Compositions Comprising Bacterial Strains for Improving Metabolic Health - Patent application

Abstract

Provided herein are, inter alia, compositions comprising one or more biologically pure strains of bacteria and methods of making and using the compositions for treating and / or preventing one or more obesity-related disorders in a subject in need thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 004,617, filed April 3, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Provided herein are bacterial compositions and methods of making and using the bacterial compositions that are useful, inter alia, for improving metabolic health in a subject. [Background technology]

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

[0004] First-generation probiotics are live microorganisms primarily derived from the genera Lactobacillus and Bifidobacterium, which are often trace components of the gastrointestinal tract or originate from their use as dairy starter cultures. Traditionally, first-generation probiotics have primarily targeted intestinal and immune health. Some, such as B. lactis B420, have also been shown to exhibit beneficial activity in metabolic health (e.g., reduction of body fat mass and some improvement in blood glucose and insulin). However, current first-generation probiotics do not appear to offer optimal solutions for glucose and insulin metabolism, i.e., as potential treatments or preventatives for type 2 diabetes, prediabetes, or metabolic syndrome. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, what is needed are additional microorganisms identified based on their natural occurrence in the gastrointestinal tract of metabolically healthy individuals and selected for 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 provides additional advantages as well.

[0007] Provided herein are, inter alia, compositions comprising one or more biologically pure strains of bacteria, as well as methods of making and using the compositions for treating and / or preventing one or more obesity-related disorders (e.g., 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, reduced resistin levels, and / or cardiovascular disease) in a subject in need thereof.

[0008] Accordingly, in some aspects, provided herein are (a) biologically pure strains of Eubacterium eligens; (b) biologically pure strains of Intestinimonas massiliensis; (c) bacterial strains having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of Prevotella copri, deposited at the German Collection of Microorganisms and Cell Cultures (DSM) under number DSM 33457; and / or (d) biologically pure strains of Akkermansia sp., wherein said Akkermansia sp. is selected from the group consisting of: (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia. and (ii) Akkermansia glycaniphilia. In some embodiments, the genome-wide average nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia is less than about 95%. In some embodiments of any of the embodiments disclosed herein, the composition comprises (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 at DSM under number 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 at DSM under number DSM 33460.In some embodiments of any of the embodiments disclosed herein, the composition comprises (a) the E. eligens strain deposited at DSM under number DSM 33458 or a viable strain having all of the identifying characteristics of the E. eligens strain deposited at DSM under number DSM 33458; (b) the I. massiliensis strain deposited at DSM under number DSM 33460 or a viable strain having all of the identifying characteristics of the I. massiliensis strain deposited at DSM under number DSM 33460; (c) the P. copri strain deposited at DSM under number DSM 33457 or a viable strain having all of the identifying characteristics of the P. copri strain deposited at DSM under number DSM 33457; and / or (d) the P. copri strain deposited at DSM under number DSM 33457. and / or (b) in combination with a culture supernatant derived from one or more of these strains. In some embodiments, the composition comprises Akkermansia sp. deposited with DSM under number DSM 33459, or a viable strain having all of the identifying characteristics of Akkermansia sp. deposited with DSM under number DSM 33459. In some embodiments, the composition comprises (b) a biologically pure strain of Intestinimonas massiliensis; and (d) a biologically pure strain of Akkermansia sp., wherein the Akkermansia sp. is not (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia. In some embodiments, the composition comprises (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 at DSM under number DSM 33460.In some embodiments of any of the embodiments disclosed herein, the genome-wide average nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia is less than about 95%. In some embodiments of any of the embodiments disclosed herein, the composition comprises (b) the I. massiliensis strain deposited with DSM under number DSM 33460 or a viable strain having all of the identifying characteristics of the I. massiliensis strain deposited with DSM under number DSM 33460; and (d) Akkermansia sp. deposited with DSM under number DSM 33459 or a viable strain having all of the identifying characteristics of the Akkermansia sp. deposited with DSM under number DSM 33459. In some embodiments of any of the embodiments disclosed herein, the composition is formulated for oral administration. In some embodiments of any of the embodiments disclosed herein, the composition is lyophilized or freeze-dried. In some embodiments of any of the embodiments disclosed herein, the composition is encapsulated or coated. In some embodiments of any of the embodiments disclosed herein, the composition is a food, a food ingredient, a dietary supplement, or a pharmaceutical. In some embodiments of any of the embodiments disclosed herein, the composition is at least about 1 x 10 4 CFU / g composition ~ at least about 1 x 10 12CFU / g composition of bacteria 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 aspect, provided herein is (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 that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of Prevotella copri, deposited at the German Collection of Microorganisms and Cell Cultures (DSM) under number DSM 33457; and / or (d) a biologically pure strain of Akkermansia sp., wherein said Akkermansia sp. is selected from the group consisting of: (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia. A composition comprising isolated bacterial extracellular vesicles (EVs) derived from at least one or more biologically pure strains of Akkermansia sp. that are not Akkermansia 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 genome-wide average nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia is less than about 95%.In some embodiments of any of the embodiments disclosed herein, the composition comprises (a) 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 E. eligens deposited with DSM under number DSM 33458; and / or (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 DSM under number DSM 33460. In some embodiments of any of the embodiments disclosed herein, the composition comprises: (a) EVs derived from the E. eligens strain deposited at DSM under number DSM 33458 or a viable strain having all of the identifying characteristics of the E. eligens strain deposited at DSM under number DSM 33458; (b) EVs derived from the I. massiliensis strain deposited at DSM under number DSM 33460 or a viable strain having all of the identifying characteristics of the I. massiliensis strain deposited at DSM under number DSM 33460; (c) EVs derived from the P. copri strain deposited at DSM under number DSM 33457 or a viable strain having all of the identifying characteristics of the P. copri strain deposited at DSM under number DSM 33457; and / or (d) EVs derived from the I. massiliensis strain deposited at DSM under number DSM 33460 or a viable strain having all of the identifying characteristics of the I. massiliensis strain deposited at DSM under number DSM 33457. The method comprises the step of depositing EVs derived from Akkermansia sp. deposited at DSM under number DSM 33459 or a viable strain having all of the identifying characteristics of Akkermansia sp. deposited at DSM under number DSM 33459, either (A) alone; and / or (b) in combination with culture supernatant derived from one or more of these strains.In some embodiments of any of the embodiments disclosed herein, the composition comprises (b) EVs derived from a biologically pure strain of Intestinimonas massiliensis; and (d) EVs derived from a biologically pure strain of Akkermansia sp., wherein the Akkermansia sp. is not (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia. In some embodiments, the composition comprises (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 DSM under DSM number 33460. In some embodiments of any of the embodiments disclosed herein, the genome-wide average nucleotide identity (gANI) between the Akkermansia sp. and (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia is less than about 95%. In some embodiments of any of the embodiments disclosed herein, the composition comprises (b) EVs derived from the I. massiliensis strain deposited with DSM under number DSM 33460 or a viable strain having all of the identifying characteristics of the I. massiliensis strain deposited with DSM under number DSM 33460; and (d) EVs derived from Akkermansia sp. deposited with DSM under number DSM 33459 or a viable strain having all of the identifying characteristics of the Akkermansia sp. deposited with DSM under number DSM 33459. In some embodiments of any of the embodiments disclosed herein, the composition is formulated for oral administration.In some embodiments of any of the embodiments disclosed herein, the composition is lyophilized or freeze dried. In some embodiments of any of the embodiments disclosed herein, the composition is encapsulated or coated. In some embodiments of any of the embodiments disclosed herein, the composition is a food, a food ingredient, a dietary supplement, or a pharmaceutical. In some embodiments of any of the embodiments disclosed herein, the composition is at least about 1 x 10 4 CFU / g composition ~ at least about 1 x 10 12 CFU / g composition of bacteria 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.

[0010] In other aspects, provided herein are tablets, extended release capsules, extended release granules, powders, sachets, or gummies comprising any of the compositions (e.g., probiotic compositions) disclosed herein.

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

[0012] In yet another aspect, provided herein is a method of 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, extended-release capsules, extended-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.

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

[0014] In another aspect, provided herein is a composition for use in preventing and / or treating one or more obesity-related disorders in a subject in need thereof, the composition comprising any of the compositions disclosed herein (e.g., probiotic compositions) or any of the tablets, extended-release capsules, extended-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 are methods of providing a source for the production of agmatine in the intestine for the treatment and / or prevention of diabetes, inflammation, oxidative stress, neurotrauma and neurodegenerative diseases, opioid addiction, mood disorders, cognitive disorders, and cancer, comprising administering to a subject any of the compositions disclosed herein (e.g., probiotic compositions) or any of the tablets, extended release capsules, extended release granules, powders, sachets, or gummies disclosed herein.

[0015] Each of the aspects and embodiments described herein may be used in combination unless expressly or specifically excluded in connection with that embodiment or aspect.

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

[0017] [Figure 1A] Differentially abundant fecal 16S rRNA operational taxonomic units (OTUs) measured in lean healthy compared to obese prediabetic individuals and corresponding associations to clinical metabolic markers using Spearman correlation coefficient analysis are shown. [Figure 1B] Taxonomically defined OTUs identified from a clinical study comparing lean healthy subjects with obese pre-diabetic subjects (high BMI, insulin, and glucose) are shown, including Intestinimonas, Prevotella, Eubacterium, and Akkermansia sp., showing clear associations with metabolic health.

[0018] [Figure 2] Phylogenetic tree of strain AF3360009 and strains of Verrucomicrobiae included in Ouwerkerk et al., 2016. The tree was reconstructed by neighbor-joining with 1000 bootstraps. Numbers represent bootstrap values. Legend bars indicate 5% sequence divergence. Chlamydia trachomatis was used as an outgroup.

[0019] [Figure 3] Scanning electron micrographs of strain AF3360009 are shown, which have oval or elongated, filament-like structures when grown in YCFA and mucin (left side) compared to YCFA (right side).

[0020] [Figure 4]FIG. 1 shows a graph illustrating the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. on insulin levels in a DIO mouse model.

[0021] [Figure 5] FIG. 1 shows a graph illustrating the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. on leptin levels in a DIO mouse model.

[0022] [Figure 6A] FIG. 1 shows a graph illustrating the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. on glucose tolerance in a DIO mouse model. [Figure 6B]FIG. 1 shows a graph illustrating the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. on glucose tolerance in a DIO mouse model.

[0023] [Figure 7] FIG. 1 shows a graph illustrating the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. on cholesterol levels in a DIO mouse model.

[0024] [Figure 8] FIG. 1 shows a graph showing the effects of Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. on resistin levels in a DIO mouse model.

[0025] [Figure 9]FIG. 1 shows an Akkermansia gANI dendrogram comparing the publicly available genomes of A. muciniphila, A. glycaniphilia, and strain AF3360009.

[0026] [Figure 10] 1 shows the effect of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​body weight in a DIO model.

[0027] [Figure 11] Figure 1 shows the effect of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization and freeze-drying) on ​​body fat mass.

[0028] [Figure 12] 1 shows the effect of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization and freeze-drying) on ​​liver weight.

[0029] [Figure 13] 1 shows the effect of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​insulin levels in a DIO model.

[0030] [Figure 14] 1 shows the measurement of insulin resistance by HOMA-IR.

[0031] [Figure 15]1 shows the effect of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​leptin levels in a DIO model.

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

[0033] [Figure 17] 1 shows the effect of Intestinimonas massiliensis and Akkermansia sp. (freezing, pasteurization, and freeze-drying) on ​​resistin levels in a DIO model.

[0034] [Figure 18] 1 shows the production of SCFAs by Akkermansia sp. and I. massiliensis.

[0035] [Figure 19] 1 shows a sample comparison of CE-TOFMS relative peak areas of agmatine.

[0036] [Figure 20] 1 shows the removal of extracellular ATP by Akkermansia strains. DETAILED DESCRIPTION OF THE INVENTION

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

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

[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, "probiotic" refers to a composition consumed by an animal (i.e., as animal feed or as an ingredient in animal feed) that contains viable (i.e., living) microorganisms (i.e., microorganisms that are viable and capable of proliferation) that, when administered in appropriate amounts, confer a health benefit to a subject (see Hill et al. 2014 Nature Revs Gastro&Hep 11, 506-514, incorporated herein by reference in its entirety). Probiotics can include one or more (e.g., one, two, three, or four) of any of the microbial strains described herein. Probiotics are distinguished from bacterial compositions that have been killed, for example, by pasteurization or heat treatment. Certain embodiments of the methods disclosed herein also contemplate the administration of nonviable bacterial compositions for the treatment of one or more metabolic disorders.

[0041] A bacterial "strain," as used herein, refers to a bacterium that remains genetically unchanged when grown or propagated. Varieties of the same bacterium are also encompassed.

[0042] "At least one strain" refers to a single strain, but also refers to a mixture of strains comprising at least two strains of a microorganism. A "mixture of at least two strains" refers to a mixture of 2, 3, 4, 5, 6, or more strains. In some embodiments of a mixture of strains, the proportions can vary from 1% to 99%. When a mixture comprises more than two strains, the strains can be present in the mixture in substantially equal proportions or in different proportions.

[0043] For purposes of this disclosure, a "biologically pure strain" means a strain that is free of other bacterial strains in an amount sufficient to prevent replication of the strain or to be detectable by conventional bacteriological techniques. "Isolated," when used in reference to the organisms and cultures described herein, encompasses 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, the strain is a mutant, variant, or derivative of a Eubacterium eligens strain, an Intestinimonas massiliensis strain, a Prevotella copri strain, and / or an Akkermansia sp. strain, wherein the Akkermansia sp. is a mutant, variant, or derivative of a Eubacterium eligens strain, an Intestinimonas massiliensis strain, a Prevotella copri strain, and / or an Akkermansia sp. strain. This Akkermansia sp. is not A. muciniphila or A. glycaniphilia, which also provide benefits equivalent to those provided by Akkermansia sp. This Akkermansia sp. is not A. muciniphila or A. glycaniphilia, which also provide benefits equivalent to those provided by Akkermansia sp.In some embodiments, the strain is a Eubacterium eligens strain, an Intestinimonas massiliensis strain, a Prevotella copri strain, and / or an Akkermansia sp. strain, wherein the Akkermansia sp. is a strain having all of the identifying characteristics of an Akkermansia sp. strain that is not A. muciniphila or A. glycaniphilia. Additionally, 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 of these strains may provide one or more of the benefits described herein. It will also be apparent that the addition of other microbial strains, carriers, additives, enzymes, etc., may also provide one or more benefits or improvements in one or more metabolic conditions in a subject and do not constitute a substantially different bacterial strain.

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

[0045] The term "sequence identity" or "sequence similarity," as used herein, means that two polynucleotide sequences (i.e., candidate and reference sequences) are identical (i.e., 100% sequence identity) or similar (i.e., similar on a nucleotide-by-nucleotide basis) over the entire length of the candidate sequence. In comparing a candidate sequence to a reference sequence, when the two sequences are optimally aligned, the candidate sequence may contain additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions). Optimal alignment of sequences to determine sequence identity may be performed using any publicly available local alignment algorithm known in the art (e.g., ALIGN or Megalign (DNASTAR)), or may be performed by inspection.

[0046] The terms "% sequence identity" or "% sequence similarity," as used herein with respect to a reference sequence, are defined as the percentage of nucleotide residues in a candidate sequence that are identical with the residues in the reference polynucleotide sequence, after optimal alignment of the sequences, with gaps inserted, if necessary, to maximize the % sequence identity.

[0047] As used herein, the term "subject" or "patient" refers to a mammal (e.g., a human). In some embodiments, the subject is suffering from a relevant disease, disorder, or condition, including, but not limited to, one or more metabolic disorders, such as 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, reduced resistin levels, and / or cardiovascular disease. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is one who has one or more characteristic characteristics of susceptibility to or risk for a 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," "preventing," "prevention," and grammatical variations thereof, refer to a method of partially or completely delaying or preventing the onset or recurrence of a disorder or condition (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, reduced resistin levels, and / or cardiovascular disease) and / or one or more of its attendant symptoms, or prohibiting a subject from acquiring or re-acquiring the disorder or condition, or reducing the risk of a subject acquiring or re-acquiring the disorder or condition or one or more of its attendant symptoms.

[0049] As used herein, the term "reducing" with respect to a particular trait, property, characteristic, biological process or phenomenon refers to a decrease in that particular trait, property, characteristic, biological process or phenomenon. The trait, property, characteristic, biological process or phenomenon may be decreased 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 "administering" refers to the act of introducing one or more compositions comprising one or more microbial strains into a subject, for example, by feeding or oral ingestion. The compositions comprising one or more microbial strains may also be administered in one or more doses.

[0051] As used herein, "effective amount" refers to an amount of a composition comprising one or more microbial strains to improve one or more metrics in a subject. The improvement in one or more metrics in a subject (e.g., but not limited to, treatment and / or prevention 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, reduced resistin levels, and / or cardiovascular disease) can be measured as described herein or by other methods known in the art.

[0052] As used herein, certain ranges are indicated by numerical values ​​preceded by the term "about." The term "about" 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 approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically recited number, the unrecited close or approximately number may be a number that, in the context in which the number is recited, provides a substantial equivalent to the specifically recited number. For example, with respect to a numerical value, the term "about" refers to a range of -10% to +10% of the numerical value, unless the term is clearly defined otherwise in the context.

[0053] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0054] It is further noted that the claims may be drafted to exclude any optional element, and as such, this statement is intended to serve as antecedent basis for using exclusive terminology such as "only," "solely," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0055] It should also be noted that the term "consisting essentially of," as used herein, refers to a composition in which the component following this term is present in the presence of other known components in total amounts that are less than 30% by weight of the total composition and that do not contribute to or interfere with the action or activity of this component.

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

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

[0058] It is intended that every numerical upper limit given throughout this specification will include every lower numerical limit, as if such lower numerical limit were expressly written herein. Every numerical lower limit given throughout this specification will include every higher numerical limit, as if such higher numerical limit were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0059] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

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

[0061] II. Composition A. Stocks The beneficial microorganism-containing compositions disclosed herein can be used as dietary supplements, food additives, and therapeutic agents for administration to subjects during periods of physiological stress (disease states, metabolic conditions, etc.), or as part of a daily nutritional regimen to prevent disease and promote healthy gut metabolism. Probiotics is another term that can be used for this composition containing viable microorganisms. The term "viable microorganisms" refers to microorganisms that are metabolically active or capable of differentiation. In some embodiments, the beneficial microorganism-containing compositions disclosed herein include viable probiotic products, and / or in certain embodiments, include compositions containing non-viable bacteria (e.g., heat-treated or pasteurized compositions).

[0062] 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, the I. massiliensis strain, the P. copri strain, and the Akkermansia sp. were deposited on March 4, 2020, at the German Collection of Microorganisms and Cell Cultures GmbH (DSM), Inhoffenstraße 7B, 38124 Braunschweig, Germany, and were assigned accession numbers DSM 33458, DSM 33460, DSM 33457, and DSM 33459, respectively. This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. One or more of the strains provided herein may be used as probiotics in one non-limiting embodiment.

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

[0065] The microorganism-containing compositions (e.g., probiotic compositions) can include compositions containing one or more strains of Intestinimonas massiliensis (e.g., about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains; e.g., I. massiliensis strain DSM 33460). I. massiliensis is a non-motile, Gram-negative bacillus with an average diameter of 0.5 μm and length of 1.8 μm that does not have sporulation activity (Durand et al., 2017, New Microbes New Infect., 15:1-2). The beneficial microorganism-containing compositions may further include compositions comprising one or more strains of I. massiliensis and one or more strains (e.g., about any of 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) of E. eligens, P. copri, and / or Akkermansia sp. In some embodiments, the beneficial microorganism-containing compositions include both I. massiliensis and an Akkermansia sp. (e.g., an 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. (e.g., Akkermansia. strain DSM 33459) exhibit one or more physiological or metabolic properties that are lacking when cultured individually. These properties may include, but are not limited to: changes in the amount and / or type of organic acids produced, changes in the metabolic profile and / or changes in the composition of the medium in which the bacteria are cultured.

[0066] The present microorganism-containing compositions (e.g., probiotic compositions) can include compositions comprising one or more strains of Prevotella copri (e.g., any of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains, such as P. copri strain DSM 33457). P. copri is a gram-negative bacterium normally found in the intestine. The beneficial microorganism-containing compositions can further include compositions comprising one or more strains of P. copri and one or more strains of I. massiliensis, E. eligens, and / or Akkermansia sp. (e.g., any of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains).

[0067] The microorganism-containing compositions (e.g., probiotic compositions) include compositions comprising one or more strains (e.g., about any of 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) of Akkermansia sp., where the Akkermansia sp. is not A. muciniphila; or A. glycaniphilia (e.g., Akkermansia strain DSM 33459). Until 2016, the genus contained a single known species (i.e., A. muciniphila). In the same year, Akkermansia glycanphila, a type of intestinal mucin-degrading bacterium, was isolated for the first time from the feces of a reticulated python (Ouworkerk, et al., 2016, International Journal of Systematic and Evolutionary Microbiology. 66(11):4614-4620). As explained in more detail below, without being bound by theory, the inventors believe they have identified a new species of Akkermansia based on genome-wide average nucleotide identity (gANI) between isolated Akkermansia species and A. muciniphila and A. glycanphila, which is below the species demarcation cutoff of 95% identity (Goris, et al., 2007, Int J Syst Evol Microbiol, 57, 81-91). In some embodiments, the Akkermansia sp. (e.g., Akkermansia strain DSM 33459) of the microorganism-containing compositions disclosed herein has a gANI of less than 95% compared to the A. muciniphila genome, 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 compositions disclosed herein has a gANI of less than 95% compared to the genome of A. glycanphila, for example, about any of 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%). The beneficial microorganism-containing compositions may further include compositions comprising one or more strains of Akkermansia sp. and one or more strains (e.g., any of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) of I. massiliensis, E. eligens, and / or P. copri.

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

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

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

[0071] In some embodiments, the microorganism-containing compositions disclosed herein comprise freeze-dried bacteria. Freeze-drying of bacteria is a well-established procedure in the art. Alternatively, the microorganism-containing compositions may comprise live, active bacterial cultures.

[0072] In some embodiments, any of the microorganism-containing compositions disclosed herein are encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from degradation until delivery to the target location, for example, by disruption by chemical or physical stimuli (e.g., physical disruption that may be caused by pressure, enzymatic activity, or pH changes). Any suitable encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption onto a solid support surface, self-aggregation by aggregating or cross-linking agents, and mechanical containment within a porous membrane or microcapsules.

[0073] The microorganism-containing compositions disclosed herein can be administered orally, and can be in the form of tablets, capsules, or powders. To improve in vivo delivery and / or partial or total colonization and survival, other ingredients (e.g., vitamin C or minerals) can be included as oxygen scavengers and prebiotic substrates. Alternatively, the microorganism-containing compositions disclosed herein (e.g., probiotic compositions) can 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 comprise a therapeutically effective amount of the bacterial strains disclosed herein. A therapeutically effective amount of a bacterial strain is sufficient to exert a beneficial effect on a patient. A therapeutically effective amount of a bacterial strain may be sufficient to result in delivery to and / or partial or total colonization of the intestine of a subject.

[0076] For example, a suitable daily dose of the bacteria for an adult is about 1 x 10 3 ~Approx. 1×10 11Colony forming units (CPU), for example, about 1 x 10 7 ~Approx. 1×10 10 CPU; in another example, approximately 1 x 10 6 ~Approx. 1×10 10 GPU; in another example, approximately 1×10 7 ~Approx. 1×10 11 CPU; in another example, approximately 1 x 10 8 ~Approx. 1×10 10 CPU; in another example, approximately 1 x 10 8 ~Approx. 1×10 11 In certain embodiments, the dose of the bacterium is at least 10 per day. 9 cells per day, e.g., at least 10 10 Pieces, at least 10 11 or at least 10 12 Each cell is an individual cell.

[0077] In certain embodiments, the microorganism-containing composition comprises about 1×10 6 ~Approx. 1×10 11 The bacterial strain is present in an amount of CFU / g, e.g., about 1 x 10 8 ~Approx. 1×10 10 Contains CFU / g. Doses 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 x 10 per gram by weight of the composition. 3 ~Approx. 1×10 11 colony-forming units.

[0079] In certain embodiments, any of the microorganism-containing compositions disclosed herein is administered in a dose of 500 mg to 1000 mg, 600 mg to 900 mg, 700 mg to 800 mg, 500 mg to 750 mg, or 750 mg to 1000 mg. In certain embodiments, the lyophilized bacteria in any of the microorganism-containing compositions disclosed herein is administered in 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 non-digestible carbohydrates (for example, oligosaccharides or 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) based on the total weight of the composition. The carbohydrate may be selected from the group consisting of fructooligosaccharides (i.e., FOS), short-chain fructooligosaccharides, insulin, isomaltooligosaccharides, pectin, xylooligosaccharides (i.e., XOS), chitosan oligosaccharides (i.e., COS), human milk oligosaccharides, beta-glucan, modified gum arabic and resistant starch, polydextrose, D-tagatose, acacia fiber, carob, oat, and citrus fiber. In one aspect, the prebiotic is a short-chain fructooligosaccharide (for simplicity, hereinafter referred to as FOSs-cc), an indigestible carbohydrate typically obtained by the conversion of sugar beet and comprising a sucrose molecule to which three glucose molecules are attached. In some embodiments, any of the prebiotics disclosed herein may be formulated 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 a pharmaceutically acceptable excipient or carrier. Carriers or diluents acceptable for use in therapy are known in the pharmaceutical arts. 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 can be selected taking into account the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may include, as or in addition to the carrier, excipient, or diluent, any suitable binder, lubricant, suspending agent, coating agent (e.g., a gastro-resistant enteric coating that does not dissolve or decompose until it reaches the small or large intestine), or solubilizing agent. 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, dyes, and even flavorings may be provided in the pharmaceutical composition. Preservatives include, but are not limited to, sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0083] The microorganism-containing compositions disclosed herein can be formulated as a food product. For example, the food product can provide nutritional benefits in addition to the therapeutic effects of the present invention, for example, in a dietary supplement. Similarly, the food product can be formulated to enhance the taste of the composition of the present invention or to make it more attractive to consume by resembling a common 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 refers to any liquid or semi-solid milk- or whey-based product with varying lipid content. Milk-based products can be, for example, cow's milk, goat's milk, sheep's milk, skim milk, whole milk, milk or processed products reconstituted from milk powder and whey without any processing, such as yogurt, curd, curd, soured milk, soured whole milk, buttermilk, and other soured milk products. Another important group includes milk-containing foods such as milk drinks, e.g., whey drinks, fermented milk, condensed milk, infant or baby milk; flavored milk, ice cream; and sweet confectionery.

[0084] In certain embodiments, the microorganism-containing compositions disclosed herein contain a single bacterial strain or species and are free of any other bacterial strains or species. Such compositions may contain only trace or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be cultures substantially free of other biological species. 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 compositions comprise 1 to 10 (e.g., 1 to 5) bacterial strains or species.

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

[0086] Optionally, the lyophilized bacterial strain is reconstituted prior to administration, optionally by use of a diluent as described herein.

[0087] The microorganism-containing compositions disclosed herein can include a pharmaceutically acceptable excipient, diluent, or carrier.

[0088] In certain embodiments, provided herein is a pharmaceutical composition comprising a bacterial strain disclosed herein; and a pharmaceutically acceptable excipient, carrier, or diluent, wherein the bacterial strain is present in an amount sufficient to treat a disease, when administered to a subject in need thereof, wherein the disorder is 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, reduced resistin levels, and / or cardiovascular disease.

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

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

[0091] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, wherein the excipient is selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, 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 the aforementioned pharmaceutical composition, further comprising at least one of a preservative, an antioxidant, and a stabilizer.

[0093] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, wherein the preservative is selected from the group consisting of sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid.

[0094] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, wherein said bacterial strain is lyophilized.

[0095] In certain embodiments, the pharmaceutical composition as described above, wherein when stored in a sealed container at about 4°C or about 25°C, and 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, survives 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 may be cultured using standard microbiological techniques such as those described in the Examples section or 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 term "extracellular vesicles" or "EVs" refers to a composition derived from bacteria that includes bacterial lipids and bacterial proteins, and / or bacterial nucleic acids, and / or carbohydrate moieties contained in nanoparticles. The EVs may include 1, 2, 3, 4, 5, 10, or more than 10 different lipid species. The EVs may include 1, 2, 3, 4, 5, 10, or more than 10 different protein species. The EVs may include 1, 2, 3, 4, 5, 10, or more than 10 different nucleic acid species. The EVs may include 1, 2, 3, 4, 5, 10, or more than 10 different carbohydrate species. As used herein, the term "purified EV composition" or "EV composition" refers to a preparation containing EVs that have been separated from at least one associated substance found in the source material (e.g., separated from at least one other bacterial component), or that have been separated from any substance associated with the EVs in any process used to make the preparation. The term also refers to a composition that is significantly enriched or concentrated. In some embodiments, the EVs are concentrated 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or greater than 10,000-fold.

[0098] The EVs described herein can be prepared using any method known in the art. In some embodiments, the 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 leaves the bacterial EVs intact, and the resulting bacterial components (e.g., EVs) are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the 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 the 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 which is incorporated herein by reference in its entirety. In some embodiments, the bacteria are grown to high optical density and then centrifuged to pellet the bacteria (e.g., 10,000 x g for 30 minutes at 4°C). In some embodiments, the culture supernatant is then passed through a filter to remove intact bacterial cells (e.g., a 0.22 μm filter). In some embodiments, the filtered supernatant is centrifuged to pellet bacterial EVs (e.g., 100,000-150,000 x g for 1-3 hours at 4°C). In some embodiments, the resulting EV pellet is resuspended (e.g., in PBS) and the EVs are further purified by applying the resuspended EVs to a sucrose gradient (e.g., a 30-60% discontinuous sucrose gradient), followed by centrifugation (e.g., 200,000 x g for 20 hours at 4°C). The EV band may be collected, washed (e.g., with PBS), and centrifuged to pellet the EVs (e.g., 150,000 x g for 3 hours at 4°C). Purified EVs may be stored, e.g., at -80°C, until use. In some embodiments, the EVs are further purified by treatment with DNase and / or proteinase K.

[0099] For example, in some embodiments, a culture of a bacterium disclosed herein may be centrifuged at 11,000 x g for 20-40 minutes at 4°C to pellet the bacteria. 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 that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, in the case of ammonium sulfate precipitation, 1.5-3 M ammonium sulfate may be slowly added to the filtered supernatant with stirring at 4°C. The precipitate may be incubated at 4°C for 8-48 hours, and then centrifuged at 11,000 x g for 20-40 minutes at 4°C. The resulting pellet contains bacterial EVs and other debris.

[0100] Using ultracentrifugation, the filtered supernatant can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C. The pellet from this centrifugation contains bacterial EVs and other debris. In some embodiments, a filtration technique (e.g., using an Amicon Ultra spin filter or by tangential flow filtration) can be used to filter the supernatant so that species with molecular weights above 50 or 100 kDa are retained.

[0101] Alternatively, EVs can be obtained from bacterial cultures continuously or at selected time points during growth by connecting the bioreactor to an alternating tangential flow (ATF) system (e.g., Repligen's XCell ATF). 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) are filtered and collected. For example, the system can then be configured to pass the <0.22 μm filtrate through a second 100 kDa filter, allowing species such as 0.22 μm-100 kDa EVs to be collected and smaller species compared to 100 kDa to be pumped back into the bioreactor. Alternatively, the system can be configured to allow the medium in the bioreactor to be replenished and / or changed during culture growth. EVs collected by this method can be further purified and / or concentrated by ultracentrifugation or filtration, as described above for the filtered supernatant.

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

[0103] In some embodiments, to confirm the sterility and isolation of EV preparations, EVs are serially diluted onto agar medium typically used for culturing the bacteria being tested and incubated under standard conditions. Non-sterile preparations are passed through a 0.22 μm filter to remove intact cells. To further enhance purity, isolated EVs may be treated with DNase or proteinase K.

[0104] III. Method A. Methods of Treating or Preventing Disease Further provided herein is a method for treating and / or preventing one or more obesity-related disorders, including 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, reduced resistin levels, and / or cardiovascular disease, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the microbe-containing and / or EV-containing compositions disclosed herein.

[0105] Body mass index (BMI) (calculated as weight in kilograms divided by height in meters squared) is the most commonly accepted measure of overweight and / or obesity. In adults, a BMI above 25 is considered overweight, obesity is defined as a BMI of 30 or greater, a BMI of 35 or greater is considered a severe comorbidity, and a BMI of 40 or greater is considered morbid obesity. For purposes of the present invention, "obesity" shall mean a BMI of 30 or greater.

[0106] One in five overweight people suffers from "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 problems, 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 one-quarter (22%) of the U.S. population, or an estimated 47 million people. Metabolic syndrome can increase a patient's risk of developing more serious health problems, such as diabetes, heart disease, and stroke.

[0107] Overweight and obese people have a higher incidence of heart disease, and therefore, they are more likely to suffer from heart attacks, congestive heart failure, sudden cardiac death, angina, and heart rhythm abnormalities than people with a healthy body mass index. Obesity adversely affects blood lipid levels, which are elevated in obese patients, often increasing the risk of heart disease due to elevated triglyceride levels and reduced high-density lipoproteins, also known as HDL. People with excess body fat not only have lower HDL cholesterol levels in their blood, but also higher triglyceride levels and low-density lipoproteins, also known as LDL or "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. Hypertension, or high blood pressure, significantly increases the risk of heart attack, stroke, and kidney failure. In fact, blood pressure increases with weight gain. Losing even 10 pounds can lower blood pressure, and weight loss is most effective in those who are overweight and already have high blood pressure.

[0109] Obesity is associated with the development of diabetes. Over 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' production of insulin is impaired or when the body's tissues and organs are insulin-resistant. When obesity reduces insulin's ability to control blood sugar (glucose), the body begins to overproduce insulin to regulate blood sugar levels, increasing the risk of developing diabetes. Over time, the body can no longer maintain blood sugar levels within a normal range. Eventually, a healthy blood sugar balance cannot be achieved, and type 2 diabetes develops. Furthermore, obesity complicates the management and treatment of type 2 diabetes by increasing insulin resistance and impaired glucose tolerance, which reduces the effectiveness of drug treatments for the disease. In many cases, weight loss to the normal range normalizes blood sugar and restores insulin sensitivity.

[0110] Childhood obesity is also a major public health problem, particularly in Western countries. Children aged 2 to 18 years are considered obese if their BMI is higher than 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. Similar to adults, childhood obesity leads to hypertension, dyslipidemia (abnormalities of lipid metabolism), chronic inflammation, increased tendency to blood clots, endothelial dysfunction, and hyperinsulinemia. This clustering of risk factors for cardiovascular disease has been identified in children as young as 5 years old.

[0111] The methods disclosed herein are directed to the prevention, inhibition, and treatment of obesity-related disorders. "Obesity-related disorders," as used herein, include, but are not limited to, obesity, unwanted weight gain, and overeating disorders (e.g., overeating, binge eating, compulsive eating, or lack of appetite control, each of which can optionally lead to unwanted weight gain or 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. "Obesity" and "obese," as used herein, refer to Class I obesity, Class II obesity, Class III obesity, and pre-obesity (e.g., being "overweight") as defined by the World Health Organization.

[0112] Reduction of body fat is expected to result in a variety of primary and / or secondary benefits in a subject (e.g., a subject diagnosed with obesity-related complications, such as an obesity-related disorder), including, for example, increased insulin responsiveness or reduced glucose intolerance (e.g., in a subject diagnosed with type II diabetes); reduced elevation of blood pressure; reduced elevation of cholesterol levels, and / or LDL and / or VLDL; reduced (or reduced risk or progression of) cardiovascular disease (e.g., ischemic heart disease, arteriovascular disease, angina pectoris, myocardial infarction and / or stroke), migraine, congestive heart failure, deep vein thrombosis, pulmonary embolism, gallstones, gastroesophageal reflux disease, obstructive sleep apnea, obesity hypoventilation syndrome, asthma, gout, decreased mobility, lower back pain, erectile dysfunction, urinary incontinence, liver damage (e.g., fatty liver disease, cirrhosis, alcoholic cirrhosis, endotoxin-mediated liver damage), chronic renal failure, leptin resistance and increased resistin levels.

[0113] In another embodiment, the disclosure relates to methods comprising administering an effective amount of any of the microbe- and / or EV-containing compositions (e.g., probiotic compositions) disclosed herein to a subject to reduce obesity. In some embodiments, the subject's obesity is reduced by about any of the following percentages (including all values ​​in between): 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​in between): compared to an obese subject not administered one or more of the microbe- and / or EV-containing compositions disclosed herein. Reduction in obesity may be measured by any method known in the art, such as a reduction in BMI.

[0114] In another embodiment, the disclosure relates to methods comprising administering to a subject an effective amount of any of the microbe- and / or EV-containing compositions (e.g., probiotic compositions) disclosed herein to reduce one or more of metabolic syndrome, diabetes (e.g., type II diabetes), insulin resistance and / or impaired glucose tolerance. In some embodiments, the rate of one or more of metabolic syndrome, diabetes (e.g., type II diabetes), insulin resistance and / or impaired glucose tolerance is reduced by any of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​in between these percentages) compared to a subject diagnosed with one or more of these conditions who is not administered one or more of the microbe- and / or EV-containing compositions disclosed herein. Reduction in one or more of metabolic syndrome, diabetes (e.g., type II diabetes), insulin resistance and / or impaired glucose tolerance may be determined by any means known in the art, such as by measuring blood glucose and determining A1C.

[0115] In another embodiment, the present disclosure relates to methods comprising administering to a subject an effective amount of any of the microbe- and / or EV-containing compositions (e.g., probiotic compositions) disclosed herein to treat one or more liver disorders (e.g., but not limited to, dyslipidemia, non-alcoholic fatty liver disease, and / or hepatic steatosis). In some embodiments, the incidence of liver damage is reduced by about any of the following percentages (including all values ​​in between): 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​in between): compared to a subject with liver damage who has not been administered one or more of the microbe- and / or EV-containing compositions disclosed herein. The reduction in one or more liver disorders may be determined by any means known in the art.

[0116] In another embodiment, the present disclosure relates to methods for treating leptin resistance and / or reduced resistin levels, comprising administering to a subject an effective amount of any of the microbe- and / or EV-containing compositions (e.g., probiotic compositions) disclosed herein. In some embodiments, leptin resistance is reduced and / or resistin levels are increased 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 with leptin resistance and / or reduced resistin levels who has not been administered one or more of the microbe- and / or EV-containing compositions disclosed herein. Reduced leptin resistance and / or reduced resistin levels may be determined by any means known in the art.

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

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

[0119] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) strains of E. eligens, I. massiliensis, P. copri, and Akkermansia sp. are cultured in a concentration of at least about 1 x 10 4 CFU / subject / day ~ at least approximately 1 x 10 12 CFU / subject / day, e.g., about 1 x 10 4 CFU / subject / day, 1 × 10 5 CFU / subject / day, 1 × 10 6 CFU / subject / day, 1 × 10 7 CFU / subject / day, 1 × 10 8 CFU / subject / day, 1 × 10 9CFU / subject / day, 1 × 10 10 CFU / subject / day, 1 × 10 11 CFU / subject / day or 1 x 10 12 CFU / subject / day (including all values ​​between these amounts) are administered to subjects.

[0120] B. Methods for Preparing Microbial Compositions Also provided herein is a method for preparing a microorganism-containing and / or EV-containing composition (e.g., a probiotic composition), comprising combining a biologically pure strain of Intestinimonas massiliensis with a biologically pure strain of Akkermansia sp., wherein the Akkermansia sp. is not A. muciniphila or A. glycaniphilia, and the Akkermansia sp. differs by at least 95% genome-wide average nucleotide identity (gANI) from other known Akkermansia sp. I. massiliensis may comprise a 16S ribosomal RNA sequence that exhibits at least 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 of I. massiliensis deposited at DSM under number DSM 33460.

[0121] Additionally, the method of preparing the composition may further comprise lyophilizing or freeze-drying the microbial composition. The method may additionally comprise the further 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 to the gastrointestinal tract to allow delivery and / or partial or total colonization of the intestine with the bacterial strains of the invention. Generally, compositions of the invention are administered orally, but may also be administered rectally, intranasally, or by buccal or sublingual routes.

[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 microbe-containing and / or EV-containing compositions of the invention disclosed herein may be administered as a suppository, such as a rectal suppository, for example in the form of cocoa butter, synthetic hard fats (e.g., suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin compositions.

[0125] In certain embodiments, the microbe-containing and / or EV-containing compositions disclosed herein are administered to the gastrointestinal tract via a tube (e.g., nasogastric tube, orogastric tube, stomach tube, jejunostomy tube (J-tube), percutaneous endoscopic gastrostomy (PEG)) or a port (e.g., chest wall ports allowing access to the jejunum, the engorged stomach, and other suitable access ports).

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

[0127] In certain embodiments of the present invention, treatment with the microbe- and / or EV-containing compositions disclosed herein according to the methods disclosed herein is accompanied by an assessment of the subject's gut microbiota. If delivery of the strains of the present invention and / or partial or total colonization of the strains is not achieved and, as a result, efficacy is not observed, treatment may be repeated; if delivery and / or partial or total colonization is successful and efficacy is observed, treatment may be stopped. In certain embodiments, compositions of the present invention may be administered to pregnant animals (e.g., mammals such as humans) to prevent the development of certain conditions in utero and / or in the offspring after birth.

[0128] The compositions of the invention can be administered to patients who have been diagnosed with a disease or condition mediated by histone deacetylase activity or who are identified as being at risk for a disease or condition mediated by histone deacetylase activity. The compositions can also be administered as a prophylactic measure to prevent the onset of a disease or condition mediated by histone deacetylase activity in healthy patients.

[0129] The microbe- and / or EV-containing compositions disclosed herein can be administered to subjects identified as having an abnormal gut microbiome, for example, patients who have reduced or absent colonization with Eubacterium eligens, Intestinimonas massiliensis, Prevotella copri, and / or Akkermansia sp., but not A. muciniphila or A. glycaniphilia.

[0130] The microorganism-containing and / or EV-containing compositions disclosed herein may be administered as a food product, such as a dietary supplement.

[0131] Generally, the microorganism-containing and / or EV-containing compositions disclosed herein are intended for the treatment of human subjects, but can 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 can be useful for enhancing the growth and performance of animals. When administered to animals, oral gavage can be used.

[0132] IV. Kit Further provided herein are kits comprising one or more of the microbial strains disclosed herein and / or EVs derived from one or more of the microbial strains. The kit may contain an Akkermansia sp. that is not A. muciniphila or A. glycaniphilia (e.g., Akkermansia strain DSM 33459), an E. eligens strain (e.g., E. eligens strain DSM 33458), an I. massiliensis strain (e.g., I. massiliensis strain DSM 33460), and / or a P. copri strain (e.g., P. copri strain DSM 33471), together with instructions for suitable storage, maintenance, and use for administration to a subject for the treatment or prevention of one or more obesity-related disorders. In one embodiment, the kit may include one or more (e.g., any one, two, three, or four) strains of the microbial strains provided herein, such as I. cerevisiae strain DSM 33457, and / or EVs derived from one or more of the microbial strains. In one embodiment, the kit may include Akkermansia strain DSM 33459 and I. massiliensis strain DSM 33460.

[0133] The present invention may be further understood by reference to the following examples, which are offered by way of illustration and are not intended to be limiting. [Example] [Example]

[0134] Strain isolation Isolation of Intestinimonas massiliensis: Clinical fecal samples, which harbored the highest population of Intestinimonas massiliensis as determined by 16S community analysis of all clinical fecal samples, were used for 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 inoculated culture was sealed in 50 ml glass vials. The vials were incubated at 37°C under anaerobic conditions. Approximately 5 ml of culture was aliquoted 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 enrichment. DNA was extracted from each sample, and 16S community analysis sequencing was performed to determine the bacterial population. The day 11 sample was determined to have the highest percentage of Intestinimonas massiliensis, so one of the day 11 isolates was diluted and plated on agar plates in 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, 5x Wolfe's trace minerals, 1x standard vitamin solution, 80 mM lactate, 80 mM acetate.

[0135] Isolation of Prevotella copri: Prevotella copri strains were isolated by plating diluted feces directly onto BHIB agar plates and incubating anaerobically for 24 hours. BHIB: Brain Heart Infusion Agar (commercially available, BD 221843) supplemented with 10% sheep blood. BHIS is used for growth in liquid broth. BHIS: Brain Heart Infusion supplemented with yeast extract, vitamin K1, and hemin.

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

[0137] Isolation of Eubacterium eligens: E. eligens strains were isolated by plating diluted feces directly onto BHIB agar plates and incubating anaerobically for 24 hours. BHIB: Brain Heart Infusion Agar (commercially available, BD 221843) supplemented with 10% sheep blood. BHIS is used for growth in liquid broth. BHIS: Brain Heart Infusion supplemented with yeast extract, vitamin K1, and hemin.

[0138] Genome sequencing: Genome sequences for all strains were obtained using the same method. Strains were grown on either BHIB or YCFA agar plates. Growth was removed from the agar plates using a large loop, taking care not to remove the agar. The number of wells to process for DNA extraction was determined based on the amount of growth. For a single well, growth was resuspended in 750 μl of PowerMag Bead Solution, the first solution used in the DNA extraction kit. If there was enough growth to spread to multiple wells, additional volume was used. Cells were resuspended and aliquoted into the desired number of wells. The DNA extraction protocol was then performed using the kit instructions. For elution, slightly less elution buffer was used per well to obtain a higher DNA concentration. Similar wells were pooled after DNA extraction was completed. DNA concentration was determined using Invitrogen's Quant-It PicoGreen DS DNA Assay kit.

[0139] Candidates were isolated from fecal samples of healthy donors and identified by whole-genome sequencing. Statistical analysis (e.g., correlation analysis values ​​(insulin, BMI, glucose, DXA, and fat)), prevalence, and number of lean samples carrying this candidate) were used for ranking. Candidates were selected based on correlation analysis and isolation availability. Four of these candidates were selected for further study (Figure 1A and Figure 1B). [Example]

[0140] Identification of a new species of Akkermansia Unless otherwise stated, all work was carried out in an anaerobic chamber using a gas mixture of N2 / CO2 / H2 (85 / 10 / 5%).

[0141] Strain AF33600009 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 previously analyzed 16S community analysis. This strain was isolated from fecal sample F015V3. The isolation method used in this round was selection on YCFA medium containing 10 g / L mucin.

[0142] Aliquots used in the isolation rounds were prepared in advance with feces from sample F015V3 mixed with glycerol to a final glycerol concentration of 25%. The aliquots were stored at -80°C until needed. One aliquot was removed from the freezer, placed in an anaerobic chamber, and thawed at room temperature for approximately 10 minutes. All work was performed in the anaerobic chamber unless otherwise specified. The test portion was removed and serially diluted in mucin-free YCFA broth. 100 μl of the aliquot was plated on YCFA + mucin agar in an Omnitrray. -4 , 10 -5 and 10 -6 The plates were plated at dilutions. The bacterial cells were spread using approximately 12 sterile glass beads to spread the diluted aliquots evenly over the agar surface. The plates were incubated at 37°C for approximately 72 hours in an anaerobic box equipped with a pouch to create an anaerobic environment.

[0143] Deoxygenated growth medium (YCFA + 10 g / L mucin) was dispensed into 1 ml deep-well plates at 350 μl per well. Single colonies were picked and inoculated into plates containing pre-dispensed medium, one colony per well. The plates were covered with a breathable cover to allow gas exchange. The plates were incubated at 37°C for approximately 216 hours. The cultures were gently mixed using a 96-well head Integra pipettor. An aliquot of the culture taken for 16S PCR analysis and the remaining culture 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 vials and stored at -80°C.

[0144] 16S Identification: An aliquot of cell culture for PCR was diluted approximately 1:100 with sterile water. This water dilution was used as a template in 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 thermocycler settings were standard for polymerase Q5. An aliquot of the 16S PCR reaction was run on a gel to confirm the presence of a 16S PCR product of the expected size. An aliquot of the 16S PCR reaction was enzymatically purified using the ExoSAP-IT Express for PCR Cleanup Kit. The sample was then sent for Sanger sequencing using an external third-party vendor. The 16C primer most commonly used for 16S Sanger sequencing was 515F: GTG CCA GCM GCC GCG GTA A.

[0145] This 16S sequence was then compared to the 16S amplicon sequences of a list of top candidates. The results revealed that the best match 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 removed from the vial and streaked onto a YCFA + 10 g / L mucin agar plate. The plate was incubated at 37°C in a pouched anaerobic box until sufficient growth was observed to generate frozen stocks and extract DNA.

[0146] Genome of strain AF33600009: The DNA extraction kit used was the Qiagen MagAttract® PowerSoil® DNA KF (King Fisher) Kit. Freshly streaked growth was scraped from the YCFA + 10 g / L mucin agar plate, and the cells were resuspended in the first solution of the DNA extraction kit. The cells were gently resuspended by gently pipetting to break up clumps of cells. This cell suspension was evenly distributed across multiple wells of a PowerMag Bead Plate. The number of wells was determined by the amount and density of cells in the cell suspension. DNA was then extracted according to the manufacturer's protocol. After DNA extraction was completed, similar wells were combined into one DNA 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] Sequencing libraries were prepared using the Nextera Flex kit (Illumina) and sequenced on a MiSeq (Illumina) with paired reads of 2 × 150 nt. Genomic sequencing data were assembled using an in-house pipeline. Briefly, reads were quality filtered and trimmed, then corrected using BFC (Li, 2015). The corrected reads were assembled using the SPAdes assembler (Bankevich et al., 2012) with the kmer length options "31, 55, 77, 99, 121." This assembly was corrected using 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 by Barrnap, and the closest species were identified using the RDP pairwise alignment tool ( Fish et al., 2013 ).

[0148] The draft genome of strain AF33600009 consists of 31 contigs with an N50 of 331,405 bp and 125x coverage. The genome size is 3.19 Mb, which is larger than the genome sizes of the type strains of 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%. As only two species of the genus Akkermansia are currently known, a phylogenetic tree was reconstructed between these three strains and several strains of the Verrucomicrobiae included in a publication describing A. glycaniphilia (Ouwerkerk et al., 2016).

[0149] From this phylogenetic analysis, strain AF33600009 is a member of the genus Akkermansia, and is classified as A. muciniphila Muc T The AF33600009 strain and A. muciniphila Muc are closely related species (Fig. 2). T The genome-wide average nucleotide identity (gANI) between strain AF33600009 and A. glycaniphilia Pyt was 87.58%, indicating a high genome-wide average nucleotide identity (gANI) between strain AF33600009 and A. glycaniphilia Pyt T Based on a gANI value below the 95% species delimitation cutoff (Goris et al., 2007), strain AF33600009 is proposed as a new species within the genus Akkermansia.

[0150] Using the publicly available genomes of two A. glycaniphilia species and the genome of strain AF33600009, along with many of the publicly available genomes closest to the A. muciniphila type strain genome GCF_000020225.1, an Akkermansia gANI dendrogram was constructed (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 from strain AF33600009 and A. muciniphila ATCC strain BAA835 by growing them in BHIA and extracting fatty acid methyl esters for identification. The samples were then loaded onto a gas chromatograph for analysis. Sherlock® pattern recognition software was used to generate FAME profiles of the samples. The samples were then compared to determine similarities. As shown in Table 1, the FAME profiles showed significant differences between strain AF3360009 and ATCC strain BAA835. [Table 1]

[0152] The cells of the AF33600009 strain were oval or elongated. The elongation was more pronounced when the cells were grown in a mucin-containing medium. Compared to YCFA alone, cells aggregated more and formed more filaments when grown in YCFA + mucin medium (Figure 3). [Example]

[0153] Mouse models for assessing candidate efficacy A diet-induced obesity (DIO) mouse model was used to evaluate the efficacy of the following candidates: Eubacterium eligens, as described in Example 2; Intestinimonas massiliensis; Prevotella copri, and Akkermansia sp.

[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 animal studies, 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 singly in polycarbonate cages with appropriate bedding. Animals were distributed to treatment groups noted in Table 2 on Study Day -1 in a manner that generated cohorts that were not significantly different in terms of body weight and non-fasting blood glucose based on Day -1 measurements. [Table 2]

[0155] The test articles 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 oral gavage on days 1 through 36. The dose volume for each animal was 100 μl. The test articles (Groups 2 through 7) were administered once daily by oral gavage on days 1 through 36. The dose volume for each animal was 100 μl. Each dose was administered using a syringe equipped with a gavage cannula.

[0156] The control article (Group 8) was administered to the appropriate animals by subcutaneous injection in the interscapular region once daily on days 1 through 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 defined area. The first day of administration was designated Day 1.

[0157] Study parameters included mortality / moribundity checks, daily observations, body weight measurements, food intake, fecal samples, blood glucose measurements, oral glucose tolerance test, qNMR assessment, cytokine assessment, and clinical chemistry parameters. Blood samples were collected for biomarker evaluation at designated time points during the treatment period and on the scheduled day of euthanasia.

[0158] Insulin levels were measured in serum from mice in various groups. The Intestinimonas massiliensis gavage group showed a 12% decrease in insulin levels compared to vehicle control. The Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. gavage groups showed a 50%, 50%, and 64% improvement in insulin levels, respectively (Figure 4).

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

[0160] After a 2-hour fast, all mice received an intraperitoneal injection of 2.0 g / kg glucose (10 mL / kg). Blood glucose was checked via tail nick using a portable blood glucose meter at the following times relative to the glucose dose: 0 (before the glucose dose), 15, 30, 60, 90, and 120 min. The groups receiving oral gavage of Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. showed a 9.5%, 10%, and 8.5% improvement in glucose tolerance, respectively (Figures 6A and 6B).

[0161] Cholesterol levels were measured in serum from mice in the various groups. The group receiving Akkermansia sp. by oral gavage showed an 11% improvement in cholesterol levels (Figure 7).

[0162] Resistin levels were measured in serum from mice in various groups. The Eubacterium eligens gavage group showed a 19% decrease in resistin levels compared to vehicle control. The Prevotella copri, Akkermansia sp., and Intestinimonas massiliensis + Akkermansia sp. gavage groups showed 14%, 11%, and 14% improvements in insulin levels (Figure 8). [Example]

[0163] Mouse models for evaluating 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 studies, 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 singly in polycarbonate cages with appropriate bedding. Animals were distributed to treatment groups noted in Table 3 on Study Day -1 in a manner that generated cohorts that were not significantly different in terms of body weight and non-fasting blood glucose based on Day -1 measurements. [Table 3]

[0164] The test articles Intestinimonas massiliensis and Akkermansia sp. (frozen, pasteurized, and lyophilized) were prepared daily and administered within 1 hour of formulation. Vehicle (Group 2) was administered once daily by oral gavage on days 1 through 84. The dose volume for each animal was 100 μl. The test articles (Groups 2 through 6) were administered once daily by oral gavage on days 1 through 84. The dose volume for each animal was 100 μl. Each dose was administered using a syringe equipped with a gavage cannula.

[0165] The control article (Group 7) was administered to the appropriate animals via subcutaneous injection in the interscapular region once daily on days 1 through 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 defined area. The first day of administration was designated Day 1.

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

[0167] Body weights were measured weekly throughout the study. As shown in Figure 10, the vehicle control group showed a significant increase in body weight compared to the chow-only group. The group receiving frozen Akkermansia sp. showed a 9% to 14% improvement in body weight from days 43 to 84.

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

[0169] At the end of the study (day 84), liver weights were determined. As shown in Figure 12, the vehicle control animals showed increased liver weight compared to the chow-only group. The group administered frozen Akkermansia sp. showed 37% lower fat accumulation compared to the control.

[0170] Insulin levels were measured in serum from mice in various groups. Measurements were performed 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 in animals compared to the chow diet. 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. The freeze-dried and pasteurized forms of Akkermansia sp. also showed decreased insulin levels (5% and 17%, respectively).

[0171] Insulin resistance was measured by 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 test. As shown in Figure 14, the vehicle control showed increased insulin resistance in the animals compared to the chow diet. 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 from mice in various groups. As shown in Figure 15, the group given Akkermansia sp. (pasteurized form) by oral gavage showed a 21% improvement in leptin levels. Akkermansia sp. in freeze-dried form also showed a decrease in leptin levels (7%).

[0173] PAI1 levels were measured in serum from mice in various groups. As shown in Figure 16, the group given Akkermansia sp. (frozen form) by oral gavage showed a 15% improvement in PAI1 levels. Akkermansia sp. (pasteurized form) showed an 8% improvement in PAI1 levels.

[0174] Resistin levels were measured in serum from mice in various groups. As shown in Figure 17, the I. massiliensis gavage group showed a 16% decrease in resistin levels compared to the vehicle control. Freeze-dried Akkermansia sp., pasteurized Akkermansia sp., and freeze-dried Akkermansia sp. showed 31%, 17%, and 32% improvements in resistin levels, respectively.

[0175] SCFA production by Akkermansia sp. and I. massiliensis was analyzed by growing the bacteria in RCM for 72 hours. The supernatant was collected, filtered, and SCFAs were detected by HPLC. In the medium, Akkermansia sp. showed propionate production, and I. massiliensis showed butyrate production (Figure 18). Both propionate and butyrate have been shown 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, adiposity, liver weight, insulin resistance, and resistin levels. A pasteurized form of Akkermansia sp. showed similar but less pronounced activity, suggesting that administering live bacteria may be preferable. Akkermansia sp. administered in lyophilized form was not as effective as the frozen form, but still showed improvements in resistin and leptin levels, and some improvements in adiposity and insulin levels. Without being bound by theory, the reason the lyophilized form was less effective may be due to insufficient hydration time of the cells when administered in this form. Since the transition period in mice is 3-4 hours, the lyophilized form may not have had sufficient hydration and transcriptional activity to achieve the same efficacy in this model. [Example]

[0177] Metabolic analysis of Akkermansia sp. strain AF3360009 Comparison of the metabolic capacity of Akkermansia sp. strains grown in YCFAC medium with that of Akkermansia muciniphila type strain BAA835. YCFAC medium was inoculated with a 1% overnight culture, and the supernatant was harvested 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 supernatants were collected and analyzed by CE-TOF-MS. Cationic conditions: Samples were 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 for 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 positive ionization mode, m / z range 50–1000. Anionic conditions: Samples were injected into a fused silica capillary (id 50 μm × 0 cm) using an Agilent CE-TOF System (Agilent Technologies Inc.) at 50 mbar for 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: 3.5 V, ESI negative ionization 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 min and identified against known standards. Quantities reported are peak areas.

[0180] Agmatine is metabolized from arginine by the enzyme arginine decarboxylase EC 4.1.1.19 (Piletz et al., 2013; Taksande et al., 2016). As shown in Figure 19, agmatine levels were 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 media levels indicate that Akkermansia sp. produces agmatine from arginine, while Akkermansia muciniphila ATCC BAA 835 consumes agmatine.

[0181] Extracellular ATP has been shown to induce inflammation (Cauwels et al., 2014). Akkermansia sp. and Akkermansia muciniphila BAA835 were evaluated for their ability to remove ATP from growth medium. YCFAC medium containing mucin (10 g / L) was inoculated with 1% overnight cultures and supplemented with 1 mM ATP. Supernatants were collected immediately after inoculation 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 supernatants were collected and analyzed for ATP levels.

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

[0183] Using whole-genome comparisons, an operon potentially encoding the enzymatic machinery involved in vitamin B12 synthesis is present in Akkermansia sp. but absent from the A. muciniphila ATCC BAA 835 type strain (Table 4). The ability to synthesize this important coenzyme suggests a broader metabolic potential of Akkermansia sp. compared to the 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 Ackermansia sp. deposited at the DSM under number DSM 33459.

2. (a) the E. eligens strain deposited at 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 at the DSM under number DSM 33458; (b) the I. massiliensis strain deposited at the DSM under number DSM 33460 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 I. massiliensis strain deposited at the DSM under number DSM 33460; (c) the P. eligens strain deposited at the DSM under number DSM 33457 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 I. massiliensis strain deposited at the DSM under number DSM 33457; 10. The composition of claim 1, further comprising: a P. copri strain or 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 at DSM under DSM number 33457; either (A) alone; and / or (B) in combination with culture supernatant derived from one or more of said strains.

3. A composition comprising isolated bacterial extracellular vesicles (EVs) derived from a biologically pure strain of Akkermansia sp. deposited at the DSM under number DSM 33459.

4. (a) EVs derived from the E. eligens strain deposited at 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 at the DSM under number DSM 33458; (b) EVs derived from the I. massiliensis strain deposited at the DSM under number DSM 33460 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 at the DSM under number DSM 33460; 4. The composition of claim 3, further comprising: (a) 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 the I. massiliensis strain; (c) EVs derived from the P. copri strain deposited at the DSM under number DSM 33457 or 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 at the DSM under number DSM 33457; either (A) alone; and / or (B) in combination with culture supernatant derived from one or more of said strains.

5. The composition of any one of claims 1 to 4, formulated for oral administration.

6. The composition of any one of claims 1 to 5, which is freeze-dried or lyophilized.

7. The composition of any one of claims 1 to 6, which is encapsulated or coated.

8. The composition of any one of claims 1 to 7, which is a food product, a food ingredient, a dietary supplement, or a pharmaceutical product.

9. At least 1 x 10 ４ CFU / g composition to at least 1 x 10 １２ 9. The composition of any one of claims 1 to 8, wherein CFU / g composition of bacteria are present in said composition.

10. The composition according to any one of claims 1 to 9, which is a probiotic.

11. The composition of any one of claims 1 to 10, which is pasteurized or heat treated.

12. The composition according to any one of claims 1 to 11, which is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient.

13. A tablet, sustained release capsule, sustained release granule, powder, sachet or gummy comprising the composition of any one of claims 1 to 12.

14. 14. A kit comprising: (a) (i) a composition according to any one of claims 1 to 12; or (ii) a tablet, sustained release capsule, sustained release granule, powder, sachet, or gummy according to claim 13; and b) instructions for administration to a subject.

15. 14. A pharmaceutical composition for the treatment and / or prevention of one or more obesity-related disorders in a subject in need thereof, comprising the composition of any one of claims 1 to 12 or the tablet, extended release capsule, extended release granules, powder, sachet or gummy of claim 13.

16. 16. The pharmaceutical composition of claim 15, wherein 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.

17. A method for producing a composition, comprising combining a biologically pure strain of Intestinimonas massiliensis with a biologically pure strain of Akkermansia sp. deposited with the DSM under number DSM 33459.

18. A method for producing a composition, comprising obtaining extracellular vesicles (EVs) from a biologically pure strain of Intestinimonas massiliensis and a biologically pure strain of Akkermansia sp. deposited with the DSM under number DSM 33459, and combining them.

19. 19. The method of claim 17 or 18, wherein the I. massiliensis comprises 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.

20. 20. The method of any one of claims 17 to 19, further comprising freezing and frying or freeze-drying the composition.

21. 13. Use of a composition according to any one of claims 1 to 12 in the manufacture of a medicament for the prevention and / or treatment of one or more obesity-related disorders in a subject in need thereof.

22. 22. The use of claim 21, wherein 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, reduced resistin levels and / or cardiovascular disease.

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