Novel microbial compositions and methods of using same

Abstract

Provided herein is a novel Akkermansia muciniphila strain. Also provided is a composition comprising the strain. Also provided is a method comprising administering the composition to a subject in need thereof. In some embodiments, a method is provided for treating a metabolic disorder in a subject, comprising administering to the subject an amount of the composition of the present disclosure that is effective for treating the metabolic disorder in the subject. Also provided is a method comprising culturing the novel strain.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 320,000, filed March 15, 2022, the entirety of which is incorporated herein by reference.

[0002] Akkermansia is a genus of Gram-negative, strict anaerobic, non-motile, non-spore-forming, oval-shaped bacteria. The cultivation and formulation of compositions containing Akkermansia can be difficult due to the strict anaerobic requirement of the bacteria. Furthermore, traditionally, Akkermansia has been cultivated under anaerobic conditions on a medium containing gastric mucin.

[0003] Provided herein are novel strains of Akkermansia that grow without mucin in plant media under anaerobic conditions. Summary of the Invention

[0004] The disclosure provides a bacterium of the Akkermansia muciniphila strain deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838. The bacterium comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or both.

[0005] The disclosure provides compositions comprising bacteria of the Akkermansia muciniphila strain deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838. The compositions provided herein include a nucleic acid encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or both.

[0006] Also provided herein is a composition for use in treating a metabolic disorder in a subject, the composition comprising bacteria of the Akkermansia muciniphila strain deposited at the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 126838 in an amount effective to treat the metabolic disorder. In one embodiment, the metabolic disorder is selected from the group consisting of or consisting essentially of insulin resistance based disorders, insulin sensitivity based disorders, type 1 diabetes, type 2 diabetes, and obesity.

[0007] In certain embodiments, the composition comprises: Anaerostipes caccae, Anaerobutyricum hallii, Bacteroides finegoldii, Bacteroides ovatus, Bacteroides stercoris, Eubactrium hallii, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Blautia hydrogenotrophica, Blautia producta, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium innocuum, Clostridium orbiscindens, Enterococcus faecium, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacteriumdolichum, Eubacterium ventriosum, Roseburia faeccis, Roseburia hominis, Roseburia intestinalis, Collinsella aerofaciens, Coprococcus comes, Eubacterium limosum, and Ruminococcus faeccis, and any combination thereof.

[0008] In certain embodiments, the composition further comprises one or more additional microorganisms selected from the group consisting of Anaerobutyricum hallii, Clostridium butyricum, Clostridium beijerinckii, Clostridium butyricum, and any combination thereof.

[0009] In certain embodiments, the composition comprises at least 10^5 AFU / g of each of the one or more additional microorganisms.

[0010] In a further embodiment, the composition comprises at least one preservative and / or an enteric coating. In some embodiments, the composition comprises a prebiotic selected from the group consisting of inulin, green banana, Ganoderma lucidum, tapioca, oats, pectin, potato or an extract thereof, complex carbohydrates, complex sugars, resistant dextrin, resistant starch, amino acids, peptides, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, lignin, psyllium, chitin, chitosan, gums (e.g., guar gum), high amylose corn starch (HAS), cellulose, b-glucan, hemicellulose, lactulose, mannooligosaccharides, mannanoligosaccharides (MOS), oligofructose-enriched inulin, oligofructose, oligodextrose, tagatose, trans-galactooligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof. In some preferred embodiments, the prebiotic used is inulin.

[0011] In some embodiments, compositions comprising lyophilized bacteria are provided herein. In some embodiments, compositions provided herein comprise lyophilized bacteria of the Akkermansia muciniphila strain deposited at the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838.

[0012] In some embodiments, the compositions provided herein comprise viable bacteria. In some embodiments, the compositions provided herein comprise viable bacteria of the Akkermansia muciniphila strain deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838.

[0013] In some embodiments, the compositions provided herein comprise non-viable bacteria. In some embodiments, the compositions provided herein comprise non-viable bacteria of the Akkermansia muciniphila strain deposited at the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838.

[0014] In some embodiments, the composition is formulated as a pharmaceutical preparation. In some embodiments, the composition is formulated as a nutritional supplement. In some embodiments, the composition is formulated as a dietary supplement. In some embodiments, the composition is formulated as a medical food.

[0015] In some embodiments, compositions are provided herein that are dairy-free. In some embodiments, the compositions do not include animal products.

[0016] In some embodiments, the composition is in the form of a pill, capsule, lozenge, food bar, or gummy ball.

[0017] Also provided herein is a method of treating a metabolic disorder in a subject, the method comprising administering to the subject a composition comprising an Akkermansia muciniphila strain deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838. The composition may further comprise Anaerobutyricum hallii, Clostridium butyricum, Clostridium beijerinckii, Clostridium butyricum, and any combination thereof.

[0018] The metabolic disorders referred to in the methods of treatment are selected from insulin resistance based disorders, insulin sensitivity based disorders, type 1 diabetes, type 2 diabetes, and obesity, gut-related disorders.

[0019] Provided herein is a method for culturing the Akkermansia muciniphila strain deposited at the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838 in / on a growth medium under anaerobic growth conditions. The growth medium used herein is VEG medium. [Brief description of the drawings]

[0020] [Figure 1] 1 shows a comparison of the growth phases of Akkermansia muciniphila strains Amuc-OG and Amuc-i09. [Diagram 2] Growth curves of three independent replicate comparisons between A. muciniphila (OG) and Amuc-i09 are shown, respectively. [Diagram 3] 1 shows comparative plots of doubling time, pH consumption, and glucose consumption between Amuc-i09 and Amuc-OG during the growth phase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0022] Where a range of values ​​is provided, unless the context dictates otherwise, it is to be understood that each intervening value between the upper and lower limits of that range is also specifically disclosed to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also included within the invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, it should be understood that the present disclosure supersedes any disclosure of the incorporated publications.

[0024] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0025] Deposit of strains The following biological material has been deposited under the terms of the Budapest Treaty at the American Type Culture Collection (ATCC®) Patent Depository-P0071744, 10801 University Boulevard Manassas, Virginia 20110, and has been assigned the following accession number: PTA-126838. [Table 3]

[0026] A. Bacteria Described herein is a new strain of anaerobic bacteria. In some embodiments, the bacteria described herein is the strain deposited with the ATCC® under ATCC® deposit number PTA-126838 (referred to herein as Amuc-i09).

[0027] Provided herein are novel strains of Akkermansia muciniphila. The strains disclosed herein are those deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838. The bacterial genome of the Akkermansia muciniphila strain deposited with the ATCC under ATCC Accession No. PTA-126838 encodes a polypeptide comprising SEQ ID NO:1 and a polypeptide comprising SEQ ID NO:2. Sequence information SEQ ID NO:1 MFLPCGGCRRAFLYPPGLFSGV SEQ ID NO:2 MNQLLSLESLRAYMIILQGEHHPKNKVAPARQTVTARNPRTMISFRYNTENNTVDAAKTGRDDDTN

[0028] B. Additional bacteria Embodiments of the compositions described herein include, but are not limited to, compositions that include Amuc-i09, alone or in combination with one or more other bacteria. Examples of bacteria that can be combined in compositions with Amuc-i09 include Clostridium butyricum, Clostridium beijerinckii, Anaerostipes caccae, Anaerobutyricum hallii, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, EubacteriumRoseburia cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0029] In certain embodiments, the composition comprising Amuc-i09 further comprises a bacterium of a genus selected from the group consisting of Clostridia, Eubacteria, Bifidobacteria, Anaerostipes, Coprococcus, Bacteroides, Blautia, Ruminococcus, Faecalibacterium, Oscillospira, Streptococcus, and Roseburia.

[0030] In one embodiment, the composition comprises Amuc-i09 and one or more additional microorganisms selected from the group consisting of Anerobutyricum hallii DSM 3353, Akkermansia muciniphila ATCC BAA83, Clostridium butyricum ATCC 1939, Clostridium beijerinckii ATCC 5174, and any combination thereof.

[0031] In one embodiment, the composition comprises Amuc-i09 and one or more additional bacterial strains selected from the group consisting of Anerobutyricum hallii, Akkermansia muciniphila, Clostridium butyricum, Clostridium beijerinckii, and any combination thereof.

[0032] In one embodiment, the additional bacterial strain in the composition is an anaerobic bacteria. In another embodiment, the additional bacteria is non-viable. In yet another embodiment, the additional bacteria is lyophilized. In another embodiment, the additional bacteria is lyophilized and viable.

[0033] In another embodiment, the present disclosure provides Amuc-i09, as well as Clostridium butyricum, Clostridium beijerinckii, Anaerostipes caccae, Anaerobutyricum hallii, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faecis, RoseburiaIn one embodiment, the composition comprises at least one additional microorganism comprising a 16S rRNA sequence that comprises at least about 85%, 87%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the full-length 16S rRNA sequence of a microorganism selected from the group consisting of: C. hominis, C. natalensis, C. cerevisiae ...

[0034] In another embodiment, described herein is a bacterium that expresses a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or both. In some embodiments, such a bacterium is a strain of Akkermansia muciniphila.

[0035] C. Properties of bacteria In embodiments, the novel strain of Akkermansia muciniphila disclosed herein, Amuc-i09, exhibits unique phenotypic characteristics, including improved viability (as measured by CFU per gram), faster and / or improved growth characteristics of Amuc-i09 compared to other Akkermansia strains, such as Amuc-OG.

[0036] The growth characteristics of the frozen or lyophilized strains are measured and compared to the base strain. Such base strains may be those deposited in a certified depository or other starting material. In a particular embodiment, the growth characteristics of the lyophilized strain(s) of Amuc-i09 are measured and compared to those of Amuc-OG.

[0037] a. Viability Viability, as defined herein, is a measure of living cells. Specifically, viability is defined as the ability to grow through binary fission under controlled conditions. Viability is measured in colony forming units (CFU), which is measured by culturing the microorganism and counting only the viable cells. Another unit used is the active fluorescent unit (AFU), which measures viability based on the results of a flow cytometer that correlates cell wall integrity with live cells.

[0038] Viability can be measured using flow cytometry (BD Accuri™ C6). Such flow cytometry measurements include staining the cell mixture and detecting the fluorescence of each cell as it flows. Staining of the cell mixture can be done using any dye that preferentially stains cells, particularly live and dead cells, or only dead cells. Examples of such stains include, but are not limited to, thiazole orange, propidium iodide, 7-AAD, DAPI, SYTO 9, fixable dyes such as Zombie Aqua™, Zombie Green™, Zombie NIR™, Zombie Red™, Zombie Violet™, Zombie UV™, and Zombie Yellow™, or any combination thereof. In one embodiment, the dye used herein for the process of the present disclosure is a combination of thiazole orange and propidium iodide.

[0039] As disclosed herein, Amuc-i09 exhibits higher viability per gram compared to the wild type variant Amuc-OG. For example, as shown in the Examples, a dry weight of 4 g of Amuc-i09 contains 8.72 x 10^11 CFU, whereas a dry weight of 21.3 g of Amuc-OG contains only 8.29 x 10^11 CFU of Amuc-OG. Calculated per gram, Amuc-i09 has 2.18 x 10^11 CFU / g of microorganisms, whereas Amuc-OG has only 0.38 x 10^11 CFU / g of Amuc-OG microorganisms. This also means that there are more live bacteria per gram of Amuc-i09 than per gram of Amuc-OG strain.

[0040] In some embodiments, Amuc-i09 is in a viable lyophilized state. In some embodiments, the bacteria of the present disclosure are non-viable.

[0041] In further embodiments, within any of the compositions described herein, one or more of the bacteria is in a lyophilized state. Further, within any of the compositions described herein, one or more of the bacteria is viable or non-viable.

[0042] In some embodiments, the bacteria of the present disclosure are at least about 1×10 3 ~1×10 14 In some embodiments, the concentration is about 1×10 9 ~1×10 13 AFU / g, approx. 1×10 5 ~1×10 7 AFU / g, or approximately 1 x 10 8 ~1×10 9 AFU / g (including any number within the range).

[0043] In certain embodiments, the compositions of the present disclosure contain at least 10^5 CFU of each strain per gram. In further embodiments, the compositions contain at least 10^6 CFU / g, or at least 10^7 CFU / g, or at least 10^8 CFU / g, or at least 10^9 CFU / g, or at least 10^10 CFU / g, or at least 10^11 CFU / g, or at least 10^12 CFU / g of at least one strain in the composition. In other embodiments where more than one strain is present, each strain may have a different viability (measured in CFU or AFU) than the other strains in the composition.

[0044] b. faster and / or improved growth characteristics The Amuc-i09 disclosed herein has faster growth characteristics compared to the wild-type strain. Growth is measured in terms of OD or optical density of the medium in which the microorganism is cultured.

[0045] As used herein, the term "faster growth" refers to the doubling time of a particular organism being shorter than another organism under otherwise identical conditions.

[0046] In some embodiments, Amuc-i09 has improved growth characteristics compared to the wild-type strain Amuc-OG. The term "improved" as used herein refers to a quantitative improvement in viable cell count after a certain time point in the bacterial growth phase. Specifically, it is shown herein that under conditions in which both bacteria are growing under the same conditions, Amuc-i09 has a higher viable cell count (measured in CFU / g) after, for example, 9 hours or 12 hours, compared to the viable cell count (measured in CFU / g) of Amuc-OG after the same time.

[0047] As described in Example 1 and FIG. 3, Amuc-i09 is characterized by a faster doubling time compared to Amuc-OG during the exponential growth phase.

[0048] c. Improved pelleting properties In some embodiments, the Amuc-i09 disclosed herein has improved pelleting properties compared to the pelleting properties of Amuc-OG.

[0049] As used herein, "pelleting characteristics" refers to the proportion of organisms in a liquid culture that are present in the recoverable cell pellet after centrifugation. An organism with "improved pelleting characteristics" has a greater ratio of organisms present in the pellet to the supernatant (pellet:supernatant) when centrifuged compared to the pellet:supernatant ratio of another organism pelleted under otherwise identical conditions. In the embodiments presented herein, Amuc-i09 has been shown to have a greater pellet:supernatant ratio compared to Amuc-OG when cultured and pelleted under otherwise identical conditions.

[0050] In certain embodiments, Amuc-i09 exhibits "improved pelleting properties" by exhibiting a more (visually) clear supernatant compared to the supernatant of Amuc-OG organisms pelleted under otherwise identical conditions.

[0051] Specifically, as described in Example 2 and shown in Table 2, Amuc-i09 exhibited improved pelleting properties compared to Amuc-OG.

[0052] D.How to use In certain embodiments, the present disclosure provides a method for treating a disorder. Such disorder can be a disorder of the intestinal system or any other disorder based on an imbalance in the bacterial population in the intestine. As a non-limiting example, such population can be the population of Akkermansia muciniphila in the intestine.

[0053] In certain embodiments, the disclosure provides methods of increasing the population of Akkermansia muciniphila in the small intestine.

[0054] In further embodiments, the compositions disclosed herein may be used to treat one or more of disorders such as irritable bowel syndrome, inflammatory bowel disease, gastric ulcers, pouchitis, Helicobacter pylori infection, diarrhea, type 1 diabetes mellitus, type 2 diabetes mellitus, and / or obesity.

[0055] In embodiments, the compositions described herein may include a pharma- ceutically acceptable carrier suitable for oral administration to a mammal, for example, as a powdered dietary supplement, various pelleted formulations, or liquid formulations. In some embodiments, the compositions are in the form of a pill, capsule, lozenge, food bar, and / or gummy ball. In some embodiments, the compositions are in the form of a suppository or injection for delivery to a mammal.

[0056] In some embodiments, the compositions described herein may be used as a nutritional or dietary supplement for a mammal. In some cases, the mammal may be a human.

[0057] E. Preparation method Embodiments include methods of producing bacteria with improved growth and pelleting characteristics, which can be obtained by a process that involves isolating the bacteria from a biological sample, inoculating and culturing it in a suitable medium under suitable anaerobic conditions, pelleting with a cryoprotectant, freezing, lyophilizing, and resuspending in the medium.

[0058] In some embodiments, the bacteria are grown in any suitable medium. Some non-limiting examples include PYG, RCM, GYT veg, BHI, nutrient medium, minimal medium, selective medium, and differential medium. In certain embodiments, the medium is a plant-derived medium. As used herein, a "plant-derived medium" is a growth medium that is essentially free of animal or dairy-derived components or derivatives. In some embodiments, the plant-derived medium is a meat-free medium that does not contain any animal-derived components. In some embodiments, the plant-derived medium is a liquid "VEG medium" having the components shown in Table 1. [Table 1-1] [Table 1-2]

[0059] Cryoprotectants useful in the methods of the invention include, but are not limited to, lactate, trehalose, glycerol, DMSO, propylene glycol, 2-methyl-2,4-pentanediol, methanamide, glycerophospholipids, proline, sorbitol, diethyl glycol, sucrose, glucose, and polymers such as polyvinyl alcohol, PEG, hydroxyethyl starch, skim milk, tapioca, polyvinylpropylene, inulin, methylcellulose, sodium alginate, gum arabic, propylene glycol, or xylitol, and any possible combinations thereof. In an embodiment, the cryoprotectant is added to the bacteria prior to freezing.

[0060] In certain embodiments, the bacterial pellet is resuspended in a cryoprotectant, hi some embodiments, the cryoprotectant used is sucrose.

[0061] The cryoprotected bacteria were then resuspended in VEG medium and centrifuged to obtain a pellet.Comparing the bacterial pellets between Amuc-OG and Amuc-i09, it was surprisingly found that Amuc-i09 had improved pelleting properties compared to Amuc-OG.

[0062] After resuspension, the frozen suspended bacterial pellet is frozen. For example, the pellet may be flash frozen in liquid nitrogen. Other freezing techniques that one of skill in the art can use herein are contemplated as part of this disclosure, including cryogenic (e.g., in ethanol and dry ice, or carbon dioxide), mechanical freezing, or flash freezing.

[0063] In certain embodiments, the mixture of cells and cryoprotectant is freeze-dried. Such freeze-drying may take place over a period of 12-48 hours. Freeze-drying may be performed under vacuum and at subzero temperatures. A non-limiting example of freeze-drying conditions is 0.008 mbar and a temperature of -84°C. Other freeze-drying conditions that can be varied include controlling the surface area of ​​the product undergoing sublimation, the pressure and temperature of the freeze-drying chamber, and varying the amount of heat applied to the product both during sublimation throughout the primary drying stage and throughout the desorption of the secondary drying stage.

[0064] In a particular embodiment, the bacterial pellet is freeze-dried in a LabConco FreeZone 2.5Plus freeze dryer at -84°C and 0.008-0.1 mbar vacuum pressure for 8-24 hours.

[0065] The lyophilized or frozen bacteria are then revived. In embodiments, the bacteria are revived by placing the bacteria in a medium and incubating under growth conditions. In a particular embodiment, bacteria at a rate of about 0.01 mg / mL are revived by placing the lyophilized cells in a vegetable medium (as defined above) and growing the culture for 12-20 hours at a temperature suitable for bacterial growth. This process selects for cells that can be quickly revived from freezing or lyophilization. This process can then be repeated through additional cycles to further select the bacterial population.

[0066] At the end of each cycle, the bacteria are revived, frozen, or lyophilized and grown in medium under appropriate conditions. A comparative study was performed to identify differences in growth characteristics between the novel Amuc-i09 strain and Amuc-OG.

[0067] As described herein, the novel Amuc-i09 strain was found to have improved growth characteristics compared to Amuc-OG, which refers to a faster growth phase as characterized by FIG. 1, which compares the growth characteristics of Amuc-i09 ("i09-3" is the third copy of Amuc-i09 grown in the above medium) and Amuc-OG when grown in the same medium as described above.

[0068] As can be seen in Figure 1, i09-3 (batch 3 of Amuc-i09) grew to a much higher OD value within 9 hours, while OG-2 (batch 2 of Amuc-OG) does not reach the same high OD value even after 12 hours.

[0069] In certain embodiments, a single colony of bacteria or a sample of bacteria is revived and grown in a plant medium as described above.The number of cycles that a selected colony can undergo can be at least 5 cycles, at least 10 cycles, at least 15 cycles, at least 20 cycles, at least 25 cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, at least 45 cycles, at least 50 cycles, at least 55 cycles, at least 60 cycles, at least 65 cycles, at least 70 cycles, at least 75 cycles, at least 80 cycles, at least 85 cycles, at least 90 cycles, at least 95 cycles, at least 100 cycles, at least 105 cycles, at least 110 cycles, at least 115 cycles, at least 120 cycles, at least 125 cycles, at least 130 cycles, at least 135 cycles, at least 140 cycles, at least 145 cycles, or at least 150 cycles.Each cycle can present one or more challenges to the bacteria. Examples of such challenges include, but are not limited to, changes in growth conditions, nutrient medium components, temperature, pressure, oxygen conditions to the microbial isolation, freezing and / or lyophilization, and / or revival processes described herein.

[0070] In one embodiment, the lyophilized powder of each of the Amuc-i09 and Amuc-OG strains was revived on an agar plate. Three replicates were cultured from a single colony of each strain in the plant medium described herein. Data showing the growth phase of each of these replicates are shown in FIG. 2.

[0071] F. Composition Compositions comprising one or more bacteria of the present disclosure are provided herein. Such compositions can be administered to a subject. In certain embodiments, such compositions can be administered as a therapeutic or dietary supplement. One or more bacteria of the present disclosure described herein can be used to make a pharmaceutical formulation comprising an effective amount of bacteria to treat a subject. The microorganism can be in any formulation known in the art. Some non-limiting examples of such formulations include topical, capsule, pill, enema, liquid, injectable formulations, and the like. In some embodiments, one or more strains disclosed herein can be included in a food or beverage product, a cosmetic product, or a dietary supplement.

[0072] In some embodiments, the subject is a mammal. Mammal as used herein refers to any mammal, including but not limited to humans, mice, cats, rats, dogs, sheep, monkeys, goats, rabbits, hamsters, horses, cows, or pigs. In a preferred embodiment, the mammal is a human.

[0073] The composition may contain one or more active ingredients, including, but not limited to, those selected from the group consisting of antibiotics, prebiotics, probiotics, glymay (e.g., as a decoy to limit the binding of certain bacteria / viruses to the intestinal wall), bacteriophages, microorganisms, etc.

[0074] In some embodiments, the composition comprises a prebiotic. The prebiotic may be a combination or a single component. In some embodiments, the prebiotic may be a source of starch, mucin, fructooligosaccharide, pectin, gum, glucan, xylan, arabinogalactan, seaweed polysaccharide, or derivatives thereof. Examples of such sources are inulin, green banana, ganoderma lucidum, tapioca, oats, pectin, raw or cooked potato, corn, rice, rice bran, cereals, porcine or human mucin sources, derivatives of L-threonine, chicory root, agave, artichoke, dandelion, lemon peel, apple peel, berries, guar gum, xanthan gum, acacia chia, barley, sorghum, corn, larch, Arabinex, kelp, dulse, or any combination or derivative thereof. In some embodiments, the prebiotic is inulin. Inulin serves as an energy source for the microbial composition.

[0075] In some embodiments, the prebiotic is selected from the group consisting of inulin, green banana, Ganoderma lucidum, tapioca, oats, pectin, potato or extracts thereof, complex carbohydrates, complex sugars, resistant dextrin, resistant starch, amino acids, peptides, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, lignin, psyllium, chitin, chitosan, gums (e.g., guar gum), high amylose corn starch (HAS), cellulose, b-glucan, hemicellulose, lactulose, mannooligosaccharides, mannanoligosaccharides (MOS), oligofructose-enriched inulin, oligofructose, oligodextrose, tagatose, trans-galactooligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof.

[0076] In some other embodiments, olive oil polyphenols, including hydroxytyrosol and its derivatives, such as oleuropein complex and tyrosol ("phenol alcohol"), are used as prebiotics.

[0077] The compositions may be administered by any suitable method for delivery to any site of the subject's gastrointestinal tract, including the oral cavity, mouth, esophagus, stomach, duodenum, small intestinal region including the duodenum, jejunum, ileum, and large intestinal region including the cecum, colon, rectum, and anal canal. In some embodiments, the compositions are formulated for delivery to the ileal and / or colonic regions of the gastrointestinal tract.

[0078] In some embodiments, the composition is administered orally, for example, via a capsule, pill, powder, tablet, gel, or liquid designed to release the composition in the gastrointestinal tract. In some embodiments, the composition is administered by injection, for example, in the case of compositions including butyrate, propionate, acetate, and short chain fatty acids. In some embodiments, the composition is administered by applying to the skin, for example, a cream, liquid, or patch. In some embodiments, the composition is administered by suppository and / or enema. In some embodiments, a combination of routes of administration is utilized.

[0079] The microbial composition may be formulated as a dietary supplement. The microbial composition may be incorporated into a vitamin supplement. The microbial composition may be formulated in a chewable form, such as a probiotic gummies. The microbial composition may be incorporated into food and / or drink forms. Non-limiting examples of foods and drinks that may incorporate the microbial composition include, for example, bars, shakes, juices, infant formulas, beverages, frozen foods, fermented foods, and cultured dairy products, such as yogurt, yogurt drinks, cheese, lactobacillus drinks, and kefir.

[0080] The composition of the present disclosure can be administered as part of a fecal transplant process.The composition can be administered to a subject by a tube, for example, a nasogastric tube, a nasojejunal tube, a nasoduodenal tube, an oral gastric tube, an oral jejunal tube, or an oral duodenal tube.The composition can be administered to a subject by colonoscopy, endoscopy, sigmoidoscopy, and / or enema.

[0081] In some embodiments, the microbial composition is formulated so that one or more microorganisms can replicate when delivered to the target habitat (e.g., the gut). In one non-limiting example, the microbial composition is formulated into a pill. Such a pill can have a shelf life of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In another non-limiting example, the microbial composition is formulated so that the microorganisms can reproduce when they enter the gut.

[0082] In some embodiments, Amuc-i09 may be formulated with other additives in a pill that may be orally administered to an individual so that the pill does not disintegrate until it reaches the intestine of the individual. In some embodiments, other ingredients may be added to aid in the shelf life of the microbial composition. In some embodiments, Amuc-i09 may be formulated to survive in a non-native environment. For example, microorganisms naturally present in the intestine may not be able to survive in an oxygen-rich environment. To overcome this limitation, the microorganisms may be formulated to reduce or eliminate exposure to oxygen. Other strategies for improving the shelf life of microorganisms may include other microorganisms (e.g., one or more strains in the composition improve the survival of one or more other strains in the composition).

[0083] In some embodiments, Amuc-i09 may be formulated with polyphenols. In some embodiments, the polyphenol may be a combination of one or more polyphenols. Commonly used polyphenols are flavonoids, including flavones, flavonols, flavanols, flavanones, isoflavones, proanthocyanidins, and anthocyanins. Flavonoids that are particularly abundant in foods are catechins (tea, fruits), hesperetin (citrus fruits), cyanidins (red fruits and berries), daidzein (soybeans), proanthocyanidins (apples, grapes, cocoa), and quercetin (onion, tea, apples). Phenolic acids include caffeic acid, while lignans are polyphenols derived from phenylalanine found in flaxseed and other cereals. In one embodiment, tannins obtained from papaya are used.

[0084] In some embodiments, Amuc-i09 is lyophilized.

[0085] In some embodiments, Amuc-i09 is lyophilized and formulated as a composition as provided herein. Such compositions can be formulated as a powder, tablet, enteric capsule (e.g., for delivery to the ileum / colon), or pill that can be administered to a subject by any suitable route. In some embodiments, the lyophilized formulation can be mixed with saline or other solution before administration.

[0086] In some embodiments, the composition comprises Amuc-i09, and the bacteria are fully viable (comprising 100% live population of cells), partially viable (comprising <50% live population of cells), or nearly non-viable (comprising >50% dead population of cells).

[0087] In some embodiments, a composition is provided comprising Amuc-i09 and Amuc-OG. In some embodiments, the composition comprises at least 50% live Amuc-i09 bacteria. In some embodiments, the composition comprises at least 50% dead Amuc-i09. In some embodiments, the composition comprises extracellular vesicles derived from Amuc-i09.

[0088] In some embodiments, the microbial compositions are formulated for oral administration, e.g., as an enteric coated capsule or pill, to deliver the contents of the formulation to the ileal and / or colonic region of the subject.

[0089] I. Capsule Ingredients In some embodiments, the microbial composition is formulated for oral administration. In some embodiments, the microbial composition is formulated as an enteric coated pill or capsule for oral administration. In some embodiments, the microbial composition is formulated to deliver the microorganism to the ileal region of the subject. In some embodiments, the microbial composition is formulated to deliver the microorganism to the colonic region (e.g., upper colon and / or lower colon) of the subject. In some embodiments, the microbial composition is formulated to deliver the microorganism to the ileal and colonic regions of the subject.

[0090] In one embodiment, a composition comprising Amuc-i09 is administered as a capsule for oral delivery.

[0091] In one embodiment, the composition comprising a combination of Amuc-i09 and Amuc-OG is administered as a capsule for oral delivery.

[0092] In one embodiment, the composition comprising a combination of Amuc-i09 and another anaerobic bacterium is administered as an oral formulation.

[0093] In one embodiment, the size of the capsule is size 0, but the size of the capsule may be any size suitable for administration to a subject. Additionally, in this embodiment, the capsule has an enteric coating. The enteric coating can protect the contents of the oral formulation from the acidity of the stomach and provide delivery to the ileum and / or upper colon region. Non-limiting examples of enteric coatings include pH-sensitive polymers (e.g., Eudragit FS30D), methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methylcellulose acetate succinate (e.g., hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, zein, other polymers, fatty acids, waxes, shellac, plastics, and vegetable fibers. In some embodiments, the enteric coating is formed by a pH-sensitive polymer. In some embodiments, the enteric coating is formed by Eudragit FS30D.

[0094] The enteric coating may be designed to dissolve at any suitable pH. In some embodiments, the enteric coating is designed to dissolve at a pH of about pH 6.5 to greater than about pH 7.0. In some embodiments, the enteric coating is designed to dissolve at a pH of greater than about pH 6.5. In some embodiments, the enteric coating is designed to dissolve at a pH of greater than about pH 7.0. The enteric coating may be designed to dissolve at a pH of greater than 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5 pH units.

[0095] In some embodiments, administration of the formulations of the present disclosure may be preceded by colonic cleansing procedures such as, for example, colonic irrigation / hydrotherapy, enemas, laxatives, dietary supplements, dietary fiber, enzymes, and administration of magnesium or derivatives thereof.

[0096] The formulations provided herein may include the addition of one or more agents to the therapeutic or cosmetic product to improve the stability and / or viability of the microbial formulation. Non-limiting examples of stabilizing agents include genetic elements, glycerin, ascorbic acid, skim milk, lactose, Tween, alginate, xanthan gum, carrageenan gum, mannitol, palm oil, and poly-L-lysine (POPL).

[0097] II. Microbial preparations and compositions in microbial consortia The compositions disclosed herein may contain a single microbial species or a combination of microbial species. In one embodiment, the formulation contains only Amuc-i09. In a further embodiment, the formulation comprises Amuc-i09 and Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium hallii, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus inf antis, StreptococcusThe present invention relates to a method for producing a bacterial strain comprising the steps of: Clostridium sporogenes, Clostridium tetani, Clostridium tetani, Clostridium mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faeccis, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0098] In another embodiment, the compositions disclosed herein include one or more recombinant microorganisms or genetically modified microorganisms. In other embodiments, the one or more microorganisms are not modified or recombinant. In some embodiments, the compositions include microorganisms that can be regulated, for example, microorganisms that include an operon or promoter for controlling the growth of the microorganism. The microorganisms disclosed herein can be generated, grown, or modified using any suitable method, including recombinant methods.

[0099] In one embodiment, the bacterial population that can be administered as part of the compositions disclosed herein is Amuc-i09, Amuc-OG, Akkermansia muciniphila, Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium hallii, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infants, StreptococcusThe bacterial strains may include any one or more of a species selected from the group consisting of: Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faeccis, Roseburia hominis, any other strain of Roseburia intestinalis, and any combination thereof.

[0100] In some embodiments, Amuc-i09, Eubacterium hallii DSM 3353, Akkermansia muciniphila ATCC BAA83, Clostridium butyricum ATCC 1939, Clostridium beijerinckii ATCC 5174, Akkermansia muciniphila, Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium hallii, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcusobeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, and any combination thereof. A therapeutic composition for treating a metabolic disorder is provided, comprising a purified microbial population of bacteria with at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to an rRNA.

[0101] In some embodiments, the metabolic disorder may be selected from an insulin resistance based disorder, an insulin sensitivity based disorder, type 1 diabetes, type 2 diabetes, and obesity.

[0102] In some embodiments, Amuc-i09, Eubacterium hallii DSM 3353, Akkermansia muciniphila ATCC BAA83, Clostridium butyricum ATCC 1939, Clostridium beijerinckii ATCC 5174, Akkermansia muciniphila, Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium hallii, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcusobeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, and any combination thereof. A therapeutic composition for treating type 2 diabetes or obesity is provided, comprising a purified microbial population of bacteria with at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to an rRNA sequence.

[0103] In some embodiments, a therapeutic composition for treating type 2 diabetes or obesity is provided comprising a purified microbial population comprising bacteria with at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to 16S rRNA and / or 23S rRNA of Amuc-i09.

[0104] In some embodiments, a therapeutic composition for treating type 2 diabetes or obesity is provided comprising a purified microbial population comprising bacteria with at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to 16S rRNA and / or 23S rRNA of Amuc-OG.

[0105] In some embodiments, Amuc-i09, Eubacterium hallii DSM 3353, Akkermansia muciniphila ATCC BAA83, Clostridium butyricum ATCC 1939, Clostridium beijerinckii ATCC 5174, Akkermansia muciniphila, Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium hallii, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcusobeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, and any combination thereof. A therapeutic composition for treating a neurological or behavioral disorder is provided, comprising a purified microbial population of bacteria with at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to an rRNA.

[0106] In some embodiments, the neurological or behavioral disorder is selected from a brain-gut disorder, anxiety, or hyperalgesia. In some embodiments, the compositions disclosed herein modulate neurotransmitter levels in the brain of a subject.

[0107] In some embodiments, a therapeutic composition for treating a neurological or behavioral disorder is provided, comprising a purified microbial population consisting of Amuc-i09.

[0108] In some embodiments, a therapeutic composition for treating a gut-related disorder is provided, comprising a purified microbial population consisting of Amuc-i09. In some embodiments, the gut-related disorder is selected from constipation, abdominal bloating, gut damage-related disorders, diarrhea, gastritis, enteritis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, or dysbiosis.

[0109] In some embodiments, the therapeutic consortium includes Akkermansia muciniphila, Bifidobacterium adolescentis, Bifidobacterium infantis, Clostridium beijerinckii, Clostridium butyricum, and Amuc-i09.

[0110] In some embodiments, the therapeutic consortium includes Akkermansia muciniphila and Eubacterium hallii or Anerobutyricum hallii.

[0111] In some embodiments, the therapeutic consortium includes Amuc-i09 and Eubacterium hallii or Anerobutyricum hallii.

[0112] In some embodiments, the therapeutic consortium includes Amuc-i09 and any one of Bifidobacterium adolescentis, Bifidobacterium infantis, Clostridium beijerinckii, and Clostridium butyricum.

[0113] A therapeutic composition may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, or at least 50, or at least 75, or at least 100 types of bacteria. A therapeutic composition may include up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35, up to 36, up to 37, up to 38, up to 39, up to 40, up to 45, or up to 50, or up to 75, or up to 100 types of bacteria.

[0114] In some embodiments, the combination of one or more microorganisms in a therapeutic composition or consortium can provide a synergistic effect when administered to an individual.

[0115] For example, administration of a first microorganism may be beneficial to a subject, and administration of a second microorganism may be beneficial to a subject, but when the two microorganisms are administered to a subject together, the benefit is greater than the benefit of either microorganism alone.

[0116] The different types of microorganisms may be present in the therapeutic composition in the same or different amounts, for example, the ratio of the two bacteria in the therapeutic composition may be about 1:1, 1:2, 1:5, 1:10, 1:25, 1:50, 1:100, 1:1000, 1:10,000, or 1:100,000.

[0117] III. Compositions Containing Probiotics In one embodiment, a composition is disclosed that comprises a single microorganism as a probiotic.In another embodiment, a composition is disclosed that comprises a bacterial consortium that comprises at least two different bacteria.Apart from the probiotic, the composition may also comprise a combination of prebiotics, antibiotics, or active agents as described herein.

[0118] In some embodiments, the composition is administered before, during, and / or after treatment with an antibacterial agent, such as an antibiotic. For example, the composition can be administered at least about 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6 months, or 1 year before and / or after treatment with an antibiotic. The formulation can be administered up to 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6 months, or 1 year before and / or after treatment with an antibiotic.

[0119] In some embodiments, the compositions disclosed herein are administered during, after, and / or prior to use of an antidiabetic drug such as insulin or an agent such as metformin. For example, the formulations can be administered at least about 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6 months, or 1 year before and / or after treatment with an antidiabetic drug.

[0120] In some embodiments, the composition is administered before, during, and / or after food ingestion by the subject. In some embodiments, the composition is administered along with food ingestion by the subject. In some embodiments, the composition is administered along with (e.g., simultaneously with) food ingestion.

[0121] In some embodiments, the composition is administered before the subject takes in food.In some embodiments, the composition is more effective or potent in treating metabolic disorders when administered before taking in food.For example, the composition can be administered about 1 minute, about 2 minutes, about 3 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, or about 1 day before the subject takes in food.

[0122] In some embodiments, the composition is administered after the subject takes in food.In some embodiments, the composition is more effective or potent in treating metabolic disorders when administered after taking in food.For example, the composition can be administered at least about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, or 1 day after the subject takes in food.For example, the formulation can be administered at most about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, or 1 day after the subject takes in food.

[0123] Compositions provided herein include those suitable for oral administration, including buccal and sublingual administration, intranasal administration, topical administration, transdermal administration, transdermal patch administration, pulmonary administration, vaginal administration, rectal administration, suppository administration, transmucosal administration, systemic administration, or parenteral administration, including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous, and intravenous administration, or in a form suitable for administration by aerosolization, inhalation, or insufflation.

[0124] In some embodiments, the composition may be a therapeutic composition, a pharmaceutical composition, or a dietary supplement.

[0125] Therapeutic compositions disclosed herein may include carriers and excipients (including, but not limited to, buffers, carbohydrates, lipids, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents, and / or preservatives), metals (e.g., iron, calcium), salts, vitamins, minerals, water, oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, saline solutions, dextrose and glycerol solutions in water, flavoring agents, coloring agents, anti-adherents, and other acceptable additives, adjuvants, or binders, other pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifiers, wetting agents, and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0126] Non-limiting examples of pharma- ceutically acceptable excipients suitable for use in the present disclosure include granulating agents, binders, lubricants, disintegrants, sweeteners, glidants, antiadherents, antistatic agents, surfactants, antioxidants, gums, coating agents, colorants, flavoring agents, dispersion enhancers, disintegrants, coating agents, plasticizers, preservatives, suspending agents, emulsifiers, plant cellulosic materials, and spherization agents, and any combination thereof.

[0127] Non-limiting examples of pharma- ceutically acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0128] The therapeutic composition may be substantially free of preservatives. In some applications, the composition may include at least one preservative.

[0129] The therapeutic composition may be encapsulated in a suitable vehicle, such as liposomes, microspheres, or microparticles. Microspheres made of polymers or proteins may be tailored to pass through the gastrointestinal tract and directly enter the bloodstream. Alternatively, the compound may be incorporated into a microsphere or a complex of microspheres and implanted for sustained release over a period ranging from several days to several months.

[0130] The therapeutic composition may be formulated as a sterile solution or suspension. The therapeutic composition may be sterilized by conventional techniques or may be sterile filtered. The resulting aqueous solution may be packaged for immediate use or lyophilized. The lyophilized preparation of the microbial composition may be packaged in a form suitable for oral administration, such as a capsule or pill. In one embodiment, the capsule may include an enteric polymer that does not disintegrate until the capsule leaves the subject's stomach.

[0131] In one embodiment, the capsule ingredients may include methylcellulose, hydroxypropylmethylcellulose phthalate, propylene glycol, and derivatives of such polymers.

[0132] The compositions may be administered topically and may be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, ointments, etc. Such pharmaceutical compositions may also contain solubilizers, stabilizers, tonicity enhancing agents, buffers, and preservatives.

[0133] The compositions can also be formulated into rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, etc. In suppository forms of the composition, a low melting wax such as a mixture of fatty acid glycerides can be used, optionally in combination with cocoa butter.

[0134] In practicing the methods of treatment or use provided herein, a therapeutically effective amount of the microbial composition described herein is administered as a pharmaceutical composition to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal, e.g., a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the formulation, and other factors. The subject can be, for example, a human, elderly, adult, adolescent, pre-adolescent, child, infant, or newborn.

[0135] The subject may be a patient. The subject may be an individual enrolled in a clinical study. The subject may be a laboratory animal, e.g., a mammal. The subject may be a human.

[0136] The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, including excipients and auxiliaries, which facilitate the processing of the microorganism into a pharma-ceutically usable preparation. The formulation can be modified according to the selected route of administration. The pharmaceutical composition described herein can be manufactured by conventional methods, for example, conventional mixing, dissolving, granulating, vitrifying, spray drying, lyophilizing, wet milling, encapsulating, encapsulating, emulsifying, or compressing processes.

[0137] In some embodiments, the pharmaceutical composition is produced in a dry form, for example by spray drying or freeze drying. In some embodiments, the formulation is prepared as a liquid capsule to maintain the liquid form of the microorganism.

[0138] The compositions provided herein can be stored at any suitable temperature. The formulations can be stored in refrigerated storage at temperatures of, for example, about -80°C, about -20°C, about -4°C, or about 4°C. The storage temperature can be, for example, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 12°C, about 14°C, about 16°C, about 20°C, about 22°C, or about 25°C. In some embodiments, the storage temperature is about 2°C to about 8°C. Storage of the microbial composition at low temperatures, for example, about 2°C to about 8°C, can keep the microorganisms viable and increase the efficiency of the composition, for example, when present in a liquid or gel formulation. Storage at freezing temperatures below 0°C using a cryoprotectant can further increase stability.

[0139] The pH of the composition can be in the range of about 3 to about 12. The pH of the composition can be, for example, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 11, or about 11 to about 12 pH units.

[0140] The pH of the composition can be, for example, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units. The pH of the composition can be, for example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 pH units. The pH of the composition can be, for example, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, or up to 12 pH units. If the pH is outside the formulator's desired range, the pH can be adjusted by using sufficient pharma- ceutically acceptable acids and bases. In some embodiments, the pH of the composition is about 4 to about 6.

[0141] Pharmaceutical compositions comprising the microorganisms described herein may be administered for prophylactic and / or therapeutic treatment. In therapeutic applications, the compositions may be administered to a subject already suffering from a disease or condition in an amount sufficient to reverse or at least partially prevent the symptoms of the disease or condition, or to reverse, cure, improve, or ameliorate the condition. The microbial compositions may also be administered to reduce the likelihood of developing, contracting, or worsening the condition. Amounts effective for this use may vary based on the severity and course of the disease or condition, medical history, the subject's health, weight, and response to the drugs, and the judgment of the treating physician.

[0142] The multiple therapeutic agents may be administered in any order or simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single unified form or in multiple forms, for example, as multiple individual pills. The compositions may be packaged together or separately in a single package or multiple packages. One or all of the therapeutic agents may be administered multiple times. If not simultaneously, the timing between multiple doses may vary by as much as about one month.

[0143] The compositions described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the composition can vary. For example, the microbial composition can be used as a prophylactic and can be administered continuously to a subject prone to a condition or disease to reduce the likelihood of the onset of the disease or condition. The microbial composition can be administered to the subject during or as soon as possible after the onset of symptoms. Administration of the microbial composition can begin within the first 48 hours of the onset of symptoms, within the first 24 hours of the onset of symptoms, within the first 6 hours of the onset of symptoms, or within the first 3 hours of the onset of symptoms. Initial administration can be via any practical route, such as any route described herein, using any formulation described herein. The microbial composition can be administered as soon as possible after the onset of a disease or condition is detected or suspected, for the period of time required to treat the disease, for example, from about 1 month to about 3 months. The treatment period can vary from subject to subject.

[0144] In one aspect, the composition may be administered in combination with another therapy, for example, immunotherapy, chemotherapy, radiation therapy, anti-inflammatory agents, anti-viral agents, anti-microbial agents, and anti-fungal agents.

[0145] In another aspect, the composition may be packaged as a kit. In some embodiments, the kit includes written instructions for administration / use of the composition. The writing may be, for example, a label. The writing may suggest conditions for administration. The instructions provide the subject and the supervising physician with the best guidance to achieve optimal clinical outcomes from administration of the treatment. The writing may be a label. In some embodiments, the label may be approved by a regulatory agency, for example, the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or other regulatory agency.

[0146] G. Treatment method The present disclosure provides a method of treating a subject. The subject may suffer from or be predisposed to symptoms associated with disorders resulting from an imbalance in the gut microbiome. Altering the composition of the microbiome of such a subject may produce desirable health effects. The compositions of the present disclosure may be administered as therapeutics and / or dietary supplements to treat health conditions. Treatments designed to alter the host microbiome(s) may result in the alleviation of patient symptoms, prevention of disease, and / or treatment of disease or health conditions. For example, modifying the gut microbiome may reduce the risk of health conditions such as metabolic, neurological, or behavioral disorders.

[0147] Thus, the present disclosure provides methods for restoring a subject's microbial habitat to a healthy state. The methods may include, for example, modifying and / or adjusting the microbiome, including replenishing native microorganisms, removing pathogenic microorganisms, administering prebiotics, and growth factors necessary for the survival of the microbiome. In some embodiments, the methods also include administering an antimicrobial agent, such as an antibiotic.

[0148] Based on the microbiome profile, the present disclosure provides a method for generalized treatment recommendation to a subject and a method for subject-specific treatment recommendation. The method for treatment may include one of the steps of determining a first ratio between the level of the subject-specific microbiome profile and the level of the second microbiome profile in a biological sample obtained from at least one subject, detecting the presence or absence of disease in the subject based on the determination, and recommending at least one generalized or subject-specific treatment to the subject to ameliorate disease symptoms.

[0149] Diseases that may be treated include diseases in which an alteration of the gut microbiome would be beneficial to a subject afflicted with the disease. Specifically, the target gut microbiome includes Akkermansia muciniphila, Clostridium butyricum, Clostridium beijerinckii, Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium rectale, Eubacterium hallii, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, EubacteriumIn one embodiment, the population of at least one live, active enterobacteria selected from the group consisting of: Eubacterium dolichum, Eubacterium ventriosum, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, and any combination thereof may be deficient.

[0150] In one embodiment, the gut microbe that is lower in subjects suffering from or prone to such disorders is Akkermansia muciniphila. In another embodiment, the gut microbe that is lower in subjects suffering from or prone to such disorders is Eubacterium hallii. In another embodiment, the gut microbe that is lower in subjects suffering from or prone to such disorders is belonging to the genera Clostridia, Eubacteria, Bifidobacteria, Anaerostipes, Coprococcus, Bacteroides, Blautia, Ruminococcus, Faecalibacterium, Oscillospira, Streptococcus, and Roseburia.

[0151] In another embodiment, the subject has a plurality of gut microbes at a level lower than that in a normal subject. Specifically, the microorganisms include Akkermansia muciniphila, Clostridium butyricum, Clostridium beijerinckii, Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium, Eubacterium rectale, Eubacterium hallii, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacteriumdolichum, Eubacterium ventriosum, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0152] Measuring the microbiome of a subject may show that a microbiome that is deficient in various microbial strains results in health and / or disease. Restoring one or more of the deficient strains (e.g., via bacterial strains such as A.muciniphila) results in a change in health. Some non-limiting examples include changing the gut microbiome so that the host has increased energy harvesting capacity, increased insulin sensitivity, and / or decreased appetite, decreased anxiety, decreased hyperalgesia, decreased behavioral problems such as anxiety, regular bowel movements, decreased abdominal bloating, and decreased constipation.

[0153] Some non-limiting health conditions that may be affected by an imbalance in microbial populations include, for example, type 2 diabetes mellitus (T2DM), preterm labor, chronic fatigue syndrome, skin conditions such as acne, allergies, autism, asthma, depression, hypertension, metabolic syndrome, obesity, lactose intolerance, oral candidiasis, ulcerative colitis, drug metabolism, vaginal diseases, atopic dermititus, psoriasis, type I diabetes mellitus (T1DM), multiple sclerosis, neurological disorders such as Parkinson's disease, Clostridium Difficile infection, heart disease, diabetic foot ulcers, bacteremia, infantile colic, cancer, cystic fibrosis, multiple sclerosis, urinary tract infections, radiation enteropathy, drug metabolism, dental pulp cavity, and halitosis.

[0154] The measurement of the subject's microbiome can be performed by analyzing a body fluid sample or by performing a microbial analysis on the subject's feces. Candidate strains can be found in scientific literature and studies. These candidate strains can be used as parameters measured from the subject's microbiome analysis. By comparing the ideal level of each microorganism in a healthy individual with the level of that microorganism in the subject, a conclusion can be made regarding the level of that microorganism in the subject's gut. Thus, the compositions disclosed herein can be administered to replenish and restore the population of deficient gut microorganisms identified by microbial analysis.

[0155] I. Administration The appropriate amount of therapeutic composition to be administered, the number of treatments, and the unit dose may vary depending on the subject and / or the disease state of the subject.

[0156] The pharmaceutical compositions described herein may be in unit dosage form suitable for single administration of a precise dose. In unit dosage form, the formulation may be divided into unit doses containing an appropriate amount of one or more microbial compositions. The unit dose may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are liquids in vials or ampoules. Aqueous suspension compositions may be packaged in single-dose non-reclosable containers. The compositions may be in multi-dose format. Multi-dose reclosable containers may be used, for example, in combination with a preservative. Preparations for parenteral injection may be presented in unit dosage form, for example, ampoules, or multi-dose containers containing a preservative.

[0157] The dosage form may be in the form of a solid, semi-solid, or liquid composition.Non-limiting examples of dosage forms include feed, food, pellets, lozenges, liquids, elixirs, aerosols, inhalants, sprays, powders, tablets, pills, capsules, gels, gel tabs, nanosuspensions, nanoparticles, microgels, suppositories, troches, aqueous or oily suspensions, ointments, patches, lotions, dentifrices, emulsions, creams, drops, dispersible powders or granules, emulsions in hard or soft gel capsules, syrups, phytoceuticals, nutraceuticals, dietary supplements, and any combination thereof.

[0158] The microorganism may be present in the pharmaceutical composition at any suitable concentration. The concentration of the microorganism may be, for example, about 10^1 to about 10^18 CFU or AFU per gram of medium. The concentration of the microorganism may be, for example, at least 10^1, at least 10^2, at least 10^3, at least 10^4, at least 10^5, at least 10^6, at least 10^7, at least 10^8, at least 10^9, at least 10^10, at least 10^11, at least 10^12, at least 10^13, at least 10^14, at least 10^15, at least 10^16, at least 10^17, or at least 10^18 CFU or AFU per gram. The concentration of the microorganisms can be, for example, up to 10^1, up to 10^2, up to 10^3, up to 10^4, up to 10^5, up to 10^6, up to 10^7, up to 10^8, up to 10^9, up to 10^10, up to 10^11, up to 10^12, up to 10^13, up to 10^14, up to 10^15, up to 10^16, up to 10^17, or up to 10^18 CFU or AFU per gram. In some embodiments, the concentration of the microorganisms is about 10^8 CFU to about 10^9 CFU or AFU per gram. In some embodiments, the concentration of the microorganisms is about 10^8 CFU or AFU per gram. In some embodiments, the concentration of the microorganisms is about 10^9 CFU or AFU per gram.

[0159] The pharmaceutical compositions provided herein can be formulated with any suitable therapeutically effective concentration of prebiotics.For example, the therapeutically effective concentration of prebiotics can be at least about 1mg / ml, about 2mg / ml, about 3mg / ml, about 4mg / ml, about 5mg / ml, about 10mg / ml, about 15mg / ml, about 20mg / ml, about 25mg / ml, about 30mg / ml, about 35mg / ml, about 40mg / ml, about 45mg / ml, about 50mg / ml, about 55mg / ml, about 60mg / ml, about 65mg / ml, about 70mg / ml, about 75mg / ml, about 80mg / ml, about 85mg / ml, about 90mg / ml, about 95mg / ml, about 100mg / ml, about 110mg / ml, about 125mg / ml, about 130mg / ml, about 140mg / ml, or about 150mg / ml. For example, a therapeutically effective concentration of a prebiotic can be up to about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 110 mg / ml, about 125 mg / ml, about 130 mg / ml, about 140 mg / ml, or about 150 mg / ml. For example, the therapeutically effective concentration of the prebiotic may be about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 110 mg / ml, about 125 mg / ml, about 130 mg / ml, about 140 mg / ml, or about 150 mg / ml. In some embodiments, the concentration of the prebiotic in the pharmaceutical composition is about 70 mg / ml.In some embodiments, the prebiotic is inulin.

[0160] The pharmaceutical compositions of the present disclosure may be administered, for example, 1, 2, 3, 4, 5 or more times per day. In one embodiment, the pharmaceutical compositions may be administered, for example, daily, every other day, three times per week, twice per week, once per week, or at other appropriate intervals for the treatment of a condition.

[0161] Although the present invention has been described in some detail for purposes of clarity and understanding, it will be apparent to one skilled in the art upon reading this disclosure that various changes in form and details can be made without departing from the true scope of the invention. For example, all of the compositions and methods described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually and individually indicated to be incorporated by reference for all purposes.

[0162] H. Working Example Example 1: Improved growth phase of Amuc-i09 vs. Amuc-OG Objective: The objective of this study was to determine the growth differences between AMUC_i09 and AMUC_OG.

[0163] Methods: AMUC colonies were grown on VEG agar plates for 5 days until single colonies were identified. Single colonies were inoculated into two replicate 8 ml tubes containing VEG medium at 37°C with shaking at 180 rpm. Optical density at 600 nm (OD600) was measured at various time intervals. As shown in Figure 3, Amuc-i09 is characterized by a faster doubling time in exponential growth compared to Amuc-OG. There was no difference in pH and glucose consumption between the two strains in the exponential growth phase.

[0164] Example 2: Improved pelleting properties of Amuc-i09 Objective: The objective of this experiment was to identify physiological differences between Amuc-i09 and Amuc-OG.

[0165] Methods: Amuc-i09 and Amuc-OG colonies were grown on VEG agar plates for 5 days until single colonies were identified. Single colonies were inoculated into two replicate 8 ml tubes containing VEG medium at 37°C with shaking at 180 rpm. Optical density at 600 nm (OD600) was measured in the 8 ml tubes at various time intervals throughout the growth phase. Once OD600 reached 1.0, 8 ml of culture was used to inoculate 100 ml bottles. The 100 ml bottles were grown to an OD600 of 1.0 and then subcultured into 500 ml bottles. The 500 ml bottles were grown to an OD600 of 1.0 and centrifuged at 7,500 rpm for 20 min. Amuc-i09 cells showed improved pelleting properties compared to Amuc-OG. This resulted in differences in dry weight when the products were lyophilized, as shown in Table 2. [Table 2]

[0166] Example 3: Treatment of metabolic disorders with a composition comprising Amuc-i09 Objective: To treat metabolic disorders in humans with a composition containing Amuc-i09.

[0167] Method: An individual suffering from a metabolic disorder is treated with a composition comprising Amuc-i09, the composition comprising Amuc-i09 in the range of about 10^9 CFU to 10^10 CFU, and inulin.

[0168] A contemplated delivery form for the oral composition is an enteric coated (e.g., pH sensitive polymer Eudragit FS30D) pill that provides protection from stomach acid and allows delivery to the ileum / upper colon region of the subject. The enteric coating is designed to dissolve at a pH of about 6.5 to above 7. In some embodiments, the oral composition is administered as a liquid capsule. The subject is administered the composition twice daily for 14 consecutive days, prior to food intake (e.g., 1 hour prior to a meal). In some cases, the composition is administered simultaneously with food intake.

[0169] The microbial composition changes the microbial habitat in the gut of the subject to that of a healthy subject. The subject loses weight. The metabolic condition of the subject, such as obesity, insulin insensitivity, T2DM, and / or T1DM, is treated by the composition.

[0170] Example 4: Sequencing of Amuc-i09 Objective: To sequence Amuc-i09 and identify genomically unique sequences.

[0171] Methods: Amuc-i09 was sequenced using PacBio RSII and assembled into one contig that appears to be a complete chromosome, as indicated by repeated overlapping ends.

[0172] After assembly, a circular contig containing the complete genome was obtained. We constructed a database of all publicly available Akkermansia muciniphila proteins by downloading all proteins from the reference sequences assigned to Akkermansia muciniphila. A regression blast search of the predicted Amuc-i09 protein sequence against this database combined with the genome of AMUC-OG revealed a total of two unique genes (diamond blastp with a maximum e-value of 1e-03), SEQ ID NO:1 and SEQ ID NO:2. Of these genes, 0 hit genes from organisms outside the Akkermansia clade (blastp against refseq).

[0173] Thus, the above merely describes the principles of the invention. It will be appreciated that those skilled in the art may devise various modifications that embody the principles of the invention and are within the spirit and scope of the invention, although not expressly described or shown herein. Furthermore, all examples and conditional terms recited herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts provided by the inventors to further the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function, regardless of structure. Thus, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. A strain of Akkermansia muciniphylla bacteria deposited with the American Type Culture Collection (ATCC) under ATCC accession number PTA-126838.

2. A bacterium of the Akkermansia muciniphila strain, comprising one or more nucleic acids encoding a polypeptide containing the amino acid sequence described in SEQ ID NO: 1, a polypeptide containing the amino acid sequence described in SEQ ID NO: 2, or both.

3. A bacterium of the Akkermansia muciniphila strain, comprising a polypeptide containing the amino acid sequence described in SEQ ID NO: 1 and a polypeptide containing the amino acid sequence described in SEQ ID NO:

2.

4. A bacterium expressing a polypeptide containing the amino acid sequence described in SEQ ID NO: 1, a polypeptide containing the amino acid sequence described in SEQ ID NO: 2, or both.

5. A composition comprising the bacteria described in any one of claims 1 to 4.

6. A composition for use in treating metabolic disorders in a subject, comprising an amount of the bacteria described in any one of claims 1 to 4 that is effective in treating the metabolic disorder.

7. Anaerotypes caccae, Anaerobutyricum hallii, Bacteroides finegoldii, Bacteroides ovatus, Bacteroides stercoris, Eubactrium hallii, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Blautia hydrogenotrophica, Blautia producta, Butyrivibrio Fibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium innocuum, Clostridium orbicindens, Enterococcus faecium, Eubacterium rectal, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Oscillospira guilliermondii, Roseburia cecicola, Roseburia inulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerotypes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, EubacteriumThe composition according to claim 5, further comprising one or more additional microorganisms having a 16S rRNA sequence having at least 97% identity to the full length of the 16S rRNA sequence of a microorganism selected from the group consisting of ventriosum, Roseburia faeccis, Roseburia hominis, Roseburia intestinalis, Collinsella aerofaciens, Coprococcus comes, Eubacterium limosum, and Ruminococcus faeccis, and any combination thereof.

8. The composition according to claim 5, further comprising at least one additional microorganism selected from the group consisting of Anaerobutyricum hallii, Clostridium butyricum, Clostridium beijerinckii, Clostridium butyricum, and any combination thereof.

9. The composition according to claim 5, wherein each of the one or more additional microorganisms comprises at least 10^5 AFU / g.

10. The composition according to claim 5, further comprising at least one preservative.

11. The composition according to claim 5, further comprising an enteric coating.

12. The composition according to claim 5, further comprising a prebiotic.

13. The composition according to claim 12, wherein the prebiotics are selected from the group consisting of inulin, green banana, reishi mushroom, tapioca, oats, pectin, potato or its extract, complex carbohydrates, complex sugars, indigestible dextrin, indigestible starch, amino acids, peptides, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, lignin, psyllium, chitin, chitosan, gums (e.g., guar gum), high-amylose corn starch (HAS), cellulose, β-glucan, hemicellulose, lactulose, manno-oligosaccharides, mannan-oligosaccharides (MOS), oligofructose-fortified inulin, oligofructose, oligodextrose, tagatose, trans-galactooligosaccharides, pectin, indigestible starch, xylooligosaccharides (XOS), and any combination thereof.

14. The composition according to claim 12, wherein the prebiotic is inulin.

15. The composition according to claim 5, wherein the bacteria are freeze-dried.

16. The composition according to claim 7, wherein one or more additional microorganisms are freeze-dried.

17. The composition according to claim 5, wherein the bacteria can survive.

18. The composition according to claim 5, wherein the bacteria are unable to survive.

19. The composition according to claim 5, which is formulated as a pharmaceutical product.

20. The composition according to claim 5, which is formulated as a nutritional supplement.

21. The composition according to claim 5, which is formulated as a health supplement.

22. The composition according to claim 5, which is formulated as a medical food.

23. The composition according to claim 5, which does not contain dairy products.

24. The composition according to claim 5, which is substantially free of animal products.

25. The composition according to claim 5, which is in the form of a pill, capsule, lollipop, food bar, or gummy ball.

26. The composition according to claim 5, characterized in that the composition is administered to a subject that requires it.

27. The composition according to claim 6, characterized in that the subject is administered the composition in a dose at least once per day.

28. The composition according to claim 6, characterized in that the subject is administered a single dose of at least 1 × 10^8 CFU of Akkermansia muciniphila strain.

29. The composition according to claim 6, wherein the metabolic disorder is selected from the group consisting of or essentially comprising insulin resistance disorders, intestinal disorders, insulin sensitivity disorders, type 1 diabetes, type 2 diabetes, and obesity.

30. A method comprising culturing the bacteria described in any one of claims 1 to 4 in / on a growth medium under anaerobic conditions.

31. The method according to claim 30, wherein the growth medium is a plant-derived medium.

32. The method according to claim 31, wherein the plant-derived culture medium is VEG medium.

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