WO2019136269A1 | 2019-07-11 | |||
WO2015003001A1 | 2015-01-08 |
US20190381115A1 | 2019-12-19 | |||
US20170367641A1 | 2017-12-28 | |||
US9701964B2 | 2017-07-11 |
CLAIMS What is claimed is: 1. An anhydrous composition comprising a microbiota, wherein the microbiota comprises a plurality of bacterial species 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 minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in a powder form, wherein the powder form has a moisture content of less than 5% wt/wt in the anhydrous composition, and the microbiota exhibits resistance to perturbational stress. 2. An anhydrous composition comprising a microbiota, wherein the microbiota comprises at least one 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; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in a powder form, wherein the powder form has a moisture content of less than 5% wt/wt in the anhydrous composition, and the microbiota exhibits resistance to perturbational stress. 3. An anhydrous composition comprising a microbiota, wherein the microbiota comprises a plurality of bacterial species, the plurality of bacterial species 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 Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in a powder form, wherein the powder form has a moisture content of less than 5% wt/wt in the anhydrous composition, and the microbiota exhibits resistance to perturbational stress. 4. An anhydrous composition comprising a microbiota, wherein the microbiota comprises a plurality of bacterial species consisting of at least one bacterial species from at least one phylum of bacteria selected from: Firmicutes; Bacteroidetes; Actinobacteria; or Verrucomicrobia, wherein the bacterial species are in a powder form, wherein the powder form has a moisture content of less than 5% wt/wt in the anhydrous composition, and the microbiota exhibits resistance to perturbational stress. 5. The anhydrous composition of claim 4, wherein 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. 6. The anhydrous composition of claim 4 or claim 5, wherein the at least one bacterial species is selected from a phylum of bacterial species selected from: Firmicutes and Verrucomicrobia. 7. An anhydrous composition comprising a microbiota, wherein the microbiota comprises at least one 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 excrementihominis, wherein the MET-5A bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 8. An anhydrous composition comprising a microbiota, wherein the microbiota comprises 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, content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 9. An anhydrous composition comprising a microbiota, wherein the microbiota comprises at least one of the MET-5C bacterial species selected from: Faecalibacterium prausnitzii; Bacteroides uniformis; Akkermansia muciniphila; Bifidobacterium longum; Christensenella minuta; Parasutterella excrementihominis; and [Clostridium] scindens, wherein the MET-5C bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 10. An anhydrous composition comprising a microbiota, wherein the microbiota comprises at least one 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 valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof that is present in each of MET-5, MET-5A, MET-5B, and MET-5C, wherein the bacterial species present in each of MET-5, MET-5A, MET-5B, and MET-5C are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 11. An anhydrous composition comprising a microbiota, wherein the microbiota comprises at least one 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 valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the microbiota comprises bacterial strains selected from: 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; 130; NB4-CNA-122; NB4-D5-120; NB4-EtOH-104; NB4-FAA-116; NB4-D5-137; NB4-NA-102; and 14LG, wherein the bacterial strains are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 12. The anhydrous composition of claim 11, wherein the microbiota comprises 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, wherein the bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 13. The anhydrous composition of claim 11, wherein the microbiota comprises 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, wherein the bacterial strains are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 14. The anhydrous composition of claim 11, wherein the microbiota comprises 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, wherein the bacterial strains are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 15. The anhydrous composition of any one of the preceding claims, wherein the microbiota comprises at least 10% (e.g., 15%, 20%, 21%, 23%, 25%, 27%, 29%, 30%, 31%, 33%, 35%, 37%, 39%, 70%, 71%, 73%, 75%, 77%, 79%, 80%) Gram-negative bacterial species. 16. The anhydrous composition of any one of the preceding claims, wherein the microbiota comprises 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-positive bacterial species. 17. The anhydrous composition of any one of the preceding claims, wherein the microbiota comprises a ratio of Gram-positive to Gram-negative bacterial species in a range of 2:1 to 1:5 (e.g., 9:5 to 2:5, 3:2 to 1:1). 18. The anhydrous composition of any one of the preceding claims, wherein the microbiota comprises at least 30 % (e.g., 31%, 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 within the Firmicutes phylum. 19. The anhydrous composition of any one of the preceding claims, wherein the microbiota comprises at least 0.5 % (e.g., 0.7%, 0.9%, 1%, 3%, 5%, 7%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%) bacterial species within the Bacteroidetes phylum. 20. The anhydrous composition of any one of the preceding claims, wherein the microbiota comprises a sub-group as set forth in TABLE 2 or TABLE 3 with respect to category and/or functional properties. 21. The anhydrous composition of any one of the preceding claims, wherein the bacterial species are in a state of suspended animation. 22. The anhydrous composition of any one of the preceding claims, further comprising a pharmaceutically acceptable carrier. 23. The anhydrous composition of claim 22, wherein the pharmaceutically acceptable carrier is cellulose. 24. The anhydrous composition of any one of the preceding claims, wherein the anhydrous composition is encapsulated in a capsule. 25. The anhydrous composition of claim 24, wherein the anhydrous composition is encapsulated in a double capsule. 26. The anhydrous composition of any one of the preceding claims, further comprising a prebiotic. 27. An anhydrous composition of any one of the preceding claims for use in treating a disease or disorder associated with metabolic syndrome, wherein an effective amount of the anhydrous composition improves relative ratios of microorganisms, thereby treating the disease or disorder associated with metabolic syndrome. 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 lyase deficiency (HMG deficiency). 29. An anhydrous composition of any one of the preceding claims for use in treating a disease or disorder associated with dysbiosis, wherein an effective amount of the anhydrous composition improves relative ratios of microorganisms, thereby treating the disease or disorder associated with dysbiosis. 30. The anhydrous composition of claim 29, wherein 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. 31. An anhydrous composition of any one of the preceding claims for use in the preparation of a medicament for treating a disease or disorder associated with metabolic syndrome, wherein an effective amount of the medicament improves relative ratios of microorganisms, thereby treating the disease or disorder associated with metabolic syndrome. 32. The anhydrous composition of claim 31, wherein 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), 2,4 dienoyl-CoA reductase deficiency, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG deficiency). 33. An anhydrous composition of any one of the preceding claims for use in the preparation of a medicament for treating a disease or disorder associated with dysbiosis, wherein an effective amount of the medicament improves relative ratios of microorganisms, thereby treating the disease or disorder associated with dysbiosis. 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. 35. An anhydrous composition comprising a plurality of bacterial species, the plurality of bacterial species 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 minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, and the bacterial species are: (a) in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the anhydrous composition when tested by a chemostat model test is: (b) suspended in a first growth media and cultured to achieve steady state growth of the plurality of bacterial species in the first growth media, wherein a relative abundance of the plurality of bacterial species at steady state growth in the first growth media is established as a first relative abundance, and (c) the plurality of bacterial species at steady state growth in the first growth media is challenged by perturbational stress, wherein the perturbational stress is a change in at least one of substrate type, substrate availability, or xenobiotic challenge, and the plurality of bacterial species exhibits robustness when challenged by the perturbational stress, wherein the robustness is exhibited by maintenance of the first relative abundance of the plurality of bacterial species after challenge by the perturbational stress. 36. An anhydrous composition comprising a plurality of bacterial species, the plurality of bacterial species 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 minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides any combination thereof, and the bacterial species are: (a) in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the anhydrous composition when tested by an ecosystem output assay is: (b) suspended in a first growth media and cultured to achieve steady state growth of the plurality of bacterial species in the first growth media, wherein a relative abundance of the plurality of bacterial species at steady state growth in the first growth media is established as a first relative abundance, and (c) the plurality of bacterial species at steady state growth in the first growth media is challenged by perturbational stress, wherein the perturbational stress is a change in at least one of substrate type, substrate availability, or xenobiotic challenge, and the plurality of bacterial species exhibits robustness when challenged by the perturbational stress, wherein the robustness is exhibited by maintenance of functional output of types and quantities of selected small molecules generated by the plurality of bacterial species after challenge by the perturbational stress. 37. An anhydrous composition comprising a plurality of bacterial species, the plurality of bacterial species 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; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the at least one bacterial species from each phylum of bacteria are: (a) in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the anhydrous composition when tested by a chemostat model test is: (b) suspended in a first growth media and cultured to achieve steady state growth of the plurality of bacterial species in the first growth media, wherein a relative abundance of the plurality of bacterial species at steady state growth in the first growth media is established as a first relative abundance, and by perturbational stress, wherein the perturbational stress is a change in at least one of substrate type, substrate availability, or xenobiotic challenge, and the plurality of bacterial species exhibits robustness when challenged by the perturbational stress, wherein the robustness is exhibited by maintenance of the first relative abundance of the plurality of bacterial species after challenge by the perturbational stress. 38. An anhydrous composition comprising a plurality of bacterial species, the plurality of bacterial species 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; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the at least one bacterial species from each phylum of bacteria are: (a) in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the anhydrous composition when tested by an ecosystem output assay is: (b) suspended in a first growth media and cultured to achieve steady state growth of the plurality of bacterial species in the first growth media, wherein a relative abundance of the plurality of bacterial species at steady state growth in the first growth media is established as a first relative abundance, and (c) the plurality of bacterial species at steady state growth in the first growth media is challenged by perturbational stress, wherein the perturbational stress is a change in at least one of substrate type, substrate availability, or xenobiotic challenge, and the plurality of bacterial species exhibits robustness when challenged by the perturbational stress, wherein the robustness is exhibited by maintenance of functional output of types and quantities of selected small molecules generated by the plurality of bacterial species after challenge by the perturbational stress. 39. The anhydrous composition of any one of the preceding claims, wherein the bacterial species are in a state of suspended animation. 40. The anhydrous composition of any one of the preceding claims, further comprising a pharmaceutically acceptable carrier. is cellulose. 42. The anhydrous composition of any one of the preceding claims, wherein the anhydrous composition is encapsulated in a capsule. 43. The anhydrous composition of any one of the preceding claims, wherein the anhydrous composition is encapsulated in a double capsule. 44. The anhydrous composition of any of the preceding claims, wherein 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. 45. The anhydrous composition of claim 44, wherein the at least one bacterial species is selected from a phylum of bacterial species selected from: Firmicutes and Verrucomicrobia. 46. The anhydrous composition of any one of the preceding claims, further comprising a prebiotic. 47. An 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 a plurality of bacterial species consisting of 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; Lactobacillus mucosae; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 48. An 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 at least one 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 valericigenes; Faecalicoccus distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 49. An 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 a plurality of bacterial species consisting of 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; Lactobacillus mucosae; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 50. An 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 at least one 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 valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. in a state of suspended animation. 52. The anhydrous composition of any one of claims 47-51, wherein the anhydrous composition further comprises a pharmaceutically acceptable carrier. 53. The anhydrous composition of claim 52, wherein the pharmaceutically acceptable carrier is cellulose. 54. The anhydrous composition of any one of claims 47-53, wherein the anhydrous composition is encapsulated in a capsule. 55. The anhydrous composition of claim 54, wherein the anhydrous composition is encapsulated in a double capsule. 56. The anhydrous composition of any one of claims 47-55, wherein the anhydrous composition further comprises a prebiotic. 57. The anhydrous composition of claim 47 or claim 48, wherein 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), 2,4 dienoyl-CoA reductase deficiency, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG deficiency). 58. The anhydrous composition of claim 49 or claim 50, wherein 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. 59. Use of an anhydrous composition comprising a microbiota for treating a disease or disorder associated with metabolic syndrome, wherein the microbiota comprises a plurality of bacterial species 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 minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 60. Use of an anhydrous composition comprising a microbiota for treating a disease or disorder associated with metabolic syndrome, wherein the microbiota comprises at least one 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 valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 61. Use of an anhydrous composition comprising a microbiota for treating a disease or disorder associated with dysbiosis, wherein the microbiota comprises a plurality of bacterial species 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 minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 62. Use of an anhydrous composition comprising a microbiota for treating a disease or disorder associated with dysbiosis, wherein the microbiota comprises at least one of the bacterial species selected from: Faecalibacterium prausnitzii; Barnsiella intestinihominis; Dorea formicigenerans; Coprococcus catus; Anaerostipes hadrus; Bacteroides uniformis; Bifidobacterium adolescentis; Bacteroides xylanisolvens; Collinsella aerofaciens; Roseburia faecis; Blautia luti; Lactobacillus mucosae; Ruminococcus albus; Bifidobacterium longum; Christensenella minuta; Oscillibacter valericigenes; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, wherein the bacterial species are in powder-form, wherein the powder-form has a moisture content of less than 5% wt/wt in the anhydrous composition, and wherein the microbiota exhibits resistance to perturbational stress. 63. The use of claim 59 or claim 60, wherein 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), 2,4 dienoyl-CoA reductase deficiency, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG deficiency). 64. The use of claim 61 or claim 62, wherein 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. 65. The use of any one of claims 59-64, wherein the bacterial species are in a state of suspended animation. 66. The use of any one of claims 59-65, wherein the anhydrous composition further comprises a pharmaceutically acceptable carrier. 67. The use of claim 66, wherein the pharmaceutically acceptable carrier is cellulose. 68. The use of any one of claims 59-67, wherein the anhydrous composition is encapsulated in a capsule. 69. The use of claim 68, wherein the anhydrous composition is encapsulated in a double capsule. 70. The use of any one of claims 59-69, wherein the anhydrous composition further comprises a prebiotic. with metabolic syndrome, the method 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. 72. The method of claim 71, wherein 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), 2,4 dienoyl-CoA reductase deficiency, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG deficiency). 73. A method for treating a mammalian subject afflicted with a disease or disorder associated with dysbiosis, the method 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. 74. The method of claim 73, wherein 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. |
[103] For most applications, washing steps that follow hybridization will also vary in stringency. 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. In another embodiment, 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. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York): and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
[104] As used herein, 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 ”). In particular, 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. [105] As used herein, 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. When presented with an appropriate nucleic acid template, 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. [106] The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, 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. For example, a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, 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. [107] Primers and/or probes may be labeled fluorescently with 6-carboxyfluorescein (6-FAM). Alternatively, primers may be labeled with 4, 7, 2', 7'-Tetrachloro-6-carboxyfluorescein (TET). Other alternative DNA labeling methods are known in the art and are contemplated to be within the scope of the invention. [108] As used herein, 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, for example, 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). [109] By “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). The gut microbiota plays a critical role in metabolism since dietary modifications may be used to treat metabolic disorders. [110] As used herein, 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. [111] As used herein, 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. In an embodiment thereof, 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. Accordingly, 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.
[112] In one embodiment, 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. Further to the above, 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) may, moreover, be used in polymerase chain reaction (PCR) assays in methods directed to determining the amount of a 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 bacterial species and/or strain comprises any one of the sequences presented here.
[113] Further to the above, a given strain’s 16S rRNA sequence 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.
Preparation of Bacterial Samples (for l6SrRNA Sanger Sequencing)
[114] Master Mix contents (per reaction or sample):
HPLC grade ddII2O (Caledon Laboratory Chemicals) - 38.5 μL dNTPs, working stock (Invitrogen) • 3 μL 10X ThermoPol Reaction Buffer (NEB) -- 5 μL
V3kl/V6r primers (IDT) - 1 μL of each
Taq DNA Polymerase High Purity (BioBasic) - 0.5 μL DNA Template - 1 μL or 1 colony PCR, Sequencing of Products, and Analysis of PCR Products
[115] 1) Determine how much of each Master Mix component is required by multiplying each by the number of samples. Add three additional reactions to account for pipetting error.
[116] 2) Bring the required amounts of ddH2O, dNTPs (stored at -20°C), buffer (stored at -20°C), primers (stored at -20°C), 2 mL flip-cap tubes (sterile, from Axygen) and a V-bottom 96-well plate (sterile, from Fisher) into the Labconco Purifier Biological Safety Cabinet (Labconco, 08018496A). UV the hood and supplies for 15 minutes.
[117] 3) Turn the UV light off. Bring Taq (stored at -20°C) and DNA template samples (if they are in liquid form) into the safety cabinet.
[118] 4) Prepare Master Mix in a 2 mL flip-cap tube, aliquoting all of the required reagents into the same tube. Mix it by gently inverting the tube several times.
[119] 5) Aliquot Master Mix into the 96-well plate, 48 μL per reaction (or sample).
[120] 6) If your DNA is in liquid or broth form, skip Steps 9) and 10). If your DNA is taken directly from colonies on media plates skip Step 7).
[121] 7) Add 1 μL of your DNA template per aliquoted reaction (i.e. one PCR reaction for each of your DNA template samples). For example, 27 DNA template samples (or 27 strains to be tested) = 27 PCR reactions.
[122] 8) Remove the aliquoted Master Mix in the 96-well plate from the biological safety cabinet.
[123] 9) Transfer the aliquoted Master Mix in the 96-well plate into the Whitley anaerobic chamber workstation.
[124] 10) Use a sterile wooden applicator (Puritan) to touch a colony of interest and, using a twisting motion, deposit the colony into one well of the 96-well plate that contains an aliquot of Master Mix. Repeat for all bacterial strains of interest.
[125] 11) Run PCR reactions in the 96-well plate in the Eppendorf Mastercycler epgradient (Eppendorf, 5340 014805):
[126] 12) Using, for example, an Eppendorf Mastercycler (a.k.a. thermocycler) run the following:
-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.
[127] 13) Sequencing is performed via Sanger sequencing methods, which are a matter of routine practice in research-based laboratories.
[128] 14) Sequences (16S rRNA full-length rRNA sequences associated with each bacterial strain) generated are compared to databases of known sequences such as, for example, those maintained by U.S. government agencies, which can be accessed via the web (e.g., blast.ncbi.nlm.nih.gov/Blast.cgi) using known programs (e.g., BLAST). [129] 15) When using, for example, a BLAST program and an alignment application thereof, a value of 99% or higher indicates that the template sequence and the query sequence are identical. If the template sequence and the query sequence are identical this indicates that the query sequence (which was obtained from a bacterial strain of interest) is the same identity as that which is associated with the template sequence. [130] 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. In a one embodiment, 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. In another embodiment thereof, 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 . [131] In an embodiment, 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); Roseburia faecis (NB4-GAM-3); Blautia luti (NB4-GAM-33); Lactobacillus mucosae (NB4-MRS-35); Ruminococcus albus (NB4-NB-2); Christensenella minuta (NB4-BHI-105); Oscillibacter valericigenes (NB4-WC-130); Faecalicoccus acidiformans (NB4-CNA-122); Anaerofustis stercorihominis (NB4-EtOH- 104); Parabacteroides distasonis (NB4-FAA-116); 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. 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. For example, 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. [132] In some embodiments, 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. 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. The risk of bowel cancer, IBD, and IBS are also all reduced with butyrate. 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. Although lactate is not technically a short chain fatty acid, it is produced by gut bacteria and contributes to the health of the colon. Like acetate, lactate nourishes butyrate-producing bacteria, regulates the immune system, and combats opportunistic bacteria. One of the main producers of lactate is Lactobacillus. [133] 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. 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. [134] In another embodiment thereof, 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 aerofaciens (NB4-FMN-6); Roseburia faecis (NB4-GAM-3); Blautia luti (NB4-GAM-33); Lactobacillus mucosae (NB4-MRS-35); Ruminococcus albus (NB4 NB 2); Christensenella minuta (NB4 BHI 105); Oscillibacter valericigenes (NB4-WC-130); Faecalicoccus acidiformans (NB4-CNA-122); Anaerofustis stercorihominis (NB4-EtOH- 104); and Parabacteroides distasonis (NB4-FAA-116); Parasutterella excrementihominis (NB4-D5-137), but does not exceed further including each and every one of the species recited in the exemplary list of TABLE 1. [135] In yet a further embodiment thereof, 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-GAM-33); Lactobacillus mucosae (NB4-MRS-35); Ruminococcus albus (NB4-NB-2); Oscillibacter valericigenes (NB4-WC-130); Parabacteroides distasonis (NB4-FAA-116); and [Clostridium] scindens (NB4-NA-102), but does not exceed further including each and every one of the species recited in the exemplary list of TABLE 1. [136] 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. [137] In another embodiment, 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. [138] 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
Faecalibacterium prausnitzii (NB4-GAM-19)
[139] 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.
[140] 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. However, in embodiments described here, this production limitation has been overcomeF.. 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). Although F. prausnitzii is unable to utilize mucin or mucopolysaccharides (Lopez-Siles et al., 2012), mucin stimulates the growth of this species (Sadaghian Sadabad et al., 2016), andF. 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). In ulcerative colitis (UC), high relapse rates are associated with low counts of F. prausnitzii (Varela et al., 2013). 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). Additionally, the gut microbiota of patients suffering from type 2 diabetes (T2D) and obesity saw an enrichment of F. prausnitzii following weight loss intervention (Remely et al., 2016). No reports on risk factors of F. prausnitzii have been published. See, e g., ‘Table 2. Summary of probiotic strain Faecalibacterium prausnitzii impact on human diseases” in /Mmeida et al., 2019.
[141] 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. Material ftom the original isolate frozen stock was streaked onto nutrient broth (NB) plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on NB for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony ftom the NB plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot plated on NB and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on NB, then cryopreserved as described herein. This stock was deep sequenced using Illumina MiSeq 16S rRNA gene amplicon sequencing (Mr. DNA (Molecular Research LP), Shallowater, TX) to ensure purity (method described in Oliphant et al., 2019; see FIG.3). [142] The polynucleotide sequence of Faecalibacterium prausnitzii (NB4-GAM-19) 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: TATTTCTACGTTCGTCAAGGGATGTCAANAANTGGTAAGGTTCTTCGCGTTGCGTCGAAT TA AACCACATACTCCACTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCG GTC GTACTCCCCAGGTGGATTACTTATTGTGTTAACTGCGGCACTGAAGGGGTCAATCCTCCA AC ACCTAGTAATCATCGTTTACGGTGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCA CAC TTTCGAGCCTCAGCGTCAGTTGGTGCCCAGTAGGCCGCCTTCGCCACTGGTGTTCCTCCC GAT ATCTACGCATTCCACCGCTACACCGGGAATTCCGCCTACCTCTGCACTACTCAAGACATA CA GTTTTGAAAGCAGTTCATGGGTTGAGCCCATGGATTTCACTTCCAACTTGTCTGCCCGCC TGC GCTCCCTTTACACCCAGTAATTCCGGACAACGCTTGTGACCTACGTTTTACCGCGGCTGC TGG CACGTAGTTAGCCGTCACTTCCTTGTTGAGTACCGTCATTATCTTCCTCAACAACAGGAG TTT ACAATCCGAAGACCTTCTTCCTCCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTG CA ATATTCCCCACTGCTGCCTNCCGTA Barnsiella intestinihominis (NB4-CNA-21) [143] NB4-CNA-21 was isolated on Fastidious Anaerobe Agar (FAA) 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 Barnsiella intestinihominis (98.57% similarity) when aligned against the NCBI database. [144] B. intestinihominis isolates are generally Gram-negative, non-motile, pleomorphic rod-shaped fastidious anaerobic bacteria (Morotomi et al., 2008). This species represents a potential “oncomicrobiotic” that ameliorates the efficacy of the most common alkylating immunomodulatory compound (Dalliere et al., 2016). Additionally, fucosy llactose, a human milk oligosaccharide, is selectively utilized by B. intestinihominis and is associated with the promotion of protection against DSS colitis in a mouse model (Weiss et al., 2014). Decreases in the average relative abundance of B. 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).
[145] 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 drag product production. Material from the original isolate frozen stock was streaked onto FAzA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on FAA, then cryopreserved as described herein. This stock was deep sequenced using Illumina MiSeq 16S rRNzX gene amplicon sequencing (Mr. DNA (Molecular Research LP), Shallowater, TX) to ensure purity (method described in Oliphant et al., 2019: see FIG. 3).
[146] The polynucleotide sequence oYBarnsiella intestinihominis (NB4-CNA-2I) 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)
[147] 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.
[148] 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).
[149] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on FAA, then cryopreserved as described herein. This stock was deep sequenced using Illumina MiSeq 16S rRNzX gene amplicon sequencing (Mr. DNA (Molecular Research LP), Shallowater, TX) to ensure purity (method described in Oliphant et al., 2019: see FIG. 3). [150] 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:
Coprococcus catus (NB4-DCM-31)
[151] 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.
[152] 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).
[153] Once this strain was chosen forthe 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on FAA, then cryopreserved as described herein. This stock was deep sequenced using Illumina MiSeq 16S rRNA gene amplicon sequencing (Mr. DNA (Molecular Research LP), Shallowater, TX) to ensure purity (method described in Oliphant et al., 2019; see FIG. 3).
[154] 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: TCACCGNNNTGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCT CC ACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCATTCTTGCGAACGTACTCCCCA GGT GGAATACTTATTGCGTTTGCTGCGGCACCGAANCCCTTATGGGCCCCGACACCTAGTATT CA TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCC TCA GCGTCATTGTCAGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCA TTT CACCGCTACACTAGGAATTCCGCTTACCTCTCCTGAAATCAAGCAGGGCAGTTTCAAAAG CC GTCCCGGGGTTGAGCCCCGGG-CTTTCACTTCTGACTTGCTCCGCCGCCTACGCTCCCTT TAC ACCCAGTAAATCCGGATAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGT TA GCCGGGGCTTCTTAGTCAGGTACCGTCATTATCTTCCCTGCTGATAGAAGTTTACATACC GAG ATACTTCTTCCTTCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCC CAC TGCTGCCTNCCGTA Anaerostipes hadrus (NB4-DCM-7) [155] NB4-DCM-7 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 Anaerostipes hadrus (99.69% similarity) when aligned against the NCBI database. [156] 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). It represents one of the 10 most dominant species in the healthy human gut (Walker et al., 2011). This species is a butyrate producer and encodes a composite inositol catabolism-butyrate biosynthesis pathway, the presence of which is associated with lower host metabolic disease risk (Zeevi et al., 2019). It is also a D-lactate utilizer relevant to short bowel syndrome (Allen-Vercoe et al., 2012). The Anaerostipes genus, based on 2 single-nucleotide polymorphisms (SNPs) from different Genome-wide Association Study (GWAS), was associated with lower risk of Type 2 diabetes (Yang et al., 2018). 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on FAA, then ctyopreserved as described previously. This stock was deep sequenced using Illumina MiSeq 16S rRNA gene amplicon sequencing (Mr. DNA (Molecular Research LP), Shallowater, TX) to ensure purity (method described in Oliphant et al ., 2019; see FIG, 3).
[158] 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:
AAGGTCCGGITAAGGACCGNNCAGAAGGATGTCAAGACCAGGTAAGG1TCITCGCGI TGCT
TCGAATTAAACCACATGCTCCACCGC1TGTGCGGGTCCCCGTCAATTCC1TTGAG1T TCATTC
TTGCG.AACGTACTCCCCAGGTGGAATACTTACTGCGTTGGCTGCGGG'ACCGAAGC CTCTACG
GCCCCGACACCTAGTATTCATCGTTTACGCiCGTGGACTACCAGGGTATCTAATCCT GTTrGCT
CCCCACGCTTTCGTGCCTCAGTGTCAGTTTCAGTCCAGTAAGGCGCCTTCGCCACTG ATGTTC
CTCCTAATATCTACGCAmCACCGCTACACTAGGAATICCGClTACCTClGCTGCACT CCAG
TCTGACAGTTrCAAAAGCAGTCCCAGAGTrAAGCCCTGGGTITrCACTTCTGACTTG CCATAC
CACCTACGCACCCTTTACACCCAGTAATTCCGGATAACGCTTGCCCCCTACGTATTA CCGCG
GCTGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTCATTTTCTrCCCT GCTGAT
AGAGCTTTACATACCGAGATACTTCTTCACTCACGCGGCGTCGCTGCATCAGGGTTr cCCCC
ATTGTGCAATATTCCCCACTGCTGCCT
Bacteroides uniformis (NB4-FAA-15)
[159] 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. 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). In experiments with wild-type C57BL-6 mice, 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). B. uniformis has also been associated with leanness in a study examining co-occurrence network topology of the gut microbiota and the relationship of short chain fatty acids with obesity (Dugas et al, 2018). This finding was supported in a study by Borgo, et al. (2017) where B. uniformis was found to be negatively correlated with BMI. [161] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[162] The polynucleotide sequence of Bacteroides uniformis (NB4-FAA-15) 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. An exemplary nucleic acid sequence of Bacteroides uniformis strain JCM 5828 16S ribosomal RNA gene, partial sequence is: CTTTATTCCCGTATAAAAGAAGTTTACAACCCATAGGGCAGTCATCCTTCACGCTACTTG GCT GGTTCAGACTCTCGTCCATTGACCAATATTCCTCACTGCTGCCTTCCCGTA Bifidobacterium adolescentis (NB4-FAA-20) [163] NB4-FAA-20 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 Bifidobacterium adolescentis (100% similarity) when aligned against the NCBI database. [164] 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). [165] 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). In the elderly, bifidobacterial probiotics have been shown to improve constipation and enhance cellular immune activity (Martinez-Martinez et al., 2017; Miller 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). [166] 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 composition or drug product production. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution basing 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. DNzX, Shallowater, TX) to ensure purity (method described in Oliphant et al., 2019; see FIG. 3).
[167] The polynucleotide sequence of Bifidobacterium adolescentis (NB4-FAA-20) 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:
Akkermansia muciniphila (NB4-FAA-24)
[168] 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).
[169] A. 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). A. 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).
[170] This species stands as one of the strongest contenders for novel bacterial probiotics, particularly for treating metabolic syndrome, liver disorders and type 2 diabetes (Cani, 2017; Zhou et al., 2019; Xu et al . 2020). A higher abundance of A. muciniphila at baseline was linked with improvement in blood glucose homeostasis, lipid profile and body fat distribution after dietary intervention (Schneeberger et al., 2015, Dao et al., 2016). In a recent randomized, double-blind placebo-controlled study in overweight/obese insulin-resistant volunteers, daily oral supplementation of A. muciniphila improved insulin sensitivity and reduced both insulinemia and plasma total cholesterol (Depommier et al., 2019). It also has a potential protective role against the development of cardiovascular disease (Li et al., 2016, see also ‘Table 1. Summary of probiotic strain Akkermansia muciniphila impact on human diseases’’ in Almeida et al., 2019).
[171] 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. Material from the original isolate frozen stock was streaked onto FMU plates and incubated anaerobically at 37° C for 48 hours. Plate culture was then restreaked on FMU for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FMU plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FMU and incubated anaerobically al 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on FMU, 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).
[172] The polynucleotide sequence of Akkermansia muciniphila (NB4-FAA-24) 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: GCTCCGGCACGCAGGGGGTCGATTCCCCGCACACXAAGCGTGCACCGITTACTGCCAGGA CT
ACAGGGGTzVrCTzXATCXXnTrCGCTCCCXTGGCCTTCGTGCCTCAGCGTCAGTTA ATGTCCAG
GAACCCGCCrrCGCCACGAGTG'ITCCTC'ICGATATCTACGCATTICACTGCTACA CCGAGAAT
TCCGGTTCCCCCTCCATTACTCTAGTCTCGCAGTATCATGTGCCGTCCGCGCiGTTG AGCCCGC
GCCTTTCACACACGACTTACGAAACAGCCTACGCACGCTTTACGCCCAGTGATrCCG AACAA
CGC'ITGAGACXTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGTCTC'nXXn X'nXjTGG
TACTATCTTTTTAATITGCTCCCACATGACAGGGGTTTACAATCCGAAGACCTFCA1 TCCCCC
ACGCGGCGTCGCACCATCAGGGTTTCCCCCATTGTGAATGATTCTCGACTGCTGCC
Phascolarctobacterium faecium (NB4-FMN-1)
[173] 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.
[174] 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).
[175] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[176] The polynucleotide sequence of Phascolarctobacterium faecium (NB4-FMN-1 ) 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:
Agathobaculum butyriciproducens (NB4-TSAB-40)
[177] 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.
[178] 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).
[179] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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). [180] The polynucleotide sequence of Agathobaculum butyriciproducens (NB4-TSAB-40) 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: CCACCTGTCACCGATGTTCCGAAGAANAGCCTTATCTCTAAGGCGGTCATCGGGATGTCA AG ACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATACTCCACTGCTTGTGCGGG CCC CCGTCAATTCCTTTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGTGGGATACTTATTGT GTT AACTGCGGCACGGAAGGGGTCAATACCTCCCACACCTAGTATCCATCGTTTACGGCGTGG AC TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTAATGT CCA GCAGGCCGCCTTCGCCACTGGTGTTCCTCCGTATATCTACGCATTTCACCGCTACACACG GA ATTCCGCCTGCCTCTCCATCACTCAAGACCAGCAGTTTTGAAAGCAGTTTATGGGTTAAG CC CATAGATTTCACTTCCAACTTACCGGCCCGCCTGCGCGCCCTTTACACCCAGTAAATCCG GAT AACGCTTGCTCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGAGCTTATTCT TCA GGTACCGTCATTTTTTTCGTCCCTGATTAAAGATCTTTACAACCCGAAGGCCTTCTTCAA TCA CGCGGCGTTGCTGCGTCAGGGTTTCCCCCATTGCGCAATATTCCCCACTGCTGCCT Bacteroides xylanisolvens (NB4-FMN-21) [181] NB4-FMN-21 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 Bacteroides xylanisolvens (99.39% similarity) when aligned against the NCBI database. [182] B. xylanisolvens isolates are generally non-motile, rod-shaped, non-spore-forming Gram- negative fastidious anaerobic bacteria (Chassard et al., 2008). This species degrades xylan and may be responsible for the production of substrates that can in-turn be readily utilized by other commensal members of the gut microbiota (Mirande et al., 2010). The use of 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. [183] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[184] The polynucleotide sequence of Bacteroides xylanisolvens (NB4-FMN-21) 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:
Collinsella aerofaciens (NB4-FMN-6)
[185] 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). 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. Plate culture was then restreaked on FzAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[188] The polynucleotide sequence of Colhnsella aerqfaciens (NB4-FMN-6) 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:
Roseburia faecis (NB4-GAM-3)
[189] 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).
[190] 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).
[191] 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 drag product production. Material from the original isolate frozen stock was streaked onto FAzX plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[192] 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:
Blautia luti (NB4-GAM-33)
[193] 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).
[194] 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). People with Crohn’s Disease (and injury) were found to have decreased levels of Blautia species in colonic biopsies as compared to healthy controls (Libertuc-ci et al., 2018). A separate-sample Mendelian randomization study showed a nominal association of Blautia presence with reduced high-density lipoprotein cholesterol in several consortia including C ARDloGRAAMplusC4D (Y ang et al., 2018) . The depletion of Blautia spp. in the microbiota of obese children has also been linked to intestinal inflammation and the worsening of the metabolic phenotype (Benitez-Paez et al., 2020). 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).
[195] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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). [196] 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: CAAGACTTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTG CG GGTCCCCGTCAATTCCTTTGAGTTTCATTCTTGCGAACGTACTCCCCAGGTGGAATACTT ACT GCGTTTGCGACGGCACCGAAGAGCTTTGCTCCCCGACACCTAGTATTCATCGTTTACGGC GT GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTTA CCG TCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACA CTA GGAATTCCGCTTACCCCTCCGGCACTCAAGTATGACAGTTTCCAATGCAGTCCACAGGTT GA GCCCATGCCTTTCACATCAGACTTGCCACACCGTCTACGCTCCCTTTACACCCAGTAAAT CCG GATAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCT TA GTCAGGTACCGTCACTATCTTCCCTGCTGATAGAAGTTTACATACCGAGATACTTCTTCC TTC ACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCACTGCTGCCT Lactobacillus mucosae (NB4-MRS-35) [197] NB4-MRS-35 was isolated on deMan Rogosa & Sharpe 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 Lactobacillus mucosae (100% similarity) when aligned against the NCBI database (see Appendix B for ab1 sequencing file). [198] 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. coli-Kl -induced colitis in mice, suggesting that it may play a more significant role in gut microbiota homeostasis (Kim et al., 2020). [199] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[200] The polynucleotide sequence of Lactobacillus mucosae (NB4-MRS-35) 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:
Ruminococcus albus (NB4-NB-2)
[201] 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).
[202] 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).
[203] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on FAA, then cry opreserved 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).
[204] The polynucleotide sequence of Ruminococcus albus (NB4-NB-2) 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). 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 Bifidobacterium longum (99.43% similarity) when aligned against the NCBI database. [206] 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). [207] 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). In the elderly, bifidobacterial probiotics have been shown to improve constipation and enhance cellular immune activity (Martinez-Martinez et al., 2017; Miller et al., 2017). Some strains of B. longum have been shown to protect mice from experimental colitis, and to improve barrier function in these animals (Srutkova et al., 2015, Elian et al., 2015). Research has indicated that a synergistic interaction between Lactobacillus mucosae and B. longum may help to alleviate symptoms of anxiety and depression by reducing gut inflammation, as examined using a mouse model (Han et al., 2019). Additionally, B. longum is depleted in the gut microbiota of those patients suffering from metabolic syndrome (Haro et al., 2016) and if taken as a probiotic may help to improve symptoms of metabolic disease, obesity, fatty liver disease and type 2 diabetes (Koutnikova et al., 2019). [208] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[209] The polynucleotide sequence of Bifidobacterium longum (NB4-TSAB-38) 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:
Bacterial Isolate-Specific Summaries - Isolates Derived from Chemostat Culture
Christensenella minuta (NB4-BHI-105)
[210] 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.
[211] 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).
[212] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 120 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 120 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 120 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on FAA, then cry opreserved 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).
[213] 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:
Oscillibacter valericigenes (NB4-SVC-130)
[214] 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.
[215] 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).
[216] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[217] The polynucleotide sequence of Oscillibacter valericigenes (NB4-WC-130) 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:
Faecalicoccus acidiformans (NB4-CNA-122)
[218] 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).
[219] 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).
[220] 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. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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. DMA, Shallowater, TX) to ensure purity (method described in Oliphant et al., 2019; see FIG. 3).
[221] The polynucleotide sequence of Faecalicoccus acidiformans (NB4-CN/V122) 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:
Anaerotignum lactatifermentans (NB4-D5-120)
[222] 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.
[223] A. 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).
[224] 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 drag product production. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on Fz\z\, 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).
[225] 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:
Anaerofustis stercorihominis (NB4-EtOH-104)
[226] 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. One hundred microliters (100uL) of this sample was then plated on Fastidious Anaerobe Agar supplemented with 5% defibrinated sheep blood (v/v) and incubated anaerobically for 48 hours. The isolate was then restreaked onto FAA, ciyopre served 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 Anaerofustis stercorihominis (100% similarity) when aligned against the NCBI database (see Appendix B for abl sequencing file).
[227] 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 .
[228] Once this strain was chosen for the MET-5 drag product, dilution to extinction stocks were produced in an anaerobic chamber to function as the seed stocks for future drug product production. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and the dilution having a single colony was used to restreak once again on FAA, then ciyopreserved 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). [229] The polynucleotide sequence of Anaerofustis stercorihominis (NB4-EtOH-104) 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: TAATCTCTTAGGCGGTCAAGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAA TT AAACCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCACTCTTGC GAG CGTACTCCCCAGGCGGAATACTTAATGTGTTAACTGCGGCACTGAGTTACCCCAACACCT AG TATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTT CGC ACCTCAGCGTCAGTTATCGTCCAGTAAGCCGCCTTCGCCACCGGTGTTCTTCCTAATCTC TAC GCATTTCACCGCTACACTAGGAATTCCGCTTACCTCTCCGATACTCAAGTTATCCAGTTT CAA GTGCACTTTTCCGGTTGAGCCGAAAACTTTCACACCTGACTTAATTAACCGCCTACGTGC TCT TTACGCCCAGTGAATCCGGACAACGCTTGCTCCCTACGTATTACCGCGGCTGCTGGCACG TA GTTAGCCGGAGCTTCCTCCTTGGGTACCGTCATTATCTTCCCCAAAGACAGAGCTTTACG ATC CGAAAACCTTCATCGCTCACGCGGCGTTGCTGCGTCAGGGTTTCCCCCATTGCGCAATAT TCC CCACTGCTGCCT Parabacteroides distasonis (NB4-FAA-116) [230] NB4-FAA-116 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 Parabacteroides distasonis (98.86% similarity) when aligned against the NCBI database. [231] 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). 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). In a subsequent study by the same group, 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).
[232] Once this strain was chosen for the MET-5 drag product, dilution to extinction stocks were produced in an anaerobic chamber to function as the seed stocks for future drug product production. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. The dilution plates were observed, and dilutthioen having a single colony was used to restreak once again on FAA, then ciyopreserved 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).
[233] The polynucleotide sequence of Parabacteroides aistasonis (NB4-FAA-116) 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:
Parasutterella excrementihominis (NB4-D5-137)
[234] 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.
[235] 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).
[236] Once this strain was chosen for the MET-5 drag product, dilution to extinction stocks were produced in an anaerobic chamber to function as the seed stocks for future drug product production. Material from the original isolate frozen stock was streaked onto FAA plates and incubated anaerobically at 37°C for 48 hours. Plate culture was then restreaked on FAA for isolated colonies and incubated anaerobically at 37°C for 48 hours. One colony from the FAA plate was used for a ten-fold serial dilution in sterile reduced 0.9% saline, and each dilution was spot-plated on FAA and incubated anaerobically at 37°C for 48 hours to allow for sufficient growth. 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).
[237] 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: GCTCGGGGATTTCACATCTGTCTTACTCAACCGCCTGCGCACCCTTTACGCCCAGTAATT CCG ATTAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTAT TCT TAGAGTACCGTCAGCAACACCCTTTTTTAAAAGGTGTCTTTTCGTTCTCTACAAAAGTGG TTT ACAACCCGAGGGCCTTCATCCCACACGCGGAATAGCTGGATCAGGCTTGCGCCCATTGTC CA AAATTCCCCACTGCTG [Clostridium] scindens (NB4-NA-102) [238] NB4-NA-102 was isolated on Nutrient 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 [Clostridium] scindens (99.69% similarity) when aligned against the NCBI database. [239] 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). [240] The polynucleotide sequence of [Clostridium] scindens (NB4-NA-102) 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: GAGCGCGTTACGCGCTTTGNCATCGGGNTGTCAAGATCAGGTAAGGTTCTTCGCGTTGCT TC GAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCATT CTT GCGAACGTACTCCCCAGGTGGACTACTTATTGCGTTTGCTGCGGCACCGAATGGCCTTGC CA CCCGACACCTAGTAGTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGC TCC CCACGCTTTCGAGCCTCAACGTCAGTCATCGTCCAGCAGGCCGCCTTCGCCACTGGTGTT CCT CCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCCTGCCTCTCCGACACTCCA GCC ACGCAGTTCCAAATGCAGTCCCGGGGTTGAGCCCCGGGCTTTCACATCTGGCTTGCATCG CC GTCTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTTGCCCCCTACGTATTACCGC GGC TGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTCATCTTCTTCCCTGCTGA TAG AAGTTTACATACCGAAATACTTCATCCTTCACGCGGCGTCGCTGCATCAGGGTTTCCCCC ATT GTGCAATATTCCCCACTGCTGCCT Bacterial isolate from a donor other than NB4 community Acidaminococcus intestini (166 I 14 LG) [241] Acidaminococcus intestini was originally isolated from a healthy, 41-year-old female donor (Petrof et al., 2013). 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. [242] 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: GACTTCACCCCAATCATNGGCCCCANTTAGACAGCTGACTCCTAAAAGGTTATCTCACCG GC TTCGGGTGTTACCAACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTAT TC ACCGCAGTATGCTGACCTGCGATTACTAGCGATTCCAACTTCACGTAGGCGGGTTGCAGC CT ACGATCCGAACTGGGGTCGGGTTTCTGGGATTTGCTCCACCTCGCGGTTTCGCTGCCCTT TGT TGCCGACCATTGTAGTACGTGTGTAGCCCAAGACATAAGGGGCATGATGACTTGACGTCA TC CCCGCCTTCCTCCAAGTTATCCCTGGCAGTCTCCTATGAGTCCCCGCCTTTACGCGCTGG TAA CATAGGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGA CG ACAGCCATGCACCACCTGTTTTCGTGTCCCCGAAGGGAGGGACCTATCTCTAGGTCTTTC ACT CAATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAACCACATACTCCACCG CTT GTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAATCTTGCGATCGTAGTCCCCAGGCGGGA TA CTTATTGCGTTAACTCCGGCACAGAAGGGGTCGATACCTCCTACACCTAGTATCCATCGT TTA CGGCCAGGACTACCGGGGTATCTAATCCCGTTTGCTACCCTGGCTTTCGCATCTCAGCGT CA GACACAGTCCAGAAAGGCGCCTTCGCCACTGGTGTTCCTCCCAATATCTACGCATTTCAC CG CTACACTGGGAATTCCCCTTTCCTCTCCTGCACTCAAGACTTCCAGTATCCAACGCCATA CGG GGTTAAGCCCCGCATTTTCACGTCAGACTTAAAAGCCCGCCTACATGCTCTTTACGCCCA AT AATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGT GG CTTCCTCGTTAGGTACCGTCAACACCATGACCTGTTCGAACACGGTGCTTTCGTCCCTAA CAA CAGAGTTTTACAATCCGAAGACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCG TCC ATTGCGGAAGATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAA TG TGGCCGTTCATCCTCTCAGACCGGCTACTGATCATCGCCTTGGTGAGCCGTTACCCCACC AAC TAGCTAATCAGACGCGGGCCCATCTTCCAGCGATAGCTTGCAAGCAGAGGCCATCTTTCC TC CCTCCTCCATGCGGAGGAGGGAGCACATTCGGTATTAGCATCCCTTTCGGAATGTTGTCC CC AACTGGAGGGCAGGTTGCCCACGCGTTACTCACCCGTTCGCCACTAAGAACTTACCGAAA TA AGTTCTCCGTTCGACTTGCATGTGTTAAGCACGCCGCCAGCGTTCGTCCTGAGCC [243] In a further embodiment, 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 bacterial populations listed herein (e.g., TABLE 2), but does not exceed further including each and every one of the MET-5 species/strains recited in, for example, TABLE 2. In another embodiment, 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. In another embodiment, 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. [244] In a particular embodiment, the number of bacterial cells may be determined using a LIVE/DEAD™ BacLight™ Bacterial Viability Kit in accordance with the manufacturer’s protocol. Live versus dead cells are distinguished using the LIVE/DEAD™ BacLight™ 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. [245] In a more particular embodiment, the number of cells is determined via flow cytometry used in conjunction with a LIVE/DEAD™ BacLight™ Bacterial Viability Kit, which combination facilitates measuring the different colors of the differentially stained cells via fluorescence detection in a plate reader. Such an approach reveals information as to relative values of live and dead cells in a sample and generally improves accuracy of cell counting. [246] 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. Using the chemostat model assay, 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. Results determined from multiple vessels run in the chemostat model assay can, in turn, be compared to determine if different bacterial seed populations respond differentially to different growth conditions and perturbational stress. Fecal Derived Bacterial Populations and Compositions Thereof [247] In one embodiment, a fecal-derived bacterial population may be isolated or derived from a healthy subject who is not suffering from dysbiosis or dysbiosis-related diseases. [248] In another embodiment, 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. obtaining a freshly voided stool sample from the healthy subject, and placing the sample in an anaerobic chamber (in an atmosphere of, for example, 90% N 2 , 5% CO 2 and 5% H 2 ); b. generating a fecal slurry by macerating the stool sample in a buffer (e.g., chemostat medium, 0.9% saline (NaCl)); and c. removing food particles and other contaminants by centrifugation, and retaining the supernatant, which comprises the bacteria isolated from fecal matter and food particles. Accordingly, the supernatant comprises a purified population of intestinal bacteria that is free of fecal matter and food particles. Given that, the purified population of intestinal bacteria is a manmade product that is fecal matter-free and food particle-free. [249] In a further embodiment, a fecal sample from a healthy donor (either fresh or frozen) 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. In another embodiment, antibiotics are added that exclude certain types of bacterial cells. In another embodiment, 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. [250] Once the strains have been curated/characterized, 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. For bacterial species/strains that grow poorly relative to other species listed in, for example, TABLEs 13, 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. [251] In some embodiments, the bacterial species/strains identified herein (e.g., TABLEs 1-3) 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). [252] In some embodiments, 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. In some embodiments, 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. In some embodiments, the culture medium is a culture medium disclosed in U.S. Patent Application Publication No. 20140342438, which is incorporated herein by reference in its entirety. Other embodiments may provide for a culture medium of TABLE 3. Compositions Containing Bacterial Species/Strains [254] 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). In some embodiments, the 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.; Agathobaculum spp.; Bacteroides spp.; Collinsella spp.; Parasutterella spp.; Lactobacillus spp.; and Bifidobacterium spp. [255] In other embodiments, 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; Phascolarctobacterium faecium; Agathobaculum butyriciproducens; Bacteroides xylanisolvens; Collinsella aerofaciens; Parasutterella excrementihominis; Lactobacillus mucosae; and Bifidobacterium longum. [256] A further embodiment of the disclosure is directed to 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. [257] In one embodiment, 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. [258] Yet another embodiment provides a 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. [259] 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; NB4-MRS-35; and NB4-TSAB-38. [260] 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. [261] In a further embodiment, 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. [262] Another embodiment provides a 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. [263] In some embodiments, the 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. [264] In some embodiments, 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. Another embodiment of may be directed to 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. [265] Other embodiments may be directed to a composition 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. In other embodiments, 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. Another embodiment provides for a composition 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. [266] One embodiment provides for 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. In further embodiments, 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. Other embodiments may provide for 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. [267] In other embodiments, 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). Some embodiments provide for 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. [268] 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 species/strains selected from at least one, a plurality of, or a combination of bacterial species/strains of TABLEs 1-3 selected from: Barnsiella intestinihominis; Anaerostipes hadrus; Bacteroides uniformis; Agathobaculum butyriciproducens; Bifidobacterium longum; Christensenella minuta; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; and Parasutterella excrementihominis. [269] In one embodiment, an anhydrous composition comprising a co-selected microbiota is presented, wherein 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 bacterial species/strains listed in TABLEs 1-3 selected from: Barnsiella intestinihominis; Anaerostipes hadrus; Bacteroides uniformis; Agathobaculum butyriciproducens; Bifidobacterium longum; Christensenella minuta; Faecalicoccus acidiformans; Anaerotignum lactatifermentans; Anaerofustis stercorihominis; Parasutterella excrementihominis and any combination thereof; and optionally a bacterial species/strain that has formulation benefits, such as but not limited to, beneficial characteristics for cryopreservation and lyophilization, e.g., Acidaminococcus intestini. 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. [270] In yet a further embodiment, an anhydrous composition comprising a microbiota is presented, wherein 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; NB4-TSAB-38; NB4-BHI-105; NB4-CNA-122; NB4-D5-120; NB4- EtOH-104; and NB4-D5-137; and optionally a bacterial species/strain that has formulation benefits, such as but not limited to, beneficial characteristics for cryopreservation and lyophilization, e.g., 14 LG. 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. [271] Another embodiment may be directed to a composition 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. In other embodiments, the 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. Further embodiments provide for compositions 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, Verrucomicrobia, Proteobacteria, and combinations thereof. [272] Some embodiments may provide 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). In other embodiments, the 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). [273] In one embodiment, 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). In other embodiments, the 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). [274] Yet a further embodiment provides 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). In another embodiment, 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). [275] 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. [276] The chemostat model allows for cultivation of microbes under controlled conditions. Depending on environmental changes, microbes may quickly modify their metabolic pathways. For example, 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). [277] In one embodiment, 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). [278] Another 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 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%, 82%-85%) of the total metabolites in the microbiota. [279] 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. [280] In yet another embodiment, 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%, 0.8%, 0.6%, 0.4%, 0.2%, 0.1%); and/or in a range of 0.01%-10% (e.g., 0.03%-8%, 0.05%-6%, 0.07%-4%, 0.09%-2%, 0.1%-1%, 0.3%-0.8%, 0.5%-0.6%) of the total metabolites in the microbiota. [281] A further embodiment provides for 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. Without being bound by theory, the 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. [282] In one embodiment, 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. [283] Another embodiment provides 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, 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), 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 (HMG deficiency). [284] 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. [285] 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. Similar to MET-5C, 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. There are also some bacterial species/strains which are unique to MET-5 that are not isolatable from donors other than NB4. For example, 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. [286] Without being bound by theory, the compositions of bacterial species/strains listed in, for example, TABLEs 1-3, may be selected in accordance to the need for treatment. For example, 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. Yet some bacterial species/strains that produce butyrate, either directly or indirectly, 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. With respect to 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. [287] A further embodiment provides 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, 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. [288] 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. In one embodiment, the compositions are formulated into a capsule (e.g., an enteric-coated microcapsule). In another embodiment, the compositions are formulated into a tablet. In yet another embodiment, the compositions are formulated into granulated or water-soluble powders. Further compositions may be formulated into liquids, creams, lotions, gels dispersions or ointments for topical administration. [289] In another embodiment, 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. With respect to oral consumption, 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. [290] 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. [291] When formulated, 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. Non-limiting examples of additional ingredients that may be incorporated in compositions described herein include: prebiotics, vitamins, minerals, nutritional supplements (e.g., fiber), sweeteners, flow aids, and fillers. When formulated for oral administration, 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. [292] 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-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 (HMG deficiency). [293] Other embodiments provide for 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. [294] 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. [295] 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. Formulations [297] A composition (e.g., anhydrous composition or drug product) 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, dysbiosis, or disbyosis- associated disease(s). 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). 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 X 10 8 to 1 X 10 13 ) colony forming units (CFU) per capsule. In other embodiments, 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. Another embodiment provides for 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. The 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. [298] As indicated above, in one example, 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. The identities of the bacterial isolates have been confirmed microbiologically as well as using 16S ribosomal RNA (rRNA) sequencing. [299] All isolates included in MET-5 (or subgroups thereof) are sensitive to imipenem, ceftriaxone, and piperacillin and have not demonstrated any resistance to some antibiotics. Susceptibility to antimicrobials was determined by antibiotic sensitivity testing or directly measuring susceptibility with e- strips and/or Kirby Bauer disks. [300] Another embodiment of the disclosure provides for bacterial species/strains listed in any of the tables described here (e.g., TABLEs 1-5) 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. In a further embodiment, 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. [301] An exemplary list of cultured isolates that have been selected for the composition or drug substance described here are provided in TABLE 4 with concentration ranges for each bacterial species/strain per capsule. TABLE 4: MET-5 Composition and Concentrations lb [302] Further to the above, 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. This accepted understanding applies to all percent identities described herein. [303] 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. For example, 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. [304] 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). In some embodiments, 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. Batch formula (MET-5, Capsule) [305] 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) containing 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
Protocol for treating humans afflicted with diseases associated with metabolic syndrome [306] As described herein, MET-5 and exemplary subgroups thereof (e.g., MET-5A, MET-5B, and MET-5C) 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 (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 (HMG deficiency), 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; and remission (whether partial or total) of cancer related to the gastrointestinal system, whether detectable or undetectable, as compared to the response obtained without administration of the bacterial population described here or composition comprising, consisting essentially of, or consisting of any one or more of the plurality of bacterial species/strains described here (e.g., MET-5, MET-5A, MET-5B, MET-5C). The bacterial isolates found in MET-5 and subgroups thereof are pure live bacterial cultures of intestinal bacteria that were isolated from a stool sample of a healthy donor. A microbial ecosystem therapeutic product may comprise of, consist essentially of, or consist of, for example, 26 lyophilized pure bacterial cultures mixed in predefined ratios. The product is delivered to the patients orally, in capsule form. [307] 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. Furthermore, 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. [308] In some embodiments of the compositions of the disclosure, the at least one, plurality of, or combination of bacterial species/strains listed in, for example, TABLEs 1-5, 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 stercorihominis; Parabacteroides distasonis; Parasutterella excrementihominis; [Clostridium] scindens; Acidaminococcus intestini and any combination thereof, may be selected to have beneficial effects for the gut microbiome, i.e., increase the bacterial species/strains that improve symptoms or diseases, including but not limited to, metabolic syndrome, disases associated with metabolic syndrome, dysbiosis, and/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. In another embodiment, 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. [309] Ulcerative colitis (UC) 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. For mild-to-moderate disease anti-inflammatory agents, e.g.5-aminosalicyclic acid (5-ASA), 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. [311] Although the pathogenesis of UC is complex, multifactorial, and not fully understood, aberrant host immune responses, and a dysfunctional intestinal barrier have been associated with this condition. [312] The human body is host to more than 10 trillion microbial cells with the majority of these residing in the gut. The collection of microorganisms, their gene products and corresponding metabolic functions in the human gastrointestinal (GI) tract is termed the gut microbiome. Recent advances in molecular microbiology have revealed the critical role of the gut microbiome in a variety of important processes including: vitamin/nutrient production, regulation of metabolism and host energy demands, intestinal epithelial cell homeostasis, protection against pathogens, and development and maintenance of normal immune function. [313] 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). [314] Bacterial strains were purified and grown in a bioreactor modeling the conditions of the human distal gut. Susceptibility to antimicrobials was determined. Isolates representing commensal species, sensitive to a range of antimicrobials, were selected for the final stool substitute formulation. Full length 16S rRNA sequences were classified using basic local alignment search tool (BLAST) with the most specific name used to report the DNA maximum likelihood score. MET-5 constituent strains were individually grown in pure culture, snap-frozen, and subjected to lyophilization. After each strain meets CFU/g specifications, lyophilized bacterial product from all strains were combined in pre-determined ratios to make the active pharmaceutical ingredient (API). [315] The composition and route of delivery of MET differ based on the indication. For example, 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, or any of its subgroups, 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. A recent non-inferiority trial showed that oral capsules are equally effective compared to colonoscopy-delivered fecal microbial therapy (FMT) for recurring C. difficile infection (rCDI). Notably, there were fewer minor adverse events in patients receiving FMT capsules compared to patients receiving FMT via colonoscopy in the above study. Additionally, several FMT studies have shown that frozen fecal material is as effective as fresh fecal material in treating rCDI. More recently, FMT has been given in lyophilized form by capsule delivery, with an 88% success rate. Accordingly, the MET-5 clinical protocol implemented changes in the route of administration with regards to these recent advances in the literature. Encapsulated lyophilized FMT material has not been studied for use in metabolic syndrome patients, although lyophilized bacterial product preparations are commonplace in the probiotic industry. [316] As described herein, 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. MET-5 capsules containing 0.5 g of MET-5 (or subgroups) (equivalent to 1 X 10 4 to 1 X 10 13 CFU) and are administered to patients via an oral route of administration. [317] 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. [318] 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. [319] 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. Methods [320] 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. [321] 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 (HMG deficiency). Symptoms of metabolic syndrome that may also be treated using any of the MET-5 (or MET-5 subgroups) compositions or drug products described here include, but are not limited to hyperglycemia, hypertension, hypercholesterolemia, hyper triglycerides, and the like. [322] The methods of treating a subject suffering from dysbiosis or diseases associated with dysbiosis, such as but not limited to, 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, 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. [323] While a number of embodiments of the present disclosure have been described, it is understood that these embodiments are illustrative only, and not restrictive, and that many modifications may become apparent to those of ordinary skill in the art. Further still, the various steps may be carried out in any desired order (and any desired steps may be added and/or any desired steps may be eliminated). [324] Reference is made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion. EXAMPLES EXAMPLE 1: COMMUNITY PROFILE ANALYSIS – DONOR FECAL SAMPLES Donor Information [325] MET-5 is composed of 26 strains of bacteria that were isolated from the stool of a healthy donor. 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. difficile), Salmonella, Shigella, Escherichia coli 0157:H7, Yersinia enterocolitica, Campylobacter, and other antibiotic-resistant bacteria. In addition, a risk assessment for high-risk behaviors (e.g., for blood-borne pathogens), a thorough detailed medical history, and a physical examination were all conducted to confirm the overall health of the stool donor. There are no specific considerations for MET-5 recipients. Empiric and targeted antibiotic therapy should be guided by routine standards of care in close consultation with appropriate experts including infectious disease or medical microbiology specialists. The isolated strains were then purified by repeated subculture, initially sequenced for identification and screened for bacterial resistance to ensure no transfer of resistant strains. Within the manufacturing process, there are multiple passaging steps, where purity is subsequently examined on plate culture. Finally, a MET-5 (e.g., 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) may be used for the treatment of inflammatory and/or metabolic diseases; for example, hypertriglyceridemia and insulin resistance. [326] A list of screening tests performed on the stool donor are provided in TABLE 6. All tests performed were within the normal range of a healthy individual, i.e., without metabolic syndrome, disease associated with metabolic syndrome, dysbiosis, dysbiosis-associated disease, or any other gastrointestinal disease or condition. TABLE 6: Donor sample screening tests BLOOD TESTS:
Stool Sample Storage
1327) Upon receipt, samples were immediately placed at -80°C until use.
[328] Seven (7) frozen fecal samples were collected from a healthy donor between a week’s time. 16S rRNA gene community profiling was performed on all 7 samples using the Illumina MiSeq platform. Fecal sample gDNA was extracted using the QIAamp Fast DNA stool kit (QIAGEN). Briefly, 0.22 g of fecal sample were added to 0.2-0.3 g of silicon beads and mixed with 1 mL of Inhibite,x buffer (QIAGEN). After vortexing, samples were bead-beat for 5 minutes at 3000 rpm and then heated at 95°C for 10 minutes. Samples were then sonicated for 5 minutes followed by a centrifugation step to pellet solids. The 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). [329] 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 (i.e., from 100% down to 0%) occurs in the order of: Tyzzerella, Sutterella, Subdoligranulum, Streptococcus, Senegalimassilia, Ruminococcus,
Ruminococcaceae, Roseburia, Romboutsia, Rhodospirillaceae-unclassified, Phascolarctobacterium, Parabacterioids, Oscillibacter, Odoribacter, Mollicutes RFO unclassified, Methanobrevibacter, Lachnospiraceae, Lachnoclostridium, Fusicatenibacter, Faecalibacterium, Escherichia/Shigella, Erysipelotrichaceae- UCG-003, Enterobacteriaceae-unclassified, Dorea, Dialister, Desulfovibrio, Coriobacteriaceae-unclassified, Coprococcus, Coprobacillus, Collinsella, Christensenella, Butyricimonas, Blautia, Bilophila, Bifidobacterium, Bamesiella, Bacteroides, Bacteroidales._S24-7 group-unclassified, Anaerostipes, Allobaculum, Alistipes, Allobaculum, Alistipes, Akkermansia, and Adlercreutzia. Based on the alpha diversity and community profile 16S rRNA gene results, sample NB4-04 was selected for subsequent bacterial isolation.
EXAMPLE 2: PROTON NUCLEAR MAGNETIC RESONANCE (1H NMR) PROFILING OF MET-5
[330] Two batches of MET-5 drug product (e.g., batch NB5-005 and NB5-003) were used to create inocula for two chemostats, respectively. 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.
TABLE 7: NB5 Donor Profile Comparison [331] Metabolites were then divided into three different groups and the concentrations added: (1) carboxylic acids and derivatives (e.g., acetate, propionate, pyruvate, succinate) (in bold text); (2) fatty acids and conjugates (e.g., butyrate); and (3) amino acids and derivatives (e.g., methionine, phenylalanine, glutamate) (in italicized text). These groups were then represented as a percentage of the total metabolite concentration and then averaged between the two batches in TABLE 8. TABLE 8: NB5 Donor Metabolite Concentration (mM) Comparison at Day 6 MET-5 % MET-5 % MET-5 (NB5-005) (NB5-003) Average [332] The average percent total for MET-5 was then compared to a MET-2 H NMR profile generated as an average of several batches of MET-2 (see, e.g., WO 2019/136269, incorporated herein by reference in its entirety) drug product (also run to day 6 in a chemostat model). The two compositions or drug product bacterial communities were compared and contrasted in TABLE 9. TABLE 9: Comparison of MET-5 and MET-2 drug product communities M ET-5 Average %Total MET-2 Average %Total [333] By day 6 in a chemostat model the average MET 5 drug product community has more fatty acids and conjugates representing the total proportion of metabolites when compared to the average MET- 2 profile. However, the average MET-2 profile has more carboxylic acids and derivatives, as well as amino acids and derivatives representing the total proportion of metabolites compared to the average MET-5 profile. Nanopore 16s rRNA Gene Community Profiling of MET 5 [334] The composition of the MET-5 drug product community is shown in TABLE 10. TABLE 10: MET-5 Bacterial Species/Strain Characterization Original ID by % Alignment Strain BLAST – Closest (to Closest Phylum Family
*14 LG was isolated from a different donor than the remainder of the strains in the MET-5 drug product and is the subject of WO 2018/197951 Al.
[335] Two capsules from a batch of MET-5 drug product (e.g., batch NB5-003) were used to create an inoculum for the chemostat by resuspending the capsule material in 20 mL of reduced 0.9% saline at
37°C under anaerobic conditions. 'Die chemostat was then run for 6 days using methods described previously (McDonald et al., 2013). Chemostat effluent from day 6 was then analyzed by Nanopore 16S rRNA gene profile sequencing and summarized in TABLE 11.
TABLE 11: Chemostat effluent profile of NB5-0003 donor on Day 6 [336] After 6 days of growth in the chemostat a subset of the ecosystem is detected by Nanopore 16S rRNA profile sequencing (above the 0.1% cut-off threshold for the technique). A longer chemostat run with more replicates may provide more informative, especially in terms of demonstrating the robustness and stability of the ecosystem. Clinical study results may also indicate which species in a composition or drug product community confer a benefit in subjects or patients versus in an in vitro model similar to the chemostat. EXAMPLE 3: STOOL SAMPLE PROCESSING AND BACTERIAL ISOLATION [337] Once 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. Direct Bacterial Isolations [339] A ten-fold serial dilution was then performed in the anaerobic chamber using reduced Wilkins- Chalgren Broth and dilutions were then plated on 16 different media types (Table 1 and certificates of analysis for media components are available upon request) and left to grow for at least 48 hours at 37°C aerobically and anaerobically. Isolated colonies were then picked from each media type, and restreaked on Fastidious Anaerobe Agar supplemented with 5% defibrinated sheep blood (FAA) for identification by 16S rRNA gene Sanger sequencing. 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. T ) * NB4 LG denotes filter-sterilized chemostat effluent obtained from the chemostat run described above. D5 denotes filter-sterilized chemostat effluent obtained from a chemostat run with a fecal sample from a healthy donor (Donor 5, consent provided, extensive medical history taken, and health status managed by family doctor). [341] Approximately 126 different species were obtained from either direct isolation or isolation from chemostat-derived culture. No obvious pathogens were cultured (e.g. Salmonella spp., Campylobacter, etc.), and isolated species with any negative clinical connotations were immediately removed during the strain selection process. 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. [342] 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. [343] During the process of purifying the bacterial isolates, the incubation time is generally 48 hours. 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, respectively 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
1345] Grown culture was scraped from plates following 24-168 hours of incubation (depending on the strain) under anaerobic conditions at 37°C. Culture was re-suspended in 1 mL of sterile 0.9% NaCl to a McFarland standard of 0.5, using a Wickerham card and pre-made 0.5 McFarland standard for reference.The bacterial suspension (100 pL) was then plated on either FAA, NB or FMU (depending on the strain). Kirby-Bauer discs were then placed on the surface of the plated culture and allowed to incubate under anaerobic conditions at 37°C for between 24 and 120 hours (depending on the strain). The results of the zone of clearance for each strain are summarized in TABLE 14.
T C P / 1 0 5 1 1 0 - 3 4 8 9 5 1 . o N 2 t 1 e 1 k c o D n o i t a c i l p p At n e t a P
3 1 1 Comparisons with MET3 [346] A 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). TABLE 15: A comparison of those strains in MET-3 and MET-5 [347] Beneficial microbes were then added to create the MET-5 drug product based on their known positive attributes in the literature and/or clinical significance, with the rationale for the inclusion of each strain described in detail above. These strains are outlined in TABLE 16 below. to the MET-3 product MET-5 (derived from NB4 fecal donor) [348] In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A Laboratory Handbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocols in Immunology” Volumes I-III [Coligan, J. E., ed. (1994)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984). [349] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. [350] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description take a variety of alternative forms without departing from the invention.
Next Patent: BIOADHESIVE MATERIALS AND MINIMALLY INVASIVE METHODS FOR ADHERING TISSUES WITH BIOADHESIVE MATERIALS