 50% glycerol + 1 g / L L-cysteine ​​to a final glycerol concentration of 25%. These cultures were pipetted into appropriate long-term storage pouches and stored at -80°C.

[0144] 16S Identification: Aliquots of cell cultures for PCR were diluted approximately 1:100 with sterile water. This aqueous dilution was used as a template for the 16S PCR reaction. The PCR primers used to amplify the 16S gene were: 8F: AGA GTT TGA TYM TGG CTC and 1492R: CGG TTA CCT TGT TAC GAC TT. PCR reaction conditions and thermocyclization settings were standard for polymerase Q5. Aliquots of the 16S PCR reaction were run on a gel to confirm the presence of a 16S PCR product of the expected size. Aliquots of the 16S PCR reaction were enzymatically purified using the ExoSAP-IT Express for PCR Cleanup Kit. These samples were then sent for Sanger sequencing using an external third-party vendor. The most frequently used 16C primer for 16S Sanger sequencing was 515F: GTG CCA GCM GCC GCG GTA A.

[0145] Next, this 16S sequence was compared with the 16S amplicon sequences of the top candidate list. The results revealed that the most suitable candidate was Akkermansia muciniphila. The vial corresponding to the well containing the desired strain was removed from the freezer and placed in an anaerobic chamber. A small portion of the frozen culture was then extracted from the vial and streaked onto a YCFA+ 10 g / L mucin agar plate. This plate was incubated at 37°C in an anaerobic box with a sachet until sufficient growth was achieved to prepare a frozen stock and extract DNA.

[0146] Genome of strain AF33600009: The DNA extraction kit used was Qiagen MagAttract® PowerSoil® DNA KF (King Fisher) Kit. Freshly streaked cells were scraped from a YCFA+ 10 g / L mucin agar plate and resuspended in the first solution of the DNA extraction kit. Cells were gently resuspended by gently pipetting to break up cell clumps. This cell suspension was uniformly dispersed into multiple wells of a PowerMag Bead Plate. The number of wells was determined by the volume and density of cells in the cell suspension. DNA was then extracted according to the manufacturer's protocol. After DNA extraction was complete, the same wells were combined into a single DAN sample, and the DNA concentration was determined using the Invitrogen Quant-iT PicoGreen dsDNA quantification kit. The DNA was then sent for whole-genome sequencing.

[0147] The sequencing library was prepared using the Nextera Flex kit (Illumina), and paired reads of 2 × 150 nt were sequenced using MiSeq (Illumina). The genome sequencing data was assembled using an in-house pipeline. Briefly, the reads were filtered and trimmed based on quality, and then corrected using BFC (Li, 2015). The corrected reads were assembled using the SPAdes assembler (Bankevich et al., 2012) with kmer length options of "31, 55, 77, 99, 121". This assembly was corrected with Pilon (Walker et al., 2014). After assembly, open reading frames (ORFs) were predicted and annotated using Prokka (Seemann, 2014). The 16S rRNA gene was predicted using Barrnap, and the closest species was identified using the RDP pairwise alignment tool (Fish et al., 2013).

[0148] The draft genome of strain AF33600009 consists of 31 contigs with N50 and 125x coverage, totaling 331,405 bp. The genome size is 3.19 Mb, which is larger than the genome sizes of the type strains of the following two other Akkermansia species: A. muciniphila (Muc). T (2.66 Mbp) and A. glycaniphilia Pyt T (3.07 Mb). The G+C content of this genomic DNA is 57.7%. Currently, only two species of the genus Akkermansia are known, so we reconstructed a phylogenetic tree using these three strains and several strains of the class Verrucomicrobiae included in the publication describing A. glycaniphilia (Ouwerkerk et al., 2016).

[0149] From this phylogenetic tree analysis, strain AF33600009 is a member of the genus Akkermansia, specifically A. muciniphila Muc T It is shown to be a closely related species (Figure 2). Strain AF33600009 and A. muciniphila Muc T The whole-genome mean nucleotide identity (gANI) between strain AF33600009 and A. glycaniphilia Pyt was 87.58%. T The gap between them is only 70.17%. Based on gANI values ​​below the 95% species boundary cutoff (Goris et al., 2007), strain AF33600009 is proposed as a new species within the genus Akkermansia.

[0150] Akkermansia gANI dendrogram was constructed using many publicly available genomes most closely related to the A. muciniphila type strain genome GCF_000020225.1, along with the publicly available genomes of two A. glycaniphilia species and the genome of strain AF33600009 (Figure 9). All genomes from A. muciniphila clustered together, the publicly available genomes of the two A. glycaniphilia species clustered together, and AF3360009 formed a separate cluster distinct from the other two species.

[0151] Fatty acid methyl ester (FAME) analysis of cellular fatty acids (Welch, 1991. Applications of cellular fatty-acid analysis. Clin. Microbiol. Rev. 4:422-438) was performed by Microbial ID Inc (DE, USA). Standard samples were prepared by growing A. muciniphila strain AF33600009 and A. muciniphila ATCC strain BAA835 in BHIA and extracting fatty acid methyl esters for identification. The samples were then loaded into a gas chromatograph for analysis. FAME profiles of the samples were created using Sherlock® pattern recognition software. The samples were then compared to determine similarity. As shown in Table 1, the FAME profiles showed significant differences between strain AF3360009 and strain ATCC BAA835. [Table 1]

[0152] Cells of strain AF33600009 were either oval or elongated. Elongation was more observed when cells were grown in a mucin-containing medium. Compared to YCFA alone, cells grown in YCFA + mucin medium aggregated more and formed more filaments (Figure 3). [Examples]

[0153] Mouse models for evaluating the effectiveness of candidates To evaluate the effectiveness of the following candidates, a diet-induced obesity (DIO) mouse model was used: Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, and Akkermansia sp., as described in Example 2.

[0154] E. eligens, I. massiliensis, P. copri, and Akkermansia sp. were evaluated for their efficacy in improving metabolic disorders in a DIO mouse model. For the animal experiment, animals in group 1 were maintained on PMI Nutrition International Certified Rodent Chow No. 5 CR4 upon arrival. Animals in groups 2-9 were maintained on Research Diet D12492. Animals were housed individually in polycarbonate cages with appropriate bedding. On day 1 of the study, animals were assigned to the treatment groups mentioned in Table 2 in a manner that generated cohorts with no significant differences in body weight and non-fasting blood glucose based on measurements taken on day 1. [Table 2]

[0155] The test substances Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. were prepared daily and administered within 1 hour of formulation. Vehicle (group 2) was administered once daily by forced oral administration from days 1 to 36. The dose volume for each animal was 100 μl. The test substances (groups 2-7) were administered once daily by forced oral administration from days 1 to 36. The dose volume for each animal was 100 μl. Each dose was administered using a syringe fitted with a forced oral cannula.

[0156] The control substance (group 8) was administered to the relevant animals once daily via subcutaneous injection into the interscapular region from days 1 to 36. The dose volume for each animal was based on the most recent body weight measurement. Each dose was administered using a syringe / needle within a partitioned area. The first day of administration was designated as day 1.

[0157] The study parameters included mortality / mortality checks, daily observations, body weight measurements, food intake, fecal samples, blood glucose measurements, oral glucose tolerance tests, qNMR assessments, cytokine assessments, and clinical chemistry parameters. Blood samples were collected for biomarker evaluation at designated points during the administration period and on the scheduled date of euthanasia.

[0158] Serum insulin levels were measured from mice of various groups. The group administered Intestinimonas massiliensis orally showed a 12% reduction in insulin levels compared to the vehicle control. The groups administered Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. orally showed improvements of 50%, 50%, and 64%, respectively, in insulin levels (Figure 4).

[0159] Leptin levels were measured in serum obtained from mice of various groups. The group administered Eubacterium eligens orally showed a 21% reduction in insulin levels compared to the vehicle control. The groups administered Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. orally showed improvements of 20%, 15%, and 25%, respectively, in leptin levels (Figure 5).

[0160] After a 2-hour fast, all mice were intraperitoneally administered 2.0 g / kg of glucose (10 mL / kg). Blood glucose levels were checked at the tail slit using a portable blood glucose meter at the following times, in relation to glucose dose: 0 (before glucose dose), 15, 30, 60, 90, and 120 minutes. The groups administered orally with Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. showed improvements in glucose tolerance of 9.5%, 10%, and 8.5%, respectively (Figures 6A and 6B).

[0161] Serum cholesterol levels were measured from mice of various groups. The group administered Akkermansia sp. orally showed an 11% improvement in cholesterol levels (Figure 7).

[0162] Serum resistin levels were measured from mice of various groups. The group administered Eubacterium eligens orally showed a 19% reduction in resistin levels compared to the vehicle control. The groups administered Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. orally showed improvements of 14%, 11%, and 14% in insulin levels, respectively (Figure 8). [Examples]

[0163] Mouse models for evaluating pharmaceutical formulations Intestinimonas massiliensis and Akkermansia sp. (frozen, pasteurized, and freeze-dried) were evaluated for their efficacy in improving metabolic disorders in a DIO mouse model. For animal experiments, animals in group 1 were maintained on PMI Nutrition International Certified Rodent Chow No. 5 CR4 upon arrival. Animals in groups 2-7 were maintained on Research Diet D12492. Animals were housed individually in polycarbonate cages with appropriate bedding. On day 1 of the study, animals were assigned to the treatment groups mentioned in Table 3 in a manner that generated cohorts with no significant differences in body weight and non-fasting blood glucose based on measurements taken on day 1. [Table 3]

[0164] The test substances Intestinimonas massiliensis and Akkermansia sp. (frozen, pasteurized, and freeze-dried) were prepared daily and administered within one hour of formulation. Vehicle (group 2) was administered once daily by forced oral administration from days 1 to 84. The dose volume for each animal was 100 μl. Test substances (groups 2-6) were administered once daily by forced oral administration from days 1 to 84. The dose volume for each animal was 100 μl. Each dose was administered using a syringe fitted with a forced oral cannula.

[0165] The control substance (group 7) was administered to the relevant animals once daily via subcutaneous injection into the interscapular region from days 1 to 84. The dose volume for each animal was based on the most recent body weight measurement. Each dose was administered using a syringe / needle within a partitioned area. The first day of administration was designated as day 1.

[0166] The test parameters included mortality / mortality checks, daily observations, body weight measurements, food intake, fecal samples, blood glucose measurements, qNMR assessment, and clinical chemistry parameters.

[0167] Body weight was measured weekly during this study. As shown in Figure 10, the vehicle control group showed a significant increase in body weight compared to the group given only solid feed. The group administered frozen Akkermansia sp. showed a 9%–14% improvement in body weight between days 43 and 84.

[0168] Body composition analysis was performed using qNMR (Bruker NMR LF90II). Body composition was determined at the start of the study (days -2 and -1) and at the end of the study (days 83 and 84). As shown in Figure 11, the vehicle control showed significant fat accumulation compared to the group given only solid feed. The group administered frozen Akkermansia sp. showed 25% lower fat accumulation compared to the control. Freeze-dried and pasteurized Akkermansia sp. also showed low fat accumulation (5.5% and 5%, respectively).

[0169] Liver weight was determined at the end of the study (day 84). As shown in Figure 12, the vehicle control group showed increased liver weight compared to the group fed only solid feed. The group administered frozen Akkermansia sp. showed 37% lower fat accumulation compared to the control group.

[0170] Serum insulin levels were measured from mice of various groups. Measurements were taken once every two weeks during the study. The area under the curve was calculated based on the values ​​obtained during the study. As shown in Figure 13, the vehicle control showed elevated insulin levels compared to solid feed. The group administered I. massiliensis showed a 17% decrease in insulin levels compared to the control, and the group administered frozen Akkermansia sp. showed a 22% decrease in insulin levels compared to the control. Freeze-dried and pasteurized Akkermansia sp. also showed decreases in insulin levels (5% and 17%, respectively).

[0171] Insulin resistance was measured using HOMA-IR. Measurements were based on fasting glucose and insulin levels. The area under the curve was calculated based on the values ​​obtained during this study. As shown in Figure 14, the vehicle control showed increased insulin resistance in animals compared to solid feed. The group administered I. massiliensis showed a 10% decrease in insulin levels compared to the control, and the group administered frozen Akkermansia sp. showed a 20% decrease in insulin levels compared to the control. Pasteurized Akkermansia sp. also showed a 12% decrease in insulin levels.

[0172] Leptin levels were measured in serum obtained from mice of various groups. As shown in Figure 15, the group that received forced oral administration of Akkermansia sp. (pasteurized form) showed a 21% improvement in leptin levels. Lyophilized Akkermansia sp. also showed a decrease in leptin levels (7%).

[0173] PAI1 levels were measured in serum obtained from mice of various groups. As shown in Figure 16, the group that received forced oral administration of Akkermansia sp. (frozen form) showed a 15% improvement in PAI1 levels. The group that received Akkermansia sp. (pasteurized form) showed an 8% improvement in PAI1 levels.

[0174] Resistin levels were measured in serum obtained from mice of various groups. As shown in Figure 17, the group that received forced oral administration of I. massiliensis showed a 16% reduction in resistin levels compared to the vehicle control. Lyophilized Akkermansia sp., pasteurized Akkermansia sp., and lyophilized Akkermansia sp. showed improvements of 31%, 17%, and 32%, respectively, in resistin levels.

[0175] SCFA production by Akkermansia sp. and I. massiliensis was analyzed by growing bacteria in RCM for 72 hours. The supernatant was collected, filtered, and SCFA was detected by HPLC. In the culture medium, Akkermansia sp. showed production of propionate, while I. massiliensis showed production of butyrate (Figure 18). Both propionate and butyrate are known to play important roles in regulating the metabolic health of the host (Chambers et al 2018, Rios-Covian et al 2016).

[0176] In this model, Akkermansia sp. (frozen form) showed significant improvements in body weight, fat accumulation, liver weight, insulin resistance, and resistin levels. Pasteurized Akkermansia sp. showed similar activity, but less significantly, suggesting that administration of live bacteria may be preferable. When administered in lyophilized form, Akkermansia sp. did not have the same effect as the frozen form, but still showed improvements in resistin and leptin levels, as well as some improvement in fat accumulation and insulin levels. While not bound by theory, the reason why the lyophilized form was less effective is likely due to insufficient hydration time of cells when administered in this form. Since the transient time in mice is 3-4 hours, in this model, the lyophilized form may not have been sufficient for adequate hydration and transcriptional activity to achieve the same efficacy. [Examples]

[0177] Metabolic analysis of Akkermansia sp. strain AF3360009 This study compared the metabolic capacity of Akkermansia sp. strains grown in YCFAC medium with that of Akkermansia muciniphila type strain BAA835. A 1% overnight culture was seeded in YCFAC medium, and the supernatant was collected after 24 hours of growth. Cells were separated by centrifugation at 10,000 rpm for 5 minutes and then filtered through a 0.2 μM filter.

[0178] Cell-free supernatant was collected and analyzed by CE-TOF-MS. Cationic conditions: The sample was injected into a fused silica capillary (id 50 μm × 0 cm) using an Agilent CE-TOF System (Agilent Technologies Inc., Santa Clara, CA, USA) at 50 mbar and 10 seconds. A cationic buffer solution (1 M formic acid) was used, and the CE voltage was 30 kV. Positive mode mass spectrometer conditions: MS capillary voltage: 4.0 V, ESI cationization mode, m / z range 50~1000. Anionic conditions: The sample was injected into a fused silica capillary (id 50 μm × 0 cm) using an Agilent CE-TOF System (Agilent Technologies Inc.) at 50 mbar and 22 seconds. Anionic buffer solution (50 mM ammonium acetate, pH 7.5) was used, and the CE voltage was 30 kV. Negative mode mass spectrometer conditions: MS capillary voltage: 3.5V, ESI anionization mode, m / z range 50~1000.

[0179] The metabolite agmatine (N-(4-aminobutyl)guanidine) was detected in cationic mode at m / z 131.130 mu with a retention time of 4.23 minutes, and identification was performed against known standards. The reported amount is the peak area.

[0180] Agmatine is metabolized from arginine by the enzyme arginine decarboxylase EC4.1.1.19 (Piletz et al 2013, Taksande et al 2016). As shown in Figure 19, agmatine levels are higher in Akkermansia sp. (8.7E10-5) compared to Akkermansia muciniphila ATCC BAA 835 (2.4E10-5) and YCFAC growth medium (4.0E10-5). The measured differences between the two strains and the medium levels indicate that Akkermansia sp. produces agmatine from arginine, while Akkermansia muciniphila ATCC BAA835 consumes agmatine.

[0181] Extracellular ATP is known to induce inflammation (Cauwels et al 2014). Akkermansia sp. and Akkermansia muciniphila BAA835 were evaluated for their ability to remove ATP from growth medium. 1% overnight cultures were seeded in YCFAC medium containing mucin (10 g / l), and 1 mM ATP was added. Supernatants were collected immediately after seeding and after 8 hours of growth. ATP levels were measured using standard techniques. Cells were separated by centrifugation at 10,000 rpm for 5 minutes and then filtered through a 0.2 μM filter. Cell-free supernatant was collected and analyzed for ATP levels.

[0182] ATP levels were detected in the supernatant of Akkermansia muciniphila ATCC BAA 835 at time 0 (Figure 20A) and after 8 hours of growth (Figure 20B). Similarly, ATP levels in the supernatant of Akkermansia sp. were estimated at time 0 (Figure 20C) and after 8 hours of growth (Figure 20D). Akkermansia sp. was able to remove ATP more efficiently than A. muciniphila ATCC BAA 835.

[0183] Using whole-genome comparisons, an operon that may encode an enzymatic mechanism involved in vitamin B12 synthesis is present in Akkermansia sp. species but absent in A. muciniphila ATCC BAA 835 type strain (Table 4). The ability to synthesize this important coenzyme suggests broader metabolic capacity in Akkermansia sp. compared to this type strain. [Table 4]

[0184] References Bankevich A,et al.SPAdes:a new genome assembly algorithm and its applications to single-cell sequencing.J Comput Biol.2012;19(5):455-77. Collado, MC, et al. (2007).Intestinal integrity and Akkermansia muciniphila, a mucindegrading member of the intestinal microbiota present in infants, adults, and the elderly.Appl Environ Microbiol 73,7767-7770. Costello,E.K.,et al.(2010).Postprandial remodeling of the gut microbiota in Burmese pythons.ISME J 4,1375-1385. Chun J.,et al.(2018).Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes.Int.J.Syst.Evol.Microbiol.68 461-466.(10.1099)ijsem.0.002516 Fish JA,et al.FunGene:thefunctional gene pipeline and repository.Front Microbiol.2013;4:291.doi:10.3389 / fmicb.2013.00291. Goris,J.,et al.(2007).DNA-DNA hybridization values and their relationship to whole-genome sequence similarities.Int J Syst Evol Microbiol 57,81-91. Li H.(2015)BFC:correcting illumina sequencing errors.arXiv:1502.03744. Lopez-Siles M,et al.2012.Cultured Representatives of Two Major Phylogroups of Human Colonic Faecalibacterium prausnitzii Can Utilize Pectin,Uronic Acids,and Host-Derived Substrates for Growth.Applied and Environmental Microbiology 78(2):420-428. Seemann T.Prokka:rapid prokaryotic genome annotation.Bioinformatics.2014;15:2068-9 Walker BJ,et al.Pilon:an integrated tool for comprehensive microbial variant detection and genome assembly improvement.PLoS One.2014;9:e112963.doi:10.1371 / journal.pone.0112963. Weizhong Li&Adam Godzik.Cd-hit:a fast program for clustering and comparing large sets of protein or nucleotide sequences”,Bioinformatics,2006;22:1658-9. Magoc T,Salzberg SL.FLASH:fast length adjustment of short reads to improve genome assemblies.Bioinformatics.2011;27:2957-63. Wang Q,Garrity GM,Tiedje JM,Cole JR.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Appl Environ Microbiol.2007 Aug;73(16):5261-7. Ouwerkerk J.P.,Aalvink S.,Belzer C.,de Vos W.M.(2016).Akkermansia glycaniphila sp.nov.,an anaerobic mucin-degrading bacterium isolated from reticulated python faeces.Int.J.Syst.Evol.Microbiol.66,4614,4620.10.1099 / ijsem.0.001399 Clinical breakpoints(Bacterial v9.0 and Fungal v9.0).The European Committee on Antimicrobial Susceptibility Testing:http: / / www.eucast.org / clinical_breakpoints.Published January 1,2019(bacteria). Guo X,et al.(2016).Different subtype strains of Akkermansia muciniphila abundantly colonize in southern China.J Appl Microbiol.2016 Feb;120(2):452-9.doi:10.1111 / jam.13022. Piletz JE,Aricioglu F,Cheng JT,Fairbanks CA,Gilad VH,Haenisch B,Halaris A,Hong S,Lee JE,Li J,Liu P,Molderings GJ,Rodrigues AL,Satriano J,Seong GJ,Wilcox G,Wu N,Gilad GM.Agmatine:clinical applications after 100 years in translation.Drug Discov Today.2013 Sep;18(17-18):880-93.doi:10.1016 / j.drudis.2013.05.017.Epub 2013 Jun 13.PMID:23769988 Taksande BG,Gawande DY,Chopde CT,Umekar MJ,Kotagale NR.Agmatine ameliorates adjuvant induced arthritis and inflammatory cachexia in rats.Biomed Pharmacother.2017 Feb;86:271-278.doi:10.1016 / j.biopha.2016.12.039.Epub 2016 Dec 19.PMID:28006753. Freitas AE,Bettio LE,Neis VB,Santos DB,Ribeiro CM,Rosa PB,Farina M,Rodrigues AL.Agmatine abolishes restraint stress-induced depressive-like behavior and hippocampal antioxidant imbalance in mice.Prog Neuropsychopharmacol Biol Psychiatry.2014 Apr 3;50:143-50.doi:10.1016 / j.pnpbp.2013.12.012.Epub 2013 Dec 24.PMID:24370459. Cauwels,A.,Rogge,E.,Vandendriessche,B.et al.Extracellular ATP drives systemic inflammation,tissue damage and mortality.Cell Death Dis 5,e1102(2014). Chambers ES,Preston T,Frost G,Morrison DJ.Role of Gut Microbiota-Generated Short-Chain Fatty Acids in Metabolic and Cardiovascular Health.Curr Nutr Rep.2018;7(4):198-206.doi:10.1007 / s13668-018-0248-8 Rios-Covian D, Ruas-Madiedo P, Margolles A, Gueimonde M, de los Reyes-Gavilan CG and Salazar N(2016) Intestinal Short Chain Fatty Acids and their Link with Diet and Human Health. Front. Microbiol. 7:185. doi:10.3389 / fmicb.2016.00185

[0185] Array

Chem.

Chem.

Chem.

Claims

1. A composition comprising a biologically pure strain of the genus Akkermansia, wherein the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila and / or (ii) Akkermansia glycaniphilia is less than approximately 95%.

2. The composition according to claim 1, wherein the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and Akkermansia muciniphylla is less than about 94%, for example less than about 93%, for example less than about 92%, for example less than about 91%, for example less than about 90%, for example less than about 89%, for example less than 88%.

3. The composition according to claim 1 or 2, wherein the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and Akkermansia glycanophilia is less than about 94%, for example less than about 93%, for example less than about 92%, for example less than about 91%, for example less than about 90%, for example less than about 89%, for example less than about 88%, for example less than about 87%, for example less than about 86%, for example less than about 85%, for example less than about 84%, for example less than about 83%, for example less than about 82%, for example less than about 81%, for example less than about 80%, for example less than about 79%, for example less than about 78%, for example less than about 77%, for example less than about 76%, for example less than about 75%, for example less than about 74%, for example less than about 73%, for example less than about 72%, for example less than about 71%.

4. The composition according to any one of claims 1 to 3, wherein the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and Akkermansia muciniphylla is about 87.58%.

5. The composition according to any one of claims 1 to 4, wherein the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and Akkermansia glycanophilia is 70.17%.

6. The composition according to any one of claims 1 to 5, wherein the whole-genome mean nucleotide identity (gANI) between the Akkermansia sp. and the Akkermansia strain deposited with the German Collection of Microorganisms and Cell Culture (DSM) under the number DSM 33459 is at least 95%.

7. The composition according to any one of claims 1 to 6, further comprising a biologically pure strain of E. eligens, I. massiliensis, or P. copri.

8. The biologically pure strain of E. eligens is a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the E. eligens strain deposited in the DSM under the number DSM 33458; I. massiliensis is a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the I. massiliensis strain deposited in the DSM under the number DSM 33460; (c) and P. The composition according to claim 7, wherein P. copri is a bacterial strain deposited with the DSM under the number DSM 33457, or has a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the P. copri strain deposited with the DSM under the number DSM 33457.

9. A composition comprising isolated bacterial extracellular vesicles (EVs) derived from a biologically pure strain of an Akkermansia species (Akkermansia sp.) as described in any one of claims 1 to 8; optionally, the composition further comprises (a) E. eligens deposited in the DSM under the nomination DSM 33458, or the aforementioned E. (b) a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the E. eligens strain; (b) Intestinimonas massiliensis, wherein the EV derived from a biologically pure strain of Intestinimonas massiliensis is the EV derived from the I. massiliensis strain deposited in the DSM under number DSM 33460; or (c) P. massiliensis deposited in the DSM under number DSM 33457 A composition comprising EV derived from a biologically pure strain of a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the P. copri strain deposited in the DSM under number DSM 33457, either alone or in combination with a culture supernatant derived from one or more of the strains.

10. The composition according to any one of claims 1 to 9, wherein the composition is formulated for oral administration.

11. The composition according to any one of claims 1 to 10, wherein the composition is freeze-dried or lyophilized and / or the composition is encapsulated or coated.

12. The composition according to any one of claims 1 to 11, wherein the composition is a food, a food ingredient, a nutritional supplement, or a pharmaceutical product.

13. At least about 1 x 10 ４ CFU / g composition - at least about 1 × 10 １２ The composition according to any one of claims 1 to 12, wherein the bacteria of the CFU / g composition are present in the composition.

14. The composition according to any one of claims 1 to 13, wherein the composition is a probiotic.

15. The composition according to any one of claims 1 to 14, wherein the composition is pasteurized or heat-treated.

16. The composition according to any one of claims 1 to 15, wherein the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient.

17. A tablet, sustained-release capsule, sustained-release granule, powder, sachet, or gummy comprising the composition according to any one of claims 1 to 16.

18. (a) (i) a composition according to any one of claims 1 to 16; or (ii) a tablet, sustained-release capsule, sustained-release granule, powder, sachet, or gummy according to claim 17; and (b) instructions for administration to a subject.

19. A method for producing a composition, Combining a biologically pure strain of Intestinimonas massiliensis, E. eligens, or P. copri with a biologically pure strain of an Akkermansia sp. species as described in any one of claims 1 to 6; or The method includes obtaining extracellular vesicles (EVs) from a biologically pure strain of Intestinimonas massiliensis, E. eligens, or P. copri and a biologically pure strain of an Akkermansia sp. as described in any one of claims 1 to 16, and combining them; The aforementioned E. eligens includes the E. eligens strain deposited in the DSM under number DSM 33458, or a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the E. eligens strain deposited in the DSM under number DSM 33458; the aforementioned I. massiliensis includes the I. massiliensis strain deposited in the DSM under number DSM 33460, or the I. A method comprising: a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the I. massiliensis strain; and the P. copri strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the P. copri strain deposited with the DSM under number DSM 33457.

20. The method according to claim 19, further comprising freeze-drying or lyophilizing the composition.

21. Use of any composition according to claim 1 to 16 in the manufacture of a pharmacopoeia for the prevention and / or treatment of one or more obesity-related disorders in a subject requiring it.

22. Use according to claim 21, wherein the obesity-related disorder is one or more disorders selected from the group consisting of obesity, metabolic syndrome, diabetes mellitus, insulin deficiency-related disorder, insulin resistance-related disorder, impaired glucose tolerance, dyslipidemia, non-alcoholic fatty liver disease, fatty liver, leptin resistance, decreased resistin levels, and / or cardiovascular disease.

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