WO2022081428A1 - Compositions bactériennes et procédés pour leur utilisation dans le traitement du syndrome métabolique - Google Patents

Compositions bactériennes et procédés pour leur utilisation dans le traitement du syndrome métabolique Download PDF

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WO2022081428A1
WO2022081428A1 PCT/US2021/054144 US2021054144W WO2022081428A1 WO 2022081428 A1 WO2022081428 A1 WO 2022081428A1 US 2021054144 W US2021054144 W US 2021054144W WO 2022081428 A1 WO2022081428 A1 WO 2022081428A1
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bacterial species
anhydrous composition
microbiota
disease
powder
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PCT/US2021/054144
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English (en)
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Emma Allen-Vercoe
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Nubiyota Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism

Definitions

  • the field of invention relates to compositions and methods for treating disorders associated with dysbiosis (i.e., an imbalance of the microbial community inhabiting a subject or inhabiting a particular tissue in a subject).
  • disorders associated with dysbiosis i.e., an imbalance of the microbial community inhabiting a subject or inhabiting a particular tissue in a subject.
  • compositions and methods for treating gastrointestinal disorders associated with dysbiosis are envisioned.
  • Metabolic Syndrome is associated with a variety of diseases and disorders resulting from sedentary lifestyles and poor diets leading to obesity. People with metabolic syndrome tend to have an increase in cardiovascular disease and type 2 diabetes mellitus, which are leading causes of morbidity and increased risk of mortality.
  • the typical diet of obese individuals includes high sugar and processed foods with additives, which lacks a normal, healthy diversity in gut microbiomes. Intestinal microbiota play an important role in metabolizing food into energy and metabolites. Accordingly, there is a need for reagents and methods for using same to restore a healthful balance of microorganisms that comprise a healthy microbiome and treating metabolic syndrome in individuals.
  • MET Microbial Ecosystem Therapeutic
  • MET-5 e.g., MET-5A, MET-5B, MET-5C
  • Additional exemplary subgroups of MET-5, MET- 5A, MET-5B, and MET-5C are set forth in, for example, TABLEs 1-3 presented herein.
  • Bacterial species of METs listed in TABLEs 1-3 and described here may be selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis ster
  • strains of MET-5 may include: NB4-GAM-19; NB4-CNA-21; NB4-D5-8; NB4-DCM- 31; NB4-DCM-7; NB4-FAA-15; NB4-FAA-20; NB4-FAA-24; NB4-FMN-1; NB4-TSAB-40; NB4-FMN- 21; NB4-FMN-6; NB4-GAM-3; NB4-GAM-33; NB4-MRS-35; NB4-NB-2; NB4-TSAB-38; NB4-BHI- 105; NB4-WC-130; NB4-CNA-122; NB4-D5-120; NB4-EtOH-104; NB4-FAA-116; NB4-D5-137; NB4- NA-102; 14 LG of TABLEs 1-3.
  • At least one bacterial species of MET-5 (including MET-5 subgroups thereof; e.g., MET-5A, MET-5B, MET-5C) and compositions comprising, consisting essentially of, or consisting of at least one of, a plurality of, or combinations of bacterial species/strains of MET-5 (including MET-5 subgroups) are encompassed, wherein the total number of bacterial species/strains of MET-5 or a subgroup thereof comprises, consists essentially of, or consists of the total number of bacterial species/strains included in MET-5 or a specific subgroup of MET-5 (e.g., MET-5A, MET-5B, MET-5C) indicated.
  • the subset of bacterial species/strains listed in TABLEs 1-3 comprises, consists essentially of, or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 bacterial species/strains.
  • an anhydrous composition comprising a microbiota, where the microbiota comprises, consists essentially of, or consists of 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, or at least 26, or a plurality of, or a combination of the bacterial species/strains listed in TABLEs 1-3.
  • At least one of, a plurality of, or a combination of bacterial species/strains of MET-5 e.g., MET-5, MET-5A, MET-5B, MET-5C
  • compositions comprising, consisting essentially of, or consisting of at least one, a plurality of, or a combination of bacterial species/strains of MET-5 or subgroups of MET-5 are presented as therapeutic agents or drug products for use in treating a metabolic syndrome, a variety of diseases or disorders associated with metabolic syndrome, dysbiosis, or dysbiosis- associated diseases or disorders.
  • a plurality of, or combination of bacterial species/strains of MET-5 or subgroups of MET-5 e.g., MET-5A, MET-5B, MET-5C
  • Such metabolic syndromes or diseases or disorders of metabolic syndromes may include but are not limited to: cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acylcarnitine translocase deficiency (CACT), carnitine palmitoyl transferase I & II (CPT I deficiency and CPT II deficiency), 2,4 dienoyl-CoA reductase deficiency, and 3- hydroxy-3-methylglutaryl-CoA lyase defici
  • Non-limiting examples of dysbiosis and diseases or disorders associated with dysbiosis may include: Clostridium difficile (Clostridioides difficile) infection, Crohn’s disease, irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, and/or AIDS enteropathy.
  • an anhydrous composition comprising a microbiota, wherein the microbiota comprises, consists essentially of, consists of a plurality of bacterial species comprising, consisting essentially of, or consisting of each of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Bifidobacterium longum; Christensenella minut
  • anhydrous composition comprising a microbiota, wherein the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of a bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscilli
  • an anhydrous composition comprising a microbiota, where the microbiota comprises, consists essentially of, or consists of a plurality or a combination of bacterial species, the plurality or combination of bacterial species comprising, consisting essentially of, or consisting of at least one bacterial species from each phylum of bacteria selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Rumi
  • anhydrous composition comprising a microbiota, wherein the microbiota comprises, consists essentially of, or consists of a plurality or combination of bacterial species comprising, consisting essentially of, or consisting of at least one bacterial species from at least one phylum of bacteria selected from: Firmicutes; Bacteroidetes; Actinobacteria; and Verrucomicrobia, where the bacterial species are in a powder form, where the powder form has a moisture content of less than 5% wt/wt (e.g., 4.5% wt/wt, 4% wt/wt, 3.5% wt/wt, 3% wt/wt, 2.5% wt/wt, 2% wt/wt, 1.5% wt/wt, 1% wt/wt, 0.5%, 0.3%, 0.1%, 0.05%) in the anhydrous composition, and the microbiota exhibits resistance to perturbational stress.
  • the microbiota comprises, consists
  • the plurality of bacterial species comprising, consisting essentially of, or consisting of at least one bacterial species from each phylum of bacterial species may be selected from: Firmicutes; Bacteroidetes; Actinobacteria; and Verrucomicrobia.
  • the bacterial species may also be selected from a phylum of bacterial species selected from: Firmicutes and Verrucomicrobia.
  • the disclosure provides an anhydrous composition
  • a microbiota comprising a microbiota, wherein the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the MET-5A bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerofustis stercorihominis; Parabacteroides distasonis; and Parasutterella excrementihomin
  • a further aspect provides for an anhydrous composition
  • a microbiota comprising a microbiota, where the microbiota comprises, consists essentially of, or consists of at least one of the MET-5B bacterial species selected from: Parabacteroides distasonis; Phascolarctobacterium faecium; Ruminococcus albus; Akkermansia muciniphila; Roseburia faecis; Oscillibacter valericigenes Dorea formicigenerans; Lactobacillus mucosae; Blautia luti; [Clostridium] scindens; Faecalibacterium prausnitzii; Bacteroides uniformis; Coprococcus catus; Bifidobacterium adolescentis; and Collinsella aerofaciens, where the MET- 5B bacterial species are in powder-form, where the powder-form has a moisture content of less than 5% wt/wt (e
  • an anhydrous composition comprising a microbiota, where the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the MET-5C bacterial species selected from: Faecalibacterium prausnitzii; Bacteroides uniformis; Akkermansia muciniphila; Bifidobacterium longum; Christensenella minuta; Parasutterella excrementihominis; and [Clostridium] scindens, where the MET-5C bacterial species are in powder-form, where the powder-form has a moisture content of less than 5% wt/wt (e.g., 4.5% wt/wt, 4% wt/wt, 3.5% wt/wt, 3% wt/wt, 2.5% wt/wt, 2% wt/wt, 1.5% wt/wt, 1% wt/wt,
  • anhydrous composition comprising a microbiota, where the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter
  • an anhydrous composition comprising a microbiota, where the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter
  • anhydrous composition comprising a microbiota, where the microbiota comprises, consists essentially of, or consists of bacterial strains selected from: NB4 GAM 19; NB4 CNA 21; NB4 FAA 15; NB4 FAA 20; NB4 FAA 24; NB4 FMN-1; NB4-FMN-6; NB4-GAM-3; NB4-GAM-33; NB4-MRS-35; NB4-NB-2; NB4-BHI-105; NB4- WC-130; NB4-CNA-122; NB4-EtOH-104; NB4-FAA-116; and NB4-D5-137, where the bacterial species are in powder-form, where the powder-form has a moisture content of less than 5% wt/wt (e.g., 4.5% wt/wt, 4% wt/wt, 3.5% wt/
  • a microbiota that comprises, consists essentially of, or consists of bacterial strains selected from: NB4-GAM-19; NB4-D5-8; NB4- DCM-31; NB4-FAA-15; NB4-FAA-20; NB4-FAA-24; NB4-FMN-1; NB4-FMN-6; NB4-GAM-33; NB4- MRS-35; NB4-NB-2; NB4-FAA-116; NB4-NA-102; NB4-GAM-3; and NB4-WC-130, where the bacterial strains are in powder-form, where the powder-form has a moisture content of less than 5% wt/wt (e.g., 4.5% wt/wt, 4% wt/wt, 3.5% wt/wt, 3% wt/wt, 2.5% wt/wt, 2% wt/wt, 1.
  • 5% wt/wt
  • a further embodiment of any one of the aforementioned aspects may be directed to an anhydrous composition
  • a microbiota comprising a microbiota
  • the microbiota comprises, consists essentially of, or consists of bacterial strains selected from: NB4-GAM-19; NB4-FAA-15; NB4-FAA-24; NB4-TSAB-38; NB4- BHI-105; NB4-D5-137; and NB4-NA-102, where the bacterial strains are in powder-form, where the powder-form has a moisture content of less than 5% wt/wt (e.g., 4.5% wt/wt, 4% wt/wt, 3.5% wt/wt, 3% wt/wt, 2.5% wt/wt, 2% wt/wt, 1.5% wt/wt, 1% wt/wt, 0.5%, 0.3%, 0.1%, 0.05%) in the anhydrous composition, and
  • an anhydrous composition may comprise a microbiota, where the microbiota comprises, consists essentially of, or consists of at least 10% (e.g., 15%, 20%, 21%, 23%, 25%, 27%, 29%, 30%, 31%, 33%, 35%, 37%, 39%, 40%, 41%, 43%, 45%, 47%, 49%, 50%, 51%, 53%, 55%, 57%, 59%, 60%, 61%, 63%, 65%, 67%, 69%, 70%, 71%, 73%, 75%, 77%, 79%, 80%) Gram-negative bacterial species; at least 10% (e.g., 15%, 20%, 21%, 23%, 25%, 27%, 29%, 30%, 31%, 33%, 35%, 37%, 39%, 40%, 41%, 43%, 45%, 47%, 49%, 50%, 51%, 53%, 55%, 57%, 59%, 60%, 61%, 61%, 61%, 69%, 69%, 80%) Gram-
  • a further embodiment of any one of the aforementioned aspects provides an anhydrous composition of any one of the preceding claims, where the microbiota comprises, consists essentially of, or consists of a sub-group as set forth in TABLE 2 or TABLE 3 with respect to category and/or functional properties.
  • the bacterial species of the anhydrous composition are in a state of suspended animation.
  • an anhydrous composition further comprising a pharmaceutically acceptable carrier, where the pharmaceutically acceptable carrier is cellulose.
  • the anhydrous composition is encapsulated in a capsule or in a double capsule.
  • anhydrous composition further comprising a prebiotic.
  • One aspect of the disclosure provides an anhydrous composition of any one of the preceding aspects for use in treating a metabolic syndrome or a disease or disorder associated with metabolic syndrome, where a therapeutically effective amount or an effective amount of the anhydrous composition that improves relative ratios of microorganisms, thereby treating the metabolic syndrome or the disease or disorder associated with metabolic syndrome.
  • the anhydrous composition may be used for treating a metabolic syndrome or a disease or disorder associated with metabolic syndrome, where the metabolic syndrome or disease or disorder associated with metabolic syndrome is selected from: cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acylcarnitine translocase deficiency (CACT), carnitine palmitoyl transferase I & II (CPT I deficiency and CPT II deficiency), 2,4 dienoyl-
  • diabetes e.g., type 2 diabetes
  • an anhydrous composition of any one of the preceding claims may be used in treating a disease or disorder associated with dysbiosis, where an effective amount of the anhydrous composition improves relative ratios of microorganisms, thereby treating the disease or disorder associated with dysbiosis.
  • anhydrous composition for treating dysbiosis or a disease or disorder associated with dysbiosis selected from: Clostridium difficile (Clostridioides difficile) infection, Crohn’s disease, irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, and AIDS enteropathy.
  • a further aspect may be directed to an anhydrous composition of any one of the aforementioned aspects for use in the preparation of a medicament for treating a metabolic syndrome or a disease or disorder associated with metabolic syndrome, where an effective amount of the medicament improves relative ratios of microorganisms, thereby treating the metabolic syndrome or disease or disorder associated with metabolic syndrome.
  • the anhydrous composition may be used in the preparation of a medicament for treating a metabolic syndrome or a disease or disorder associated with metabolic syndrome, where the metabolic syndrome or the disease or disorder associated with metabolic syndrome is selected from: cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acylcarnitine translocase deficiency (CACT), carnitine palmitoyl transferase I & II (CPT I deficiency and CPT II deficiency),
  • cardiovascular disease e.g., type 2
  • Another aspect provides for an anhydrous composition of any one of the aforementioned aspects for use in the preparation of a medicament for treating dysbiosis or a disease or disorder associated with dysbiosis, where a therapeutically effective amount or an effective amount of the medicament improves relative ratios of microorganisms, thereby treating dysbiosis or the disease or disorder associated with dysbiosis.
  • the dysbiosis or disease or disorder associated with dysbiosis is selected from: Clostridium difficile (Clostridioides difficile) infection, Crohn’s disease, irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, and AIDS enteropathy.
  • anhydrous composition comprising, consisting essentially of, or consisting of at least one, a plurality of, or a combination of bacterial species, where the at least one of, the plurality of, or the combination of bacterial species comprising, consisting essentially of, or consisting of at least one of or each of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococc
  • an anhydrous composition may comprise, consist essentially of, or consist of at least one of, a plurality of, or a combination of bacterial species, where the at least one of, the plurality of, or the combination of bacterial species comprising, consisting essentially of, or consisting of at least one of or each of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae;
  • anhydrous composition comprising, consisting essentially of, or consisting of at least one of, a plurality of, or a combination of bacterial species, the at least one of, the plurality of, or the combination of bacterial species comprising, consisting essentially of, or consisting of at least one, a plurality of, or a combination of bacterial species from at least one or each phylum of bacteria selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia
  • an anhydrous composition may comprise, consist essentially of, or consist of at least one of, a plurality of, or a combination of bacterial species, the at least one of, the plurality of, or the combination of bacterial species comprising, consisting essentially of, or consisting of at least one, a plurality of, or a combination of bacterial species from at least one or each phylum of bacteria selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti
  • anhydrous composition comprising, consisting essentially of, or consisting of the bacterial species that are in a state of suspended animation. Any one of the aforementioned aspects directed to anhydrous compositions further comprising a pharmaceutically acceptable carrier, where the pharmaceutically acceptable carrier is cellulose. In other embodiments of any one of the aforementioned aspects, where the anhydrous composition is encapsulated in a capsule or encapsulated in a double-capsule.
  • a further embodiment of any one of the aforementioned aspects provides for anhydrous compositions where the plurality of bacterial species consisting of at least one bacterial species from each phylum of bacterial species selected from: Firmicutes; Bacteroidetes; Actinobacteria; and Verrucomicrobia or selected from a phylum of bacterial species selected from: Firmicutes and Verrucomicrobia.
  • Any of the aforementioned aspects may further comprise, consist essentially of, or consist of a prebiotic.
  • anhydrous composition comprising a microbiota for use in the preparation of a medicament for treating a disease or disorder associated with metabolic syndrome, wherein the microbiota comprises, consists essentially of, or consists of a plurality of bacterial species comprising, consisting essentially of, or consisting of at least one or each of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae;
  • a further aspect of the disclosure may be directed to an anhydrous composition
  • a microbiota for use in the preparation of a medicament for treating a metabolic syndrome or a disease or disorder associated with metabolic syndrome
  • the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Rum
  • anhydrous composition comprising a microbiota for use in the preparation of a medicament for treating a disease or disorder associated with dysbiosis, wherein the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of bacterial species comprising, consisting essentially of, or consisting of at least one of or each of the bacterial species listed in Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti
  • anhydrous composition comprising a microbiota for use in the preparation of a medicament for treating a dysbiosis or a disease or disorder associated with dysbiosis, where the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococc
  • an anhydrous composition comprises, consists essentially of, or consists of bacterial species that are in a state of suspended animation. Another embodiment of any one of the aforementioned aspects provides for an anhydrous composition further comprising a pharmaceutically acceptable carrier, where the pharmaceutically acceptable carrier is cellulose. In a further embodiment of any one of the aforementioned aspects, the anhydrous composition is encapsulated in a capsule or encapsulated in a double capsule. In yet another embodiment of any one of the aforementioned aspects, the anhydrous composition may further comprise a prebiotic.
  • metabolic syndrome or the disease or disorder associated with metabolic syndrome that is selected from: cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acylcarnitine translocase deficiency (CACT), carnitine palmitoyl transferase I & II (CPT I deficiency and CPT II deficiency), 2,4 dienoyl-CoA reductase deficiency, and 3-hydroxy-3-methylglutaryl- Co
  • diabetes e.g., type 2 diabetes, diabetes
  • treating dysbiosis or the disease or disorder associated with dysbiosis is selected from: Clostridium difficile (Clostridioides difficile) infection, Crohn’s disease, irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, or AIDS enteropathy.
  • a further aspect provides for a use of an anhydrous composition
  • a microbiota for treating metabolic syndrome or a disease or disorder associated with metabolic syndrome
  • the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Bifidobacter
  • anhydrous composition comprising a microbiota for treating dysbiosis or a disease or disorder associated with dysbiosis, where the microbiota comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of bacterial species comprising, consisting essentially of, or consisting of at least one of or each of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti
  • One embodiment may provide the use of any one of the aforementioned aspects, where the metabolic syndrome or disease or disorder associated with metabolic syndrome is selected from: cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acylcarnitine translocase deficiency (CACT), carnitine palmitoyl transferase I & II (CPT I deficiency and CPT II deficiency), 2,4 dienoyl-CoA reductase deficiency, and 3-hydroxy-3-methylglutaryl
  • dysbiosis or the disease or disorder associated with dysbiosis is selected from: Clostridium difficile (Clostridioides difficile) infection, Crohn’s disease, irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, and AIDS enteropathy.
  • Clostridium difficile Clostridioides difficile
  • Crohn’s disease irritable bowel syndrome
  • spastic colon idiopathic ulcerative colitis
  • mucous colitis mucous colitis
  • collagenous colitis inflammatory bowel disease in general
  • microscopic colitis antibiotic-associated colitis
  • idiopathic or simple constipation diverticular disease
  • diverticular disease and AIDS enteropathy.
  • Yet another embodiment of any one of the aforementioned aspects provide for uses where the bacterial species are in a state
  • anhydrous composition further comprises a pharmaceutically acceptable carrier, where the pharmaceutically acceptable carrier is cellulose.
  • An embodiment of any one of the aforementioned aspects may be directed to uses where the anhydrous composition is encapsulated in a capsule or encapsulated in a double capsule.
  • uses where the anhydrous composition may further comprise a prebiotic.
  • One aspect of the disclosure provides for a method for treating a mammalian subject afflicted with metabolic syndrome or a disease or disorder associated with metabolic syndrome, where the method comprises: administering a therapeutically effective amount of an anhydrous composition of any one of the preceding aspects to the mammalian subject, where the therapeutically effective amount improves relative ratios of microorganisms in the mammalian subject, thereby treating the mammalian subject suffering from the metabolic syndrome or the disease or disorder associated with metabolic syndrome.
  • the metabolic syndrome or disease or disorder associated with metabolic syndrome is selected from: cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acylcarnitine translocase deficiency (CACT), carnitine palmitoyl transferase I & II (CPT I deficiency and CPT II deficiency), 2,4 dienoyl-CoA reductase deficiency, and 3-hydroxy-3-methylglutaryl- CoA
  • a method for treating a mammalian subject afflicted with dysbiosis or a disease or disorder associated with dysbiosis comprising: administering a therapeutically effective amount of an anhydrous composition of any one of the preceding claims to the mammalian subject, wherein the therapeutically effective amount improves relative ratios of microorganisms in the mammalian subject, thereby treating the mammalian subject.
  • a further embodiment of any one of the aforementioned aspects provides methods, where dysbiosis or the disease or disorder associated with dysbiosis is selected from: Clostridium difficile (Clostridioides difficile) infection, Crohn’s disease, irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, and AIDS enteropathy.
  • Clostridium difficile Clostridioides difficile
  • Crohn’s disease irritable bowel syndrome
  • IBS irritable bowel syndrome
  • spastic colon idiopathic ulcerative colitis
  • mucous colitis mucous colitis
  • collagenous colitis inflammatory bowel disease in general
  • microscopic colitis antibiotic-associated colitis
  • idiopathic or simple constipation diverticular disease
  • diverticular disease and AIDS enteropathy
  • FIG. 1 shows the pooled alpha diversity results for all seven fecal samples using the Shannon diversity index.
  • sample 4 (NB4-04) had the highest alpha (within sample) diversity and therefore afforded the best opportunity to capture culturable diversity from the donor.
  • Increasing class numbers correspond to data graphed from left to right.
  • FIG. 2 illustrates a 16S rRNA gene community profile analysis of a stool sample (NB4-04). Analysis was conducted at the genus level and depicts all genera present above 0.1% abundance of the total community composition. Decreasing genus names (i.e., from top to bottom) correspond to decreasing percentages of NB4-0416S rRNA gene profile (i.e., from 100% to 0%). DETAILED DESCRIPTION OF THE INVENTION [40] Among those benefits and improvements that have been disclosed, other objects and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying figures.
  • Patent law can mean “ includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • all ranges of numeric values include the endpoints and all possible values disclosed between the disclosed values. The exact values of all half-integral numeric values are also contemplated as specifically disclosed and as limits for all subsets of the disclosed range.
  • a range of from 0.1% to 3% specifically discloses a percentage of 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%.
  • a range of 0.1 to 3% includes subsets of the original range including from 0.5% to 2.5%, from 1% to 3%, from 0.1% to 2.5%, etc. It will be understood that the sum of all weight % of individual components will not exceed 100%.
  • various components may be identified having specific values or parameters, however, these items are provided as exemplary embodiments. Indeed, the exemplary embodiments do not limit the various aspects and concepts of the present disclosure as many comparable parameters, sizes, ranges, and/or values may be implemented.
  • the terms “first,” “second,” and the like, “primary,” “secondary,” and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
  • ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise.
  • the phrases “in one embodiment” and “in some embodiments” as used herein do not necessarily refer to the same embodiment(s), though it may.
  • the phrases “in another embodiment” and “in some other embodiments” as used herein do not necessarily refer to a different embodiment, although it may.
  • Detect refers to identifying the presence, absence or amount of an analyte to be detected.
  • detecttable label is meant any molecule or composition that when linked to a molecule of interest (e.g., analyte, detector reagent, analog, or binding partner) renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
  • useful labels include enzymes (for example, as commonly used in an ELISA), enzyme substrates, radioactive isotopes, magnetic beads, metallic beads, colloidal particles (e.g., colloidal gold particles), chemiluminescent or fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenin, or haptens, and the like.
  • enzymes for example, as commonly used in an ELISA
  • enzyme substrates for example, as commonly used in an ELISA
  • radioactive isotopes for example, as commonly used in an ELISA
  • magnetic beads for example, as commonly used in an ELISA
  • metallic beads metallic beads
  • colloidal particles e.g., colloidal gold particles
  • chemiluminescent or fluorescent dyes e.g., chemiluminescent or fluorescent dyes
  • electron-dense reagents e.g., enzymes, biotin, digoxigenin, or haptens, and the like.
  • a compound e.g., an antibody
  • the attachment of a compound (e.g., an antibody) to a label may be through covalent bonds, adsorption processes, hydrophobic and/or electrostatic bonds, as in chelates and the like, or combinations of these bonds and interactions and/or may involve a linking group.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. As used herein, “disease” may be used interchangeably with disorder or condition.
  • the term “dysbiosis” as used herein refers to an imbalance of a microbial community or population inhabiting a subject or inhabiting a particular tissue or organ in a subject.
  • metabolic syndrome typically refers to a decrease in beneficial microbes relative to deleterious microbes or a change in the ratio of microbes such that microbes that are normally only present in small numbers proliferate to a degree whereby they are present at elevated numbers, which may be deleterious for the subject.
  • metabolic syndrome is a term that emphasizes traits having an increased risk of disease, such as, for example, cardiovascular disease and type 2 diabetes mellitus. Conditions primarily responsible for most cases of metabolic syndrome are obesity and insulin resistance. Other names for metabolic syndrome include insulin resistance syndrome or syndrome X.
  • Metabolic syndrome may include, for example, obesity, hypertension, dyslipidemia and hyperglycemia and is associated with insulin resistance and the risk of diabetes mellitus in addition to non- alcoholic fatty liver disease.
  • metabolic syndrome may be linked to the etiology and progression of some cancers.
  • diabetes and obesity have separately been affiliated with cancers of the breast, endometrium, pancreas, bowel, colon (and rectal parts of the large intestine), liver, kidney, and uterus.
  • Individuals suffering from metabolic syndrome may include those who exhibit three or more of the following cardiovascular risk factors: increased waist-to-hip and waist-to-thigh ratios, waist circumference, and sagittal abdominal diamter all associated with central obesity; hypertension; hypertriglyceridaemia; low HDL-C; and elevated fasting blood glucose.
  • Central obesity is defined by the World Health Organization (WHO) as a waist circumference of more than 102 cm for men and more than 88 cm for women.
  • WHO World Health Organization
  • a hypertriglyceridaemia definition is defined as triglyceride levels of more than 150 mg/dL.
  • Reduced high-density lipoprotein cholesterol (HDL-C) may be defined as 85 mmHg.
  • Elevated fasting blood glucose in individuals may have a fasting plasma glucose of more than 100 mg/dL.
  • Recommended treatments include lifestyle changes of exercise and diet resulting in weight loss and drugs for treating conditions characterized by high blood sugar (e.g., fasting at least 8 hrs with blood sugar greater than 130 mg/dL; two hours after-meal blood sugar greater than 140 mg/dL); high blood pressure (e.g., greater than a normal, healthy blood pressure of 120/80; 130/80 or greater; systolic blood pressure 120 or greater; diastolic blood pressure 80 or higher); high levels of triglycerides (e.g., 150 mg/dL or greater; 1.7 mmol/L or greater); high levels of low-density lipoproteins (LDLs; “bad cholesterol;” 100 mg/dL or greater; 130 mg/dL or greater), while having low levels of high-density lipoproteins (HDLs; “good cholesterol;” less than 50 mg/dL or 40 mg/dL).
  • the term “effective amount” or “therapeutically effective amount” of a composition or drug product is that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied.
  • the bacterial species/strains and/or compositions comprising, consisting essentially of, or consisting of the at least one bacterial species/strain or a plurality of or a combination of bacterial species/strains are administered in an effective amount for the treatment or prophylaxis of a disease, disorder, or condition, where disease, disorder, and condition are used interchangeably here.
  • compositions or drug products of the disclosure in an effective amount to treat metabolic syndrome, diseases associated with metabolic syndrome, dysbiosis, or diseases associated with dysbiosis.
  • an effective amount of the bacterial species/strains and/or compositions comprising, consisting essentially of, or consisting of the bacterial species/strain or plurality of bacterial species/strains is, for example, an amount sufficient to achieve alleviation or amelioration or prevention or prophylaxis of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the metabolic syndrome or disease, disorder, or condition associated with metabolic syndrome (e.g., cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia,
  • isolated refers to material (e.g., bacterial strains and/or species) that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” microorganism e.g., bacteria, bacterial strains, bacterial species) is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the microorganism or cause other adverse consequences.
  • a microorganism e.g., bacterial species/strains
  • the bacterial species/strains may be present in an amount of at least 5% wt/wt (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%) in the composition.
  • a population of bacterial species/strains may be in an amount of at least 1x10 3 CFU/g (e.g., 1x10 4 CFU/g, 1x10 5 CFU/g, 1x10 6 CFU/g, 1 x10 7 CFU/g, 1x10 8 CFU/g) in the composition disclosed here.
  • the bacteria present in the bacterial populations described here are viable or alive, where the bacteria are active or functional, and the bacterial population may be in a liquid or dry, powder form.
  • the term “OTU” refers to an operational taxonomic unit, defining a species, or a group of species via similarities in nucleic acid sequences, including, but not limited to 16S rRNA gene sequences.
  • the term “state of suspended animation” as used herein with respect to a population of bacteria refers to a population of bacteria that is metabolically quiescent, but capable of resuming normal metabolic activity and proliferating in response to suitable growth promoting conditions.
  • the term “prebiotic” as used herein refers to “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health”. See Roberfroid (2007, J Nutri 137:8305-8375. Particular prebiotics may be chosen for optimal results when used in conjunction with compositions described herein based on the mode of administration to the subject and the target tissue/s needing treatment.
  • compositions described herein may be food grade.
  • Particular prebiotics envisioned for use in combination with compositions described herein include: inulin, fructo- oligosaccharides, or gluco-oligosaccharides and mixtures thereof.
  • Solutions of bacterial species/strains may be freeze dried/lyophilized to generate anhydrous compositions comprising a plurality of bacterial species/strains having a moisture content of less than 25% (e.g., 20%, 15%, 10% 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%).
  • anhydrous compositions described herein are freeze dried to a moisture content of less than 5 % (e.g., 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%).
  • freeze dried/lyophilized refers to a laboratory method (e.g., shelf lyophilizer, manifold) where live microbes in aqueous suspension are rapidly frozen to less than 20°C ( ⁇ 20°C) (e.g., 10°C, 5°C, 4°C, 2°C, 1°C, -15°C, -20°C, -25°C, -40°C, -80°C, -120°C, -150°C), and then the majority of the frozen water content is forced to sublime under vacuum conditions (e.g., less than 200 millitorr (mtorr), 100 mtorr, 0.133 mbar, 13.3 Pascal, 99.99% vacuum), allowing this water to be efficiently removed in
  • lyophilized bacterial strains or species have a moisture content of less than 5 % (e.g., 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 0.05%), or a moisture content ranging from 0%-5% (e.g., 0.08%-4.5%, 0.15%-4.75%, 0.25%-4.25%, 0.4%-4%, 0.75%-3.75%, 1.25%- 3.5%, 1.5%-3.25%, 2.25%-3%, 2.5%-2.75%).
  • 0%-5% e.g., 0.08%-4.5%, 0.15%-4.75%, 0.25%-4.25%, 0.4%-4%, 0.75%-3.75%, 1.25%- 3.5%, 1.5%-3.25%, 2.25%-3%, 2.5%-2.75%.
  • anhydrous composition comprising a plurality of bacterial species/strains refers to a manmade, freeze dried/lyophilized population of bacterial species/strains substantially free of moisture (e.g., water), such as for example, having a moisture content of less than 25% wt/wt (e.g., 20%, 15%, 10% 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.25%, 0.1%) in the anhydrous composition.
  • moisture e.g., water
  • a plurality of bacterial species/strains is isolated from the fecal matter of a single, healthy individual wherein the plurality of bacterial species/strains has been selected and adapted as a bacterial population.
  • one or more of the pluralities of bacterial species or strains (i.e., bacterial species/strains) in the composition described herein may be isolated from the fecal matter of multiple (i.e., more than one) healthy individuals.
  • Yet further embodiments may provide for a bacterial suspension of cultured isolates from at least one healthy individual, and in some instances from at least two healthy individuals.
  • Anhydrous compositions of the disclosure may comprise a bacterial suspension of a plurality of bacterial species or strains, where the bacterial species or strains may be freeze-dried and/or lyophilized to a powder form, and the powder form has a moisture content of less than 5% wt/wt in the anhydrous composition.
  • Technologies typically used for moisture determination include, without limitation: thermogravimetric analysis (oven drying, halogen/IR drying, microwave drying, etc.); chemical analysis (Karl Fischer titration, calcium carbide testing); spectroscopic analysis (IR spectroscopy, microwave spectroscopy, proton nuclear magnetic resonance spectroscopy); and other analyses (e.g. gas chromatography, density determination, refractometry, etc.).
  • thermogravimetric analysis moisture content is derived from the loss of product weight during drying by measuring the change in mass of a sample while being heated at a controlled rate until no more change in weight is observed.
  • microbiota refers to microorganisms that typically inhabit a host, such as a human body, particularly of the gut. In particular, the gut has a densely colonized population of microorganisms or microbiota, which plays a role in digestion.
  • “Microbiota ecosystem therapeutics” (METs) as used herein, is an alternative treatment approach to “fecal microbial therapy” (FMT), which has risks and challenges.
  • microbiota refers to a plurality of bacterial strains or bacterial species that has collectively undergone selection and adaptation in an individual subject (e.g., a healthy subject not suffering from dysbiosis).
  • the selected microbiota has collectively undergone selection and adaptation in the intestines of a single, healthy subject, but could also refer to a microbiota comprising a plurality of bacterial strains or species selected from a pooled sample from more than one healthy subject who does not suffer from dysbiosis or dysbiosis-associated diseases or disorders.
  • MET compositions may have a defined mixture of pure live cultures of intestinal bacteria isolated from a stool sample of a healthy donor and/or a synthetic mixture of such bacteria.
  • METs One of the benefits of METs is the standardization of the manufacturing process and the ability to create a quality-controlled preparation, which is a requirement for biologic drug regulation.
  • the MET treatment by oral administration of a live, anhydrous or freeze-dried preparation is simpler and safer as opposed to FMT’s donor stool infusion into a recipient subject’s gastrointestinal tract.
  • the composition of the disclosure comprising a population of bacterial species/strains described here may be administered in a liquid formulation.
  • “MET-5” as used herein refers to a plurality of bacterial species/strains that were isolated from the stool of a healthy donor.
  • MET-5 also has subgroups identified herein as “MET-5A”, “MET-5B”, and “MET-5C,” which may be used interchangeably with MET-5 unless specifically distinguished.
  • MET-5 may be directed to 26 bacterial species or bacterial strains identified here.
  • MET-5A may provide 17 bacterial species or bacterial strains selected from those identified in MET-5.
  • MET-5A is a representative subset of MET-5 at the family taxonomic level, and this subset can form a stable ecosystem in a subject’s gut microbiome or chemostat model.
  • MET-5B may be directed to 15 bacterial species or bacterial strains selected from those identified in MET-5 that aligns taxonomically with MET-3 (see, e.g., MET-2A at WO 2019/136269, incorporated herein by reference in its entirety). While MET-5C may provide 7 bacterial species or bacterial strains selected from those identified in MET 5 and form a stable ecosystem in a subject or chemostat model.
  • composition or “pharmaceutical composition” represents a composition containing, comprising, consisting essentially of, or consisting of a microbiota having at least a plurality of bacterial strains or bacterial species described herein and formulated with a pharmaceutically acceptable carrier, excipient, or diluent.
  • a composition may comprise a microbiota, where the microbiota comprises, consists essentially of, or consists of at least a plurality of MET-5 bacterial species or strains thereof.
  • MET-5 may be used interchangeably with any of the MET-5 subgroups, namely, MET-5A, MET-5B, MET-5C, unless otherwise indicated.
  • compositions may comprise, for example, a microbiota, wher, for example, the microbiota may comprise, consist essentially of, or consist of at least a plurality of MET-5A, MET-5B, or MET-5C bacterial species or strains thereof.
  • the microbiota may comprise, consist essentially of, or consist of at least a plurality of MET-5A, MET-5B, or MET-5C bacterial species or strains thereof.
  • One embodiment may provide an anhydrous and/or lyophilized composition comprising a microbiota of at least a plurality of MET-5, MET-5A, MET-5B, or MET-5C bacterial species or strains, or combinations thereof, which may further contain a pharmaceutically acceptable carrier, excipient, or diluent.
  • compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gel cap, etc.); or in any other formulation described herein.
  • unit dosage forms also referred to as unitary dosage forms, often denote those forms of medication supplied in a manner that does not require further weighing or measuring to provide the dosage (e.g., tablet, capsule, caplet, etc.).
  • a unit dosage form may refer to a physically discrete unit suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with any suitable pharmaceutical excipient or excipients.
  • Exemplary, non-limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, and gel cap.
  • the compounds described herein, including crystallized forms, polymorphs, and solvates thereof, may be present in a unit dosage form.
  • pharmaceutically acceptable means generally safe for ingestion or contact with biologic tissues at the levels employed. Pharmaceutically acceptable is used interchangeably with physiologically compatible. It will be understood that the pharmaceutical compositions of the disclosure include nutraceutical compositions (e.g., dietary supplements) unless otherwise specified.
  • phrases “pharmaceutically-acceptable excipient” may include pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • the term “subject” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • Typical subjects or “patients” include any animal (e.g., mammals such as murine, canine, feline, non-human primates, and humans, etc.).
  • non-limiting examples of animal subjects or patients include mice, rats, cows, pigs, horses, chickens, cats, dogs, etc., and is in some instances a mammal, such as a primate, and in other instances, a human.
  • a subject in need thereof is typically a subject for whom it is desirable to treat a disease, disorder, or condition as described herein.
  • a subject in need thereof may seek or be in need of treatment, require treatment, be receiving treatment, may be receiving treatment in the future, or a human or animal that is under care by a trained professional for a particular disease, disorder, or condition.
  • the term “treat,” “treating,” or “treatment” of any disease or disorder refers, in one embodiment, to reducing or ameliorating the disease or disorder (i.e., arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof) and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the treatment of a disease, disorder, or condition e.g., the conditions described herein such as metabolic syndrome or dysbiosis
  • beneficial or desired results such as clinical results.
  • Exemplary metabolic syndrome diseases include, but are not limited to, cardiovascular disease, diabetes, high blood pressure, high blood sugar, high triglycerides,
  • diseases associated with dysbiosis include Clostridium difficile (Clostridioides difficile) infection, Crohn’s disease, irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, and AIDS enteropathy.
  • Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable.
  • “Palliating” a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.
  • “treat,” “treating,” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject.
  • “treat,” “treating,” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both.
  • “treat,” “treating,” or “treatment” relates to slowing the progression of the disease.
  • the term “prevent,” “preventing,” “prevention,” or “prophylactic treatment” refers to a reduction in probability or risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to a disease- causing agent, or predisposed to the disease in advance of disease onset).
  • the term “prophylaxis” is related to “prevention” and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease.
  • Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.
  • an anti-malarial agent such as chloroquine
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • the phrase therapeutically effective amount is used to refer to an amount of an agent (e.g., a therapeutic agent) sufficient to reduce a pathological feature of a disease or condition by at least about 30 percent, by at least 50 percent, or by at least 90 percent.
  • a “therapeutically effective amount” of an agent results in a clinically significant reduction in at least one pathological feature (e.g., a clinical symptom) of a disease or condition.
  • the term “robustness” as it relates to a microbial community refers to the resistance and resilience of the community towards external perturbation(s) relative to the state of the microbial community absent or prior to exposure to the external perturbation(s). Robustness may, for example, be reflected in the ability of the microbial community to maintain relative ratios of representation (numbers) of each of the different species or phylums wherein the species are classified post-perturbation as compared to pre-perturbation.
  • Robustness may also, for example, be reflected in the ability of the microbial community to maintain metabolic output post-perturbation relative to pre-perturbation.
  • a healthy gastrointestinal/gut microbiome is known to impact an individual’s health by providing important benefits, including but not limited to, developing the immune system, utilizing and metabolizing nutrients, preventing infections, and even potentially affecting the function of the brain and nervous system.
  • the relative ratios of different bacterial species/strains in the gut microbiome of an individual suffering from a metabolic syndrome, a disease associated with metabolic syndrome, dysbiosis, or a dysbiosis-associated disease may be improved to those ratios found in the gut microbiome of a healthy individual (i.e., one who is not suffering from disease).
  • the term “perturbational stress” refers to a change in at least one of substrate type, substrate availability, and xenobiotic challenge in the culturing conditions in which a population of bacterial cells is grown.
  • substrate refers to a substance or compound present in the culture medium in which a population of bacterial cells is grown that is utilized metabolically by the bacterial cells.
  • xenobiotic challenge refers to the introduction of a chemical substance into an ecosystem, wherein the chemical substance is not naturally produced or expected to be present within the ecosystem or is present at a much higher concentration than in the natural situation.
  • reference is meant a standard or control condition.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Such a sequence is at least 60% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 99%) identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.
  • BLAST Altschul et al.
  • BESTFIT Altschul et al.
  • GAP Garnier et al.
  • PILEUP/PRETTYBOX programs Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • a BLAST program may be used, with a probability score between e -3 and e -100 indicating a closely related sequence.
  • nucleic acid or a “nucleic acid molecule” refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.
  • nucleic acid molecules a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction.
  • isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated.
  • an “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
  • isolated nucleic acid refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined here. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it is generally associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • nucleic acid and “polynucleotide” as used herein refer to an oligonucleotide, polynucleotide or nucleotide and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double stranded, and represent the sense or anti sense strand.
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiments being described.
  • the DNA of most bacteria are in a single circular molecule, i.e., bacterial chromosome, although some bacterial DNA may be linear.
  • bacterial DNA also encodes proteins via messenger RNA (mRNA).
  • mRNA messenger RNA
  • naturally allelic variants “mutants”, and “derivatives” of particular sequences of nucleic acids refer to nucleic acid sequences that are closely related to a particular sequence but which may possess, either naturally or by design, changes in sequence or structure. By closely related, it is meant that at least about 60%, but often, more than 85%, of the nucleotides of the sequence match over the defined length of the nucleic acid sequence referred to using a specific sequence identifier (i.e., SEQ ID NO).
  • Changes or differences in nucleotide sequence between closely related nucleic acid sequences may represent nucleotide changes in the sequence that arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence.
  • Other changes may be specifically designed and introduced into the sequence for specific purposes, such as to change an amino acid codon or sequence in a regulatory region of the nucleic acid.
  • Such specific changes may be made in vitro using a variety of mutagenesis techniques or produced in a host organism placed under particular selection conditions that induce or select for the changes.
  • sequence variants generated specifically may be referred to as “mutants” or “derivatives” of the original sequence.
  • the terms “percent similarity”, “percent identity” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program and are known in the art.
  • the phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given sequence. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
  • a “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control.
  • a replicon may be either RNA or DNA and may be single or double stranded.
  • a “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
  • an “expression vector” or “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • transcriptional and translational control sequences such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • the term “operably linked” refers to a regulatory sequence capable of mediating the expression of a coding sequence and which are placed in a DNA molecule (e.g., an expression vector) in an appropriate position relative to the coding sequence so as to effect expression of the coding sequence.
  • oligonucleotide refers to primers and probes described herein, which are defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
  • the term “probe” refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre- determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
  • hybridize is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringency See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
  • stringent salt concentration will ordinarily be less than 750 mM NaCl and 75 mM trisodium citrate, less than 500 mM NaCl and 50 mM trisodium citrate, and less than 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least 35% formamide, and at least 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least 30° C, of at least 37° C, and of at least 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than 30 mM NaCl and 3 mM trisodium citrate, and less than 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least 25° C, of at least 42° C, and of at least 68° C. In one embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet a further embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al.
  • the term “specifically hybridize” refers to the association between two single- stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary ”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • the term “primer” refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.
  • suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH
  • the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.
  • the primer may vary in length depending on the particular conditions and requirement of the application.
  • the oligonucleotide primer is typically 15-25 or more nucleotides in length.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal to the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template.
  • a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer.
  • non- complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • Primers and/or probes may be labeled fluorescently with 6-carboxyfluorescein (6-FAM).
  • primers may be labeled with 4, 7, 2', 7'-Tetrachloro-6-carboxyfluorescein (TET).
  • TERT 4, 7, 2', 7'-Tetrachloro-6-carboxyfluorescein
  • the term “ecosystem output assay” refers to a method whereby the composition of a microbial ecosystem may be determined from its functional output in terms of types and quantities of selected small molecule metabolites.
  • Small molecule metabolites are known in the art and include, without limitation: organic acids (e.g., carboxylic acids and derivatives thereof), amino acids, alcohols (e.g., polyols), phenols, and fatty acids and conjugates thereof. Metabolites are typically measured in the range of millimolar concentrations.
  • Carboxylic acid derivatives as used here may include, but are not limited to, carboxylates (deprotonated carboxylic acids), amides, esters, thioesters, and acyl phosphates.
  • Fatty acids as used here may include hydrophobic molecules containing an aliphatic hydrocarbon chain terminating in a carboxylic acid moiety.
  • a conjugate of a fatty acid may be the conjugate base of the fatty acid, which results from deprotonation of the carboxylic acid group of the corresponding fatty acid.
  • Amino acid derivatives as used here may include resultants from amino acid reactions at the amino group, carboxy group, side-chain functional group, or from the replacement of any hydrogen by a heteroatom.
  • a microbiota ecosystem therapeutics community exhibits a metabolic profile that comprises tartrate and urea and significantly elevated levels of glutamate, pyroglutamate, asparagine, glycolate, choline, thymine, and formate when compared to the metabolic profiles of bacterial communities isolated from different donors. See Yen et al. (2015, J Proteome Res 14:1472-1482).
  • metabolic disease or disorder or “metabolic syndrome,” used interchangeably here, is meant a disease, disorder, or condition having abnormal chemical reactions in a subject that disrupt the process of metabolism to make or obtain energy, which include, but are not limited to, for example, acid- base imbalance; calcium metabolism disorders; glucose metabolism disorders; hyper lactatemia; iron metabolism disorders; lipid metabolism disorders; malabsorption syndromes; phosphorus metabolism disorders; and the like.
  • the metabolic disease results when a subject’s metabolism fails or induces the subject to have either too much or too little of essential substances necessary for good health.
  • Non-limiting examples of metabolic diseases include, but not limited to hypertriglyceridemia, insulin resistance, obesity, diabetes (e.g., type 2 diabetes, diabetes mellitus), hypertension, dyslipidemia, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acylcarnitine translocase deficiency (CACT), carnitine palmitoyl transferase I & II (CPT I deficiency and CPT II deficiency), 2,4 dienoyl-CoA reductase deficiency, 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG deficiency).
  • hypertriglyceridemia insulin resistance, obesity, diabetes (e.g., type 2 diabetes
  • the term “microbial ecosystem” refers to a plurality of different bacterial species/strains that have been grown together either in an in vitro assay or in a biological setting such as, for example, a subject’s gut. In a particular embodiment, the subject may be a human.
  • the term “chemostat model assay” refers to an assay wherein a plurality of bacterial species/strains is seeded into a vessel compatible with bacterial proliferation, wherein the vessel is maintained under growth promoting conditions and comprises culture medium comprising growth factors suitable for promoting proliferation of the plurality of bacterial species/strains.
  • the proliferation of each of the bacterial species/strains seeded into the vessel may be determined after a defined time period of incubation in the chemostat model assay. Such a determination may be made using techniques known in the art such as cell counting via automated or manual means and may be facilitated by cell staining using various dyes that are taken up by cells. Such dyes may be taken up differentially by live versus dead cells and thus, provide for distinguishing viable cells from dead or dying cells.
  • the relative proliferation of each of the bacterial species/strains seeded into tlie vessel may also be determined and total numbers of each bacterial species/strains determined after a defined time period of incubation in the chemostat model assay.
  • the chemostat model assay may be used to determine proliferation and/or proliferation rate of different bacterial species/strains in the plurality of bacterial species/strains seeded into the vessel and thus, provide an assay for comparing proliferation and/or proliferation rate among the different bacterial species/strains seeded into the vessel under various growth promoting conditions.
  • oligonucleotides according to the present invention that hybridize to nucleic acid sequences identified as specific for one of the bacterial species and/or strains (i.e., bacterial species/strains) described herein, are at least about 10 nucleotides in length, more particularly at least 15 nucleotides in length, more particularly at least about 20 nucleotides in length.
  • fragments of nucleic acid sequences identified as specific for one of the bacterial species and/or strains described herein represent aspects of the present invention.
  • Such fragments and oligonucleotides specific for same may be used as primers or probes for determining the amount of the particular bacterial species and/or strain in a bacterial sample generated in vitro or in a biological sample obtained from a subject, wherein the particular species or strain may be identified by the presence of any one of the sequences presented here.
  • Primers such as those described herein e.g., V3kl and V6r
  • PCR polymerase chain reaction
  • a given strain is species specific and, in many cases, depending on the species, strain specific as well. Further to this point, some bacterial species/strains are highly conserved and thus, different strains may have extremely similar or even identical sequences. Most species, however, include strains wherein sequence differences are detected.
  • -Cycle parameters are 94°C for (the initial) 10 minutes, (94°C for 30s, 60° C for 30s, 72°C for 30s) for 30 cycles, then 72°C for 5 minutes, and 4°C for indefinite time.
  • TABLE 1 presents a list of MET-5 strains, which is an exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described herein if the bacterial species/strains are derived from a microbiota.
  • an exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described here comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the following strains listed in, for example, TABLE 1, but does not exceed including each and every one of the species recited in the exemplary list of TABLE 1.
  • the exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described here may comprise, consist essentially of, or consists of at least one, a plurality of, a combination of, or each of the bacterial species/strains listed in TABLE 1.
  • TABLE 1 MET-5 Bacterial Strains/Species Strain name Closest species match Gram Status NB4 MRS 35 L t b ill G iti nd is t e subject o WO 0 8/ 9795 .
  • an exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described herein comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the following species/strains listed in TABLE 1: Faecalibacterium prausnitzii (NB4-GAM-19); Barnsiella intestinihominis (NB4-CNA-21); Dorea formicigenerans (NB4- D5-8); Coprococcus catus (NB4-DCM-31); Bacteroides uniformis (NB4-FAA-15); Bifidobacterium adolescentis (NB4-FAA-20); Akkermansia muciniphila (NB4-FAA-24); Phascolarctobacterium faecium (NB4-FMN-1); Bacteroides xylanisolvens (NB4-FMN-21); Collinsella aerofaciens (NB4-FMN-6);
  • Subgroups of MET-5 may comprise, consist essentially of, or consist of at least one, a plurality of, or a combination of bacterial species/strain identified in TABLE 1.
  • Various bacterial populations may be, for example, selected for removing or adding functionally redundant bacterial species/strains, optimized for increasing or decreasing certain metabolites, or optimized for positively- or negatively-associated characteristics.
  • certain bacterial species/strains may be positively- associated with or correlated with decreasing risks of various metabolic syndromes, diseases associated with metabolic syndromes, dysbiosis, or dysbiosis-associated diseases.
  • bacterial populations may be selected for producing short chain fatty acids (SCFAs), which are the primary products of the breakdown of non-digestible carbohydrates, for example acetate, butyrate, and propionate.
  • SCFAs short chain fatty acids
  • Acetate is known to regulate the pH level in the gut, control appetite, protect against pathogens, and nourish butyrate-producing bacteria.
  • Acetate is produced mainly by Bifidobacteria and Lactobacilli, but also Akkermansia muciniphila, Prevotella spp., and Ruminococcus spp.
  • Butyrate is another important SCFA for digestive health and healthy gut. It can control inflammation and prevent disease. Butyrate is also known to neutralize free radicals in the gut, which is linked to inflammation.
  • butyrates may also prevent gut inflammation and improve obesity and type 2 diabetes.
  • Firmicutes family are known for making this SCFA.
  • the main producers of butyrate are anaerobic bacteria like Faecalibacterium prausnitzii, Eubacterium rectale, and Roseburia spp.
  • Another SCFA propionate, regulates appetite, combats inflammation, and helps protect against cancer.
  • Propionate forms when carbohydrates are broken down by bacteria, including those from the Bacteroidetes, Firmicutes, and Lachnospiraceae phyla.
  • lactate is not technically a short chain fatty acid, it is produced by gut bacteria and contributes to the health of the colon.
  • lactate Like acetate, lactate nourishes butyrate-producing bacteria, regulates the immune system, and combats opportunistic bacteria.
  • lactobacillus One of the main producers of lactate is Lactobacillus.
  • Amino acids and their metabolites may regulate energy and immune balance in organisms. For example, in metabolic reactions, alanine (Ala), leucine (Leu), isoleucine (Ile), valine (Val), and histidine (His) may act as hydrogen donors; whereas, glycine (Gly), proline (Pro), ornithine (Orn), arginine (Arg), and tryptophan (Trp) may function as hydrogen receptors.
  • Amino acids arginine, proline, and cysteine may be used to produce metabolites; whereas, enterocytes may use glutamine (Gln), glutamic acid (Glu), and aspartic acid (Asp) as fuel and may advance epithelial cell renewal and assists with nutrient absorption.
  • enterocytes may use glutamine (Gln), glutamic acid (Glu), and aspartic acid (Asp) as fuel and may advance epithelial cell renewal and assists with nutrient absorption.
  • the genera: Bacteroides, Clostridium, Propionibacterium, Fusobacterium, Streptococcus, and Lactobacillus may metabolize amino acids to ammonia, nitrogen-compounds, and toxic metabolites, such as but not limited to, amines, phenols, and indoles.
  • Non-limiting examples of disorders that may affect the metabolism of amino acids include: phenylketonuria, non-ketotic hyperglycinemia, homocystinuria, tyrosinemia, and maple syrup urine disease.
  • the exemplary list of bacterial species/strains that exhibit robustness in chemostat model test assays described herein comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the following bacterial species/strains listed in TABLE 1: Faecalibacterium prausnitzii (NB4-GAM-19); Barnsiella intestinihominis (NB4-CNA-21); Bacteroides uniformis (NB4-FAA-15); Bifidobacterium adolescentis (NB4-FAA-20); Akkermansia muciniphila (NB4- FAA-24); Phascolarctobacterium faecium (NB4-FMN-1); Collinsella aerof
  • the exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described herein comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the following species/strains listed in TABLE 1: Faecalibacterium prausnitzii (NB4-GAM-19); Dorea formicigenerans (NB4-D5-8); Coprococcus catus (NB4-DCM-31); Bifidobacterium adolescentis (NB4-FAA-20); Akkermansia muciniphila (NB4-FAA-24); Phascolarctobacterium faecium (NB4-FMN-1); Bacteroides xylanisolvens (NB4-FMN-21); Collinsella aerofaciens (NB4-FMN-6); Roseburia faecis (NB4-GAM-3); Blautia luti (NB4
  • Another embodiment provides the exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described here comprises, consists essentially of, or consists of at least one of, a plurality of, or a combination of the following strains listed in TABLE 1: Faecalibacterium prausnitzii (NB4-GAM-19); Bacteroides uniformis (NB4-FAA-15); Akkermansia muciniphila (NB4-FAA-24); Bifidobacterium longum (NB4-TSAB-38); Christensenella minuta (NB4- BHI-105); Parasutterella excrementihominis (NB4-D5-137); and [Clostridium] scindens (NB4-NA-102), but does not exceed further including each and every one of the species recited in this exemplary list.
  • the exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described herein comprises, consists essentially of, or consists of at least one of the following strains listed in TABLE 1: NB2B-20-GAM, NB2B-6-CNA, NB2A-9-NA, 14 LG, NB2A-8-WC, NB2A-12-BBE, NB2A-3-NA, NB2A-17-FMU, NB2B-19-DCM, NB2B-10-FAA, NB2B- 26-FMU, but does not exceed further including each and every one of the species recited in the exemplary list of TABLE 1.
  • TABLE 2 sets forth additional exemplary microbiotic communities comprising the indicated bacterial strains. These exemplary MET-5 microbiotic communities are subgroups of MET-5 designated herein MET-5A, MET-5B, and MET-5C. Each of MET-5, MET-5A, MET-5B, and MET-5C comprises a stable ecosystem of bacterial species/strains identified in TABLE 2.
  • TABLE 2 E l i biti MET5 b t il lti Bacterial Isolate-Specific Summaries ⁇ Isolates Derived Directly from Donor Stool
  • NB4-GAM-19 was isolated on Gifu Anaerobic Medium (GAM) agar. The isolate was then restreaked onto Fastidious Anaerobe Agar (FAA), cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Faecalibacterium prausnitzii (97.24% similarity) when aligned against the NCBI database having the /Accession numbers identified here.
  • FAA Fastidious Anaerobe Agar
  • F. prausnitzii isolates are generally rod-shaped, non-motile, non-spore-fonning, Gram-positive fastidious anaerobic bacteria (Duncan et al., 2002; Maier et al., 2017). This species has emerged as a relevant probiotic candidate (Miquel et al., 2013) and it is one of the most abundant human gut microorganisms, representing around 5 -20% of the total microbiota in stools of healthy subjects (Tap et al., 2009; Walker et al, 2011). F. prausnitzii is extremely oxygen sensitive (Stewart et al, 2002) and so was not previously well exploited as a novel probiotic.
  • F.. prausnitzii produces a wide array of short-chain fatty acids (SCFAs) such as D-lactate, acetate, formate, and butyrate (Khan et al., 2012; Heinken et al., 2014).
  • SCFAs short-chain fatty acids
  • mucin stimulates the growth of this species (Sadaghian Sadabad et al., 2016)
  • F. prausnitzii also stimulates the synthesis of mucin and tight-j unction proteins (Martin et al., 2015).F.
  • prausnitzii has been shown to upregulate the production and secretion of IL- 10 while inhibiting the secretion of IL-8 (Chen et al., 2014, Qiu et al., 2013; Sarrabayrouse et al., 2014).
  • UC ulcerative colitis
  • F. prausnitzii is one of three species associated with colonization in a successful fecal microbiota transplant (FMT) procedure in a UC patient where disease resolution was shown (zkngelburger et al., 2013).
  • the polynucleotide sequence of Faecalibacterium prausnitzii has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR_028961 having activity or function.
  • An exemplary nucleic acid sequence of Faecalibacterium prausnitzii strain ATCC 2776816S ribosomal RNA gene, partial sequence is: TATTTCTACGTTCGTCAAGGGATGTCAANAANTGGTAAGGTTCTTCGCGTTGCGTCGAATTA AACCACATACTCCACTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTC GTACTCCCCAGGTGGATTACTTATTGTGTTAACTGCGGCACTGAAGGGGTCAATCCTCCAAC ACCTAGTAATCATCGTTTACGGTGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACAC TTTCGAGCCTCAGCGTCAGTTGGTGCCCAGTAGGCCGCCTTCGCCACTGGTGTTCCTCCCGAT ATCTACGCATTCCACCGCTACACCGGGAATTCCGCCTACCTCTGCACTACTCAAGACATACA GTTTTGAAAGCAGTTCATGGGTTGAGCCCATGGATTTCACTTCCAACTTGTCTGCCCGCCTGC GCT
  • B. intestinihominis isolates are generally Gram-negative, non-motile, pleomorphic rod-shaped fastidious anaerobic bacteria (Morotomi et al., 2008).
  • intestinihominis are also observed in the gut microbiota of children suffering from Autism Spectrum Disorder (ASD) (Averina et al., 2020), as well as in those patients who are more prone to developing colorectal anastomatic leakage following suigery (Palmisano et al., 2020).
  • ASSD Autism Spectrum Disorder
  • the polynucleotide sequence oYBarnsiella intestinihominis has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75% 80%, 85%, 90% 95%, 97% 98% 99%) to the sequence provided at NCB1 Accession No. NR_113073 having activity or function.
  • An exemplary nucleic acid sequence of Bamsiella intestinihominis strain JCM 15079 16S ribosomal RNA gene, partial sequence is: Dorea forrnicigenerans (NB4-D5-8)
  • NB4-D5-8 was isolated on Fastidious Anaerobe Agar (FAA) supplemented with 5% defibrinated sheep blood and 3% filter-sterilized Donor 5 (D5) chemostat effluent. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Dorea forrnicigenerans (97.10% similarity) when aligned against the NCB1 database.
  • FAA Fastidious Anaerobe Agar
  • D5 filter-sterilized Donor 5
  • D. forrnicigenerans isolates are generally rod-shaped, non-motile, non-spore forming Gram- positive fastidious anaerobic bacteria (Kaur et al., 2014). Species from the Dorea genus are found in decreased abundance in patients with hepatocellular carcinoma (Pinero et al, 2019), as well as in the gut microbiota of patients suffering from rheumatoid arthritis (Mena-Vazquez et al, 2020). Dorea species are potentially involved in cholic acid dehydroxylation (Martin et al., 2018). They are also acetate, lactate, and formate producers. Dorea is one of the genera found to comprise part of tlie core microbiome in a survey of 17 healthy individuals using stool samples flap et al., 2009).
  • the polynucleotide sequence of Dorea forrnicigenerans (NB4-D5-8) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75% 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCB1 Accession No. NR_044645 having activity or function.
  • An exemplary nucleic acid sequence of Dorea forrnicigenerans strain ATCC 27755 16S ribosomal RNA, partial sequence is:
  • NB4-DCM-31 was isolated on Differential Clostridium agar. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Coprococcus catus (98.59% similarity) when aligned against the NCBI database.
  • C. catus isolates are generally elongated cocci-shaped, non-motile Gram-positive fastidious anaerobic bacteria (Holeman et al., 1974).
  • the Coprococcus genus is associated with higher levels of circulating indole propionic acid, in turn associated with lower risk for metabolic syndrome parameters (Menni et al., 2019).
  • Members of this genus produce the short chain fatty acids butyrate, acetate, lactate, formate, and propionate (Reichardt et al., 2014).
  • the polynucleotide sequence of Coprococcus catus (NB4-DCM-31) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75% 80%, 85% 90% 95%, 97% 98%, 99%) to the sequence provided at NCBI Accession No. NR 024750 having activity or function.
  • An exemplary nucleic acid sequence of Coprococcus catus strain VPI C66116S ribosomal RNA gene, partial sequence is: TCACCGNNNTGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCC ACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCATTCTTGCGAACGTACTCCCCAGGT GGAATACTTATTGCGTTTGCTGCGGCACCGAANCCCTTATGGGCCCCGACACCTAGTATTCA TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCA GCGTCATTGTCAGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTT CACCGCTACACTAGGAATTCCGCTTACCTCTCCTGAAATCAAGCAGGGCAGTTTCAAAAGCC GTCCCGGGGTTGAGCCCCGGG-CTTTCACTTCTGACTTGCTCCGCCGCCTACGCTTTAC ACCCAGTAAAT
  • A. hadrus isolates are generally rod-shaped, non-motile, non-spore-forming Gram-positive fastidious anaerobic bacteria (Allen-Vercoe et al., 2012; Kant et al., 2015).
  • Anaerostipes caccae The closely related species Anaerostipes caccae has been associated with protection against the development of food allergy in infants (Feehley et al., 2019). Anaerostipes is known to convert dietary gallate (a polyphenol) to the bioactive, beneficial metabolite pyrogallol (Esteban-Torres et al., 2018). It is also thought to be syntrophic with Akkermansia muciniphila, which is another strain present in the MET-5 composition (Belzer et al., 2017). [157] Once this strain was chosen for the MET-5 drug product, dilution to extinction stocks were produced in an anaerobic chamber to function as the seed stocks for future drug product production.
  • the polynucleotide sequence of Anaeroslipes hadrus (NB4-DCM-7) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR_117139 having activity or function.
  • An exemplary nucleic acid sequence of Anaeroslipes hadrus strain DSM 3319 16S ribosomal RNA, partial sequence is:
  • NB4-FAA-15 was isolated on Fastidious Anaerobe Agar (FAA) supplemented with 5% defibrinated sheep blood (v/v). The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1 % v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Bacteroides unifonnis (99.55% similarity) when aligned against the NCBI database. [160] B.
  • B. uniformis isolates are generally rod-shaped, non-motile, non-spore-forming Gram-negative fastidious anaerobic bacteria (Bakir et al., 2006).
  • B. uniformis is a propionate producer; propionate is suggested to associate with interleukin 10-producing regulatory T cell differentiation in gut associated lymphoid tissues (Smith et al., 2013).
  • the administration of B. uniformis ameliorated the impact of high-fat-diet-induced metabolic and immune dysfunction, suggesting the potential beneficial effect of tills species in obesity (Cano et al., 2012).
  • the polynucleotide sequence of Bacteroides uniformis has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR 112945 having activity or function.
  • NB4-FAA-20 Bacteroides uniformis strain JCM 5828 16S ribosomal RNA gene, partial sequence is: CTTTATTCCCGTATAAAAGAAGTTTACAACCCATAGGGCAGTCATCCTTCACGCTACTTGGCT GGTTCAGACTCTCGTCCATTGACCAATATTCCTCACTGCTGCCTTCCCGTA Bifidobacterium adolescentis (NB4-FAA-20) [163] NB4-FAA-20 was isolated on Fastidious Anaerobe Agar supplemented with 5% defibrinated sheep blood (v/v).
  • B. adolescentis isolates are generally rod-shaped, non-motile, non-spore-forming Gram- positive anaerobic bacteria (Duranti et al., 2016). Members of this genus have been considered health- promoting for decades and some strains have been used widely as probiotics.
  • Bifidobacteria are among the founder-species of the infant gut and frequently one of the major taxa or even the most abundant genera of the infant gut microbiota in the western world (Turroni et al., 2012). In vaginally born infants, Bifidobacteria are found within three days after birth, and after one week they may compose over 90% of the overall microbiota in breast-fed infants (Duranti et al., 2017; Makino et al., 2013). The abundance of Bifidobacteria in adults is estimated to be 1–2% in many westernized countries, and approximately 7% in the Japanese population (Odamaki et al., 2016).
  • Bifidobacterial genomes show greater evidence for genes related to carbohydrate metabolism than most other gut microbial species, and also demonstrate cross-feeding abilities that develop synergistic relationships in the gut (Milani et al., 2015).
  • the effects of probiotic Bifidobacteria on the health of pre- term infants is widely studied and several systematic reviews and meta-analyses have found them effective in reducing the risk of necrotizing enterocolitis and sepsis (AlFaleh et al., 2014; Deshpande et al., 2017; Sun et al., 2017).
  • B. adolescentis has also been connected with metabolic syndrome, where B. adolescntis supplementation was shown to ameliorate visceral fat accumulation and insulin sensitivity in rats fed a high-fat diet (Chen et al., 2012). Additionally, B. adolescentis is depleted in the gut microbiota of those patients suffering from metabolic syndrome (Haro et al., 2016), with a potential modulatory impact on blood lipid level regulation (Zhu et al., 2018).
  • the polynucleotide sequence of Bifidobacterium adolescentis has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85% 90%. 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR 074802 having activity or function.
  • An exemplary nucleic acid sequence of Bifidobacterium adolescentis strain ATCC 15703 16S ribosomal RNA, complete sequence is:
  • NB4-FAA-24 was isolated on Fastidious Anaerobe Agar supplemented with 5% defibrinated sheep blood (v/v). The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Akkermansia muciniphila (99.85% similarity) when aligned against the NCBI database (see Appendix B for abl sequencing file).
  • muciniphila isolates are generally non-motile, non-spore-forming Gram-negative fastidious anaerobic bacteria (Demen et al., 2004). Although it exhibits Gram-negative staining, there is no correlation between the onset of endotoxaemia and the presence of LPS that has been identified for this species (Everard et al., 2013).
  • a muciniphila was originally classified as a strict anaerobe (Derrien et al., 2004) but has been shown to be able to tolerate small amounts of oxygen (Reunanen et al., 2015). It is also a mucin degrader (Derrien et al., 2004; Derrien et at, 2017).
  • muciniphila is a common resident of the human gut, representing approximately 1-3% of the total gut microbiota (Derrien et al., 2008). This microbe can be used as a biomarker of a healthy host metabolic profile and has been suggested as a prognostic tool to anticipate the success of dietary- interventions (Dao et al., 2016).
  • the polynucleotide sequence of Akkermansia muciniphila has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97% 98%, 99%) to the sequence provided at NCBI Accession No. NR_074436 having activity or function.
  • An exemplary nucleic acid sequence of Akkermansia muciniphila strain ATCC BAA-835 16S ribosomal RNA, partial sequence is: GCTCCGGCACGCAGGGGGTCGATTCCCCGCACACXAAGCGTGCACCGITTACTGCCAGGACT
  • Phascolarctobacterium faecium (NB4-FMN-1)
  • NB4-FMN-1 was isolated on Fastidious Anaerobe Agar supplemented with 4g/L of mucin. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Phascolarctobacterium faecium (99.58% similarity) when aligned against the NCBI database.
  • P. faecium isolates are generally non-motile, non-spore-forming Gram-negative fastidious anaerobic bacteria (Wu et al., 2017). Members of this genus have been found to be substantial producers of the short chain fatty acids acetate and propionate and have been shown to abundantly colonize the gut of healthy individuals (Wu et al., 2017). Members of this genus are also known succinate-consumers (Watanabe et al ., 201 1; Ogata et al., 2019), with increased levels of circulating succinate implicated in overactive bladder syndrome, which is itself associated with metabolic syndrome (Mossa et al., 2017).
  • the polynucleotide sequence of Phascolarctobacterium faecium has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90% 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR 0261 11 having activity or function.
  • An exemplary nucleic acid sequence of Phascolarctobactenum faecium strain ACM 3679 16S ribosomal RNA gene, partial sequence is:
  • NB4-TSAB-40 was isolated on Tryptic Soy Agar supplemented with 5% defibrinated sheep blood (v/v). The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity asAgathobaculum butyriciproducens (98.83% similarity) when aligned against the NCBI database.
  • A. butyriciproducens isolates are generally short rod-shaped, non-motile, non-spore-forming Gram-positive fastidious anaerobic bacteria (Ahn et al., 2016). It is known to produce butyrate, and members of this genus are very closely related to Faecalibacterium prausnitzii (Ahn et al., 2016).
  • the polynucleotide sequence of Agathobaculum butyriciproducens has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR_151982 having activity or function.
  • An exemplary nucleic acid sequence of Agathobaculum butyriciproducens strain SR79 16S ribosomal RNA, partial sequence is: CCACCTGTCACCGATGTTCCGAAGAANAGCCTTATCTCTAAGGCGGTCATCGGGATGTCAAG ACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATACTCCACTGCTTGTGCGGGCCC CCGTCAATTCCTTTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGTGGGATACTTATTGTGTT AACTGCGGCACGGAAGGGGTCAATACCTCCCACACCTAGTATCCATCGTTTACGGCGTGGAC TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTAATGTCCA GCAGGCCGCCTTCGCCACTGGTGTTCCTCCGTATATCTACGCATTTCACCGCTACACACGGA ATTCCGCCTGCCTCTCCATCACTCAAGACCAGCAGTTTTGAAAGCAGTTTATGG
  • xylanisolvens isolates are generally non-motile, rod-shaped, non-spore-forming Gram- negative fastidious anaerobic bacteria (Chassard et al., 2008).
  • B. xylanisolvens as a probiotic supplement has also been extensively discussed in the literature, particularly regarding its ability to produce a plethora of short- chain fatty acids and its potential immunomodulatory impact (Ulsemer et al., 2012; Tan et al., 2019). However, the use of B. xylanisolvens as a probiotic in a clinical setting has remained largely unexplored.
  • the polynucleotide sequence of Bacteroides xylanisolvens has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%. 80%, 85% 90% 95%, 97% 98% 99%) to the sequence provided at NCBI Accession No. NR_112947 having activity or function.
  • An exemplaiy nucleic acid sequence of Bacteroides xylanisolvens strain XBLX 16S ribosomal RNA gene, partial sequence is:
  • NB4-FMN-6 was isolated on Fastidious Anaerobe Agar supplemented with 4g/L of mucin. The isolate was then restreaked onto FAA, cryopreserved in freezing medium ( 12% w/v Skim milk powder, 1 % v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Collinsella aerofaciens (99.84% similarity) when aligned against the NCBI database. [186] C.
  • aerqfaciens isolates are generally coccoid-shaped, non-motile, non-spore-forming Gram- positive fastidious anaerobic bacteria (Kageyama et al., 1999). It is the most abundant member of the Actinobacteria in the gut of healthy humans (Bag et al., 2017). It is a propionate and bile salt hydrolase producer known to help reduce C. difficile colonization (Mullish et al., 2019). It utilizes a broad range of animal and plant fibers for fermentative activities, producing short-chain fatty acids and lactate in the colon (Kageyama et al., 1999).
  • Colhnsella A high abundance of Colhnsella was also associated with longevity in centenarians in Korea (Kim et al ., 2019), as well as associated with responders to anti-PD-1 immunotherapy in the treatment of metastatic cancer (Matson et al., 2018). It has also been implicated in co-occurrence networks with other commensal members of the healthy human gut microbiota (Malinen et al., 2010). [187] Once this strain was chosen for the MET-5 drug product, dilution to extinction stocks were produced in an anaerobic chamber to function as the seed stocks for future drug product production. Materia] from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours.
  • the polynucleotide sequence of Colhnsella aerqfaciens has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75? ⁇ >, 80%, 85%, 90? ⁇ >, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR 113316 having activity or function.
  • An exemplary nucleic acid sequence of Collinsella aerojaciens strain JCM 10188 16S ribosomal RNA gene, partial sequence is:
  • NB4-GAM-3 was isolated on GAM agar. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Roseburia faecis (99.26% similarity) when aligned against the NCBI database (see Appendix B for abl sequencing file).
  • R. faecis isolates are generally rod-shaped, motile, Gram-variable fastidious anaerobic bacteria (Duncan et al., 2006). Species from the Roseburia genus are major butyrate producers (Hold et al., 2003). The abundance of Roseburia has been found to be decreased in many intestinal disorders suggesting the bacterium has an important role in maintaining the gut homeostasis, e.g., by producing short chain fatty acids (Tamanai-Shacoori et al., 2017). A significant reduction in the abundance of Roseburia species has been observed in patients with UC (Machiels et al., 2014, Rajilic-Stojanovic et al., 2013).
  • the polynucleotide sequence of Roseburia faecis (NB4-GAM-3) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90% 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR 042832 having activity or function.
  • An exemplary nucleic acid sequence of Roseburia faecis strain M72/1 16S ribosomal RNA gene, partial sequence is:
  • NB4-GAM-33 was isolated on GAM agar. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C '. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Blautia luti (99.35% similarity) when aligned against the NCBI database (see Appendix B for abl sequencing file).
  • B. luti isolates are generally cocci-shaped, non-motile, non-spore-forming. Gram -positive fastidious anaerobic bacteria (Simmering et al., 2002). Blautia species are lactate and acetate producers, minor producers of ethanol, hydrogen and succinate. Gut microbiota hydrogen production has been linked to reduced risk of Parkinson’s Disease (Suzuki et al., 2018). Consumption of whole grain foods was found to promote general Blautia abundance as well as health metrics of volunteers (including serum 11,-6 concentration and postprandial glucose response) in an associative study (Martinez et at, 2013).
  • Blautia Some strains of Blautia are also thought to convert dietary- gallate (a polyphenol) to the bioactive, beneficial metabolite pyrogallol (Esteban-Torres et al., 2018). This species is also the dominant gut species in healthy Japanese subjects (Touyama et al., 2015).
  • the dilution plates were observed, and the dilution having a single colony was used to restreak once again on FAA, then cryopreserved as described previously.
  • This stock was deep sequenced using Illumina MiSeq 16S rRNA gene amplicon sequencing (Mr. DNA, Shallowater, TX) to ensure purity (method described in Oliphant et al., 2019; see FIG.3).
  • the polynucleotide sequence of Blautia luti (NB4-GAM-33) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No.
  • NR_114315 having activity or function.
  • An exemplary nucleic acid sequence of Blautia luti strain DSM 14534 16S ribosomal RNA gene, partial sequence is: CAAGACTTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCG GGTCCCCGTCAATTCCTTTGAGTTTCATTCTTGCGAACGTACTCCCCAGGTGGAATACTTACT GCGTTTGCGACGGCACCGAAGAGCTTTGCTCCCCGACACCTAGTATTCATCGTTTACGGCGT GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTTACCG TCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTA GGAATTCCGCTTACCCCTCCGGCACTCAAGTATGACAGTTTCCAATGCAGTCCACAGGTTGA GCCCATGCCTTTCACATCAGACTTGCCACACCGTCTACGCTTTACA
  • L. mucosae isolates are generally rod-shaped, non-motile, non-spore-forming Gram-positive aerotolerant bacteria (Roos et al., 2000). Lactic acid bacteria are common probiotic microorganisms in the food industry, though L.
  • mucosae in particular has recently been targeted as a novel probiotic microorganism because of its ability to adhere to intestinal mucous as well as inhibit pathogens in the gastrointestinal tract (Valeriano et al., 2014; Dias de Morales et al., 2017). Research has indicated that a synergistic interaction between L. mucosae and Bifidobacterium longum may help to alleviate symptoms of anxiety and depression by reducing gut inflammation, as examined using a mouse model (Han et al., 2019). This observation was supported in a study where L. mucosae alleviated E.
  • the polynucleotide sequence of Lactobacillus mucosae has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at MCBI Accession No. NR_024994 having activity or function.
  • An exemplary nucleic acid sequence of lactobacillus mucosae strain S32 16S ribosomal RNA, partial sequence is:
  • NB4-NB-2 was isolated on Fastidious Anaerobe Agar. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1 % v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Ruminococcus albus (95.13% similarity) when aligned against the NCBI database (see Appendix B for abl sequencing file).
  • R. albus isolates are generally cocci-shaped, non-motile, Gram-positive fastidious anaerobic bacteria (Chassard et al., 2012). This species is a member of the Ixichnospiraceae, generally considered to be core members of the human gut microbiota (Rajilic-Stojanovic et al., 2012). R. albus is generally able to degrade cellulose, with the main products of fermentation being ethanol and acetate (Christopherson et al., 2013). In vitro models have demonstrated the potential neuroprotective effect of R. albus colonization, where it may help to promote neuronal proliferation and reduce reactive oxygen species levels (Park et al., 2017).
  • the polynucleotide sequence of Ruminococcus albus has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85% 90% 95% 97% 98% 99%) to the sequence provided at NCBI Accession No. NR_1 13032 having activity or function.
  • An exemplary nucleic acid sequence of Ruminococcus albus strain JCM 14654 16S ribosomal RNA gene, partial sequence is: [205] NB4-TSAB-38 was isolated on Tryptic Soy Agar supplemented with 5% defibrinated sheep blood (v/v).
  • B. longum isolates are generally rod-shaped, non-motile, non-spore-forming Gram-positive anaerobic bacteria (Duranti et al., 2016). Members of this genus have been considered health-promoting for decades and some strains have been used widely as probiotics.
  • Bifidobacteria are among the founder- species of the infant gut and frequently one of the major taxa or even the most abundant genera of the infant gut microbiota in the western world (Turroni et al., 2012). In vaginally born infants, Bifidobacteria are found within three days after birth, and after one week they may compose over 90% of the overall microbiota in breast-fed infants (Duranti et al., 2017; Makino et al., 2013). The abundance of Bifidobacteria in adults is estimated to be 1–2% in many westernized countries, and approximately 7% in the Japanese population (Odamaki et al., 2016).
  • Bifidobacterial genomes show greater evidence for genes related to carbohydrate metabolism than most other gut microbial species, and also demonstrate cross-feeding abilities that develop synergistic relationships in the gut (Milani et al., 2015).
  • the effects of probiotic Bifidobacteria on the health of pre- term infants is widely studied and several systematic reviews and meta-analyses have found them effective in reducing the risk of necrotizing enterocolitis and sepsis (AlFaleh et al., 2014; Deshpande et al., 2017; Sun et al., 2017).
  • the polynucleotide sequence of Bifidobacterium longum has a nucleic acid sequence having at least 65% nucleic acid sequence identity' (e.g., 70% 75%. 80%, 85% 90% 95%, 97% 98% 99%) to the sequence provided at NCBI Accession No. NR_145535 having activity or function.
  • An exemplary nucleic acid sequence of Bifidobacterium longum subsp. suillum strain Su 851 16S ribosomal RNA, partial sequence is:
  • NB4-BHM05 was isolated on Brain Heart Infusion agar. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Christensenella minuta (98.81% similarity) when aligned against the NCBI database.
  • C. minuta isolates are generally rod-shaped, non-motile, non-spore-forming, Gram-negative fastidious anaerobic bacteria (Morotomi et al ., 2012). C. minuta has gained recent interest for use as a novel probiotic, particularly pertaining to metabolic disease and obesity (Chang et al., 2019). This push was sparked by research demonstrating that C. minuta is enriched in the gut microbiota of those individuals with a low body mass index and can reduce weight gain in mice (Goodrich et al., 2014). It is also seen to form heritable co-occurrence networks with other bacterial and archaea! members of the gut microbiome, suggesting that it is possible that heritable groups of microbes are partly responsible for driving many human phenotypes (Goodrich et al., 2014, Goodrich etal., 2015).
  • the polynucleotide sequence of Christensenella minuta (NB4-BHI-105) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75% 80%, 85% 90% 95%, 97% 98% 99%) to the sequence provided at NCBI Accession No. NR_112900 having activity or function.
  • An exemplary nucleic acid sequence of Christensenella minuta strain YIT 12065 16S ribosomal RNA gene, partial sequence is:
  • NB4-WC-130 was isolated on Wilkins-Chalgren agar. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1 % v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Oscillibacter valericigenes (94.86% similarity) when aligned against the NCBI database.
  • O. valericigenes isolates are generally rod-shaped, motile, non-spore-forming, Gram-negative fastidious anaerobic bacteria (lino et al., 2007). O. valericigenes produces valerate, a less abundant but integral short chain fatty acid (lino et al, 2007). It is also seen to be reduced in the gut microbiota of patients suffering from Crohn's disease, when compared to healthy controls (Mondot et al., 2011).
  • the polynucleotide sequence of Oscillibacter valericigenes has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR_074793 having activity or function.
  • An exemplary nucleic acid sequence of Oscillibacter valericigenes strain Sjm 18-20 16S ribosomal RNA, complete sequence is:
  • NB4-CNA-122 was isolated on Fastidious Anaerobe Agar supplemented with 5% defibrinated sheep blood (v/v), 10mg/L colistin and 15mg/L nalidixic acid. The isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Faecalicoccus acidiformans (92.71% similarity) when aligned against the NCBI database (see Appendix B for abl sequencing file).
  • F. acidiformans isolates are generally cocci-shaped, non-motile, non-spore-forming, Gram- positive fastidious anaerobic bacteria (De Maesschalck et al., 2014). F. acidiformans could serve as a novel probiotic organism, as it is a lactic acid-producing bacterium (De Maesschalck et al, 2014).
  • the polynucleotide sequence of Faecalicoccus acidiformans has a nucleic- acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85% 90% 95% 97% 98%, 99%) to the sequence provided at NCB1 Accession No. NR_134029 having activity or function.
  • An exemplary nucleic acid sequence of Faecalicoccus acidiformans strain 37-2a 16S ribosomal RNA, partial sequence is:
  • NB4-D5-120 was isolated on Fastidious Anaerobe Agar supplemented with 5% defibrinated sheep blood and 3% filter-sterilized Donor 5 chemostat effluent of the Donor 5 (D5) fecal sample. The isolate was then restreaked onto FAA, cryopreserved in freezing medium ( 12% w/v Skim milk powder, 1 % v/v DMSO, 1% v/v Glycerol), and stored at -80°C. This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Anaerotignum lactatifermentans (95.91% similarity) when aligned against the NCBI database.
  • lactatifermentans isolates are generally rod-shaped, non-motile, non-spore-forming, Gram- positive fastidious anaerobic bacteria (van der Widen et al., 2002).
  • A. lactatifermentans was chosen because it is a member of the Lxtchnospiraceae, generally considered to be com members of the human gut microbiota (Rajilic-Stojanovic et al., 2012).
  • the polynucleotide sequence of [Clostridium] lactatifermentans (NB4_D5_120) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85% 90%. 95%, 97%, 98%, 99%) to the sequence provided at NCBI /Accession No. NR 025651 having activity or function.
  • An exemplary nucleic acid sequence of [Clostridium] lactatifermentans strain G17 16S ribosomal RNA gene, partial sequence is:
  • NB4-EtOH-104 was isolated by first subjecting chemostat culture to an ethanol shock treatment. Two milliliters (2mL) of culture was centrifuged at 14,000 rpm for 10 minutes. The pellet was then resuspended in 2mL of 100% ethanol and incubated at room temperature for one hour. The sample was centrifuged again as previous, and the pellet was resuspended in ImL of tryptic soy broth supplemented with 5mg/mL hemin and 1mg/mL menadione.
  • A. stercorihominis isolates are generally rod-shaped, non-motile, non -spore-forming, Gram- positive fastidious anaerobic bacteria (Finegold et al., 2004).
  • A. stercorihominis is amember of the bacterial family Eubacteriaceae, which is an integral family in the composition of the core gut microbiota, and its inclusion bolsters the taxonomic diversity of the defined ecosystem .
  • the polynucleotide sequence of Anaerofustis stercorihominis has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR_027562 having activity or function.
  • An exemplary nucleic acid sequence of Anaerofustis stercorihominis strain WAL 1456316S ribosomal RNA gene, partial sequence is: TAATCTCTTAGGCGGTCAAGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATT AAACCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCACTCTTGCGAG CGTACTCCCCAGGCGGAATACTTAATGTGTTAACTGCGGCACTGAGTTACCCCAACACCTAG TATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGC ACCTCAGCGTCAGTTATCGTCCAGTAAGCCGCCTTCGCCACCGGTGTTCTTCCTAATCTCTAC GCATTTCACCGCTACACTAGGAATTCCGCTTACCTCTCCGATACTCAAGTTATCCAGTTTCAA GTGCACTTTTCCGGTTGAGCCGAAAACTTTCACACCTGACTTAATTAACCGCCTACGTG
  • the isolate was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C.
  • This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Parabacteroides distasonis (98.86% similarity) when aligned against the NCBI database.
  • P. distasonis isolates are generally rod-shaped, non-motile, non-spore-forming, Gram-negative fastidious anaerobic bacteria (Sakomoto et al., 2006).
  • the species in the Parabacteroides genus are consumers of dietary carbohydrate, protein and succinate.
  • This species transforms the bile acid profile, producing lithocholic acid and ursodeoxycholic acid, which may act through the RXR pathway to help repair gut barrier integrity, demonstrated in a mouse model (Wang et al., 2019).
  • P. distasonis In vitro studies with P. distasonis have shown that its cell membrane can suppress pro-inflammatory cytokines, possibly through blocking TLR-4 activation (Koh et al., 2018).
  • P. distasonis was also demonstrated to offer protection against colonic tumorigenesis and maintenance of the intestinal epithelial barrier in a mouse model (Koh et al., 2020). It is likely to produce phenylacetic acid and 4- hydroxylphenylacetic acid from fermentation of aromatic amino acids: these phenolic acids are considered beneficial for human health (Russell et al., 2013).
  • the polynucleotide sequence of Parabacteroides aistasonis has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97% 98%, 99%) to the sequence provided at MCBI Accession No. NR 041342 having activity or function.
  • An exemplary nucleic acid sequence of Parabacteroides distasonis strain JCM 5825 16S ribosomal RNA gene, partial sequence is:
  • NB4-D5-137 was isolated on Fastidious Anaerobe Agar supplemented with 5% defibrinated sheep blood and 3% filter-sterilized Donor 5 chemostat effluent.
  • the isolate from Donor 5 (D5) fecal sample was then restreaked onto FAA, cryopreserved in freezing medium (12% w/v Skim milk powder, 1% v/v DMSO, 1% v/v Glycerol), and stored at -80°C.
  • This isolate was analyzed by 16S rRNA gene Sanger sequencing to the closest species identity as Parasutterella excrementihominis (99.15% similarity) when aligned against the NCBI database.
  • P. excrementihominis isolates are generally cocci-shaped, non-motile, non-spore-forming. Gram-negative fastidious anaerobic bacteria (Nagai et al., 2009). Members of the genus Parasutterella are thought to be core members of the human gut microbiome, contributing to bile acid maintenance and cholesterol metabolism (Ju et al., 2019). The relative abundance of this genus has also been shown to be inversely associated with a high-fat diet in mice (Zhang et al., 2012). As well, it had an increased relative abundance in those mice who were fed a bran-enriched versus a basal diet, suggesting a positive association with a balanced diet (Koistinen et al., 2019).
  • the polynucleotide sequence eft Parasutterella excrementihominis (NB4-D5-137) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85% 90%. 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR 041667 having activity or function.
  • An exemplary nucleic acid sequence of Parasutterella excrementihominis strain YIT 11859 16S ribosomal RNA gene, partial sequence is: GCTCGGGGATTTCACATCTGTCTTACTCAACCGCCTGCGCACCCTTTACGCCCAGTAATTCCG ATTAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCT TAGAGTACCGTCAGCAACACCCTTTTTTAAAAGGTGTCTTTTCGTTCTCTACAAAAGTGGTTT ACAACCCGAGGGCCTTCATCCCACACGCGGAATAGCTGGATCAGGCTTGCGCCCATTGTCCA AAATTCCCCACTGCTG [Clostridium] scindens (NB4-NA-102) [238] NB4-NA-102 was isolated on Nutrient Agar.
  • C. scindens isolates are generally rod-shaped, non-motile, Gram-positive fastidious anaerobic bacteria (Morris et al., 1985). A core member of the normal gut microbiota, C.
  • scindens is one of the few bacteria that can form deoxycholate from cholic acid, which is known to inhibit growth of pathogens such as C. difficile (Sorg et al., 2010). It may also produce peptide antibiotics (Devendran et al., 2019). It is also indicated as a potential new probiotic in the treatment of C. difficile infection (Mills et al., 2018).
  • the polynucleotide sequence of [Clostridium] scindens has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. NR_028785 having activity or function.
  • An exemplary nucleic acid sequence of [Clostridium] scindens strain ATCC 35704 16S ribosomal RNA, partial sequence is: GAGCGCGTTACGCGCTTTGNCATCGGGNTGTCAAGATCAGGTAAGGTTCTTCGCGTTGCTTC GAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCATTCTT GCGAACGTACTCCCCAGGTGGACTACTTATTGCGTTTGCTGCGGCACCGAATGGCCTTGCCA CCCGACACCTAGTAGTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCC CCACGCTTTCGAGCCTCAACGTCATCGTCCAGCAGGCCGCCTTCGCCACTGGTGTTCCT CCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCCTGCCTCTCCGACACTCCAGCCACACTGGTGTTCCT CCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCCTGCCTCTC
  • A. intestini are non-motile, non-spore-forming, gram-negative, and strictly anaerobic. This strain was included because it has been shown to have protective attributes for cryopreservation and lyophilization of certain bacterial strains from a community other than NB4. This phenomenon has been described in International Publication No. PCT/US2018/29920 and U.S. Publication No. US 2014/0363397, both of which are incorporated herein by reference in their entirety for teaching these protective attributes. This strain is not present in the NB4 donor, and A. intestini was added to the MET-5 ecosystem to enhance ecosystem survivability.
  • the polynucleotide sequence of Acidaminococcus intestini (16-6-I 14 LG) has a nucleic acid sequence having at least 65% nucleic acid sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%) to the sequence provided at NCBI Accession No. LR698962 having activity or function.
  • An exemplary nucleic acid sequence of Acidaminococcus intestini is: GACTTCACCCCAATCATNGGCCCCANTTAGACAGCTGACTCCTAAAAGGTTATCTCACCGGC TTCGGGTGTTACCAACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC ACCGCAGTATGCTGACCTGCGATTACTAGCGATTCCAACTTCACGTAGGCGGGTTGCAGCCT ACGATCCGAACTGGGGTCGGGTTTCTGGGATTTGCTCCACCTCGCGGTTTCGCTGCCCTTTGT TGCCGACCATTGTAGTACGTGTGTAGCCCAAGACATAAGGGGCATGATGACTTGACGTCATC CCCGCCTTCCTCCAAGTTATCCCTGGCAGTCTCCTATGAGTCCCCGCCTTTACGCTGGTAA CATAGGATAGGTTGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACG ACAGCCATGCACCACCTGTTTTCGTGTCCCCGAAGGGAGGGACCTATCTCTATCTA
  • the exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described herein comprises at least one of the MET-5, MET-5A, MET-5B, and MET-5C strains listed in TABLE 2, but does not exceed further including each and every one of the species recited in the exemplary list of TABLE 2.
  • the exemplary list of bacterial species/strains that exhibits robustness in chemostat model test assays described herein consists of the one of the MET-5, including subgroups (e.g., MET-5A, MET-5B, MET-5C) bacterial populations listed in TABLE 2.
  • the number of bacterial cells may be determined using a LIVE/DEADTM BacLightTM Bacterial Viability Kit in accordance with the manufacturer’s protocol. Live versus dead cells are distinguished using the LIVE/DEADTM BacLightTM Bacterial Viability Kit, which differentially stains dead and dying cells with compromised membranes red and live cells having intact membranes green. The differential staining facilitates an accurate assessment of viable cells in a given sample.
  • the number of cells is determined via flow cytometry used in conjunction with a LIVE/DEADTM BacLightTM Bacterial Viability Kit, which combination facilitates measuring the different colors of the differentially stained cells via fluorescence detection in a plate reader.
  • the chemostat model assay provides an assay wherein the growth of the plurality of bacterial species/strains initially seeded into a vessel (i.e., bacterial seed population) may be determined at different defined time periods of incubation in the chemostat model assay.
  • a vessel i.e., bacterial seed population
  • multiple vessels can be seeded with different bacterial seed populations and the growth of the different bacterial seed populations and particular species in the different bacterial seed populations can be determined at different defined time periods of incubation.
  • a fecal-derived bacterial population may be isolated or derived from a healthy subject who is not suffering from dysbiosis or dysbiosis-related diseases.
  • a fecal-derived bacterial population may be derived from a subject (e.g., a healthy subject who is not suffering from dysbiosis or dysbiosis-related diseases) by a method comprising: a.
  • the supernatant comprises a purified population of intestinal bacteria that is free of fecal matter and food particles.
  • a fecal sample from a healthy donor is diluted in saline and plated onto a series of different media types (e.g., 10-30 media types), each tailored to the isolation of particular types of species.
  • the fecal sample may also be used undiluted as inoculum to seed a chemostat, which is grown to steady state, and then an aliquot of the steady state culture is diluted in saline and subsequently plated onto a series of different media types (e.g., 10-30), each tailored to the isolation of particular types of species.
  • a diluted sample of bacteria may, for example, be treated with ethanol to select for sporulating bacteria.
  • antibiotics are added that exclude certain types of bacterial cells.
  • filter-sterile spent chemostat medium is added to provide growth substrates that promote proliferative or provide a selective advantage for certain types of bacterial cells. Following transfer into the different media types, bacterial cell cultures are incubated for days (e.g., 3-10), streaked onto agar plates, and individual colonies are picked, re-streaked to purity, and then frozen down. Frozen stocks are grown in culture to curate/characterize the strain by conducting a 16S rRNA gene sequencing read using Sanger chemistry and the obtained trace compared to the RDP database.
  • each bacterial species/strains listed in, for example, TABLEs 1-3 or a subset thereof is cultured individually to expand the population of each bacterial species/strains to reach a threshold of biomass for each bacterial species/strains.
  • a larger volume of bacterial culture is grown so as to achieve a biomass equivalent to that of faster growing species/strains.
  • the strains/strains are all grown separately in Wilkins-Chalgren broth under anaerobic conditions at 37°C.
  • the cultured bacterial population of each species/strains (e.g., TABLES 1-3) is then concentrated by centrifugation, resuspended in medium optionally containing a cryoprotectant/lyoprotectant (inulin and riboflavin), and then rapidly frozen at -80°C. Frozen material is placed into a lyophilizer instrument and the cycle run to sublimate and remove the water content, leaving a fine powder representing a matrix of preserved bacterial cells and optionally cryo-lyoprotectant.
  • the individual powders from each individual isolate are tested for purity and if pure, may be combined into desired combinations as powders via thorough mixing to generate an anhydrous composition comprising a desired plurality of bacterial species/strains.
  • the bacterial species/strains identified herein may be in an anhydrous composition, where the bacterial species/strains have a moisture content of less than 25% wt/wt (e.g., 20%, 15%, 10% 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.25%, 0.1%) in the anhydrous composition.
  • the bacterial strains may be in a powder form or a liquid form, where moisture has been removed or substantially removed, and the bacterial species/strains are stable and active and/or functional.
  • the bacterial species/strains population disclosed here may be in an amount having a concentration of at least 1x10 3 colony forming unit per gram (CFU/g) (e.g., 1x10 4 , 2x10 4 , 1x10 5 , 2x10 5 , 1x10 6 , 2x10 6 , 1 x10 7 , 2x10 7 , 1x10 8 , 2x10 8 , 1x10 9 , 2x10 9 , 1x10 10 , 2x10 10 ) as measured by the method disclosed in the United States Pharmacopeia (USP) 61-62 (accessed online September 2020).
  • USP United States Pharmacopeia
  • an anhydrous composition comprising a population of bacterial species/strains may be derived from fecal matter in accordance with methods disclosed in U.S. Patent Nos. 8,906,668 and 9,511,099 and in U.S. Patent Application Publication No.20140342438, the entire contents of each of which are incorporated herein by reference. Culture Methods According to Certain Embodiments [253] In some embodiments, an anhydrous composition comprising a plurality of bacterial species/strains is cultured in a chemostat vessel. In some embodiments, the chemostat vessel is the vessel disclosed in U.S. Patent Application Publication No.20140342438.
  • the chemostat vessel is the vessel described in WO 2019/0136269, which is incorporated herein by reference.
  • the chemostat vessel may be converted from a fermentation system to a chemostat by turning off or obstructing the condenser.
  • the culture pH of the chemostat culture may be maintained using, for example, 5% (v/v) HCl (Sigma) and 5% (w/v) NaOH (Sigma) and the pressure forces the waste out of a metal tube (formerly a sampling tube) at a set height and allows for the maintenance of given working volume of the chemostat culture.
  • the chemostat vessel may be kept anaerobic by bubbling filtered nitrogen gas through the chemostat vessel.
  • Temperature and pressure may be automatically controlled and maintained.
  • the culture medium of the chemostat vessel is continually replaced. In some embodiments, the replacement occurs over a period of time equal to the retention time of the distal gut. Consequently, in some embodiments, the culture medium is continuously fed into the chemostat vessel at a rate of 400 mL/day (16.7 mL/hour) to give a retention time of 24 hours, a value set to mimic the retention time of the distal gut.
  • An alternate retention time can be 65 hours (approximately 148 mL/day, 6.2 mL/hour). In some embodiments, the retention time can be as short as 12 hours.
  • the culture medium is a culture medium disclosed in U.S. Patent Application Publication No.
  • compositions Containing Bacterial Species/Strains
  • Embodiments of the disclosure may provide for compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of at least one or more, a plurality, or combinations of bacterial species and/or bacterial strains (i.e., bacterial species/strains) disclosed herein (e.g., TABLEs 1-4, 9-10, 12-13).
  • compositions of the disclosure comprising a microbiota, where the microbtiota contains at least one or more, a plurality, or combinations of bacterial species selected from: Faecalibacterium spp.; Roseburia spp.; Acidaminococcus spp.; Clostridium spp.; Ruminococcus spp.; Blautia spp.; Christensenella spp.; Oscillibacter spp.; Faecalicoccus spp.; Barnsiella spp.; Anaerotignum spp.; Dorea spp.; Coprococcus spp.; Anaerostipes spp.; Anaerofustis spp.; Parabacteroides spp.; Bacteroides spp.; Bifidobacterium spp.; Akkermansia spp.; Phascolarctobacterium spp.; Agathobac
  • the composition of the disclosure comprising a microbiota, where the microbtiota contains at least one or more, a plurality, or combinations of bacterial species (e.g., MET-5) selected from: Faecalibacterium prausnitzii; Roseburia faecis; Acidaminococcus intestini; Clostridium scindens; Ruminococcus albus; Blautia luti; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Barnsiella intestinihominis; Anaerotignum lactatifermentans; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Anaerofustis stercorihominis; Parabacteroides distasonis; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Ph
  • a further embodiment of the disclosure is directed to a composition
  • a composition comprising a microbiota, where the microbtiota contains at least one or more, a plurality, or combinations of bacterial species (e.g., MET-5A) selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Bacteroides uniformis; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerofustis stercorihominis; Parabacteroides distasonis; and Parasutterella excrementihominis.
  • a composition of the disclosure comprises a microbiota, where the microbiota contains at least one or more, a plurality, or combinations of bacterial species (e.g., MET-5B) selected from: Parabacteroides distasonis; Phascolarctobacterium faecium; Ruminococcus albus; Akkermansia muciniphila; Roseburia faecis; Oscillibacter valericigenes; Dorea formicigenerans; Lactobacillus mucosae; Blautia luti; [Clostridium] scindens; Faecalibacterium prausnitzii; Bacteroides uniformis; Coprococcus catus; Bifidobacterium adolescentis; and Collinsella aerofaciens.
  • bacterial species e.g., MET-5B
  • composition of the disclosure comprising a microbiota, where the microbiota contains at least one or more, a plurality, or combinations of bacterial species (e.g., MET-5C) selected from: Faecalibacterium prausnitzii; Bacteroides uniformis; Akkermansia muciniphila; Bifidobacterium longum; Christensenella minuta; Parasutterella excrementihominis; and [Clostridium] scindens.
  • a microbiota contains at least one or more, a plurality, or combinations of bacterial species (e.g., MET-5C) selected from: Faecalibacterium prausnitzii; Bacteroides uniformis; Akkermansia muciniphila; Bifidobacterium longum; Christensenella minuta; Parasutterella excrementihominis; and [Clostridium] scindens.
  • Faecalibacterium prausnitzii Bacteroides
  • a further embodiment provides a composition comprising a microbiota, where the microbtiota contains at least one or more, a plurality, or combinations of bacterial strains (e.g., MET-5) selected from: NB4-GAM-19; NB4-GAM-3; 14 LG; NB4-NA-102; NB4-NB-2; NB4-GAM-33; NB4-BHI-105; NB4- WC-130; NB4-CNA-122; NB4-CNA-21; NB4-D5-120; NB4-D5-8; NB4-DCM-31; NB4-DCM-7; NB4- EtOH-104; NB4-FAA-116; NB4-FAA-15; NB4-FAA-20; NB4-FAA-24; NB4-FMN-1; NB4-TSAB-40; NB4-FMN-21; NB4-FMN-6; NB4-D5-137;
  • One embodiment provides a composition comprising a microbiota, where the microbiota contains at least one or more, a plurality, or combinations of bacterial strains selected from: NB4-GAM- 19; NB4-CNA-21; NB4-FAA-15; NB4-FAA-20; NB4-FAA-24; NB4-FMN-1; NB4-FMN-6; NB4-GAM- 3; NB4-GAM-33; NB4-MRS-35; NB4-NB-2; NB4-BHI-105; NB4-WC-130; NB4-CNA-122; NB4-EtOH- 104; NB4-FAA-116; and NB4-D5-137.
  • a composition of the disclosure comprises a microbiota, where the microbiota contains at least one or more, a plurality, or combinations of bacterial strains (e.g., MET-5B) selected from: NB4 FAA 116; NB4 FMN 1; NB4 NB 2; NB4 FAA 24; NB4 GAM 3; NB4 WC 130; NB4-D5-8; NB4-MRS-35; NB4-GAM-33; NB4-NA-102; NB4-GAM-19; NB4-FAA-15; NB4-DCM-31; NB4-FAA-20; and NB4-FMN-6.
  • a plurality, or combinations of bacterial strains e.g., MET-5B
  • composition of the disclosure comprising a microbiota, where the microbiota contains at least one or more, a plurality, or combinations of bacterial strains (e.g., MET- 5C) selected from: NB4-GAM-19; NB4-FAA-15; NB4-FAA-24; NB4-TSAB-38; NB4-BHI-105; NB4-D5- 137; and NB4-NA-102.
  • a microbiota contains at least one or more, a plurality, or combinations of bacterial strains (e.g., MET- 5C) selected from: NB4-GAM-19; NB4-FAA-15; NB4-FAA-24; NB4-TSAB-38; NB4-BHI-105; NB4-D5- 137; and NB4-NA-102.
  • a plurality, or combinations of bacterial strains e.g., MET- 5C
  • composition described here containing a microbiota where the microbiota comprises, consists essentially of, or consists of at least one or more, a plurality, or combinations of bacterial strains selected from: Faecalibacterium prausnitzii and Akkermansia muciniphila, and optionally Acidaminococcus intestini and/or Roseburia faecis and any combination thereof.
  • compositions of the disclosure may comprise, consist essentially of, or consist of functionally redundant bacterial species/strains. Some embodiments may provide compositions comprising, consisting essentially of, or consisting of some bacterial species/strains that work synergistically.
  • compositions described here that do not comprise, do not consist essentially of, or do not consist of functionally redundant bacterial species/strains, thereby offering an efficient bacterial population composition.
  • Further embodiments may comprise, consist essentially of, or consist of at least one, a plurality, or a combination of functionally redundant bacterial species/strains.
  • compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of Gram-negative bacteria selected from the bacterial species/strains disclosed herein (e.g., TABLEs 1-3) in at least 10% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%) of the total bacterial species/strains of the microbiota.
  • the composition of the disclosure comprises a microbiota containing, comprising, consisting essentially of, or consisting of Gram-negative bacteria selected from the bacterial species/strains of TABLEs 1-3 in 99% or less (e.g., 97%, 93%, 91%, 89%, 87%, 83%, 81%, 79%, 77%, 73%, 71%, 69%, 67%, 63%, 61%, 59%, 57%, 53%, 51%, 49%, 47%, 43%, 41%, 39%, 37%, 33%, 31%, 29%, 27%, 23%, 21%, 19%, 17%, 13%, 11%) of the total bacterial species/strains of the microbiota.
  • compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of Gram-negative bacterial species/strains selected from the bacterial species/strains of TABLEs 1-3 in at least 30% of the total bacterial species/strains of the microbiota.
  • a composition comprising a microbiota containing, comprising, consisting essentially of, or consisting of Gram-positive bacteria selected from the bacterial species/strains of TABLEs 13 in at least 10% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%) of the total bacterial species/strains of the microbiota.
  • the composition of the disclosure comprises a microbiota containing, comprising, consisting essentially of, or consisting of Gram-positive bacterial species/strains selected from the bacterial species/strains of TABLEs 1-3 in 99% or less (e.g., 97%, 93%, 91%, 89%, 87%, 83%, 81%, 79%, 77%, 73%, 71%, 69%, 67%, 63%, 61%, 59%, 57%, 53%, 51%, 49%, 47%, 43%, 41%, 39%, 37%, 33%, 31%, 29%, 27%, 23%, 21%, 19%, 17%, 13%, 11%) of the total bacterial species/strains of the microbiota.
  • compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of Gram-positive bacterial species/strains selected from the bacterial species/strains of TABLEs 1-3 in at least 25% of the total bacterial species of the microbiota.
  • compositions of the disclosure comprise microbiota containing, comprising, consisting essentially of, or consisting of bacterial species/strains selected from bacterial species of TABLEs 1-3 in a ratio of Gram-positive to Gram-negative bacterial species/strains in a range of 2:1 to 1:5 (e.g., 9:5 to 2:5, 3:2 to 1:1).
  • compositions comprising microbiota containing, comprising, consisting essentially of, or consisting of bacterial species/strains selected from bacterial species of TABLEs 1-3 in a ratio of Gram-positive to Gram-negative bacterial species/strains selected from: 9:5, 3:2, 1:1, and 2:5.
  • a further embodiment may provide a composition of the disclosure comprise microbiota containing, comprising, consisting essentially of, or consisting of bacterial species/strains selected from at least one, a plurality of, or a combination of bacterial species/strains of TABLEs 1-3 selected from: Faecalibacterium prausnitzii; Dorea formicigenerans; Coprococcus catus; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Oscillibacter valericigenes; Parabacteroides distasonis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, in combination with bacterial
  • an anhydrous composition comprising a co-selected microbiota
  • the co-selected microbiota comprises, consists of, or consists of a plurality of bacterial species/strains consisting of at least one of the bacterial species/strains listed in TABLEs 1-3 selected from: Faecalibacterium prausnitzii; Dorea formicigenerans; Coprococcus catus; Bifidobacterium adolescentis; Akkermansia muciniphila; Phascolarctobacterium faecium; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Oscillibacter valericigenes; Parabacteroides distasonis; [Clostridium] scindens and any combination thereof; at least one
  • the bacterial species/strains in the anhydrous compositions of the disclosure may be presented in a powder-form, where the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and the co-selected microbiota exhibits benefits for treating a subject suffering from a metabolic syndrome, disease associated with a metabolic syndrome, dysbiosis, disease-associated with dysbiosis, or symptoms thereof, and/or exhibits resistance to perturbational stress.
  • an anhydrous composition comprising a microbiota
  • the microbiota comprises, consists of, or consists of a plurality of bacterial species/strains consisting of at least one of the bacterial species/strains listed in TABLEs 1-3 selected from: NB4-GAM- 19; NB4-D5-8; NB4-DCM-31; NB4-FAA-20; NB4-FAA-24; NB4-FMN-1; NB4-FMN-21; NB4-FMN-6; NB4-GAM-3; NB4-GAM-33; NB4-MRS-35; NB4-NB-2; NB4-WC-130; NB4-FAA-116; NB4-NA-102; at least one bacterial species/strains listed in TABLEs 1-3 selected from: NB4-CNA-21; NB4-DCM-7; NB4-FAA-15; NB4-TSAB-40;
  • the bacterial species/strains in the anhydrous compositions of the disclosure may be presented in a powder- form, where the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and the microbiota exhibits benefits for treating a subject suffering from a metabolic syndrome, disease associated with a metabolic syndrome, dysbiosis, disease-associated with dysbiosis, or symptoms thereof, and/or exhibits resistance to perturbational stress.
  • compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of bacterial species/strains selected from the bacterial species/strains of TABLEs 1-3 within phylums selected from: Firmicutes, Bacteroidetes, Actinobacteria, Verrucomicrobia, Proteobacteria, and combinations thereof.
  • compositions of the disclosure comprising a microbiota containing, comprising, consisting essentially of, or consisting of bacterial species/strains selected from the bacterial species/strains of TABLEs 1-3 within phylums selected from: Firmicutes, Bacteroidetes, Actinobacteria, Verrucomicrobia, and combinations thereof.
  • compositions of the disclosure comprising a microbiota containing, comprising, consisting essentially of, or consisting of at least 30 % (e.g., 33%, 35% 37%, 39%, 41%, 43%, 45%, 47%, 49%, 51%, 53%, 55%, 57%, 59%, 61%, 63%, 65%, 67%, 69%, 71%, 73%, 75%, 77%, 79%, 81%, 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%) bacterial species/strains within the Firmicutes phylum (see, e.g., TABLE 3).
  • compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of 99% or less (e.g., 98%, 96%, 94%, 92%, 90%, 88%, 86%, 84%, 82%, 80%, 78%, 76% 74%, 72%, 70%, 68%, 66%, 64%, 62%, 60%, 58%, 56%, 54%, 52%, 50%, 48%, 46%, 44%, 42%, 40%, 38%, 36%, 34%, 32%, 30%, 28%, 26%, 24%, 22%, 20%, 18%, 16%, 14%, 12%, 10%) bacterial species/strains within the Firmicutes phylum (see, e.g., TABLE 3).
  • compositions of the disclosure comprising a microbiota containing, comprising, consisting essentially of, or consisting of at least 0.5 % (e.g., 0.7%, 0.9%, 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%) bacterial species/strains within the Bacteroidetes phylum (see, e.g., TABLE 3).
  • compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of 75% or less (e.g., 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 0.8%, 0.6%) bacterial species/strains within the Bacteroidetes phylum (see, e.g., TABLE 3).
  • compositions of the disclosure comprising a microbiota containing, comprising, consisting essentially of, or consisting of 0% or greater (e.g., 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 21%, 23%, 25%, 27%, 29%) bacterial species/strains within the Actinobacteria phylum (see, e.g., TABLE 3).
  • compositions of the disclosure comprise a microbiota containing, comprising, consisting essentially of, or consisting of 30% or less (e.g., 28%, 26%, 24%, 22%, 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%) bacterial species/strains within the Actinobacterial phylum (see, e.g., TABLE 3).
  • Other embodiments of the disclosure are directed to any of the compositions described here comprising, consisting essentially of, or consisting of at least one, a plurality of, or a combination of bacterial species/strains listed in, e.g., TABLEs 1-3, where the compositions are anhydrous compositions.
  • Such anhydrous compositions may comprise, consist essentially of, or consist of bacterial species/strains in powder form, where the dry, powder form has a moistures content of less than 5% wt/wt (e.g., 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 0.05%) in the anhydrous composition.
  • the chemostat model allows for cultivation of microbes under controlled conditions. Depending on environmental changes, microbes may quickly modify their metabolic pathways.
  • Escherichia coli presents differences in ribosome content depending on different nutrient conditions (see, e.g., Scott et al., 2010 “causes of growth inhibition have been suggested, including diversion of metabolites;” Li et al. 2018 “bacteria tune ribosome usage across different limiting nutrients to enable balanced nutrient limited growth”), where constraints dictate the distribution of resources for cell proliferation and gene expression.
  • Different MET-5 and MET-2 donors are analyzed and compared in chemostat models (see, e.g., EXAMPLE 2; TABLES 7-9).
  • the composition of the disclosure comprises a microbiota containing, comprising, consisting essentially of, or consisting of at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3, where the microbiota contains, comprises, consists essentially of, or consists of metabolites selected from: (1) carboxylic acids and derivatives (e.g., acetate, propionate, pyruvate, succinate); (2) fatty acids and conjugates (e.g., butyrate); and (3) amino acids and derivatives (e.g., methionine, phenylalanine, glutamate).
  • carboxylic acids and derivatives e.g., acetate, propionate, pyruvate, succinate
  • fatty acids and conjugates e.g., butyrate
  • amino acids and derivatives e.g., methionine, phenylalanine, glutamate
  • compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3, where the microbiota contains, comprises, consists essentially of, or consists of metabolites, wherein metabolites of carboxylic acids and derivatives are present in at least 70% wt/wt (e.g., 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%); in 97% wt/wt or less (e.g., 95%, 93%, 91%, 89%, 87%, 85%, 83%, 81%, 79%, 77%, 75%, 73%, 71%); and/or in a range of 70%-97% (e.g., 72%-95%, 74%-93%, 76%-91%, 78%-89%, 80%-87%
  • a further embodiment of the disclosure provides for compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3, where the microbiota contains, comprises, consists essentially of, or consists of metabolites, wherein metabolites of fatty acids and conjugates are present in at least 5% (e.g., 6%, 8%, 10%, 12%, 14%); in 20% or less (e.g., 19%, 17%, 15%, 13%, 11%, 9%, 7%); and/or in a range of 5%-20% (e.g., 7%-18%, 9%-16%, 11%-14%) of the total metabolites in the microbiota.
  • compositions of the disclosure comprise a microbiota containing, comprising, consisting essentially of, or consisting of at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3, where the microbiota contains, comprises, consists essentially of, or consists of metabolites, wherein metabolites of amino acids and derivatives are present in at least 0.01% (e.g., 0.03%, 0.05%, 0.07%, 0.09%, 0.11%, 0.13%, 0.15%, 0.17%, 0.19%, 0.21%, 0.23%, 0.25%, 0.27%, 0.29%, 0.31%, 0.33%, 0.35%, 0.37%, 0.39%, 0.41%, 0.43%, 0.45%, 0.47%, 0.49%, 0.51%, 0.53%, 0.55%, 0.57%, 0.59%); in 10% or less (e.g., 8%, 6%, 4%, 2%, 1%
  • compositions of the disclosure comprising a microbiota containing, comprising, consisting essentially of, or consisting of at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3, where the microbiota contains, comprises, consists essentially of, or consists of metabolites of: (1) carboxylic acids and derivatives present in a range of 70%-97% of the total metabolites in the microbiota; (2) fatty acids and conjugates present in a range of 5%-20% of the total metabolites in the microbiota; and (3) amino acids and derivatives present in a range of 0.01%-10% of the total metabolites in the microbiota.
  • compositions of the disclosure may be used in the treatment of dysbiosis, as well as metabolic disorders by replacing the gut microbiome of an abnormal or unhealthy subject, such that the treatment provides sufficient or appropriate nutrients or metabolites which may supplement a deficiency or reduce an excess, thereby treating any diseases that are deficient in or in excess of metabolites.
  • a composition comprising a microbiota containing, comprising, consisting essentially of, or consisting of at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3 (e.g., MET-5, MET-5A, MET-5B, MET-5C), where the microbiota contains a percentage of carboxylic acids of the range of 70%-97% of the total metabolites in the microbiota for treating metabolic disorders.
  • TABLEs 1-3 e.g., MET-5, MET-5A, MET-5B, MET-5C
  • compositions comprising a microbiota containing, comprising, consisting essentially of, or consisting of at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3 (e.g., MET-5, MET-5A, MET-5B, MET-5C), where the microbiota contains, comprises, consists essentially of, or consists of a percentage of fatty acids of the range of 5%-20% of the total metabolites in the microbiota for treating metabolic syndrome or disorders such as but not limited to cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenyl
  • the human gut microbiome has been implicated in metabolic syndrome because of the ability of gut microbes to modulate nutrient uptake, energy regulation, and inflammation. There is a strong body of pre-clinical and clinical data which suggest that the gut microbiomes of those patients suffering from metabolic syndrome differs from those of healthy subjects who do not suffer from metabolic syndrome.
  • the MET-5 compositions comprising, consisting essentially of, or consisting of the bacterial species/strains identified in TABLEs 1-3 may modulate the host microbiome in subjects or individuals suffering from diseases, such as but not limited to, metabolic syndrome, diseases associated with metabolic syndrome, dysbiosis, and dysbiosis-associated diseases.
  • MET-5A is comprises, consists essentially of, or consists of 17 bacterial species/strains and is a representative subset of MET-5 at the family taxonomic level. This subset could form a stable ecosystem in a chemostat model and/or a subject’s microbiome.
  • MET-5B comprises, consists essentially of, or consists of 15 bacterial species/strains and aligns taxonomically with a MET-3 product (also referred to as MET-2A in WO 2019/136269, incorporated herein by reference in its entirety).
  • MET-5C is another subset of MET-5 and comprises, consists essentially of, or consists of 7 bacterial species/strains and represents a small subset of bacterial species/strains that could form a stable ecosystem in a chemostat model and/or a subject’s microbiome.
  • a composition of the disclosure may comprise, consist essentially of, or consist of at least one strain from each phylum identified in, for example, TABLE 3.
  • NB4-BHI-105 Christensenella munita
  • NB4-BHI-105 is such a bacterial species/strain that is isolated from the NB4 donor and which is associated with leanness and a healthy gut microbiome.
  • the compositions of bacterial species/strains listed in, for example, TABLEs 1-3 may be selected in accordance to the need for treatment.
  • bacterial species/strains that produce acetate either directly or indirectly, may be selected to regulate the pH of the gastrointestinal system, provide nutrition to butyrate-producing bacteria, and/or protect against pathogens.
  • Other bacterial species/strains that produce propionate, either directly or indirectly, may be selected to regulate appetite, reduce inflammation, and/or protect against cancer.
  • bacterial species/strains that produce butyrate may be selected to control inflammation by, for example, neutralizing free radicals in the gut; improve digestive health; prevent or improves disease (e.g., obesity, type 2 diabetes); and/or reduce the risk of, for example, bowel cancer, inflammatory bowel disease (IBD), and/or irritable bowel syndrome (IBS).
  • Further bacterial species/strains that produce lactate, either directly or indirectly, may be selected to regulate the immune system, combat opportunistic bacteria, or provide nutrition to butyrate-producing bacteria.
  • amino acids bacteria in the gastrointestinal system or gut play a critical role in the metabolism and reusing nitrogenous compounds, including amino acids.
  • These bacterial species/strains may use amino acids from dietary food or produced by the individual, where the amino acids are the foundation of protein synthesis or metabolize nutrients by converting or fermenting the nutrients into amino acid metabolites (e.g., ammonia, hydrogen sulfide, nitric oxide, polyamines, phenols, and/or indols), which may be responsible for affecting an individual’s metabolism, immune system, and/or nervous system.
  • amino acid metabolites e.g., ammonia, hydrogen sulfide, nitric oxide, polyamines, phenols, and/or indols
  • a further embodiment provides a composition
  • a microbiota containing comprising, consisting essentially of, or consisting of at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3 (e.g., MET-5, MET-5A, MET-5B, MET-5C), where the microbiota contains, comprises, consists essentially of, or consists of a percentage of amino acids of the range of 0.01%-10% of the total metabolites in the microbiota for treating metabolic syndrome or disorders or symptoms thereof.
  • compositions comprising, consisting essentially of, or consisting of any of the at least one, a plurality, or combinations of bacterial species/strains selected from, for example, TABLEs 1-3 (e.g., MET- 5, MET-5A, MET-5B, MET-5C), described herein may be formulated for oral administration as capsules, powders, tablets, granulates, chewable foods, liquids, and beverages.
  • the compositions are formulated into a capsule (e.g., an enteric-coated microcapsule).
  • the compositions are formulated into a tablet.
  • the compositions are formulated into granulated or water-soluble powders.
  • compositions may be formulated into liquids, creams, lotions, gels dispersions or ointments for topical administration.
  • a composition described herein is anhydrous, e.g., a dry powder.
  • a powder may be administered as such or may be dissolved in a fluid, for example, for oral consumption (e.g., via capsule or double capsule) or for rectal administration via an enema.
  • a powder composition may be provided in a palatable form for reconstitution as a drink or for reconstitution as a food additive.
  • a powder composition may also be dissolved in a fluid for rectal administration via an enema (e.g., colonoscopic infusion).
  • the powder may also be reconstituted to be infused via naso-duodenal infusion.
  • Exemplary fluids for such purposes include physiological saline solutions.
  • Methods described here are applicable to animals in general (e.g., mammals), such as but not limited to, humans and economically significant domestic animals, such as, for example, dogs, cats, cows, pigs, horses, sheep, goats, mice, rats, and monkeys.
  • the compositions of the disclosure may contain additional ingredients, including, but not limited to, ingredients that confer properties relating to healthfulness, flavor, formulating, or tableting.
  • compositions described herein include: prebiotics, vitamins, minerals, nutritional supplements (e.g., fiber), sweeteners, flow aids, and fillers.
  • the compositions comprise, consist essentially, or consist of at least 0.1% w/w or more (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%) of at least one, a plurality, or combination of bacterial species/strains of a composition (e.g., an anhydrous composition) described herein.
  • Compositions of the disclosure are useful in methods for treating various diseases and disorders characterized by metabolic syndrome and/or dysbiosis.
  • compositions described here may be used to promote digestive health, metabolism (e.g., nutritional heath), and weight management when administered (e.g., orally or rectally).
  • Compositions described here may be used to treat or alleviate metabolic syndrome or a positive indicator or symptom of a metabolic syndrome including but not limited to: cardiovascular disease, hypertension, stroke, diabetes (e.g., type 2 diabetes mellitus), insulin resistance (e.g., obesity in type 1 diabetes mellitus patients, Cushing’s disease, lipodystrophy syndromes), insulin resistance- associated complications, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acy
  • compositions described herein that may be used to treat or alleviate a positive indicator or symptom of a digestive disorder or dysbiosis including, but not limited to: irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, Crohn's disease, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, and AIDS enteropathy.
  • IBS irritable bowel syndrome
  • spastic colon idiopathic ulcerative colitis
  • mucous colitis mucous colitis
  • collagenous colitis Crohn's disease
  • inflammatory bowel disease in general
  • microscopic colitis antibiotic-associated colitis
  • idiopathic or simple constipation idiopathic or simple constipation
  • diverticular disease AIDS enteropathy
  • compositions described herein are also envisioned for use in treating or alleviating a positive indicator or symptom of a digestive disorder including but not limited to: irritable bowel syndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucous colitis, collagenous colitis, Crohn's disease, inflammatory bowel disease in general, microscopic colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, and AIDS enteropathy.
  • Treatment regimens may comprise administration of compositions described herein to a subject in need thereof on, for example, a daily basis (typically, e.g., once or twice per day), twice or thrice weekly, bi-weekly, or once per month.
  • Treatment regimens may also be altered as the subject’s condition changes and may, furthermore, be intermittent.
  • a suitable treatment regimen may be determined by a medical practitioner and/or may be established based on empirical results as evaluated by a medical practitioner and/or the subject being treated. [296] Unless stated otherwise, all percentages referred to here are by weight based on the total weight of the composition. For example, the bacterial species/strains of the compositions of the disclosure are presented by percent by weight (i.e., % wt/wt) of the total weight of the composition.
  • a composition e.g., anhydrous composition or drug product
  • a composition comprising, consisting essentially of, or consisting of at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26), a plurality of, or combinations of bacterial species/strains of MET-5 or any of the MET-5 subgroups (i.e., MET-5A, MET-5B, MET-5C) identified in, for example, TABLEs 1-3, comprises, consists essentially of, or consists of, in some embodiments, a lyophilized mixture of a predetermined ratio of pure cultures of any one or more of the 26 diverse intestinal bacteria of MET-5 (e.g., 17 bacteria of MET-5A; 15 bacteria of MET-5B; 7 bacteria of MET-5C), derived from a stool sample of one or more healthy donors (e.g., two different healthy donors), where the healthy donor does not suffer from metabolic syndrome, disease(s) associated with metabolic syndrome
  • Each capsule may contain, comprise, consist essentially of, or consist of 0.5 g of at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26), a plurality of, or combinations of bacterial species/strains of MET-5 or any of the MET-5 subgroups (i.e., MET-5A, MET-5B, MET-5C) identified in, for example, TABLEs 1-3, comprises in some embodiments, a lyophilized mixture of a predetermined ratio of pure cultures of any one or more of the 26 diverse intestinal bacteria, derived from a stool sample of one or more healthy donors (e.g., two different healthy donors).
  • a lyophilized mixture of a predetermined ratio of pure cultures of any one or more of the 26 diverse intestinal bacteria derived from a stool sample of one or more healthy donors (e.g., two different healthy donors).
  • Each capsule may contain, consist essentially of, or consist of 0.5 g of at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), a plurality of, or combinations of bacterial species/strains of MET-5 or any of the MET-5 subgroups (i.e., MET-5A, MET-5B, MET-5C) identified in, for example, TABLEs 1-3, with a bacterial population concentration per capsule of at least 1 X 102 (e.g., 1.87 X 10 4 ; 2 X 10 4 ; 1 X 10 10 ; 1.87 X 10 10 ; 2 X 10 10 ; 1 X 10 2 to 1 X 10 14 ; 1 X 10 4 to 1 X 10 13 ; 2.97 X 10 4 to 2.87 X 10 10 ; 1 X 10 4 to 3 X 10 10 ; 1 X 10 5 to 1 X 10 14 ; 1 X 10 6 to 1 X 10 14 ; 1 X 10 7 to 1 X 10 14 ; 1
  • each bacterial species/strain concentration per capsule may be in an amount of at least 1 X 10 2 CFU (e.g., 1 X 10 3 , 1 X 10 4 , 1 X 10 5 , 1 X 10 6 , 1 X 10 7 , 1 X 10 8 , 1 X 10 9 , 1 X 10 10 ; 1 X 10 11 ; 1 X 10 12 ; 1 X 10 13 ; 1 X 10 2 -1 X 10 10 , 2 X 10 2 -2 X 10 10 , 3 X 10 2 -3 X 10 10 ), where the composition of the disclosure comprises, consists essentially of, or consists of at least one, a plurality, or combinations of any of the bacterial species/strains in, for example, TABLEs 1-3.
  • each bacterial species/strains in an amount of 12 g – 50 g per capsule, where each batch produces 1,000 – 4,000 capsules, where cellulose, if present, may be in an amount from 0% to 5% per gram of bacterial species/strain in powder form totaling 372g – 1,550 g.
  • a batch may be formulated in 1,000 - 4,000 capsules in hard V cap enteric coated, size 0 or hard V cap enteric coated, size 00.
  • composition or drug product of the disclosure comprising, consisting essentially of, or consisting of at least one, a plurality of, or a combination of the bacterial species/strains of, for example, TABLEs 1-3 may be shipped and maintained at room temperature; the capsule may be sealed in anaerobic packaging and is opened only immediately prior to the subject/patient swallowing the capsule.
  • 26 pure bacterial culture isolates have been selected for a MET-5 composition or drug product derived from a stool sample of primarily a single donor, or where at least one of the pure bacterial culture isolates may be derived from another donor different from the donor of the other one or more bacterial species/strains.
  • bacterial species/strains listed in any of the tables described here comprising, consisting essentially of, or consisting of a purity of: at least 95% (e.g., 96%, 97%, 98%, 99%, 100%); 100% or less (e.g., 99.5%, 98.5%, 97.5%, 96.5%, 95.5%, 94.5%, 93.5%); or a range of 90%-100% (e.g., 91%-99%, 92%-98%, 93%-97%, 94%-96%) as measured by any known and commonly used methods, including, for example, the methods described in Oliphant et al., 2019.
  • compositions of the disclosure comprising, consisting essentially of, or consisting of at least one, a plurality of, or a combination of bacterial species/strains listed in any of the tables described here (e.g., TABLEs 1-5), where each of the bacterial species/strains may have a purity of: at least 95% (e.g., 96%, 97%, 98%, 99%, 100%); 100% or less (e.g., 99.5%, 98.5%, 97.5%, 96.5%, 95.5%, 94.5%, 93.5%); or a range of 90%-100% (e.g., 91%-99%, 92%-98%, 93%-97%, 94%-96%) as measured by any known and commonly used methods, including, for example, the methods described in Oliphant et al., 2019.
  • any potential bacterial species/strain having equal to or greater than 97% (e.g., 97.5%, 98%, 98.5%, 99%, 99.5%) identity or 94% or greater (e.g., 94.5%, 95%, 95.5%, 96%, 96.5% 97%, 97.5%, 98%, 98.5%, 99%, 99.5%) identity to its closest neighbor by 16S rRNA gene sequence identity is considered in the art to be of the same species.
  • Microcrystalline cellulose may be added as a flow aid to the mixture of lyophilized drug substances comprising, consisting essentially of, or consisting of bacterial species/strains identified in TABLE 2.
  • Two-piece hard Vcaps ® Enteric Capsules (Capsugel), composed of hypromellose/hypromellose AS and titanium dioxide, may be used to encapsulate the MET-5 drug substance mixture (including microcrystalline cellulose). Any of the drug products described here (e.g., MET-5, MET-5A, MET-5B, MET-5C) may be double-encapsulated.
  • MET-5 lyophilized material may be filled into a size 0 enteric capsule, sealed, and then placed in a size 00 enteric capsule, which is then sealed again.
  • MET-5 or MET-5 subgroups capsules may be administered orally in an enteric capsule, for delivery of the live bacteria to the large intestine.
  • MET-5 capsules are to be stored at room temperature, and packaging should be opened only immediately before administration to patients in order to preserve the nitrogen atmosphere within the packages.
  • the MET-5 or MET-5 subgroup (e.g., MET-5A, MET-5B, MET-5C) composition or drug product may be in a capsule formulation for oral administration, where the concentration comprises 0.5 g per capsule (e.g., 1 x 10 2 -10 11 ; 1.87 x 10 4 -10 10 CFU/capsule) of bacterial species/strains disclosed here (e.g., TABLE 2).
  • the MET-5 or MET-5 subgroup composition may be for oral administration for treating a subject suffering from metabolic syndrome, a metabolic syndrome-associated disease, dysbiosis, or a dysbiosis-associated disease.
  • the batch formula for MET-5 (or MET-5 subgroup) capsules is provided in TABLE 5.
  • the manufacturing process for making MET-5 capsules runs continuously from culturing each drug substance strain through lyophilization and continues directly into the encapsulation process. Generally, enough dry weight of each microbe is manufactured at a strength in CFU/g within the range in TABLE 5. They are then combined to generate a batch for encapsulation, which has been found to yield approximately 1,000- 4,000 capsules.
  • the batch formula is provided in TABLE 5.
  • MET-5 or any one of its subgroups i.e., MET-5A, MET-5B, MET-5C
  • anhydrous (e.g., lyophilized) pure bacterial cultures mixed in predefined ratios, with strengths, for example, as detailed in TABLE 5.
  • TABLE 5 MET-5 Capsule Batch Formula Strength (Label Claim) 1.87 X 10 4 -10 10 CFU/capsule
  • MET-5 and exemplary subgroups thereof formulated in compositions or drug products are described as therapeutic agents for treating metabolic syndrome, diseases associated with metabolic syndrome, dysbiosis, and dysbiosis-associated gastrointestinal diseases in subjects afflicted with such diseases and/or symptoms, including but not limited to, cardiovascular disease, coronary heart disease, type 2 diabetes mellitus, insulin resistance, dyslipidemia, elevated blood pressure, prothrombotic state, proinflammatory state, non-alcoholic fatty liver disease, hypertension, stroke, diabetes (e.g., type 2 diabetes, diabetes mellitus), insulin resistance, glucose intolerance, non-alcoholic fatty liver disease, dyslipidemia, hypertriglyceridemia, hypercholesteremia, obesity, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease
  • ASA argininosuccinic acidemia
  • CIT homocystinuria
  • MET-5 comprises, consists essentially of, or consists of 26 strains of lyophilized bacteria, originally purified from a healthy stool donor and further selected based on, for example, their functional characteristics and their favorable safety profile.
  • a MET-5 (including its subgroup, for example, MET-5A, MET-5B, MET-5C) composition or drug product administered to a subject suffering from metabolic syndrome, diseases associated with metabolic syndrome, dysbiosis, or a dysbiosis-associated disease will be well-tolerated with no serious adverse events related to treatment with this therapeutic.
  • treatment with this the composition of the disclosure comprising, consisting essentially of, or consisting of a MET-5 or MET-5 subgroup bacterial population disclosed here will not result in, multi-drug resistance, bacteremia, sepsis, or invasive infections in subjects or patients suffering from metabolic syndrome, diseases associated with metabolic syndrome, dysbiosis, or dysbiosis-associated diseases undergoing treatment.
  • MET-5 may have modifications that reflect and incorporate novel information that has emerged from the rapidly evolving field of gut microbiota research in the context of metabolic syndrome, diseases associated with metabolic syndrome, dysbiosis, or dysbiosis-associated diseases.
  • Selection may also take into consideration bacterial functionality, where some overlapping functions or redundancy may be beneficial potentially providing synergistic effects.
  • some bacteria may be selected for not having overlapping functions which may improve the efficiency of the bacterial population.
  • yet a further embodiment may provide for bacteria selected to reduce the risk of harmful side effects, such as but not limited to, bacterial overgrowth or antibiotic resistance.
  • Some embodiments may provide for the selection of bacteria that are producers of favorable or desired metabolites or consumers of disadvantageous or toxic metabolites.
  • Ulcerative colitis is a chronic, relapsing, idiopathic, inflammatory disease of the colorectum. In the last decade, there has been an increase in the incidence and prevalence of UC, making it an important emerging global disease.
  • the main symptoms of UC include bloody diarrhea, abdominal pain, urgency, tenesmus, and incontinence, which cause a reduction in patient quality of life.
  • the severity of UC symptoms ranges from mild disease ( ⁇ 4 stools per day with or without blood) to severe disease (>10 stools per day with intense cramping and continuous bleeding). Depending on the clinical severity of intestinal disease, patients may also develop systemic symptoms and other life-threatening complications. [310] Management of UC is determined by the clinical severity of disease, and current treatment strategies are focused on regulating the immune system with anti-inflammatory and immunosuppressive drugs.
  • ⁇ -ASA ⁇ -aminosalicyclic acid
  • immunomodulators are the main treatment options with use of immunomodulators as a steroid sparing agent. While these therapies are able to maintain remission in many cases, current medical treatments are imperfect and there is a subset of patients that do not respond to topical 5-ASA alone or in combinational therapy with corticosteroids. Additionally, 20-30% of UC patients require colectomy to manage acute complications and medically intractable disease. Thus, there is a need for more efficacious drugs with a greater favorable safety profile for the treatment of UC.
  • Gut dysbiosis can be defined as a pathological imbalance in a microbial community characterized by a shift in the composition, diversity or function of microbes, which can result in disease. Antibiotics, toxic compounds, diet, medical interventions, and disease can all influence the gut microbiome. However, defining gut microbial dysbiosis is difficult due to the variability in bacterial composition across individuals in both in healthy and disease-states. The gut microbiome has been associated with a multitude of disease indications including, but not limited to: C. difficile infection (CDI), inflammatory bowel disease (IBD), and irritable bowel syndrome (IBS).
  • CDI C. difficile infection
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • MET-5 including its subgroups, for example, MET-5A, MET-5B, and MET-5C of TABLEs 1-3, may be a lyophilized bacterial drug product or composition administered or given orally, in encapsulated form for treating metabolic syndrome, diseases or symptoms associated with metabolic syndrome, dysbiosis, or dysbiosis-associated diseases.
  • MET-5, or any of its subgroups, for treatment may be supplied in 1-2 dosage forms: 1) lyophilized powder in capsules for oral ingestion and 2) lyophilized powder for rectal administration by colonoscopy (e.g., dry powder resuspended in 0.9% saline).
  • MET-5 may comprise, consist essentially of, or consist of live bacterial species/strains in a composition or drug product (resuspended in 0.9% saline) that may be administered orally or by colonoscopy.
  • FMT colonoscopy-delivered fecal microbial therapy
  • rCDI C. difficile infection
  • MET-5 is a therapeutic composition composed of, consisting essentially of, or consisting of a defined microbial community of 26 bacterial strains derived from the stool of at least one healthy fecal donor.
  • the bacteria are prepared as a mixture in a predetermined ratio of pure lyophilized intestinal bacteria.
  • the bacteria may be double encapsulated in enteric capsules.
  • the donor from where MET-5 strains were derived has been rigorously screened for infectious materials and blood-borne pathogens. Stool from a healthy donor has also been previously used as an FMT donor to successfully treat rCDI. There is no upper toxicity limit is expected due to the safety profile of the MET-5 bacterial community.
  • a multi-species derivative community such as that described herein may be more generally useful than a single organism probiotic or a mixed culture of such probiotic species.
  • the microbes in MET- 5 are derived from a community and are expected to retain community structure to a degree that enables them to colonize the colonic environment.
  • a defined microbial community isolated from a single healthy donor, may be sufficiently robust to withstand further perturbations by antibiotics demonstrating augmented robustness responsiveness to perturbations.
  • the different MET-5 compositions comprise, consist essentially of, or consist of various bacterial populations isolated from a healthy donor, where each composition may be beneficial for treating specific symptoms of or metabolic syndromes, diseases associated with metabolic syndromes, dysbiosis, or dysbiosis-associated diseases.
  • Some embodiments of the disclosure provide methods for treating a subject (e.g., mammalian, human, domestic animal) suffering from a metabolic syndrome, a disease associated with metabolic syndrome, dysbiosis, or dysbiosis-associated diseases, by administering a therapeutically effective amount of a composition comprising, consisting essentially of, or consisting of at least one, a plurality of, or a combination of bacterial species/strains identified in, for example, TABLEs 1-3, where the therapeutically effective amount reduces or improves symptoms of the metabolic syndrome, the disease associated with metabolic syndrome, dysbiosis, or the dysbiosis-associated disease.
  • Non-limiting examples of metabolic syndrome or metabolic diseases treated using any of the MET-5 (or MET-5 subgroups) compositions or drug products described here include, but not limited to hypertriglyceridemia, insulin resistance, obesity, diabetes (e.g., type 2 diabetes, diabetes mellitus), hypertension, dyslipidemia, argininosuccinic acidemia (ASA), citrullinemia (CIT), homocystinuria (HCY), maple syrup urine disease (MSUD), phenylketonuria (PKU), tyrosinemia type I (TYR I), fatty carnitine transport defect, carnitine-acylcarnitine translocase deficiency (CACT), carnitine palmitoyl transferase I & II (CPT I deficiency and CPT II deficiency), 2,4 dienoyl-CoA reductase deficiency, and 3-hydroxy-3- methylglutaryl-CoA lyase deficiency
  • RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS RESULTS, triglycerides, and the like.
  • the methods of treating a subject suffering from dysbiosis or diseases associated with dysbiosis may occur by administering to the subject a therapetically effective amount of any of the compositions or drug products described here comprising, consisting essentially of, or consisting of MET-5 (or MET-5 subgroup) bacterial species/strains.
  • the fecal donor was screened extensively for viral, bacterial, and medical diseases. A detailed list of blood and stool tested is listed below. Briefly, the MET-5 donor (NB4; Donor 5) was screened for pathogenic organisms and tested negative for extended-spectrum beta-lactamases (ESBL), carbapenemase- producing organisms (CPO), carbapenem-resistant Enterobacteriaceae (CRE), vancomycin-resistant Enterococcus (VRE), vancomycin resistant Staphylococcus aureus (VRSA), methicillin resistant Staphylococcus aureus (MRSA), drug-resistant Streptococcus pneumoniae, multi-drug resistant Pseudomonas aeruginosa, multi-drug resistant Mycobacterium tuberculosis (MDR-TB), multi-drug resistant Acinetobacter, drug-resistant Neisseria gonorrhoeae, toxigenic Clostridium difficile (C.
  • CPO extended-spectrum beta-
  • a MET-5 e.g., MET-5A, MET-5B, MET-5C
  • MET-5A, MET-5B, MET-5C composition or drug product release only occurs when each bacterial culture tests negative for impurities or any bacterial contaminant (e.g. pathogenic organisms) as determined by Sanger sequencing of the 16S rRNA gene (specific to bacteria).
  • MET-5 and/or any of its subgroups e.g., MET- 5A, MET-5B, MET-5C
  • sample supernatant 200 pL was then mixed with 15 pl. of proteinase K and 200 pL of lysis buffer solution and incubated at 70°C for 10 minutes. After incubation, samples were mixed with 200 pL of EtOH, vortexed, transferred to the QIAamp spin column and bound. Samples were then washed twice prior to elution in ATE buffer (i.e., a low-EDTA elution buffer optimized for long-term storage of DNA; QIAGEN). Duplicate samples were subjected to Illumina MiSeq 16S rRNA gene sequencing (Mr. DNA, Molecular Research LP; Shallowater, Texas).
  • the criteria for sample selection was based upon the likelihood that various bacterial species/strains of interest could be isolated from a particular sample, including clinically relevant microorganisms such as Christensenella spp., Faeciilibacterium prausnitzii and Akkermansia rnuciniphila, among others.
  • the pooled alpha diversity results (based on the Shannon diversity index) comparing all seven fecal samples are depicted in FIG. 1, along with the community profile results from the selected sample (FIG. 2).
  • the genera from the top to the bottom occurs in the order of: Tyzzerella, Sutterella, Subdoligranulum, Streptococcus, Senegalimassilia, Ruminococcus,
  • MET-5 drug product e.g., batch NB5-005 and NB5-003
  • two capsules from each batch were resuspended in 20 mL of reduced 0.9% saline at 37°C under anaerobic conditions.
  • the chemostats were then run for 6 days using methods described previously (McDonald et al., 2013). Chemostat effluent from day 6 was then analyzed by 1H NMR and the profiles in TABLE 7 were produced.
  • EXAMPLE 3 STOOL SAMPLE PROCESSING AND BACTERIAL ISOLATION
  • stool sample NB4-04 was selected from the catalog, it was partially thawed in the anaerobic chamber to obtain material for both direct bacterial isolation as well as an inoculum for the chemostat, an in vitro model of the distal colon (McDonald et al., 2013).
  • Chemostat Inoculum Preparation [338] In an anaerobic chamber, 5 g of the fecal sample was placed in a sterile Stomacher® bag along with 50 mL of sterile reduced chemostat medium. The fecal sample was homogenized by hand for 3 minutes.
  • the fecal slurry was centrifuged for 10 minutes at 1,500 rpm, and the supernatant was collected in an anaerobic chamber. This supernatant (20 mL) was used to inoculate a 200 mL chemostat vessel.
  • Chemostat protocol (LAB 021 – Operation of the Infors Chemostat, and LAB 024 – Preparation of Chemostat Media) was used for the chemostat run.
  • Chemostat Bacterial Isolations [340] The chemostat was run for 28 days to reach steady state (McDonald et al., 2013), followed by bacterial strain isolations. The chemostat was sampled on Day 28 and a ten-fold serial dilution was performed in an anaerobic chamber using reduced Wilkins-Chalgren Broth. Dilutions were then plated on 16 different media types (TABLE 12) and left to grow for at least 48 hours at 37°C aerobically or anaerobically. Isolated colonies were then picked from each media type, and re-streaked on fastidious anaerobic agar (FAA) for identification by 16S rRNA gene Sanger sequencing.
  • FAA fastidious anaerobic agar
  • the remaining strains were then compared to the clinically effective MET-3 (see, e.g., MET-2A at WO 2019/136269, incorporated herein by reference in its entirety) community, which was used as a guide in strain selection for the MET-5 community.
  • Beneficial microbes were added based on their known positive attributes in the literature, with the rationale for the inclusion of each strain described in detail below.
  • the closest species identity for each strain was defined in 2018/2019 using the NCBI 16S ribosomal RNA sequences (Bacteria and Archaea) database.
  • the V3-V6 region of the 16S rRNA gene was used to determine the closest species identity via Sanger sequencing. By comparison, whole genome sequencing allows for more definitive speciation.
  • Speciation using 16S rRNA gene sequencing is a moving target, especially given the use of NCBI as the 16S rRNA gene taxonomy database.
  • the best way to remain contemporary is to utilize multiple tools available through such sites as bacterio.net (LPSN), the International Journal of Systematic and Evolutionary Microbiology and published updates (e.g. Bergey’s) to validate and redefine closest species over time. It is important to note that name changes happen constantly in the field of human gut microbiology and rather than rely solely on the closest species identity, strain names are also used and remain consistent here.
  • LPSN bacterio.net
  • the International Journal of Systematic and Evolutionary Microbiology and published updates e.g. Bergey’s
  • the strains were grown anaerobically (atmosphere of 10% carbon dioxide, 10% hydrogen and 80% nitrogen) at 37°C.
  • Anaerobes generally grow slower than aerobes, because oxygen is the optimal electron acceptor in nature, and when oxygen is not present, other methods of energy conservation must be carried out. Yet, this metabolism generally does not produce as many moles of ATP as aerobic metabolism. Hence, a generally slower rate of growth is observed.
  • An incubation time of 48 hours is conservative and allows for the visualization of colonies on agar media plates. This has also historically served as the incubation time to facilitate bacterial strain isolations from other donors for the purpose of creating compositions for treating dysbiosis among other gut microbiota-related diseases or disorders.
  • EXAMPLE 4 MET-5 SUMMARY DATA Original Strain ID Data [344] PCR primers, 16S rRNA primers, were used to amplify the 16S rRNA gene from each isolate. Full length 16S rRNA sequences corresponding to each bacterial strain presented in TABLE 13.
  • Exemplary 16S rRNA primers with T3 and T7 tails are as follows: Forward (V3kl): ATTAACCCTCACTAAAGTACGG[AG]AGGCAGCAG Reverse (V6r): AATACGACTCACTATAGGGAC[AG]ACACGAGCTGACGAC TABLE 13: Identification of closest match by Basic Local Alignment Search Tool (BLAST) Original ID by BLAST – Closest % Identification (to Strain Code S i Id tit Cl t S i ) subject of WO 2018/197951 A1. Antibiotic Susceptibility Testing
  • MET-3 (see, e.g., MET-2A at WO 2019/136269, incorporated herein by reference in its entirety) composition, which was derived from an NB2 healthy fecal donor (25 years old male), was used in a Canadian Phase I clinical study to treat metabolic syndrome. Due to the preliminary clinical success of MET-3, it was used as a guide in strain selection for the MET-5 drug product community. Those strains with a taxonomically similar closest species identity (using the All-Species Living Tree [The All Species Living Tree Project Release s128_SSU Full Tree] as a taxonomic means of comparison) to those in MET-3 were selected first (as outlined in TABLE 15).

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Abstract

La présente invention concerne des compositions comprenant un microbiote et des procédés permettant de les utiliser pour traiter des troubles associés à des maladies métaboliques et/ou à une dysbiose (un déséquilibre de la communauté microbienne habitant un sujet ou habitant un tissu particulier chez un sujet). En particulier, des compositions anhydres comprenant un microbiote ayant au moins un, une pluralité, ou des combinaisons d'espèces bactériennes/stmines et des procédés de traitement du syndrome métabolique, de la dysbiose, de maladies associées au syndrome métabolique et/ou de la dysbiose, des troubles de l'aride/ou gastro-intestinal par l'administration de la composition de l'invention. L'utilisation de telles compositions ou compositions anhydres comprenant le microbiote pour traiter des troubles métaboliques, une dysbiose ou des maladies associées à une dysbiose (par exemple, des troubles gastro-intestinaux associés à des troubles métaboliques ou une dysbiose) et l'utilisation de telles compositions ou compositions anhydres pour traiter de telles maladies ou troubles dans la préparation d'un médicament pour traiter de telles maladies ou troubles.
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Citations (5)

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WO2015003001A1 (fr) * 2013-07-01 2015-01-08 The Washington University Procédés pour identifier des compléments qui augmentent la colonisation intestinale par une espèce bactérienne isolée, et compositions dérivées
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WO2015003001A1 (fr) * 2013-07-01 2015-01-08 The Washington University Procédés pour identifier des compléments qui augmentent la colonisation intestinale par une espèce bactérienne isolée, et compositions dérivées
US20170367641A1 (en) * 2014-10-21 2017-12-28 uBiome, Inc. Method and system for microbiome-derived diagnostics and therapeutics for conditions associated with microbiome taxonomic features
US20190381115A1 (en) * 2014-11-13 2019-12-19 Institut National De La Recherche Agronomique (Inra) Faecalibacterium prausnitzii strains for treating and preventing gastrointestinal pain
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