Compositions comprising bacterial strains for improving metabolic health

Abstract

[0013] Provided herein, inter alia, are compositions comprising one or more biologically pure bacterial strains and methods of making and using these compositions to treat and / or prevent one or more obesity-related disorders in a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 004,617, filed on April 3, 2020, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] In particular, provided herein are bacterial compositions useful for improving the metabolic health of a subject and methods of making and using the same. Background Art

[0004] The human gastrointestinal tract contains a complex and diverse microbial ecosystem. Not only are intestinal bacteria commensal, but they have also co-evolved in a symbiotic relationship with their hosts. The interaction between the intestinal microbiota and the host is complex. Beneficial intestinal bacteria have many important functions, and they directly or indirectly affect various physiological functions of the host, for example, they provide nutrition to their host, prevent infections caused by intestinal pathogens, and regulate normal immune responses. It has been determined that an imbalance in the composition of the microbiota can lead to various disease states in the host. Therefore, changes in the intestinal microbiota for the purpose of achieving, restoring and maintaining a favorable balance in the ecosystem, as well as the activity of the microorganisms present in the gastrointestinal tract, are necessary for maintaining and improving the health of the host.

[0005] The first generation of probiotics is a live microorganism mainly derived from the genus Lactobacillus and the genus Bifidobacterium, which are usually minor components of the digestive tract or obtained by use, as a dairy starter culture. Traditionally, the first generation of probiotics is mainly for intestinal and immune health. It has been shown that some probiotics (such as Bifidobacterium lactis (B.lactis) B420) also play a beneficial activity related to metabolic health, for example, in the reduction of body fat mass and some improvements to blood sugar and insulin. However, the current first generation of probiotics does not seem to provide the best solution in terms of glucose and insulin metabolism, that is, as a potential therapeutic agent or preventive agent for type 2 diabetes, prediabetes or metabolic syndrome.

[0006] Therefore, what is needed are additional microorganisms that are identified based on their natural occurrence in the digestive tract of metabolically healthy individuals and selected based on their ability to maintain and optimize metabolic health and prevent disease.

[0007] The subject matter disclosed herein addresses these needs and provides additional benefits as well. Summary of the invention

[0008] In particular, provided herein are compositions comprising one or more biologically pure bacterial strains and methods for preparing these compositions, as well as methods for treating and / or preventing one or more obesity-related disorders in a subject in need thereof, such as, but not limited to, obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, glucose intolerance, abnormal lipid metabolism, non-alcoholic fatty liver disease, hepatic steatosis, leptin resistance, decreased resistin levels, and / or cardiovascular disease.

[0009] Thus, in some aspects, provided herein is a composition comprising at least one or more of: (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 with the German Collection of Microorganisms (DSM) under the number DSM 33457; and / or (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 Akkermansia species has a genome-wide average nucleotide identity (gANI) of less than about 95% with (i) Akkermansia muciniphila; or (ii) Akkermansia glycanophila. 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 in DSM and numbered 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 in DSM and numbered DSM 33460. In some embodiments of any of the embodiments disclosed herein, the composition comprises (a) a strain of Eubacterium fastidious deposited in DSM and numbered DSM 33458, or a live strain having all the identifying characteristics of the strain of Eubacterium fastidious deposited in DSM and numbered DSM 33458; (b) a strain of Enteromonas massiae deposited in DSM and numbered DSM 33460, or a live strain having all the identifying characteristics of the strain of Enteromonas massiae deposited in DSM and numbered DSM 33460; (c) a strain of P. copri deposited in DSM and numbered DSM 33457, or a live strain having all the identifying characteristics of the strain of P. copri deposited in DSM and numbered DSM 33457; and / or (d) a species of Akkermansia deposited in DSM and numbered DSM 33459, or a live strain having all the identifying characteristics of the strain of Akkermansia deposited in DSM and numbered DSM 33459. Viable strains of all the identifying characteristics of Akkermansia species of 33459, (A) alone; and / or (B) in combination with culture supernatants derived from one or more of these strains.In some embodiments, the composition comprises (b) a biologically pure strain of Enteromonas massiae; and (d) a biologically pure strain of Akkermansia spp., wherein the Akkermansia spp. 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 Enteromonas massiae deposited in DSM and numbered DSM 33460. In some embodiments of any of the embodiments disclosed herein, the whole genome average nucleotide identity (gANI) between the Akkermansia spp. 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) a live strain of Enteromonas massiae deposited in DSM and numbered DSM 33460 or having all the identifying characteristics of the Enteromonas massiae strain deposited in DSM and numbered DSM 33460; and (d) a live strain of Akkermansia species deposited in DSM and numbered DSM 33459 or having all the identifying characteristics of the Akkermansia species deposited in DSM and numbered 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 product, a food ingredient, a dietary supplement, or a medicament. In some embodiments of any of the embodiments disclosed herein, at least about 1x10. 4 CFU / g composition to at least about 1x10 12 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 has been 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 a further aspect, provided herein is a composition comprising isolated bacterial extracellular vesicles (EVs) derived from at least one or more of: (a) a biologically pure strain of Eubacterium fastidiosa; (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 with the German Collection of Microorganisms (DSM) under the number DSM 33457; and / or (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 further comprises one or more bacteria from (a), (b), (c) and / or (d). In some embodiments of any of the embodiments disclosed herein, the whole genome average nucleotide identity (gANI) between the Akkermansia species and (i) Akkermansia muciniphila; or (ii) Akkermansia glycanophila 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 Eubacterium fastidiosa deposited in DSM and numbered 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 Enteromonas massiliense deposited in DSM and numbered DSM 33460.In some embodiments of any of the embodiments disclosed herein, the composition comprises (a) EVs derived from a strain of Eubacterium fastidiosa deposited in DSM and numbered DSM 33458, or a live strain having all the identifying characteristics of the strain of Eubacterium fastidiosa deposited in DSM and numbered DSM 33458; (b) EVs derived from a strain of Enteromonas massiae deposited in DSM and numbered DSM 33460, or a live strain having all the identifying characteristics of the strain of Enteromonas massiae deposited in DSM and numbered DSM 33460; (c) EVs derived from a strain of Prevotella hominis deposited in DSM and numbered DSM 33457, or a live strain having all the identifying characteristics of the strain of Prevotella hominis deposited in DSM and numbered DSM 33458. 33457; and / or (d) EVs derived from or having all the identifying characteristics of the Akkermansia species deposited in DSM and numbered DSM 33459, (A) alone; and / or (B) in combination with culture supernatants 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 Enteromonas massii; and (d) EVs derived from a biologically pure strain of Akkermansia species, wherein the Akkermansia species 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 Enteromonas massii deposited in DSM under the number DSM 33460. In some embodiments of any of the embodiments disclosed herein, the genome-wide average nucleotide identity (gANI) between the Akkermansia species and (i) Akkermansia muciniphila; or (ii) Akkermansia glycanophila is less than about 95%. In some embodiments of any of the embodiments disclosed herein, the composition comprises (b) EVs derived from a strain of Enteromonas massiae deposited in DSM and numbered DSM 33460, or a live strain having all the identifying characteristics of the strain of Enteromonas massiae deposited in DSM and numbered DSM 33460; and (d) EVs derived from a species of Akkermansia deposited in DSM and numbered DSM 33459, or a live strain having all the identifying characteristics of the species of Akkermansia deposited in DSM and numbered 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 product, a food ingredient, a dietary supplement, or a medicament. In some embodiments of any of the embodiments disclosed herein, at least about 1 x 10. 4 CFU / g composition to at least about 1 x 10 12 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 has been 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.

[0011] In other aspects, provided herein is a tablet, extended release capsule, extended release granule, powder, sachet, or sticky formulation comprising any of the compositions disclosed herein (eg, a probiotic composition).

[0012] In a further aspect, provided herein is a kit comprising (a) (i) any composition disclosed herein (such as a probiotic composition); or (ii) any of the tablets, extended-release capsules, extended-release granules, powders, sachets or adhesives disclosed herein, and b) written instructions for administration to a subject.

[0013] On the other hand, there is provided herein a method for treating and / or preventing one or more obesity-related disorders in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein or any of the tablets, extended release capsules, extended release granules, powders, sachets, or adhesives disclosed herein comprising the pharmaceutical compositions 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, glucose intolerance, abnormal lipid metabolism, nonalcoholic fatty liver disease, hepatic steatosis, leptin resistance, decreased resistin levels, and / or cardiovascular disease.

[0014] In another aspect, provided herein is a method for preparing a composition, the method comprising combining a biologically pure strain of Enteromonas massiae and a biologically pure strain of Akkermansia spp., wherein the Akkermansia spp. is not (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia. In some embodiments, the whole genome average nucleotide identity (gANI) between the Akkermansia spp. and (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphilia is less than about 95%. In some embodiments of any of the embodiments disclosed herein, Enteromonas massiae comprises a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of Enteromonas massiae deposited in DSM and numbered DSM 33460. In some embodiments of any of the embodiments disclosed herein, Enteromonas massiae comprises an Enteromonas massiae strain deposited in DSM, numbered DSM 33460, or a live strain having all the identifying characteristics of an Enteromonas massiae strain deposited in DSM, numbered DSM 33460; and wherein the Akkermansia spp. comprises an Akkermansia spp. deposited in DSM, numbered DSM 33459, or a live strain having all the identifying characteristics of an Akkermansia spp. deposited in DSM, numbered DSM 33459. In some embodiments of any of the embodiments disclosed herein, the method further comprises freeze-drying or lyophilizing the composition.

[0015] In other aspects, a pharmaceutical composition for preventing and / or treating one or more obesity-related disorders in a subject in need thereof is provided herein, the composition comprising any pharmaceutical composition disclosed herein (such as a probiotic composition) for a subject or comprising any of the tablets disclosed herein, extended release capsules, extended release granules, powders, sachets, or adhesives disclosed herein of the pharmaceutical composition 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, glucose intolerance, abnormal lipid metabolism, nonalcoholic fatty liver disease, hepatic steatosis, leptin resistance, decreased resistin levels, and / or cardiovascular disease. In other aspects, provided herein is a method for providing a source for the production of agmatine in the intestine to treat and / or prevent the following diseases, which are diabetes, inflammation, oxidative stress, neurotrauma and neurodegenerative diseases, opioid addiction, mood disorders, cognitive disorders and cancer, the method comprising administering to a subject any pharmaceutical composition disclosed herein (such as a probiotic composition) or any of the tablets, extended release capsules, extended release granules, powders, sachets or adhesives disclosed herein containing the pharmaceutical composition disclosed herein.

[0016] Each of the aspects and embodiments described herein can be used together unless explicitly or clearly excluded from the context of an embodiment or aspect.

[0017] Throughout the specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. The disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A Depicted are the differentially abundant fecal 16S rRNA operational taxonomic units (OTUs) measured in lean healthy individuals versus obese prediabetic individuals and their corresponding associations with clinical metabolic markers using Spearman correlation coefficient analysis. Figure 1B Depicted are OTUs taxonomically defined as Enteromonas, Prevotella, Eubacterium, and Akkermansia species identified from a clinical study comparing lean healthy individuals with obese prediabetic (elevated BMI, insulin, and glucose) individuals, showing that these species were positively associated with metabolic health.

[0019] Figure 2 Depicted is a phylogenetic tree consisting of strain AF3360009 and strains from the class Verrucomicrobiae included in Ouwerkerk et al., 2016. The phylogenetic tree was reconstructed using the neighbor-joining method with 1000 bootstraps. Numbers represent bootstrap values. Legend bars indicate 5% sequence divergence. Chlamydia trachomatis was used as an outgroup.

[0020] Figure 3 Depicted are scanning electron microscopy images of strain AF3360009. They are oval or elongated with filamentous structures when grown on (left) YCFA and mucin compared to (right) YCFA.

[0021] Figure 4 Depicted are graphs showing the effects of Eubacterium fastidiosa, Enteromonas massiae, Prevotella hominis, Akkermansia spp., and Enteromonas massiae + Akkermansia spp. on insulin levels in the DIO mouse model.

[0022] Figure 5 Depicted are graphs showing the effects of Eubacterium fastidiosa, Enteromonas massiae, Prevotella hominis, Akkermansia spp., and Enteromonas massiae + Akkermansia spp. on leptin levels in the DIO mouse model.

[0023] Fig. 6A and Figure 6BDepicted are graphs showing the effects of Eubacterium fastidiosa, Enteromonas massiae, Prevotella hominis, Akkermansia spp., and Enteromonas massiae + Akkermansia spp. on glucose tolerance in the DIO mouse model.

[0024] Figure 7 Depicted are graphs showing the effects of Eubacterium fastidiosa, Enteromonas massiae, Prevotella hominis, Akkermansia spp., and Enteromonas massiae + Akkermansia spp. on cholesterol levels in the DIO mouse model.

[0025] Figure 8 Depicted are graphs showing the effects of Eubacterium fastidiosa, Enteromonas massiae, Prevotella hominis, Akkermansia spp., and Enteromonas massiae + Akkermansia spp. on resistin levels in the DIO mouse model.

[0026] Fig. 9 Depicted is an Akkermansia gANI dendrogram comparing the publicly available genomes of A. muciniphila, A. glycaniphilia, and strain AF3360009.

[0027] Fig.10 Effects of Enteromonas massiliense and Akkermansia species (frozen, pasteurized, and lyophilized) on body weight in the DIO model are depicted.

[0028] Fig.11 The effects of Enteromonas massiliense and Akkermansia species (frozen, pasteurized and freeze-dried) on body fat mass are depicted.

[0029] Fig.12 The effects of Enteromonas massiliense and Akkermansia species (frozen, pasteurized and freeze-dried) on liver weight are depicted.

[0030] Fig.13 The effects of Enteromonas massiliense and Akkermansia species (frozen, pasteurized, and lyophilized) on insulin levels in the DIO model are depicted.

[0031] Fig.14 Measurement of insulin resistance by HOMA-IR is depicted.

[0032] Fig.15 Effects of Enteromonas massiliense and Akkermansia species (frozen, pasteurized, and freeze-dried) on leptin levels in the DIO model are depicted.

[0033] Fig.16 The effects of Enteromonas massiliense and Akkermansia species (frozen, pasteurized, and lyophilized) on plasminogen activator inhibitor-1 (PAI 1) levels in the DIO model are depicted.

[0034] Fig.17 Effects of Enteromonas massiliense and Akkermansia species (frozen, pasteurized, and lyophilized) on resistin levels in the DIO model are depicted.

[0035] Fig.18 SCFA production by Akkermansia species and Enteromonas massiliense is depicted.

[0036] Fig.19 Sample comparison of CE-TOFMS relative peak areas of agmatine is depicted.

[0037] Fig. 20 The removal of extracellular ATP by Akkermansia strains is depicted. DETAILED DESCRIPTION

[0038] Many previous studies have shown that probiotic bacteria (e.g. from the genera Lactobacillus and Bifidobacterium) support the growth of beneficial intestinal bacterial flora, but it appears that certain beneficial probiotic strains can also positively alter host metabolic pathways. Bioactive substances produced by microbial organisms influence carbohydrate and lipid metabolism and modulate intestinal and systemic inflammatory processes. Therefore, there is growing interest in identifying nutritional supplements and probiotic foods that are effective in controlling obesity.

[0039] The inventors of the present application have surprisingly found that microorganisms other than the commonly used probiotics Lactobacillus and Bifidobacterium can successfully change intestinal metabolism and alleviate the conditions associated with obesity. These beneficial microorganisms are found to be enriched in the digestive system of healthy people of normal weight, but lacking in individuals suffering from one or more obesity-related disorders. Supplementing one or more beneficial microorganisms in the diet of mice simulating human obesity can significantly improve one or more indicators associated with obesity-related negative conditions.

[0040] I. Definitions

[0041] As used herein, "microorganism" or "microbe" refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0042] As used herein, the term "probiotic" refers to a composition for animal consumption (i.e., as an animal feed or a component of an animal feed) containing viable (i.e., live) microorganisms, i.e., microorganisms that are capable of survival and reproduction, which, when administered in sufficient amounts, confer health benefits on a subject (see Hill et al. 2014 Nature Revs Gastro&Hep [Natural Review Gastroenterology and Hepatology] 11, 506-514, which is incorporated herein by reference in its entirety). Probiotics may include one or more of any of the microbial strains described herein (e.g., any of 1, 2, 3, or 4). Probiotics are distinguished from bacterial compositions that have been killed, for example, by pasteurization or heat treatment. In certain embodiments of the methods disclosed herein, administration of non-viable bacterial compositions to treat one or more metabolic disorders is also contemplated.

[0043] As used herein, a bacterial "strain" refers to a bacterium that remains genetically unchanged when grown or reproduced, including a plurality of identical bacteria.

[0044] "At least one strain" means a single strain, but also refers to a strain mixture comprising at least two microbial strains. "A mixture of at least two strains" means a mixture of two, three, four, five, six or even more strains. In some embodiments of the strain mixture, the ratio may vary between 1% and 99%. When the mixture comprises more than two strains, the strains may be present in the mixture in substantially equal proportions or in different proportions.

[0045] For purposes of this disclosure, a "biologically pure strain" means a strain that does not contain other bacterial strains in an amount sufficient to interfere with the replication of the strain or that can be detected by normal bacteriological techniques. When used in conjunction with the organisms and cultures described herein, "isolate" includes not only biologically pure strains, but also any culture of an organism grown or maintained other than those found in nature. In some embodiments, these strains are mutants, variants or derivatives of the following strains: Eubacterium fastidiosa, Enteromonas massiae, Prevotella hominis, and / or Akkermansia species, wherein the Akkermansia species is not Akkermansia muciniphila or Akkermansia glycanophila that also provide benefits comparable to those provided by the following strains: Eubacterium fastidiosa, Enteromonas massiae, Prevotella hominis, and / or Akkermansia species, wherein the Akkermansia species is not Akkermansia muciniphila or Akkermansia glycanophila. In some embodiments, the strains are strains having all the identifying characteristics of the following strains: Eubacterium fastidious, Enteromonas massiae, Prevotella hominis, and / or Akkermansia species, wherein the Akkermansia species is not Akkermansia muciniphila or Akkermansia glycanophila. In addition, each individual strain (Eubacterium fastidious, Enteromonas massiae, Prevotella hominis, and / or Akkermansia species, wherein the Akkermansia species is not Akkermansia muciniphila or Akkermansia glycanophila) or any combination of these strains may also provide one or more of the benefits described herein. It will also be clear that the addition of other microbial strains, carriers, additives, enzymes, yeasts, etc. will also provide one or more benefits or improve one or more metabolic disorders in a subject and will not constitute substantially different bacterial strains.

[0046] The term "16S rRNA" or "16S ribosomal RNA" means the rRNA that constitutes the small subunit of the ribosome in prokaryotes. In bacteria, this sequence can be used to identify and characterize operational taxa.

[0047] As used herein, the term "sequence identity" or "sequence similarity" means that two polynucleotide sequences (candidate sequence and reference sequence) are identical (i.e., 100% sequence identity) or similar (i.e., on a nucleotide-by-nucleotide basis) over the length of the candidate sequence. When comparing a candidate sequence to a reference sequence, the candidate sequence may contain additions or deletions (i.e., gaps) as compared to a reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). Optimal sequence alignment for determining sequence identity can be performed using any number of publicly available local alignment algorithms known in the art (e.g., ALIGN or Megalign (DNASTAR Corporation)), or by inspection.

[0048] The terms "percent (%) sequence identity" or "percent (%) 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 a reference polynucleotide sequence, after the sequences have been optimally aligned and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0049] As used herein, the term "subject" or "patient" means a mammal (e.g., a person). In some embodiments, the subject suffers from a related disease, disorder or condition, such as, but not limited to, one or more metabolic disorders, such as obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, glucose intolerance, abnormal lipid metabolism, nonalcoholic fatty liver disease, hepatic steatosis, leptin resistance, decreased levels of resistin, and / or cardiovascular disease. In some embodiments, the subject is susceptible to a disease, disorder or condition. In some embodiments, the subject shows one or more symptoms or features of a disease, disorder or condition. In some embodiments, the subject does not show any symptoms or features of a disease, disorder or condition. In some embodiments, the subject is a person with one or more of the following characteristics, the typical features of which are susceptible to disease, disorder or condition or have the risk of the disease, disorder or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who receives and / or has received diagnosis and / or therapy administration.

[0050] 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 (such as one or more metabolic disorders, for example obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, glucose intolerance, abnormal lipid metabolism, non-alcoholic fatty liver disease, hepatic steatosis, leptin resistance, decreased resistin levels and / or cardiovascular disease) and / or one or more of its associated symptoms, or preventing a subject from acquiring or reacquiring a disorder or condition, or reducing the risk of a subject acquiring or reacquiring a disorder or condition or one or more of its associated symptoms.

[0051] As used herein, the term "reduce" with respect to a particular trait, feature, characteristic, biological process, or phenomenon refers to a reduction in a particular trait, feature, characteristic, biological process, or phenomenon. A trait, feature, characteristic, biological process, or phenomenon can be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%.

[0052] As used herein, "administer" or "administering" means the action of introducing one or more compositions containing one or more microbial strains into a subject, such as by feeding or oral administration. Compositions containing one or more microbial strains can also be administered in one or more doses.

[0053] As used herein, "effective amount" means that the composition containing one or more microbial strains improves the amount of one or more indicators of the subject. The improvement of one or more indicators of the subject can be measured as described herein or by other methods known in the art (such as but not limited to the treatment and / or prevention of obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, glucose intolerance, abnormal lipid metabolism, non-alcoholic fatty liver disease, hepatic steatosis, leptin resistance, resistin level decline and / or any one of cardiovascular disease).

[0054] Certain ranges are presented herein with numerical values ​​preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that provides a substantial equivalent of the specifically recited number in the context in which it is presented. For example, with respect to numerical values, the term "about" refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.

[0055] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0056] It is also noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0057] Still, it should be noted that the term "consisting essentially of" as used herein refers to a composition wherein the component(s) following the term, in the presence of other known component(s), is a total amount of less than 30% by weight of the total composition and does not affect or interfere with the action or activity of the component(s).

[0058] It is further noted that the term "comprising" as used herein is intended to include but is not limited to one or more components following the term "comprising". The one or more components following the term "comprising" are required or mandatory, but the composition comprising the one or more components may further include other non-mandatory or optional one or more components.

[0059] It is also noted that the term "consisting of" as used herein is meant to include and be limited to the component(s) following the term "consisting of." Thus, the component(s) following the term "consisting of" are required or mandatory, and one or more other components are not present in the composition.

[0060] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as if such lower numerical limits were expressly written herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as if such higher numerical limits were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range that falls within such wider numerical range, as if such narrower numerical ranges were all expressly written herein.

[0061] 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.

[0062] Additional definitions of terms may appear throughout this specification.

[0063] II. Composition

[0064] A. strain

[0065] In some embodiments, provided herein are pharmaceutical compositions containing beneficial microorganisms, which can be used as therapeutic agents for application to subjects in physiological stress periods (disease states, metabolic states, etc.) to prevent disease and promote healthy intestinal metabolism. In other embodiments, provided herein are compositions containing beneficial microorganisms as supplements or food additives, which can be used as part of a daily nutritional regimen for application to subjects in physiological stress periods (metabolic states, etc.) to promote healthy intestinal metabolism. Probiotics are another term that can be used for these compositions containing viable microorganisms. The term "viable microorganisms" means microorganisms that are metabolically active or capable of differentiation. In some embodiments, the compositions containing beneficial microorganisms disclosed herein include viable probiotic products and / or, in specific embodiments, compositions containing inactive bacteria (such as heat-treated or pasteurized compositions).

[0066] The strains provided herein include a biologically pure strain of Eubacterium fastidiosa, a biologically pure strain of Enteromonas massiliense; a biologically pure strain of Prevotella hominis, and a biologically pure strain of Akkermansia sp., wherein the Akkermansia sp. is not Akkermansia muciniphila; or Akkermansia glycanophila.

[0067] Eubacterium fastidious strains, Enteromonas massi strains, Prevotella human strains and Akkermansia species strains were deposited in the German Collection of Microorganisms (DSM) (Inhoffenstraβe 7B, 38124 Braunschweig, GERMANY) on March 4, 2020, and the deposit numbers are DSM 33458, DSM33460, DSM 33457 and DSM 33459, respectively. The deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. In a non-limiting embodiment, one or more strains provided herein can be used as probiotics.

[0068] Compositions containing microorganisms (such as probiotic compositions) can include those containing one or more strains of Eubacterium fastidiosa (such as any of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains) (such as Eubacterium fastidiosa strain DSM33458). Eubacterium fastidiosa is a Gram-positive bacterium in the family Eubacteriaceae characterized by a rigid cell wall. Compositions containing beneficial microorganisms can further include those containing one or more strains of Eubacterium fastidiosa and one or more strains of Enteromonas massiliense, Prevotella hominis and / or Akkermansia species (such as any of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains).

[0069] Compositions containing microorganisms (such as probiotic compositions) can include those containing one or more strains of Enteromonas massiae (such as any of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains) (such as Enteromonas massiae strain DSM33460). Enteromonas massiae is a non-motile Gram-negative rod with an average diameter of 0.5 μm and a length of 1.8 μm, and no spore-forming activity (Durand et al., 2017, New Microbes New Infect. [New Microorganisms and New Infections], 15: 1-2). Compositions containing beneficial microorganisms can further include those containing the following compositions: one or more strains of Enteromonas massiae and one or more strains of Eubacterium fastidious, Prevotella hominis and / or Akkermansia species (such as any of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains). In some embodiments, the composition containing beneficial microorganisms includes both Enteromonas massiae and Akkermansia species (e.g., an Akkermansia species that is not Akkermansia muciniphila or Akkermansia glycanophila, such as Akkermansia strain DSM 33459). In addition, when cultured or administered together, one or more Enteromonas massiae strains (e.g., Enteromonas massiae strain DSM 33460) and one or more Akkermansia species (e.g., Akkermansia strain DSM 33459) exhibit one or more physiological or metabolic properties that are not present in Enteromonas massiae (e.g., Enteromonas massiae strain DSM 33460) and Akkermansia species (e.g., Akkermansia strain DSM 33459) cultured alone. These properties may include, but are not limited to, changes in the amount and / or type of organic acids produced, changes in metabolic profiles, and / or changes in the composition of the culture medium in which the bacteria are cultured together.

[0070] Compositions containing microorganisms (such as probiotic compositions) can include those containing one or more strains of human Prevotella (such as any of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains) (such as human Prevotella strain DSM33457). Human Prevotella is a gram-negative bacterium commonly found in the intestine. Compositions containing beneficial microorganisms can further include those containing one or more strains of human Prevotella and one or more strains of Enteromonas massiliense, Eubacterium fastidiosa and / or Akkermansia species (such as any of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains).

[0071] Microorganism-containing compositions (e.g., probiotic compositions) may include those containing one or more strains (e.g., any of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) of an Akkermansia species, wherein the Akkermansia species is not Akkermansia muciniphila or Akkermansia sacchariphila (e.g., Akkermansia strain DSM 33459). Until 2016, the genus contained only a single known species, Akkermansia muciniphila. That year, an intestinal mucin-degrading bacterium, Akkermansia sacchariphila, was first isolated from the feces of a reticulated python (Ouworkerk et al., 2016, International Journal of Systematic and Evolutionary Microbiology. 66(11):4614-4620). As will be described in more detail below, without being bound by theory, the inventors of the present invention believe that a new species of Akkermansia has been identified based on the fact that the genome-wide average nucleotide identity (gANI) between the isolated Akkermansia species and Akkermansia muciniphila and Akkermansia sacchariphila is below the species boundary cutoff of 95% identity (Goris et al., 2007, Int J Syst Evol Microbiol, 57, 81-91). In some embodiments, the Akkermansia species (e.g., Akkermansia strain DSM 33459) in the microorganism-containing compositions disclosed herein has a gANI of less than 95% (e.g., any of about 94%, 93%, 92%, 91%, 90%, 89%, or 88% (e.g., 87.58%)) compared to the genome of Akkermansia muciniphila. In another embodiment, the Akkermansia species (e.g., Akkermansia strain DSM 33459) in the microorganism-containing composition disclosed herein has a gANI of less than 95% (e.g., 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%, such as 70.17%) compared to the genome of Akkermansia glycanophila. Compositions containing beneficial microorganisms can further include those containing one or more strains of Akkermansia species and one or more strains of Enteromonas massiliense, Eubacterium fastidiosa and / or Prevotella hominis (such as about any of 1, 2, 3, 4, 5, 6, 7, or 8 or more strains).

[0072] The microorganism-containing compositions disclosed herein (such as probiotic compositions) may include one or more strains of Eubacterium fastidiosa having a 16S ribosomal RNA sequence that exhibits at least about 97.0% sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence comprising SEQ ID NO: 1. The beneficial microorganism-containing compositions (such as probiotic compositions) may include one or more strains of Enteromonas massiense having a 16S ribosomal RNA sequence that exhibits at least about 97.0% sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence comprising SEQ ID NO: 2. A composition containing beneficial microorganisms (such as a probiotic composition) can include one or more strains of Prevotella hominis having a 16S ribosomal RNA sequence that exhibits at least about 97.0% sequence similarity (such as 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.

[0073] The microorganism-containing compositions disclosed herein (such as probiotic compositions) may include one or more strains of Eubacterium fastidiosa (such as Eubacterium fastidiosa strain DSM 33458), one or more strains of Enteromonas massiae (such as Enteromonas massiae strain DSM 33460), one or more strains of Akkermansia spp., and / or one or more strains of Prevotella hominis (such as Prevotella hominis strain DSM 33457) (i.e., these compositions include actual bacteria (viable or non-viable) from these strains) and / or one or more culture supernatants derived from the cultivation (alone or co-cultivation) of these strains, wherein the Akkermansia spp. is not Akkermansia muciniphila or Akkermansia glycanophila (e.g. Akkermansia strain DSM 33459).

[0074] B. Preparations

[0075] Typically, the microorganism-containing compositions disclosed herein (e.g., probiotic compositions) include bacteria, such as one or more bacterial strains. In some embodiments of the invention, the composition is formulated into a freeze-dried or lyophilized form. For example, the microorganism-containing composition can include granules or gelatin capsules, such as hard gelatin capsules, which include bacterial strains disclosed herein.

[0076] 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.

[0077] In some embodiments, any microorganism-containing composition disclosed herein is encapsulated to enable bacterial strains to be delivered to the intestine. Encapsulation protects the composition from degradation before delivery to the target location, and the degradation pathway is, for example, rupture due to chemical or physical stimulation (such as pressure, enzymatic activity or physical disintegration, which may be triggered by pH changes). Any suitable encapsulation method can be used. Exemplary encapsulation techniques include entrapment in a porous matrix, attachment or adsorption on a solid carrier surface, self-aggregation by flocculation or with a cross-linking agent, and mechanical containment of a microporous membrane or microcapsule.

[0078] The compositions containing microorganisms disclosed herein can be administered orally and can be in the form of tablets, capsules or powders. Other ingredients (such as, for example, vitamin C or minerals) can be included as oxygen scavengers and prebiotic substrates to improve delivery and / or partial or complete colonization and survival in vivo. Alternatively, the compositions containing microorganisms disclosed herein (such as probiotic compositions) can be taken orally as food or nutritional products (such as milk or whey-based fermented dairy products).

[0079] 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, such as pasteurized or heat-treated bacterial compositions.

[0080] In some embodiments, disclosed herein are pharmaceutical compositions containing microorganisms, which include a therapeutically effective amount of a bacterial strain disclosed herein. The therapeutically effective amount of the bacterial strain is sufficient to produce a beneficial effect on the patient. The therapeutically effective amount of the bacterial strain may be sufficient to cause delivery to the subject's intestine and / or partial or complete colonization of the subject's intestine.

[0081] For example, for an adult, a suitable daily dose of bacteria may be about 1 x 10 3 About 1X10 11 Colony forming unit (CPU); for example, about 1x 10 7 About 1x 10 10 CPU; in another example, about 1x 10 6 About 1x 10 10 GPU; in another example, about 1x 10 7 About 1x 10 11 CPU; in another example, about 1x 10 8 About 1x 10 10CPU; in another example, about 1x 10 8 About 1x 10 11 CPU. In certain embodiments, the dosage of the bacteria is at least 10 9 cells per day, such as at least 10 10 , at least 10 11 or at least 10 12 cells.

[0082] In certain embodiments, the composition containing microorganisms contains about 1 x 10 6 About 1x10 11 CFU / g of bacterial strain; for example, about 1 x 10 8 About 1x 10 10 CFU / g. The dosage may be, for example, 1 g, 3 g, 5 g and 10 g.

[0083] In certain embodiments, the amount of the bacterial strain is about 1 x 10 3 About 1x10 11 colony forming units.

[0084] In certain embodiments, any microorganism-containing composition disclosed herein is administered at a dosage of between 500 mg and 1000 mg, between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and 750 mg, or between 750 mg and 1000 mg. In certain embodiments, the lyophilized bacteria in any microorganism-containing composition disclosed herein are administered at a dosage of between 500 mg and 100 mg, between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and 750 mg, or between 750 mg and 1000 mg.

[0085] Typically, probiotics are optionally combined with at least one suitable prebiotic compound. Prebiotic compounds are typically indigestible carbohydrates, such as oligosaccharides or polysaccharides or sugar alcohols, that are not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and trans-galacto-oligosaccharides.

[0086] In certain embodiments, the probiotic composition disclosed herein is formulated to include a prebiotic compound in an amount of about 1 wt % to about 30 wt % (e.g., 5 wt % to 20 wt %) relative to the total weight of the composition. Carbohydrates may be selected from the group consisting of: oligofructose (or FOS), short-chain oligofructose, inulin, isomaltooligosaccharide, pectin, oligoxylose (or XOS), chitosan oligosaccharide (or COS), human milk oligosaccharides, β-glucan, gum arabic modified starch and resistant starch, polydextrose, D-tagatose, gum arabic fiber, carob, oat and citrus fiber. In one aspect, the prebiotic is a short-chain oligofructose (shown as FOSs-cc herein below for simplicity); the FOSs-cc is not a digestible carbohydrate, is generally obtained by conversion of beet sugar and includes a sucrose molecule bound to three glucose molecules. In some embodiments, any of the probiotics disclosed herein may be formulated with additional probiotics derived from Lactobacillus and Bifidobacterium (such as Bifidobacterium lactis B420).

[0087] In some embodiments, disclosed herein is a pharmaceutical composition containing a microorganism, which may further include a pharmaceutically acceptable excipient or carrier. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field. Examples of suitable carriers include, but are not limited to, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include, but are not limited to, ethanol, glycerol, and water. The selection of pharmaceutical carriers, excipients, or diluents may be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include or in addition to carriers, excipients, or diluents, any suitable adhesive, lubricant, suspending agent, coating agent (such as a gastrointestinal resistant coating agent that will not dissolve or degrade before reaching the small intestine or large intestine) or solubilizing agent. Examples of suitable adhesives include, but are not limited to, starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition. Examples of 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.

[0088] In some embodiments, disclosed herein is a composition containing microorganisms that can be formulated as a food product. For example, in addition to the effects of the present invention (such as in nutritional supplements), food products can also provide nutritional benefits. Similarly, food products can be formulated to enhance the taste of the composition of the present invention, or to make the composition more consumer-attractive by being more similar to ordinary food rather than a pharmaceutical composition. In certain embodiments, the composition containing microorganisms is formulated as a milk-based product. As used herein, the term "milk-based product" means any liquid or semisolid milk-based or whey-based product with different fat contents. The milk-based product can be, for example, milk, goat milk, sheep milk, skim milk, whole milk, milk without any processing formed by milk powder and whey reorganization, or processed products, such as yogurt, coagulated milk, curd, acid milk, acid whole milk, buttermilk and other acidic milk products. Another important group includes milk beverages, such as whey beverages, fermented milk, condensed milk, baby or toddler milk; flavored milk, ice cream; candy and other milk-containing foods.

[0089] In certain embodiments, the compositions containing microorganisms disclosed herein contain a single bacterial strain or species, and do not contain any other bacterial strains or species. Such compositions can only contain trace or biologically irrelevant other bacterial strains or species. Such compositions can be cultures that are substantially free of other organism species. In certain embodiments, the compositions of the present invention are composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 bacterial strains or species. In certain embodiments, these compositions are composed of 1 to 10 (such as 1 to 5) bacterial strains or species.

[0090] Microorganism-containing compositions used in accordance with the methods disclosed herein may or may not require marketing approval.

[0091] In some cases, the lyophilized bacterial strain is reconstituted prior to administration. In some cases, reconstitution is performed by using a diluent described herein.

[0092] In certain embodiments, disclosed herein are pharmaceutical compositions containing a microorganism, which may include a pharmaceutically acceptable excipient, diluent, or carrier.

[0093] 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 amount of the bacterial strain is sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of: obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, glucose intolerance, abnormal lipid metabolism, non-alcoholic fatty liver disease, hepatic steatosis, leptin resistance, decreased resistin levels and / or cardiovascular disease.

[0094] In certain embodiments, the present invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol and sorbitol.

[0095] In certain embodiments, the present invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water.

[0096] In certain embodiments, the present invention provides the above-mentioned pharmaceutical composition, which comprises an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweetener, gum arabic, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.

[0097] In certain embodiments, the present invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant, and a stabilizer.

[0098] In certain embodiments, the present invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.

[0099] In certain embodiments, the present invention provides the above-mentioned pharmaceutical composition, wherein the bacterial strain is lyophilized.

[0100] In certain embodiments, the pharmaceutical composition described above, wherein when the composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere with 50% relative humidity, at least 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the bacterial strains as measured in colony forming units are still present 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.

[0101] The bacterial strains disclosed herein can be cultured using standard microbiological techniques, such as those described in the Examples section or otherwise well known in the art.

[0102] In further 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 vesicle" or "EV" refers to a composition derived from bacteria, comprising bacterial lipids contained in nanoparticles and bacterial proteins and / or bacterial nucleic acids and / or carbohydrate moieties. These EVs may contain 1, 2, 3, 4, 5, 10 or more than 10 different lipid species. EVs may contain 1, 2, 3, 4, 5, 10 or more than 10 different protein species. EVs may contain 1, 2, 3, 4, 5, 10 or more than 10 different nucleic acid species. EVs may contain 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 comprising EVs that have been separated from at least one associated substance found in any material associated with EVs in a source material or any method used to produce the preparation (e.g., separated from at least one other bacterial component). It also refers to a composition that has been 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 more than 10,000-fold.

[0103] The EVs described herein can be prepared using any method known in the art. In some embodiments, EVs are prepared without an EV purification step. For example, in some embodiments, bacteria containing EVs described herein are killed using a method that keeps bacterial EVs intact, and the resulting bacterial components, including EVs, are used in the methods and compositions described herein. In some embodiments, bacteria are killed using antibiotics (e.g., using antibiotics described herein). In some embodiments, bacteria are killed using UV irradiation. In some embodiments, EVs described herein are purified from one or more other bacterial components. Methods for purifying EVs from bacteria are known in the art. In some embodiments, EVs are prepared from bacterial cultures using methods described in S. Bin Park et al. PLoS ONE. [Public Library of Science · Comprehensive] 6(3): el7629 (2011) or G. Norheim et al., PLoS ONE. [Public Library of Science · Comprehensive] 10(9): e0134353 (2015), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to precipitate the bacteria (e.g., centrifuged at 10,000 x g for 30 minutes at 4°C). In some embodiments, the culture supernatant is then passed through a filter to exclude intact bacterial cells (e.g., a 0.22 μm filter). In some embodiments, the filtered supernatant is centrifuged to precipitate bacterial EVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C). In some embodiments, the EVs are further purified by resuspending the resulting EV pellet (e.g., in PBS) and applying the resuspended EVs to a sucrose gradient (e.g., a discontinuous sucrose gradient of 30%-60%) followed by centrifugation (e.g., at 200,000 x g for 20 hours at 4°C). The EV band can be collected and washed (e.g., with PBS) and centrifuged to precipitate the EVs (e.g., at 150,000 x g for 3 hours at 4°C). The purified EVs can be stored at, for example, -80°C until use. In some embodiments, the EVs are further purified by treatment with DNase and / or proteinase K.

[0104] For example, in some embodiments, the bacterial culture disclosed herein can be centrifuged at 11,000 x g for 20-40 minutes at 4°C to precipitate the bacteria. The culture supernatant can be passed through a 0.22 μm filter to exclude intact bacterial cells. The filtered supernatant can then be concentrated using a method that may include, but is not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, in the case of ammonium sulfate precipitation, 1.5-3M ammonium sulfate can be slowly added to the filtered supernatant while stirring at 4°C. The precipitate can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 minutes at 4°C. The resulting precipitate contains bacterial EVs and other debris.

[0105] Using ultracentrifugation, the filtered supernatant can be centrifuged at 100,000-200,000 x g at 4°C for 1-16 hours. The centrifuged pellet contains bacterial EVs and other debris. In some embodiments, the supernatant can be filtered using filtration techniques, such as by using an Amicon Ultra spin filter or by tangential flow filtration, so as to retain species with a molecular weight >50 or 100 kDa.

[0106] Alternatively, EVs can be obtained from bacterial cultures continuously during growth or at selected time points during growth by connecting the bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen). The ATF system retains intact cells (>0.22 μm) in the bioreactor and allows smaller components (e.g., EVs, free proteins) to pass through the filter for collection. For example, the system can be configured so that the <0.22 μm filtrate is then passed through a second filter of 100 kDa, so that species such as EVs between 0.22 μm and 100 kDa are collected, and species less than 100 kDa are pumped back into the bioreactor. Alternatively, the system can be configured to allow the culture medium in the bioreactor to be supplemented 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.

[0107] The EVs obtained by the methods provided herein can be further purified by size-based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, using sucrose gradients or Optiprep gradients. In brief, when using the sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatant, the precipitate is resuspended in 60% sucrose, 30mM Tris (pH 8.0). If filtration is used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30mM Tris (pH 8.0) using an Amicon Ultra column. The sample is applied to a 35%-60% discontinuous sucrose gradient and centrifuged at 200,000xg for 3-24 hours at 4°C. In brief, when using the Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatant, the precipitate is resuspended in 35% Optiprep in PBS. In some embodiments, if filtration is 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 xg at 4°C for 3-24 hours.

[0108] In some embodiments, to confirm the sterility and isolation of the EV preparation, EVs are serially diluted onto agar medium (which is used for routine culture of the bacteria under test) and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 μm filter to exclude intact cells. To further increase purity, isolated EVs can be treated with DNase or proteinase K.

[0109] III. Methods

[0110] A. Methods for treating or preventing disease

[0111] Further provided herein are methods for treating and / or preventing one or more obesity-related disorders in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of any microorganism- and / or EV-containing pharmaceutical composition disclosed herein, the one or more obesity-related disorders comprising obesity, metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, glucose intolerance, abnormal lipid metabolism, non-alcoholic fatty liver disease, hepatic steatosis, leptin resistance, decreased resistin levels, and / or cardiovascular disease.

[0112] 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 of more than 25 is considered overweight, while obesity is defined as a BMI of 30 or above, with a BMI of 35 or above being considered a serious comorbidity and a BMI of 40 or above being considered morbid obesity. For purposes of the present invention, "obesity" shall mean a BMI of 30 or above.

[0113] One in five overweight people is affected by "metabolic syndrome." Metabolic syndrome is one of the fastest growing obesity-related health problems in the United States and is characterized by a cluster of health problems, including obesity, high blood pressure, abnormal blood fat levels, and high blood sugar. According to the Centers for Disease Control and Prevention (CDC), metabolic syndrome affects nearly a quarter (22%) of the U.S. population, an estimated 47 million people. The cluster of problems that characterize metabolic syndrome can increase a person's risk for more serious health problems, such as diabetes, heart disease, and stroke.

[0114] Overweight and obese people have an increased incidence of heart disease and are therefore more likely to suffer heart attacks, congestive heart failure, sudden cardiac death, angina, and abnormal heart rhythms than those who maintain a healthy body mass index. Obesity generally increases the risk of heart disease due to its negative impact on blood lipid levels, which increase in obese patients, which then in turn increases triglyceride levels and decreases high-density lipoprotein (also known as HDL). People with excess body fat have higher levels of triglycerides and low-density lipoprotein (also known as LDL or "bad cholesterol") in their blood, as well as lower levels of HDL cholesterol. This combination creates optimal conditions for the development of atherosclerotic heart disease.

[0115] Being overweight or obese increases your risk of developing high blood pressure. Hypertension, or high blood pressure, greatly increases your risk of heart attack, stroke, and kidney failure. In fact, blood pressure increases as you gain weight. Even losing 10 pounds can lower blood pressure—and weight loss has the biggest impact on people who are overweight and already have high blood pressure.

[0116] Obesity is associated with the development of diabetes. More than 80% of people who develop type 2 diabetes, the most common form of the disease, are obese or overweight. Type 2 diabetes develops when the production of insulin by the pancreas is impaired in the presence of insulin resistance in tissues and organs in the body. Because obesity reduces the ability of insulin to control blood sugar (glucose), the risk of developing diabetes increases because the body begins to overproduce insulin to regulate blood sugar levels. Over time, the body is no longer able to keep blood sugar levels within the normal range. Ultimately, the inability to achieve a healthy blood sugar balance leads to the development of type 2 diabetes. In addition, obesity complicates the management and treatment of type 2 diabetes by increasing insulin resistance and glucose intolerance, which makes drug treatments for the disease less effective. In many cases, reducing weight to a normal range normalizes blood sugar and restores insulin sensitivity.

[0117] Childhood obesity is also a major public health problem, especially in Western countries. Children aged 2-18 years are considered obese if their BMI is greater than the 95th percentile. Despite policies aimed at reducing its prevalence, childhood obesity has more than doubled in the past 30 years and has tripled in adolescents. As in adults, childhood obesity causes hypertension, dyslipidemia (abnormal lipid metabolism), chronic inflammation, increased clotting tendency, endothelial dysfunction, and hyperinsulinemia. This constellation of cardiovascular disease risk factors has been identified in children as young as 5 years old.

[0118] Method disclosed herein relates to prevention, inhibition and treatment of obesity-related disorders. As used herein, "obesity-related disorders" include but are not limited to obesity, undesirable weight gain and excessive eating disorders (e.g., overeating, bulimia, compulsive eating or lack of appetite control, each of which can optionally cause undesirable weight gain or obesity), metabolic syndrome, diabetes, insulin deficiency-related disorders, insulin resistance-related disorders, glucose intolerance, abnormal lipid metabolism, nonalcoholic fatty liver disease, hepatic steatosis, leptin resistance, resistin level decline and / or cardiovascular disease. As used herein, "obesity" refers to class I obesity, class II obesity, class III obesity and pre-obesity (e.g., "overweight") defined by the World Health Organization.

[0119] It is expected that the reduction of body fat will provide various primary and / or secondary benefits in a subject (e.g., in a subject diagnosed with obesity-related complications such as obesity-related disorders), such as, for example, increased insulin responsiveness or reduced glucose intolerance (e.g., in a subject diagnosed with type II diabetes); reduced high blood pressure; reduced cholesterol levels and / or elevated LDL and / or VLDL; reduction in (or reduced risk or progression of) cardiovascular disease (including ischemic heart disease, arterial vascular disease, angina, 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, poor mobility, 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 elevated resistin levels.

[0120] In another embodiment, the present disclosure relates to a method comprising administering to a subject an effective amount of any microorganism-containing and / or EV-containing composition disclosed herein (such as a probiotic composition) to reduce obesity. In some embodiments, the subject's obesity is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, including all values ​​falling between these percentages. The reduction of obesity can be measured by any method known in the art, such as a reduction in BMI.

[0121] In another embodiment, the present disclosure relates to a method comprising administering to a subject an effective amount of any microorganism- and / or EV-containing pharmaceutical composition (such as a probiotic composition) disclosed herein to alleviate metabolic syndrome, diabetes (including type II diabetes), insulin resistance and / or glucose intolerance. In some embodiments, the rate of one or more of these conditions is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, including all values ​​falling between these percentages. The reduction of one or more of metabolic syndrome, diabetes (including type II diabetes), insulin resistance and / or glucose intolerance can be determined by any method known in the art, such as blood glucose measurement and A1C determination.

[0122] In another embodiment, the present disclosure relates to a method comprising administering to a subject an effective amount of any microorganism-containing and / or EV-containing pharmaceutical composition disclosed herein (such as a probiotic composition) to treat one or more liver disorders (including but not limited to abnormal lipid metabolism, non-alcoholic fatty liver disease and / or hepatic steatosis). In some embodiments, the incidence of the liver disorder is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, including all values ​​falling between these percentages. The alleviation of one or more liver disorders can be determined by any method known in the art.

[0123] In another embodiment, the present disclosure relates to a method comprising administering to a subject an effective amount of any microorganism- and / or EV-containing composition disclosed herein (such as a probiotic composition) to treat leptin resistance and / or decreased resistin levels. 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% relative to a subject with leptin resistance and / or decreased resistin levels who is not administered one or more microorganism- and / or EV-containing compositions disclosed herein, including all values ​​falling between these percentages. Leptin resistance and / or decreased resistin levels can be determined by any method known in the art.

[0124] In another embodiment, the present disclosure relates to a method comprising administering to a subject an effective amount of any microorganism- and / or EV-containing pharmaceutical composition disclosed herein (such as a probiotic composition) to treat one or more cardiovascular disease-related disorders (including but not limited to ischemic heart disease, arterial vascular disease, angina, myocardial infarction and / or stroke). In some embodiments, the incidence of liver disorders is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% relative to subjects with one or more cardiovascular disease-related disorders who are not administered one or more microorganism- and / or EV-containing pharmaceutical compositions disclosed herein, Any of the percentages, including all values ​​falling between these percentages. The reduction of one or more disorders associated with cardiovascular disease can be determined by any method known in the art.

[0125] In yet another embodiment, any microorganism- and / or EV-containing composition disclosed herein (such as a probiotic composition) administered to a subject comprises one or more strains of Eubacterium fastidious having a 16S ribosomal RNA sequence that exhibits at least about 97.0% sequence similarity (such as any one 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: 1. The beneficial microorganism- and / or EV-containing composition (such as a probiotic composition) may include one or more strains of Enteromonas massiense having a 16S ribosomal RNA sequence that exhibits at least about 97.0% sequence similarity (such as any one 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: 2. A composition containing beneficial microorganisms and / or EVs (such as a probiotic composition) may include one or more strains of Enteromonas massii having a 16S ribosomal RNA sequence that exhibits at least about 97.0% sequence similarity (such as 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.

[0126] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) strains of Eubacterium fastidiosa, Enteromonas massiliense, Prevotella hominis, and Akkermansia spp. are administered to a subject at a ratio of at least about 1×10 4 CFU / subject / day to at least about 1 x 10 12 CFU / subject / day, such as about 1×10 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 9 CFU / subject / day, 1×10 10 CFU / subject / day, 1×10 11 CFU / subject / day or 1×10 12 Any of CFU / subject / day, including all values ​​falling between these measurements.

[0127] B. Method for preparing microbial composition

[0128] Also provided herein are methods for preparing compositions containing microorganisms and / or EVs (such as probiotic compositions), which methods include combining a biologically pure strain of Enteromonas massiae with a biologically pure strain of Akkermansia species, wherein the Akkermansia species is not Akkermansia muciniphila or Akkermansia polysaccharophyla. The Akkermansia species is different from other known Akkermansia species by a genome-wide average nucleotide identity (gANI) of at least 95%. The Enteromonas massiae may comprise a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity (such as any one of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% sequence similarity) to the 16S ribosomal RNA sequence of Enteromonas massiae deposited in DSM and numbered DSM 33460.

[0129] Furthermore, the method for preparing the composition may further comprise lyophilizing or freeze-drying the microbial composition.The method may further comprise the further step of packaging the feed additive composition for storage or transport.

[0130] C. Application

[0131] Preferably, the microorganism-containing and / or EV-containing compositions disclosed herein will be administered to the gastrointestinal tract so as to enable the bacterial strains of the invention to be delivered to the intestine and / or partially or completely colonized in the intestine. Typically, the compositions of the invention are administered orally, but they may also be administered by rectal, intranasal or buccal or sublingual routes.

[0132] In certain embodiments, the microorganism-containing and / or EV-containing compositions disclosed herein can be administered as a foam, spray, or gel.

[0133] In certain embodiments, the microorganism-containing and / or EV-containing compositions disclosed herein can be administered as suppositories, such as rectal suppositories, for example, in the form of cocoa butter (cocoa butter), synthetic hard fats (e.g., suppocire, witepsol), glycerinated gelatin, polyethylene glycol, or glycerin soap compositions.

[0134] In certain embodiments, the microorganism- and / or EV-containing compositions disclosed herein are administered to the gastrointestinal tract via a tube (e.g., a nasogastric tube, an orogastric tube, a gastric tube, a jejunostomy tube (J tube), a percutaneous endoscopic gastrostomy (PEG)) or a port (e.g., a chest wall port with access to the tirestomach, jejunum, and other suitable access ports).

[0135] The microorganism-containing and / or EV-containing compositions disclosed herein can be administered once, or for the pharmaceutical compositions disclosed herein they can be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention will be administered daily.

[0136] In certain embodiments of the present invention, treatment with a microorganism-containing and / or EV-containing pharmaceutical composition disclosed herein is accompanied by an assessment of the intestinal microbiota of the subject according to the methods disclosed herein. If the strain of the present invention is not delivered successfully and / or partial or complete colonization is not achieved, so that no efficacy is observed, the treatment can be repeated, or if delivery and / or partial or complete colonization is successful, and efficacy is observed, the treatment can be stopped. In certain embodiments, the pharmaceutical composition of the present invention can be administered to pregnant animals, such as mammals (such as humans), to prevent their children from developing symptoms in utero and / or after birth.

[0137] The pharmaceutical compositions of the present invention can be administered to patients who have been diagnosed with, or have been identified as being at risk of, a disease or condition mediated by histone deacetylase activity. These compositions can also be administered as a preventive measure to prevent the occurrence of a disease or condition mediated by histone deacetylase activity in healthy patients.

[0138] The microorganism-containing and / or EV-containing compositions disclosed herein can be administered to a subject who has been identified as having an abnormal gut microbiota. For example, the subject may have reduced or no colonization with Eubacterium fastidiosa, Enteromonas massiliense, Prevotella hominis, and / or Akkermansia species, wherein the Akkermansia species is not Akkermansia muciniphila or Akkermansia glycanophila.

[0139] In some embodiments, disclosed herein are microorganism-containing and / or EV-containing compositions administered as food products (e.g., nutritional supplements).

[0140] Typically, the microorganism-containing and / or EV-containing pharmaceutical compositions disclosed herein are used to treat human subjects, although they can be used to treat animals, including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits or polygastric animals (such as ruminants). The compositions of the invention can be used to enhance the growth and performance of animals. When administered to animals, oral gavage can be used.

[0141] IV. Kit

[0142] Further provided herein are kits containing one or more microbial strains and / or EVs derived from one or more microbial strains disclosed herein. These kits may include one or more (such as any one of 1, 2, 3 or 4) strains and / or EVs derived from one or more microbial strains provided herein and instructions for proper storage, maintenance and administration to a subject to treat or prevent one or more obesity-related disorders, wherein the one or more microbial strains include Akkermansia species, Eubacterium fastidiosa strains (e.g., Eubacterium fastidiosa strain DSM 33458), Enteromonas massiae strains (e.g., Enteromonas massiae strain DSM 33460) and / or Prevotella humanis strains (e.g., Prevotella humanis strain DSM33457), wherein the Akkermansia species is not Akkermansia muciniphila or Akkermansia glycanophila (e.g., Akkermansia strain DSM 33459). In one embodiment, the kit may include Akkermansia strain DSM 33459 and Enteromonas massiae strain DSM 33460.

[0143] The invention may be further understood by reference to the following examples, which are provided by way of illustration and not limitation.

[0144] Examples

[0145] Example 1: Isolation of strains

[0146] Isolation of Enteromonas masseille: Clinical stool samples with higher Enteromonas masseille populations, as determined by 16S community analysis of all clinical stool samples, were used for the enrichment protocol, and all work was performed under anaerobic conditions. The samples were diluted 1:100 in basal bicarbonate buffered medium, and a total of 25 ml of inoculated medium was sealed in a 50 ml glass vial. The vials were incubated under anaerobic conditions at 37°C. Approximately 5 ml of culture was removed each day and distributed into 4 tubes, 2 for DNA extraction and 2 for strain isolation. Samples were taken on days 4, 5, 6, 7, 8, and 11 after the start of enrichment. DNA was extracted from the samples every day, and 16S community analysis sequencing was performed to determine the bacterial population. It was determined that the sample on day 11 had the highest percentage of Enteromonas masseille, so one of the strain isolation samples from day 11 was used for dilution and plated on basal bicarbonate buffered medium agar plates. Basal bicarbonate buffered medium: 0.53 g / L disodium hydrogen phosphate, 0.41 g / L potassium dihydrogen phosphate, 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.

[0147] Isolation of Human Prevotella: Human Prevotella strains were isolated by plating diluted fecal material directly on BHIB agar plates and incubating anaerobically for 24 hours. BHIB: Brain Heart Infusion Agar supplemented with 10% sheep blood. (Commercially available, BD221843) When grown in liquid broth, BHIS was used. BHIS: Brain Heart Infusion Supplemented with Yeast Extract, Vitamin K1 and Hemin.

[0148] Isolation of Akkermansia Species: Akkermansia strains were isolated from clinical stool samples by directly plating diluted fecal material on YCFA medium containing 10 g / L mucin.

[0149] Isolation of Eubacterium fastidiosa: Eubacterium fastidiosa strains were isolated by plating diluted fecal material directly on BHIB agar plates and incubating anaerobically for 24 hours. BHIB: Brain Heart Infusion agar supplemented with 10% sheep blood. (Commercially available, BD221843) When grown in liquid broth, BHIS was used. BHIS: Brain Heart Infusion supplemented with yeast extract, vitamin K1 and hemin.

[0150] Genome Sequencing: All strains were provided with genome sequences by the same method. The strains were grown on BHIB or YCFA agar plates. The growth was removed from the agar plates with a large loop, being careful not to remove the agar. The amount of growth determined the number of wells processed for DNA extraction. For 1 well, the growth was resuspended in 750 μl of the first solution used in the DNA extraction kit, PowerMag bead solution. If there was enough growth to amplify to multiple wells, an additional volume was used. The cells were resuspended and the resuspended cells were distributed to the desired number of wells. The DNA extraction protocol was followed using the kit instructions. For elution, slightly less elution buffer was used per well to obtain a higher DNA concentration. After the DNA extraction was complete, similar wells were combined. DNA concentration was determined using the Quant-It PicoGreen DSDNA Detection Kit from Invitrogen.

[0151] Candidates were isolated from fecal samples of healthy donors and identified by whole genome sequencing. Ranking was performed by statistical analysis including relevant assay values ​​(insulin, BMI, glucose, DXATotFAT), percent abundance, and number of lean samples carrying the candidate. Candidates were selected based on the correlation analysis and isolate availability. Four of these candidates were selected for further study ( Figure 1A and Figure 1B ).

[0152] Example 2: Identification of new species of Akkermansia

[0153] Unless otherwise stated, all work was performed in an anaerobic chamber using a gas mixture of N2 / CO2 / H2 (85 / 10 / 5%).

[0154] Strain AF33600009 was isolated during the second round of isolation, which was performed after a round of usual isolation, where the target was any top-ranked candidate bacteria. The clinical samples selected for this round of isolation showed a higher abundance of top-ranked candidates based on the 16S community analysis analyzed previously. The strain was isolated from stool sample F015V3. The isolation method used in this round was selection on YCFA medium containing 10 g / L mucin.

[0155] The samples used in this round of separation were previously made from fecal material from sample F015V3 mixed with glycerol to a final glycerol concentration of 25%. These samples were stored at -80°C until needed. One sample was removed from the freezer, placed in an anaerobic chamber, and left to thaw at room temperature for approximately 10 minutes. All work was performed in the anaerobic chamber unless otherwise stated. Measured portions were removed and serially diluted in mucin-free YCFA broth. From 10 -4 , 10 -5 and 10 -6 A 100 μl sample of the dilution was plated onto YCFA + mucin agar in an omni dish. The bacterial cells were spread using about twelve sterile glass beads to evenly spread the dilution sample on the agar surface. The plate was incubated in an anaerobic environment formed by an anaerobic chamber with a sachet at 37°C for about 72 hours.

[0156] Deoxygenated growth medium, i.e., YCFA + 10 g / L mucin, was dispensed into 1 ml deep well plates, 350 μl per well. Single colonies were picked and inoculated into plates with pre-dispensed medium, 1 colony per well. Plates were covered with a breathable cover to allow gas exchange. Plates were incubated at 37°C for approximately 216 hours. The cultures were gently mixed using a 96-well tip Integra pipette. An aliquot of the culture was taken for 16S PCR analysis and the remaining culture was mixed with sterile, deoxygenated 50% glycerol + 1 g / L L-cysteine ​​to a final glycerol concentration of 25%. These cultures were transferred to appropriate long-term storage vials and stored at -80°C.

[0157] 16S Identification: The cell culture sample 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 TGGCTC and 1492R: CGG TTA CCT TGT TAC GAC TT. The PCR reaction conditions and thermal cycler settings for polymerase Q5 were standard. The sample of the 16S PCR reaction was run on a gel to confirm the presence of a 16S PCR product of the expected size. The sample of the 16S PCR reaction was enzymatically purified using the ExoSAP-IT Express for PCR Purification Kit. The sample was then sent to an external third-party vendor for Sanger sequencing. The 16C primer most commonly used for 16S Sanger sequencing is 515F: GTG CCA GCM GCC GCG GTA A.

[0158] The 16S sequence was then compared to the 16S amplicon sequences in the top candidate list. These results indicated that the closest matching candidate was Akkermansia muciniphila. The vials corresponding to the wells containing the desired strains were removed from the freezer and placed in an anaerobic chamber so that a small portion of the frozen culture could be removed from the vials and streaked onto a YCFA + 10 g / L mucin agar plate. The plates were incubated at 37°C in an anaerobic chamber with a sachet until there was enough growth to prepare a frozen stock and extract DNA.

[0159] The genome of strain AF33600009: The DNA extraction kit used was from Qiagen DNA KF (King Fisher) kit. Scrape the most recently streaked growth from the YCFA + 10g / L mucin agar plate and resuspend the cells in the first solution from the DNA extraction kit. Gently resuspend the cells by gently pipetting to break up cell clumps. Evenly distribute the cell resuspension into multiple wells of the PowerMag bead plate. The number of wells is determined based on the amount and density of cells in the cell suspension. Then follow the manufacturer's protocol to extract DNA. After DNA extraction is complete, similar wells are combined into one DNA sample and the DNA concentration is determined using the Invitrogen Quant-iT PicoGreendsDNA Quantification Kit. The DNA is then sent for whole genome sequencing.

[0160] Sequencing libraries were prepared with Nextera Flex kit (Illumina), and sequencing was performed on MiSeq (Illumina) with paired reads of 2x150nt. Genome sequencing data were assembled using internal approaches. In short, reads were filtered and trimmed based on quality, and then corrected using BFC (Li, 2015). Corrected reads were assembled using SPAdes assembler (Bankevich et al., 2012) with kmer length options "31, 55, 77, 99, 121". Compilation was corrected using Pilon (Walker et al., 2014). After assembly, open reading frames (ORFs) were predicted and annotated by Prokka (Seemann, 2014). 16S rRNA genes were predicted by Barrnap, and the closest species identification was performed by RDP paired comparison tools (Fish et al., 2013).

[0161] The draft genome of strain AF33600009 consists of 31 contigs with an N50 of 331,405 bp and a coverage of 125x. The genome size is 3.19 Mb, which is larger than that of two other Akkermansia species: Akkermansia muciniphila and Akkermansia spp. T (2.66Mbp) and Akkermansia saccharophila Pyt T The genome size of the type strain is 3.07 Mb. The G+C content of the genomic DNA is 57.7%. Since only two species are currently known in the genus Akkermansia, a phylogenetic tree was reconstructed with these three strains and some strains from the class Verrucomicrobia included in the publication describing Akkermansia saccharophila (Ouwerkerk et al., 2016).

[0162] Phylogenetic analysis showed that strain AF33600009 was a member of the genus Akkermansia, which was closely related to Akkermansia muciniphila T The closest relative ( Figure 2 ). Strain AF33600009 and Akkermansia muciniphila T The average genome-wide nucleotide identity (gANI) between strain AF33600009 and Akkermansia saccharophila Pyt T The average nucleotide identity of the whole genome between AF33600009 and AF33600009 was only 70.17%. Based on the gANI value below the species boundary cutoff of 95% (Goris et al., 2007), strain AF33600009 was proposed as a new species within the genus Akkermansia.

[0163] A gANI dendrogram of Akkermansia was generated using a number of publicly available genomes that are closest to the A. muciniphila model strain genome, GCF_000020225.1, as well as two publicly available genomes of A. sacchariphila and the genome of strain AF33600009 ( Fig. 9 ). All genomes from A. muciniphila were clustered together, the two A. glycanophila publicly available genomes were clustered together, while AF3360009 formed a separate cluster distinct from the other two species.

[0164] 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) with strain AF33600009 and Akkermansia muciniphila ATCC strain BAA835 grown on BHIA for standard sample preparation to extract fatty acid methyl esters for identification. The samples were then loaded onto a gas chromatograph for analysis. Pattern recognition software was used to generate FAME profiles of the samples. The samples were then compared to determine similarity. As shown in Table 1, the FAME profiles showed significant differences between strain AF3360009 and ATCC strain BAA835.

[0165] Table 1: Comparative FAME profiles of strains BAA835 and AF3360009.

[0166]

[0167]

[0168] The cells of strain AF33600009 were oval or elongated. When the cells were grown on medium containing mucin, more elongated shapes were observed. When grown on YCFA+mucin medium, the cells were more aggregated and formed more filaments ( Figure 3 ).

[0169] Example 3: Mouse model for evaluating the efficacy of candidate bacteria

[0170] The diet-induced obesity (DIO) mouse model was used to evaluate the efficacy of the following candidate bacteria: Eubacterium fastidiosa, Enteromonas massiliense, Prevotella hominis, and Akkermansia species described in Example 2.

[0171] The efficacy of Eubacterium fastidiosa, Enteromonas massi, Prevotella hominis, and Akkermansia species in improving metabolic disorders was evaluated in the DIO mouse model. For animal studies, upon arrival, Group 1 animals were maintained on PMI Nutrition International, LLC, certified rodent food No. 5 CR4. Groups 2-9 animals were maintained on Research Diet D12492. Animals were housed individually in polycarbonate cages containing appropriate bedding. On day -1 of the study, animals were assigned to the treatment groups mentioned in Table 2 to produce cohorts that had no significant differences in body weight and non-fasting blood glucose (based on measurements on day -1).

[0172] Table 2: Experimental design

[0173]

[0174] The test articles Eubacterium fastidiosa, Enteromonas massi, Prevotella hominis, Akkermansia spp., and Enteromonas massi+Akermansia spp. were prepared daily and administered within one hour of preparation. Vehicle was administered once daily by oral gavage from day 1 to day 36 (Group 2). The dose volume was 100 μl per animal. The test articles were administered once daily by oral gavage from day 1 to day 36 (Groups 2-7). The dose volume was 100 μl per animal. Each dose was administered using a syringe with an attached gavage cannula.

[0175] From Day 1 to Day 36, the control (Group 8) was administered once daily by subcutaneous injection into the interscapular region of the appropriate animals. The dose volume for each animal was based on the latest body weight measurement. Each dose was administered into the demarcated area using a syringe / needle. The first day of dosing was designated as Day 1.

[0176] Study parameters included mortality / moribund status examinations, daily observations, body weight measurements, food consumption, stool samples, blood glucose measurements, oral glucose tolerance test, qNMR assessments, cytokine assessments, and clinical chemistry parameters. Blood samples were collected at designated time points throughout the dosing period and on the scheduled euthanasia day for biomarker evaluation.

[0177] Insulin levels were measured in serum obtained from different groups of mice. The group fed with E. massiense showed a 12% decrease in insulin levels compared to vehicle control. The groups fed with Prevotella hominis, Akkermansia species, and E. massiense + Akkermansia species showed improvements in insulin levels of 50%, 50%, and 64%, respectively ( Figure 4 ).

[0178] Leptin levels were measured in serum obtained from different groups of mice. The Eubacterium fastidiosa gavage group showed a 21% decrease in insulin levels compared to vehicle controls. The Prevotella hominis, Akkermansia species, and Enteromonas massiliense + Akkermansia species gavage groups showed 20%, 15%, and 25% improvements in leptin levels, respectively ( Figure 5 ).

[0179] After a 2-hour fast, all mice were intraperitoneally administered 2.0 g / kg glucose (10 mL / kg). Blood glucose was checked by tail snip using a handheld glucometer at the following times relative to the glucose dose: 0 (pre-glucose dose, 15, 30, 60, 90, and 120 minutes). The human Prevotella, Akkermansia species, and Enteromonas massiliense + Akkermansia species gavage groups showed improvements in glucose tolerance by 9.5%, 10%, and 8.5%, respectively ( Fig. 6A and Figure 6B ).

[0180] Cholesterol levels were measured in serum obtained from different groups of mice. The group fed with Akkermansia spp. showed an 11% improvement in cholesterol levels ( Figure 7 ).

[0181] Resistin levels were measured in serum obtained from different groups of mice. The Eubacterium fastidiosa gavage group showed a 19% decrease in resistin levels compared to vehicle controls. The human Prevotella, Akkermansia species, and Enteromonas massiliense + Akkermansia species gavage groups showed improvements in insulin levels of 14%, 11%, and 14% ( Figure 8 ).

[0182] Example 4: Mouse Model for Evaluation of Formulations

[0183] The efficacy of Enteromonas massiliense and Akkermansia species (frozen, pasteurized and lyophilized) in improving metabolic disorders was evaluated in the DIO mouse model. For animal studies, upon arrival, Group 1 animals were maintained on PMI Nutrition International LLC certified rodent food No. 5 CR4. Groups 2-7 animals were maintained on research diet D12492. Animals were housed individually in polycarbonate cages with appropriate bedding. On day -1 of the study, animals were assigned to the treatment groups mentioned in Table 3 to produce cohorts that were not significantly different in body weight and non-fasting blood glucose (based on measurements on day -1).

[0184] Table 3: Experimental design

[0185]

[0186] The test articles Enteromonas massiliense and Akkermansia species (frozen, pasteurized, and lyophilized) were prepared daily and administered within one hour of preparation. Vehicle was administered once daily by oral gavage from day 1 to day 84 (Group 2). The dose volume was 100 μl per animal. The test articles were administered once daily by oral gavage from day 1 to day 84 (Groups 2-6). The dose volume was 100 μl per animal. Each dose was administered using a syringe with an attached gavage cannula.

[0187] From Day 1 to Day 84, the control article (Group 7) was administered once daily by subcutaneous injection into the interscapular region of the appropriate animals. The dose volume for each animal was based on the latest body weight measurement. Each dose was administered into the demarcated area using a syringe / needle. The first day of dosing was designated as Day 1.

[0188] Study parameters included mortality / moribund examinations, daily observations, body weight measurements, food consumption, stool samples, blood glucose measurements, qNMR assessments, and clinical chemistry parameters.

[0189] During the study period, body weight was measured weekly. Fig.10 As shown, the vehicle control showed a significant increase in body weight compared to the food only group. From day 43 to day 84, the group receiving the frozen form of Akkermansia spp. showed a 9% to 14% improvement in body weight.

[0190] Body composition analysis was performed by qNMR (Bruker NMR LF90II). Body composition was determined at the beginning (day -2 and day -1) and end (day 83 and day 84) of the study. Fig.11 As shown, the vehicle control showed significant fat accumulation in the animals compared to the food only group. The group that received the frozen form of Akkermansia spp. showed a 25% reduction in fat accumulation compared to the control. Freeze-dried and pasteurized forms of Akkermansia spp. also showed a reduction in fat accumulation (5.5% and 5%, respectively).

[0191] Liver weights were measured at the end of the study (Day 84). Fig.12 As shown, the vehicle control showed an increase in liver weight in the animals compared to the food only group. The group that received the frozen form of Akkermansia spp. showed a 37% reduction in fat accumulation compared to the control.

[0192] Insulin levels were measured in serum obtained from mice of different groups. Measurements were performed every 2 weeks during the study. The area under the curve was calculated based on the values ​​obtained during the study. Fig.13As shown, the vehicle control showed an increase in insulin levels in the animals compared to the food group. The group receiving Enteromonas massii showed a 17% decrease in insulin levels compared to the control, while the group receiving the frozen form of Akkermansia species showed a 22% decrease in insulin levels. Freeze-dried and pasteurized forms of Akkermansia species also showed a decrease in insulin levels (5% and 17%, respectively).

[0193] Insulin resistance was measured by HOMA-IR. The measurement was based on fasting blood glucose and insulin values. The area under the curve was calculated based on the values ​​obtained during the study. Fig.14 As shown, the vehicle control showed an increase in insulin resistance in the animals compared to the food group. The group that received Enteromonas massiliense showed a 10% decrease in insulin levels compared to the control, while the group that received the frozen form of Akkermansia species showed a 20% decrease in insulin levels. The pasteurized form of Akkermansia species also showed a 12% decrease in insulin levels.

[0194] Leptin levels were measured in serum obtained from different groups of mice. Fig.15 As shown, the group gavaged with Akkermansia spp. (pasteurized form) showed a 21% increase in leptin levels. The lyophilized form of Akkermansia spp. also showed a decrease in leptin levels (7%).

[0195] The levels of PAI1 in the sera obtained from the different groups of mice were measured. Fig.16 As shown, the Akkermansia spp. (frozen form) gavage group showed a 15% improvement in PAI1 levels. Akkermansia spp. (pasteurized form) showed an 8% improvement in PAI1 levels.

[0196] Resistin levels were measured in sera obtained from different groups of mice. Fig.17 As shown, the Enteromonas massiliense gavage group showed a 16% decrease in resistin levels compared to the vehicle control. The frozen, pasteurized, and lyophilized forms of Akkermansia species showed 31%, 17%, and 32% improvements in resistin, respectively.

[0197] The production of SCFAs by Akkermansia species and Enteromonas massiense was analyzed by growing the bacteria in RCM for 72 hours. The supernatant was collected, filtered and the SCFAs were detected by HPLC. In the culture medium, Akkermansia species showed the production of propionate and Enteromonas massiense showed the production of butyrate ( Fig.18 ). Both propionate and butyrate have been shown to play key roles in regulating host metabolic health (Chambers et al. 2018, et al. 2016).

[0198] In this model, Akkermansia species (frozen form) showed significant improvements in body weight, fat accumulation, liver weight, insulin resistance and resistin levels. Akkermansia species in pasteurized form showed similar but less obvious activity, indicating that the administration of live bacteria may be preferred. Although Akkermansia species is not as effective as frozen form when administered in lyophilized form, it still shows improvements in resistin and leptin levels, as well as some improvements in fat accumulation and insulin levels. Without being bound by theory, the reason why the lyophilized form cannot be as effective may be due to the insufficient hydration time of cells when administered in this form. Due to the short time in mice is 3-4 hours, and in this model, the lyophilized form may not be enough to carry out enough hydration and transcriptional activity to drive the same effect.

[0199] Example 5: Metabolic analysis of Akkermansia sp. strain AF3360009

[0200] To compare the metabolic capacity of strains Akkermansia species and Akkermansia muciniphila model strain BAA835 grown in YCFAC medium. YCFAC medium was inoculated with 1% overnight culture and the supernatant was collected after 24 hours of growth. Cells were separated by centrifugation at 10,000 rpm for 5 minutes and then filtered through a 0.2 μM filter.

[0201] Cell-free supernatant was harvested and analyzed by CE-TOF-MS. Cation conditions: 50 mbar was used for 10 seconds to inject the sample into a fused silica capillary (inner diameter 50 μm x 0 cm) on an Agilent CE-TOF system (Agilent Technologies Inc., Santa Clara, CA, USA). A cation buffer solution (1 M formic acid) and a CE voltage of 30 kV were used. Positive mode mass spectrometer conditions: MS capillary voltage: 4.0 V, ESI positive ionization mode, m / z range 50-1000. Anion conditions: 50 mbar was used for 22 seconds to inject the sample into a fused silica capillary (inner diameter 50 μm x 0 cm) on an Agilent CE-TOF system (Agilent Technologies Inc.). Anion buffer solution (50 mM ammonium acetate, pH 7.5) and a CE voltage of 30 kV were used. Negative mode mass spectrometer conditions: MS capillary voltage: 3.5 V, ESI negative ionization mode, m / z range 50-1000.

[0202] The metabolite agmatine (N-(4-aminobutyl)guanidine) was detected in positive ion mode at m / z 131.130 mu, retention time 4.23 minutes and was identified based on known standards. Quantities reported are peak areas.

[0203] Agmatine is metabolized from arginine by the enzyme arginine decarboxylase EC 4.1.1.19 (Piletz et al., 2013, Taksande et al., 2016). Fig.19 As shown, the level of agmatine was 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 difference between the two strains and media levels measured indicates that Akkermansia sp. is producing agmatine from arginine, while Akkermansia muciniphila ATCC BA835 is consuming agmatine.

[0204] Extracellular ATP has been shown to induce inflammation (Cauwels et al. 2014). The ability of Akkermansia species and Akkermansia muciniphila BAA835 to remove ATP from growth media was evaluated. YCFAC medium containing mucin (10 g / l) was inoculated with 1% of an overnight culture and spiked 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 harvested and analyzed for ATP levels.

[0205] At time 0( Fig. 20 A) and after 8 hours of growth ( Fig. 20 B), the amount of ATP in the supernatant was measured for Akkermansia muciniphila ATCC BAA 835. Similarly, at time 0 ( Fig. 20 C) and after 8 hours of growth ( Fig. 20 D) Estimation of ATP in supernatants from Akkermansia spp. Akkermansia spp. was able to remove ATP more efficiently compared to Akkermansia muciniphila ATCC BAA 835.

[0206] Using whole genome comparisons, possible operons encoding the enzyme machinery involved in vitamin B12 synthesis were present in Akkermansia species and absent in the type strain Akkermansia muciniphila ATCC BAA835 (Table 4). The ability to synthesize this important cofactor suggests that Akkermansia species have a broader metabolic capacity compared to the type strain.

[0207] Table 4: Genome comparison between Akkermansia species and Akkermansia muciniphila ATCC BAA835 to determine the presence of the corrin ring biosynthetic gene cluster.

[0208]

[0209] References

[0210] Bankevich A,et al.SPAdes:a new genome assembly algorithm and itsapplications to single-cell sequencing.J Comput Biol.2012;19(5):455-77.

[0211] Collado,M.C.,et al.(2007).Intestinal integrity andAkkermansiamuciniphila,a mucindegrading member of the intestinal microbiotapresentin infants,adults,and the elderly.Appl Environ Microbiol 73,7767-7770.

[0212] Costello,E.K.,et al.(2010).Postprandial remodeling of thegutmicrobiota in Burmese pythons.ISME J 4,1375-1385.

[0213] Chun J.,et al.(2018).Proposed minimal standards for the use ofgenomedata for the taxonomy of prokaryotes.Int.J.Syst.Evol.Microbiol.68 461-466.10.1099 / ijsem.0.002516

[0214] Fish JA,et al.FunGene:thefunctional gene pipeline andrepository.Front Microbiol.2013;4:291.doi:10.3389 / fmicb.2013.00291.

[0215] Goris,J.,et al.(2007).DNA-DNA hybridization values andtheirrelationship to whole-genome sequence similarities.Int J SystEvolMicrobiol 57,81-91.

[0216] Li H.(2015)BFC:correcting illumina sequencing errors.arXiv:1502.03744.

[0217] Lopez-Siles M,et al.2012.Cultured Representatives of TwoMajorPhylogroups of Human Colonic Faecalibacterium prausnitzii CanUtilizePectin,Uronic Acids,and Host-Derived Substrates for Growth.AppliedandEnvironmental Microbiology 78(2):420-428.

[0218] Seemann T.Prokka:rapid prokaryotic genomeannotation.Bioinformatics.2014;15:2068-9

[0219] Walker BJ,et al.Pilon:an integrated tool for comprehensivemicrobialvariant detection and genome assembly improvement.PLoS One.2014;9:e112963.doi:10.1371 / journal.pone.0112963.

[0220] Weizhong Li&Adam Godzik.Cd-hit:a fast program for clusteringandcomparing large sets of protein or nucleotide sequences",Bioinformatics,2006;22:1658-9.

[0221] T,Salzberg SL.FLASH:fast length adjustment of short readstoimprove genome assemblies.Bioinformatics.2011;27:2957-63.

[0222] Wang Q,Garrity GM,Tiedje JM,Cole JR.Naive Bayesian classifierforrapid assignment of rRNA sequences into the new bacterialtaxonomy.ApplEnviron Microbiol.2007 Aug;73(16):5261-7.

[0223] Ouwerkerk J.P.,Aalvink S.,Belzer C.,de Vos W.M.(2016).Akkermansiaglycaniphila sp.nov.,an anaerobic mucin-degrading bacteriumisolated fromreticulated python faeces.Int.J.Syst.Evol.Microbiol.66,4614-4620.10.1099 / ijsem.0.001399

[0224] Clinical breakpoints(Bacterial v9.0 and Fungal v9.0).TheEuropeanCommittee on Antimicrobial Susceptibility Testing:http: / / www.eucast.org / clinical_breakpoints.Published January 1,2019(bacteria).

[0225] Guo X,et al.(2016).Different subtype strains ofAkkermansiamuciniphila abundantly colonize in southern China.J ApplMicrobiol.2016Feb;120(2):452-9.doi:10.1111 / jam.13022.

[0226] 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:clinicalapplications after 100 yearsin translation.Drug Discov Today.2013Sep;18(17-18):880-93.doi:10.1016 / j.drudis.2013.05.017.Epub 2013 Jun 13.PMID:23769988

[0227] Taksande BG,Gawande DY,Chopde CT,Umekar MJ,Kotagale NR.Agmatineameliorates adjuvant induced arthritis and inflammatory cachexiainrats.Biomed Pharmacother.2017 Feb;86:271-278.doi:10.1016 / j.biopha.2016.12.039.Epub 2016 Dec 19.PMID:28006753.

[0228] 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.

[0229] Cauwels, A., Rogge, E., Vandendriessche, B. et al. Extracellular ATP drives systemic inflammation, tissue damage and mortality. Cell Death Dis 5, e1102 (2014).

[0230] 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

[0231] D, Ruas-Madiedo P, Margolles A, Gueimonde M, de los Reyes-Gavilán 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

[0232] Sequence

[0233]

[0234]

Claims

1. A composition comprising biologically pure Akkermansia species deposited in the German Collection of Microorganisms (DSM) with the accession number DSM 33459 ( Akkermansia sp. ) strain.

2. The composition of claim 1, further comprising a biologically pure strain of Enteromonas massii deposited in DSM under the deposit number DSM 33460.

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

4. The composition of claim 3, further comprising EVs derived from a biologically pure strain of Enteromonas massii deposited in DSM with the accession number DSM33460.

5. The composition of any one of claims 1-4, wherein the composition is formulated for oral administration.

6. The composition of any one of claims 1-4, wherein the composition is lyophilized.

7. The composition of any one of claims 1-4, wherein the composition is encapsulated or coated.

8. The composition of any one of claims 1-4, wherein the composition is a food product, a food ingredient, a dietary supplement or a medicament.

9. The composition of claim 1, wherein 1 x 10 4 CFU / g composition to 1 x 10 12 CFU / g composition of bacteria present in said composition.

10. The composition of any one of claims 1-4, wherein the composition is a probiotic composition.

11. The composition of any one of claims 1-4, wherein the composition has been pasteurized.

12. The composition of any one of claims 1-4, wherein the composition has been heat treated.

13. The composition of any one of claims 1-4, wherein the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient.

14. A tablet, extended release capsule, extended release granules, powder, sachet or sticky tablet comprising the composition of any one of claims 1 to 13.

15. A kit comprising (a) (i) the composition of any one of claims 1-13; or (ii) the tablet, extended release capsule, extended release granules, powder, sachet or adhesive of claim 14, and b) written instructions for administration to a subject.

16. Use of the composition of any one of claims 1-7 and 9-13 in the preparation of a medicament for treating and / or preventing one or more obesity-related disorders in a subject in need thereof.

17. The use as claimed in claim 16, wherein the medicament is formulated as a tablet, extended release capsule, extended release granules, powder, sachet or sticky tablet comprising the composition.

18. The use of claim 16, 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, glucose intolerance, abnormal lipid metabolism, non-alcoholic fatty liver disease, hepatic steatosis, leptin resistance, decreased resistin levels and / or cardiovascular disease.

19. A method for preparing a composition, the method comprising combining a biologically pure strain of Enteromonas massii deposited in DSM with the accession number DSM 33460 and a biologically pure strain of Akkermansia sp., wherein the Akkermansia sp. strain is an Akkermansia sp. strain deposited in DSM with the accession number DSM 33459.

20. A method for preparing a composition, the method comprising deriving extracellular vesicles (EVs) from a biologically pure strain of Enteromonas massii deposited in DSM with accession number DSM 33460 and deriving extracellular vesicles (EVs) from a biologically pure strain of Akkermansia sp. and combining them, and wherein the Akkermansia sp. strain is an Akkermansia sp. strain deposited in DSM with accession number DSM 33459.

21. The method of any one of claims 19-20, further comprising lyophilizing the composition.

Images