Compositions and methods for reducing endogenous sulfide in inflammatory bowel disease

Abstract

The present invention relates to a composition for treating inflammatory bowel disease. The present invention also relates to a dosage form and a method for treating inflammatory bowel disease, wherein the composition is administered to a patient in need thereof to treat inflammatory bowel disease. In a first aspect, the present invention broadly relates to a composition for preventing or treating a gastrointestinal disorder in a subject in need thereof, the composition comprising at least one strain of a microorganism, wherein the microorganism is selected from the group consisting of bacteria, archaea, and yeast.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a composition for treating inflammatory bowel disease. The present invention also relates to a dosage form and a method for treating inflammatory bowel disease by administering the composition to a patient in need thereof. [Background technology]

[0002] background The following background discussion is intended merely to facilitate understanding of the present invention and is not an acknowledgment or admission that any of the material mentioned is or was part of the common general knowledge as of the priority date of this application.

[0003] The human gut microbiota consists of trillions of microorganisms, including at least 100 dominant microbial species and at least 1,000 less common microbial species, with 100 times more genes than those present in the human genome. The gut microbiota is primarily composed of bacteria, but also includes archaea, protozoa, fungi, and viruses. The microbiota performs vital functions essential for maintaining health, including food processing, digestion of complex indigestible polysaccharides, and vitamin synthesis, and secretes bioactive metabolites with diverse functions ranging from inhibiting pathogens and metabolizing toxic compounds to modulating host metabolism.

[0004] Inflammatory bowel disease (IBD) is an increasingly prevalent and currently incurable condition thought to be caused by an abnormal immune response to the resident gut microbiome in genetically predisposed patients. The term IBD encompasses ulcerative colitis (UC), Crohn's disease (CD), and pouchitis. UC is characterized by chronic non-granulomatous inflammation limited to the colonic mucosa, typically involving the rectum and various proximal areas of the colon. CD is characterized by transmural, often granulomatous, inflammation that can involve any part of the gastrointestinal tract from the mouth to the anus.

[0005] The clinical presentation of UC is characterized by diarrhea accompanied by urgency, fecal urgency, and bloody bowel movements accompanied by lower abdominal pain. CD most frequently involves the terminal ileum and perianal region, and structural and fistulizing disease are common. Clinical presentation of CD varies widely depending on disease distribution but typically involves abdominal pain, diarrhea, and weight loss. Extraintestinal manifestations (EIMs) of IBD affect up to 50% of patients, typically parallel disease activity, and are more common in CD than in UC. The spectrum of EIMs in IBD is broad and includes arthropathy, venous thromboembolic disease, metabolic bone disease, uveitis and scleritis, and skin diseases (pyoderma gangrenosum, erythema nodosum). Primary sclerosing cholangitis (PSC), the hepatobiliary manifestation of IBD, affects up to 5% of patients and carries a high risk of progression to cholangiocarcinoma and liver transplantation. IBD patients are also at increased risk of intestinal dysplasia and malignancies, especially those with extensive colitis for which colonoscopic surveillance is recommended.

[0006] The etiology of IBD is complex, multifactorial, and incompletely understood. Factors that are thought to play a role in the pathogenesis of IBD are host genetics, immune dysregulation, abnormal microbiome composition and function, and environmental factors.

[0007] Clinical trials have demonstrated the efficacy and safety of microbiome-based therapies for several diseases, including but not limited to Clostridioides difficile infection, UC, and irritable bowel syndrome (IBS). Thus, there is a need to identify additional bacteria for use in bacteriotherapy.

[0008] UC is a relapsing-remitting inflammatory bowel disease characterized by superficial colonic mucosal inflammation extending proximally from the rectum in a continuous manner. Current treatments are hampered by incomplete efficacy and have the potential for unacceptable side effects, including allergies, intolerance, serious infections, and malignancies, due to long-term immunosuppression. Current immune-targeted therapies for UC are inadequate because they only address secondary immune responses rather than the primary drivers of the disease.

[0009] Evidence suggests that UC results from an energy deficiency state in colonocytes (colonic epithelial cells) induced by high levels of sulfide and nitric oxide (NO), which deplete coenzyme A (CoA) and inhibit the β-oxidation of butyrate. Without energy in the form of adenosine triphosphate (ATP), colonocytes are unable to maintain the normal mucosal barrier that separates the luminal contents of the colon from the mucosal immune system. Loss of mucus and tight junction barriers then allows activation of the mucosal immune system in an IL-13- and IL-5-dependent, T2-mediated immune response driven by natural killer T cells.

[0010] The process is as follows: (1) high levels of NO damage human tissue, (2) the source of NO in the colon comes from anaerobic bacterial respiration or activated immune cells, (3) nitrosation of CoA subsequently produces S-nitroso-CoA (inactive), which (a) prevents acetylation required for lipogenesis and (b) reduces β-oxidation of butyrate in colonocytes, (4) energy deprivation of colonocytes subsequently leads to an inability to synthesize mucus, lipids, and proteins, (5) loss of the barrier subsequently leads to bacterial activation of the immune system, (6) sulfide inhibits NO reductase, resulting in high intracellular NO levels, and (7) the combination of NO and sulfide reproduces the colonocyte lesions seen in ulcerative colitis. This relationship between NO and sulfide was further confirmed in experimental colonocyte cell cultures, where NO alone does not replicate the lesions. See FIG. 1 for a diagrammatic representation of the inhibition of cellular respiration in colonocytes by intraluminal sulfide and NO in UC.

[0011] The role of sulfide and nitric oxide in the pathogenesis of ulcerative colitis.

[0012] Butyrate is the primary energy source for colonocytes.

[0013] Uniquely among human cells, colonocytes rely exclusively on bacterial metabolites for their energy, primarily in the form of butyrate, a short-chain fatty acid produced by fermentation of dietary fiber in the right colon by members of the colonic microbiota.

[0014] Butyrate oxidation is impaired in ulcerative colitis

[0015] Impaired butyrate oxidation in colonocytes from UC patients is observed by measuring overall oxygen consumption and the contribution to oxygen consumption by butyrate and glucose in quiescent, active, and severe colitis.

[0016] Sulfide and nitric oxide levels are increased in ulcerative colitis

[0017] In UC, measured levels of anionic sulfide in the colon are elevated compared with healthy controls. Additionally, sulfide production capacity in fecal samples has been found to be 3-4 times higher in UC patients than in controls. This is likely due to the relatively high levels and active metabolism of sulfate-reducing bacteria, putrefactive bacteria, protein-fermenting bacteria, and amino acid-utilizing bacteria in patients with UC in both active and dormant disease states. Furthermore, NO is produced by both colonocytes and the colonic microbiota in inflammatory states.

[0018] Sulfidogenic microbiota are more abundant and active in ulcerative colitis

[0019] It has been demonstrated that sulfide-producing microbiota are more abundant and active in UC than in healthy individuals. Specifically, sulfate-reducing bacteria (SRB) of the genus Desulfovibrio exhibit higher diversity, abundance, and metabolic activity in the feces of patients with UC than in healthy individuals. SRB, such as Desulfovibrio, are responsible for the reduction of sulfur oxides (SO ). x ) to produce sulfide. SRBs are more abundant in active than dormant UC and significantly correlate with disease severity, indicating a potential causal relationship. The increased abundance of sulfidogenic microbiota, paralleled by increased intraluminal sulfide and disease severity, suggests a pathogenesis involving microbial sulfide production. In this context, sulfidogenic microbiota levels have also been observed to be increased in patients with Crohn's disease, suggesting the involvement of these metabolic pathways in this indication.

[0020] Butyrate oxidation is impaired by the combination of nitric oxide and sulfide

[0021] Neither sulfide nor NO alone reproduces the biochemical lesions of UC in human colonocytes. Normal human colonocytes in the presence of sulfide and NO show that the combination of sulfide and NO reduced butyrate oxidation, ketogenesis, and CoA levels, which are observed in the biochemical lesions of ulcerative colitis.

[0022] Impaired butyrate oxidation leads to an energy deficiency state in colonocytes

[0023] High levels of NO nitrosate the sulfhydryl group of CoA to form S-nitroso-CoA, thereby inhibiting the acyl transfer and energy-forming functions of acetyl-CoA. In addition, exposure of colonocytes to NO inactivates butyryl-CoA, which is essential for β-oxidation. The failure of butyrate β-oxidation leads to low levels of ATP and energy deficiency, reducing the ability of colonocytes to maintain the mucosal barrier. Attempts to treat UC with butyrate have been unsuccessful, and butyrate levels in UC patients have been found to be either normal or elevated compared to healthy controls.

[0024] Delayed NO toxicity due to glutathione depletion in ulcerative colitis

[0025] Both glutathione levels and reduced glutathione levels are altered in UC, suggesting either a decrease in glutathione synthesis or excessive consumption. However, a significant increase in nitrosothiol groups in UC indicates excessive glutathione consumption. This glutathione consumption and depletion occurs over time and induces gradual lesions identical to those seen in experimental models using human colonocytes exposed to sulfide and NO.

[0026] Defects in colonocyte tight junction assembly and mucus production

[0027] Abnormalities of tight junctions and mucosal barriers are present in UC. These occur in both active and dormant disease, indicating that they are not primarily due to active inflammation. Scanning electron microscopy of freeze-fractured UC colonocytes shows a decrease in tight junction "meshing" between colonocytes. Another electron microscopic finding in acute colitis is cellular blebbing in colonocytes. Cell blebbing is a hallmark of cellular energy deficiency via ATP and glutathione depletion in many cell lines and is reversible. Colonocytes are the primary producers of mucus, and their production is dependent on butyrate metabolism, the inhibition of which by exogenous agents also reduces mucus production.

[0028] Loss of colonic barrier leads to microbial-triggered inflammation

[0029] The gastrointestinal tract contains a powerful immune system separated from luminal microorganisms by a mucus layer and an epithelial barrier. The innate immune system provides additional nonspecific defense against invading organisms, assisted by specific responses to antigens provided by adaptive immune cells. Current evidence from human studies indicates that the mucosal inflammatory infiltrate in UC consists of a complex mixture of innate and adaptive immune cells and their products.

[0030] Conventional Management of Ulcerative Colitis

[0031] Management of UC includes both induction therapy (to induce remission) and maintenance therapy (to prevent further flares). The goal of treatment is to maintain remission without steroids. Induction therapy is usually high-dose oral 5-aminosalicylate (5-ASA) with or without topical 5-ASA via enema or suppository. More severe flares require systemic corticosteroids, which are tapered and discontinued over time. Maintenance therapy options in UC are determined by the extent, severity, frequency of flares, and previous treatment history. The mainstay of maintenance therapy is oral or topical 5-ASA. For patients with recurrent flares, thiopurines should be used. In recent years, new biologic agents have shown efficacy in maintaining remission in UC. These include the anti-TNFα agents infliximab, adalimumab, and golimumab, and the anti-integrin agent vedolizumab. However, these drugs are expensive and have limited effectiveness.

[0032] Inadequacies of current treatments for ulcerative colitis

[0033] Current treatments are hampered by incomplete efficacy and have the potential for unacceptable side effects, including allergies, intolerance, serious infections, and malignancies due to long-term immunosuppression. Current treatments for UC are not suitable for maintaining long-term remission in the majority of patients. Many patients experience chronic or recurrent colon inflammation that causes work and personal disability, and up to 30% require colectomy despite current therapies. Thiopurines and anti-TNF agents induce systemic immunosuppression, reducing the occurrence and severity of flares, but at the expense of an increased risk of serious infections, malignancies, and especially lymphoma. The primary cause of the low therapeutic efficacy of current therapies is that most target the mucosal immune system rather than the trigger of the disease.

[0034] Thus, there is a need in the art for effective treatments for inflammatory bowel disease.It is an object of the present invention to overcome one or more of the problems presented by the prior art. Summary of the Invention [Means for solving the problem]

[0035] Summary of the Invention In a first aspect, the present invention broadly relates to a composition for preventing or treating a gastrointestinal disorder in a subject in need thereof, the composition comprising at least one strain of a microorganism, wherein the microorganism is selected from the group consisting of bacteria, archaea, and yeast.

[0036] In a preferred embodiment, the microorganisms are responsible for reducing endogenous sulfide levels in the colon of a patient in need thereof; sulfide consumption; reducing the sulfide and nitric oxide load of epithelial cells affecting cellular respiration, causing metabolic pathology; reducing the relative abundance and / or metabolic activity of sulfidogenic microbiota; reducing sulfide levels in the colon directly through consumption / assimilation / degradation; reducing sulfide levels, the relative abundance and / or metabolic activity of sulfidogenic microbiota in the colon by metabolic substrate competition; reducing sulfide levels, the relative abundance and / or metabolic activity of sulfidogenic microbiota in the colon by diverting metabolic substrates away from sulfide production; by consuming H2. reducing sulfide levels, the relative abundance of sulfidogenic microbiota, and / or metabolic activity in the colon by reducing the release of metabolizable sulfur substrates; reducing sulfide levels, the relative abundance of sulfidogenic microbiota, and / or metabolic activity by reducing sulfur amino acid release (methionine, cysteine, homocysteine, and / or taurine) into the colon; reducing colonic protein fermentation; reducing nitric oxide production in the colon; and reducing nitric oxide levels in the colon.

[0037] In a preferred embodiment, the microorganism comprises a phenotype and / or genes responsible for reducing endogenous sulfide levels in the colon of a patient in need thereof.

[0038] In a preferred embodiment, the microorganism comprises a phenotype and / or genes responsible for sulfide consumption.

[0039] In a preferred embodiment, the microorganism comprises a phenotype and / or gene responsible for reducing sulfide and nitric oxide load in epithelial cells, affecting cellular respiration and resulting in metabolic pathology.

[0040] In a preferred embodiment, the microorganism comprises a phenotype and / or gene responsible for reducing the taxonomic diversity, number of species, relative abundance, and / or metabolic activity of the sulfidogenic microbiota.

[0041] In a preferred embodiment, the microorganism comprises a phenotype and / or genes that are responsible for directly reducing sulfide levels in the colon through consumption / assimilation / degradation.

[0042] In a preferred embodiment, the microorganism comprises a phenotype and / or genes responsible for reducing sulfide levels in the colon, the taxonomic diversity, number of species, relative abundance and / or metabolic activity of the sulfidogenic microbiota through metabolic substrate competition.

[0043] In a preferred embodiment, the microorganism comprises a phenotype and / or gene that is responsible for reducing sulfide levels in the colon, the taxonomic diversity, number of species, relative abundance, and / or metabolic activity of the sulfidogenic microbiota by diverting metabolic substrates away from sulfide production.

[0044] In a preferred embodiment, the microorganism comprises a phenotype and / or gene that is responsible for reducing sulfide levels in the colon, the taxonomic diversity, number of species, relative abundance, and / or metabolic activity of the sulfidogenic microbiota by consuming H2.

[0045] In a preferred embodiment, the microorganism comprises a phenotype and / or gene that is responsible for reducing the release of metabolizable sulfur substrates, thereby reducing sulfide levels, the taxonomic diversity, number of species, relative abundance, and / or metabolic activity of the sulfidogenic microbiota.

[0046] In a preferred embodiment, the microorganism comprises a phenotype and / or gene responsible for reducing sulfur amino acid release (methionine, cysteine, homocysteine, and / or taurine) into the colon, thereby reducing sulfide levels, taxonomic diversity, species numbers, relative abundance, and / or metabolic activity of the sulfidogenic microbiota.

[0047] In preferred embodiments, the microorganism comprises a phenotype and / or gene responsible for reducing colonic protein fermentation; reducing nitric oxide production in the colon; and reducing nitric oxide levels in the colon.

[0048] In a preferred embodiment, the microorganism comprises a phenotype or gene responsible for a sulfide consumption phenotype.

[0049] In a further preferred embodiment, the sulfide is present in the form of a sulfide selected from the group consisting of: sulfide ions (S 2- ), hydrogen sulfide (HS - ) ion, bisulfide ion (SH - ), hydrogen sulfide (HS), organic compounds containing R-SH groups (thiols), metal sulfides (including but not limited to iron, copper, and molybdenum sulfides).

[0050] In a further preferred embodiment, the microorganism comprises a gene or several genes selected from the group consisting of genes responsible for the utilization of sulfide in the biosynthesis of methionine and cysteine.

[0051] In a further embodiment, the microorganism comprises a gene or several genes selected from the group consisting of genes responsible for the anaerobic oxidation of sulfide.

[0052] In a further embodiment, the microorganism comprises a gene or several genes selected from the group consisting of genes responsible for sulfide utilization in the anaerobic ethanolamine utilization pathway.

[0053] In a further preferred embodiment, the microorganism comprises a gene or several genes selected from the group consisting of genes encoding enzymes 2.3.1.30, 2.5.1.47, and / or 2.5.1.65 of Figure 4, genes responsible for pathways listed in Table 6 related to sulfur and sulfide metabolism and regulation in the colon, and genes responsible for pathways listed in Table 7 related to homoacetogenesis and hydrogen cycling in the colon.

[0054] In a further preferred embodiment, the microorganism is a species selected from the group consisting of a species selected from the group listed in Figure 5, and any combination of these species.

[0055] In a further preferred embodiment, the microorganism is selected from a phylum, genus, species, or isolate selected from the group consisting of: a species selected from the group listed in Figure 5 and any combination of these species; an isolate selected from the group listed in Table 1 and any combination of these isolates; a phylum selected from the group listed in Table 2 and any combination of these phyla; a genera selected from the group listed in Table 3 and any combination of these genera; a species selected from the group listed in Table 4 and any combination of these species; an isolate selected from the group listed in Table 5 and any combination of these isolates; an isolate selected from the group listed in Table 8 and any combination of these isolates; and an isolate selected from the group listed in Table 9 and any combination of these isolates.

[0056] In a further preferred embodiment, the microorganism comprises a 16S ribosomal RNA (rRNA) gene or a contiguous whole genome sequence having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-9193.

[0057] In a further preferred embodiment, the microorganism comprises a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-206.

[0058] In a further preferred embodiment, the microorganism comprises a complete genome sequence having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 207-412.

[0059] In a further preferred embodiment, the microorganism comprises a complete genome sequence having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 413-9193.

[0060] In a further preferred embodiment, the microorganism comprises a 16S ribosomal RNA (rRNA) gene or contiguous whole genome sequence having sequence identity to one or more of SEQ ID NOs: 1-9193, wherein the sequence identity is at least 99.9%, at least 99.5%, at least 99%, at least 98.5%, at least 98%, at least 97.5%, at least 97%, at least 96.5%, at least 96%, at least 95.5%, at least 95%, at least 94.5%, at least 94%, at least 93.5%, at least 93%, at least 92.5%, at least 92%, at least 91.5%, at least 91%, at least 90.5%, and at least 90%.

[0061] In a further preferred embodiment, the microorganism is a fecal or colonic microorganism.

[0062] In a further preferred embodiment, the microorganism is non-inflammatory.

[0063] In a more preferred embodiment, the microorganisms are cultured from feces or colon biopsy samples.

[0064] In a further preferred embodiment, the microorganisms comprise a microbial cell community derived from stool or biopsies of one or more human donors.

[0065] In a further preferred embodiment, the microbial cell community comprises cultured microbial cells.

[0066] In a further preferred embodiment, the cultured microbial cells are derived from multiple human donors.

[0067] In a further preferred embodiment, the microbial cell community comprises uncultured microbial cells.

[0068] In a further preferred embodiment, the uncultured microbial cells are derived from a single human donor.

[0069] In a further preferred embodiment, it is a fecal transplant microbiota composition.

[0070] In a further preferred embodiment, the composition is lyophilized.

[0071] In a more preferred embodiment, the composition is a liquid.

[0072] In a more preferred embodiment, after storage at room temperature for at least 4 weeks, the composition is capable of maintaining at least 50% cell viability compared to the initial cell viability immediately prior to storage.

[0073] In a more preferred embodiment, after at least 4 weeks of storage at room temperature, the composition is capable of maintaining about 60% to about 80% cell viability compared to the initial cell viability immediately prior to the initiation of storage.

[0074] In a further preferred embodiment, the composition comprises a prebiotic.

[0075] In a further preferred embodiment, the composition comprises a carrier.

[0076] In further preferred embodiments, the composition comprises insoluble fiber, a buffering agent, an antioxidant, an osmolality adjusting agent, an anti-foaming agent, and / or a preservative.

[0077] In a further preferred embodiment, the composition comprises a chemostat medium.

[0078] In a further preferred embodiment, the composition comprises a saline composition.

[0079] In a further preferred embodiment, the composition comprises resistant starch.

[0080] In a further preferred embodiment, the composition is lyophilized together with pharmaceutically acceptable excipients.

[0081] In a further preferred embodiment, the composition comprises a stabilizing agent and / or a cryoprotectant.

[0082] In a further preferred embodiment, the cryoprotectant is selected from the group consisting of trehalose, mannitol, sucrose, glycerol, sorbitol, DMSO, propylene glycol, ethylene glycol, sucrose, galactose-lactose, inulin, maltodextrin, glutathione, and any combination thereof.

[0083] In a further preferred embodiment, the cryoprotectant further comprises a compound selected from the group consisting of glycerol, polyethylene glycol (PEG), glycerin, erythritol, arabitol, xylitol, sorbitol, glucose, lactose, ribose, and any combination thereof.

[0084] In a more preferred embodiment, the cryoprotectant is trehalose at a concentration of 2% to 15% in the lyophilized formulation.

[0085] In a further preferred embodiment, said cryoprotectant is trehalose at a concentration of at least 5% in said lyophilized formulation.

[0086] In a further preferred embodiment, said cryoprotectant is trehalose at a concentration of at least 10% in said lyophilized formulation.

[0087] In a further preferred embodiment, said composition is a pharmaceutical composition.

[0088] In a further preferred embodiment, at least one strain of microorganism is diluted with an inert powdered diluent.

[0089] In a further preferred embodiment, the aforementioned compositions comprise one or more pharmaceutically acceptable carriers or excipients.

[0090] In a further preferred embodiment, the aforementioned compositions are formulated as gel tablets, pills, enemas, microcapsules, capsules, or tablets.

[0091] In further preferred embodiments, the capsule or tablet is enteric coated, pH dependent, slow release, and / or gastric acid resistant.

[0092] In a more preferred embodiment, the composition is adapted for oral or rectal administration.

[0093] In a more preferred embodiment, for every 200 mg of the composition, 10 1 ~10 14 , 10 1 ~10 16 , 10 2 ~10 14 , 10 3 ~10 14 , 10 4 ~10 14 , 10 5 ~10 14 , 10 6 ~10 14 , 10 7 ~10 14 , 10 8 ~10 14 , 10 4 ~10 13 , 10 5 ~10 12 , 10 6 ~10 11 , 10 7 ~10 10 , 10 8 ~10 9 , 10 3 ~10 13 , 10 3 ~10 12 , 10 3 ~10 11 , 10 3 ~10 10 , 10 3 ~10 9 , 10 3 ~10 8 , 10 3 ~10 7 , 103 ~10 6 , 10 3 ~10 5 , and 10 3 ~10 4 A pharmacologically active dose of microbial cells or spores selected from the group consisting of colony forming units (cfu) or total cell count.

[0094] In a further preferred embodiment, the composition contains 1 cfu / mL to 10 cfu / mL, 100 cfu / mL to 1000 cfu / mL, 10000 cfu / mL to 100000 cfu / mL, 10 cfu / mL to 10 6 cfu / mL, 100cfu / mL to 10 6 cfu / mL, 1000cfu / mL ~ 10 6 cfu / mL, 10000cfu / mL~10 6 cfu / mL, 100000cfu / mL~10 6 cfu / mL, 10 1 cfu / mL ~10 6cfu / mL, 1 cfu / mL to 10 cfu / mL, 100 cfu / mL to 1,000 cfu / mL, 10,000 cfu / mL to 100,000 cfu / mL, 1 million cfu / mL to 10 million cfu / mL, 10 million cfu / mL to 100 billion cfu / mL, 10 million to 50 million cfu / mL, more preferably 50 million to 100 million cfu / mL, 100 million to 500 million cfu / mL, 500 million to 1 billion cfu / mL, 1 billion to 5 billion cfu / mL, 5 billion to 10 billion cfu / mL, 10 billion to 15 billion cfu / mL, 15 billion to 20 billion cfu / mL, 20 billion to 25 billion cfu / mL, 25 billion to 30 0 billion cfu / mL, 30 to 35 billion cfu / mL, 35 to 40 billion cfu / mL, 40 to 45 billion cfu / mL, 45 to 50 billion cfu / mL, 50 to 55 billion cfu / mL, 55 to 60 billion cfu / mL, 60 to 65 billion cfu / mL, 65 to 70 billion cfu / mL, 70 to 75 billion cfu / mL, 75 to 80 billion cfu / mL, 80 to 85 billion cfu / mL, 85 to 90 billion cfu / mL, 90 to 95 billion cfu / mL, and 95 to 100 billion cfu / mL.

[0095] In further preferred embodiments, the composition comprises a pharmacologically active dose of microbial cells or spores, wherein the concentration of the microbial cells or spores as dry microbial mass is selected from the group consisting of 5-50% w / w, 1-75% w / w, 0.1-100% w / w, and 1-100% w / w.

[0096] In a more preferred embodiment, the gastrointestinal disorder is gastrointestinal mucosal inflammation.

[0097] In a further preferred embodiment, the gastrointestinal disorder is a dysbiosis.

[0098] In a further preferred embodiment, the inflammation is associated with one or more of the disorders selected from the group consisting of inflammatory bowel disease (IBD), pouchitis, irritable bowel syndrome (IBS), enterobacterial infection, metabolic disease, neuropsychiatric disorder, autoimmune disease, allergic disorder, hepatic encephalopathy, or cancer.

[0099] In a more preferred embodiment, the gastrointestinal disorder is inflammatory bowel disease.

[0100] In a more preferred embodiment, the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, gastroenteritis, enteritis, and pouchitis.

[0101] In a more preferred embodiment, the gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, gastrointestinal ulcers, and gastrointestinal cancer.

[0102] In a further preferred embodiment, the composition reduces endogenous sulfide levels in the colon of a patient in need thereof.

[0103] In a further preferred embodiment, the composition reduces sulfide and NO load in epithelial cells that cause metabolic pathology through inhibition of cellular respiration.

[0104] In further preferred embodiments, the composition reduces the taxonomic diversity, species number, relative abundance, and / or metabolic activity of sulfidogenic microbiota, reduces sulfide levels in the colon directly through consumption / assimilation / degradation, reduces sulfide levels in the colon through metabolic substrate competition, and / or reduces sulfide levels in the colon by consuming hydrogen.

[0105] In further preferred embodiments, the composition reduces the taxonomic diversity, species number, relative abundance, and / or metabolic activity of the sulfidogenic microbiota by reducing metabolizable sulfur substrates, and reduces sulfur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

[0106] In a further preferred embodiment, the composition reduces the taxonomic diversity, number of species, relative abundance, and / or metabolic activity of the sulfidogenic microbiota.

[0107] In a more preferred embodiment, the composition reduces sulfide levels in the colon directly through consumption / assimilation / degradation.

[0108] In a further preferred embodiment, the composition reduces sulfide levels in the colon by consuming hydrogen. In a further preferred embodiment, the composition reduces the taxonomic diversity, species number, relative abundance, and / or metabolic activity of the sulfidogenic microbiota by reducing metabolizable sulfur substrates.

[0109] In a further preferred embodiment, the composition reduces sulfur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

[0110] In a further preferred embodiment, the composition reduces sulfide levels in the colon through metabolic substrate competition, hi a further preferred embodiment, the composition reduces unwanted inflammation.

[0111] In a further preferred embodiment, the composition prevents or reduces activation of the mucosal immune system in an IL-13 and IL-5 dependent, TH2-mediated immune response driven by natural killer T cells.

[0112] In a further preferred embodiment, the composition reduces inflammation in the subject as measured by a parameter selected from the group consisting of TNFα signaling via NF-κB, IFNα signaling, IFNγ signaling, IL6 JAK STAT3 signaling, activation of pro-apoptotic pathways, and initiation of the unfolded protein response.

[0113] In a further preferred embodiment, the composition downregulates genes associated with pro-apoptotic pathways and the unfolded protein response, including genes selected from the group consisting of CHAC1, CEBPB, TRIB3, PPP1R15A, DDIT3, ATF4, and XBP1.

[0114] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Flavinofractor spp., Flavinofractor plautii, Christensenella spp., Christensenella minuta, Anaerobutyricum spp., Anaerobutyricum hallii, Escherichia-Shigella spp., and Escherichia coli, or a combination of two or more strains thereof. Preferably, the composition is used to treat or prevent ulcerative colitis. Preferably, the composition is used to treat or prevent IBD.

[0115] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Flavinofractor spp.

[0116] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Flavinofractor plautii.

[0117] In a further preferred embodiment, the composition comprises at least one strain of microorganism selected from the group consisting of Christensenella spp.

[0118] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Christensenella minuta.

[0119] In a further preferred embodiment, the composition comprises at least one strain of microorganism selected from the group consisting of Anaerobutyricum spp.

[0120] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Anaerobutyricum hallii.

[0121] In a further preferred embodiment, the composition comprises at least one strain of microorganism selected from the group consisting of Escherichia-Shigella spp.

[0122] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Escherichia coli.

[0123] In a further preferred embodiment, the composition further comprises dietary fiber, preferably selected from the group consisting of FOS, inulin, maltodextrin, and starch.

[0124] In a second aspect, the present invention broadly relates to a biotherapeutic composition comprising the composition of the first aspect of the invention together with an acceptable diluent or carrier. In one embodiment, the composition further comprises dietary fiber. Preferably, the dietary fiber is selected from the group consisting of FOS, inulin, maltodextrin, and starch.

[0125] In a third aspect, the present invention broadly relates to a pharmaceutical composition comprising the composition of the first aspect of the present invention together with a pharmaceutically acceptable diluent or carrier. In one embodiment, the composition further comprises dietary fiber. Preferably, the dietary fiber is selected from the group consisting of FOS, inulin, maltodextrin, and starch.

[0126] In a fourth aspect, the present invention broadly relates to an isolated non-inflammatory strain of a microorganism comprising a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-9193.

[0127] In a fifth aspect, the present invention broadly relates to an isolated non-inflammatory strain of a microorganism comprising a contiguous entire genome sequence having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-9193.

[0128] In a further preferred embodiment, an isolated non-inflammatory strain of a microorganism according to the fourth aspect of the invention, at least one strain of the microorganism comprises a 16S ribosomal RNA (rRNA) gene or contiguous whole genome sequence having sequence identity to one or more of SEQ ID NOs: 1-9193, wherein the sequence identity is selected from the group consisting of at least 99.9%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, and at least 90%.

[0129] In a fifth aspect, the present invention broadly relates to a method for treating and / or preventing a gastrointestinal disorder in a patient in need thereof, comprising administering to the patient in need thereof a composition of the present invention.

[0130] In a more preferred embodiment, the gastrointestinal disorder is gastrointestinal mucosal inflammation.

[0131] In a further preferred embodiment, the gastrointestinal disorder is a dysbiosis.

[0132] In a further preferred embodiment, the inflammation is associated with one or more of the disorders selected from the group consisting of inflammatory bowel disease (IBD), pouchitis, irritable bowel syndrome (IBS), enterobacterial infection, metabolic disease, neuropsychiatric disorder, autoimmune disease, allergic disorder, hepatic encephalopathy, or cancer.

[0133] In a more preferred embodiment, the gastrointestinal disorder is inflammatory bowel disease.

[0134] In a more preferred embodiment, the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, gastroenteritis, enteritis, and pouchitis.

[0135] In a more preferred embodiment, the gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, gastrointestinal ulcers, and gastrointestinal cancer.

[0136] In a more preferred embodiment, the compositions are administered orally or rectally.

[0137] In a further preferred embodiment, the composition is administered to the patient using a dosing regimen selected from the group consisting of once, hourly, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 12 hours, once daily, twice daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, once per week, twice per week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, once per month, twice per month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, once per year, twice per year, every 2 years, every 3 years, every 4 years, and every 5 years.

[0138] In a further preferred embodiment, the composition reduces endogenous sulfide levels in the colon of a patient in need thereof.

[0139] In a further preferred embodiment, the composition reduces the sulfide and nitric oxide load of epithelial cells that cause metabolic pathology through inhibition of cellular respiration.

[0140] In a further preferred embodiment, the composition reduces nitric oxide production and / or reduces nitric oxide levels in the colon.

[0141] In further preferred embodiments, the composition reduces the taxonomic diversity, reduces the number of species, reduces the relative abundance, and / or reduces the metabolic activity of sulfidogenic microbiota; reduces sulfide levels in the colon directly through consumption / assimilation / degradation; reduces sulfide levels, taxonomic diversity, number, relative abundance, and / or metabolic activity of sulfidogenic microbiota through metabolic substrate competition; and / or reduces sulfide levels, taxonomic diversity, number, relative abundance, and / or metabolic activity of sulfidogenic microbiota by consuming hydrogen.

[0142] In a further preferred embodiment, the composition reduces the taxonomic diversity, reduces the number of species, reduces the relative abundance, and / or reduces the metabolic activity of the sulfidogenic microbiota.

[0143] In a more preferred embodiment, the composition reduces sulfide levels in the colon directly through consumption / assimilation / degradation by consuming hydrogen.

[0144] In a further preferred embodiment, the composition reduces the taxonomic diversity, reduces the number of species, reduces the relative abundance, and / or reduces the metabolic activity of the sulfidogenic microbiota through metabolic substrate competition.

[0145] In a further preferred embodiment, the composition reduces sulfide levels, reduces the taxonomic diversity, reduces the number of species, reduces the relative abundance, and / or reduces the metabolic activity of sulfidogenic microbiota by consuming hydrogen.

[0146] In a further preferred embodiment, the composition reduces the taxonomic diversity, reduces the number of species, reduces the relative abundance, and / or reduces the metabolic activity of the sulfidogenic microbiota by reducing metabolizable sulfur substrates in the colon, and / or reduces the taxonomic diversity, number of species, relative abundance, and / or metabolic activity of the sulfidogenic microbiota by reducing protein fermentation and thereby reducing sulfur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon.

[0147] In a further preferred embodiment, the composition reduces the rate or concentration of sulfide produced in the colon by increasing the taxonomic diversity, species number, relative abundance, and / or metabolic activity of sulfide-consuming / assimilating / degrading isolates.

[0148] In a further preferred embodiment, the composition reduces the rate or concentration of sulfide produced in the colon by increasing the taxonomic diversity, species number, relative abundance, and / or metabolic activity of sulfide-consuming / assimilating / degrading isolates relative to the species number, taxonomic diversity, or relative abundance of the sulfide-producing microbiota.

[0149] In a further preferred embodiment, the method reduces unwanted inflammation.

[0150] In a further preferred embodiment, the composition comprises a strain selected from the group consisting of Flavinofractor spp., Flavinofractor plautii, Christensenella spp., Christensenella minuta, Anaerobutyricum spp., Anaerobutyricum hallii, Escherichia-Shigella spp., and Escherichia coli.

[0151] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Flavinofractor spp.

[0152] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Flavinofractor plautii.

[0153] In a further preferred embodiment, the composition comprises at least one strain of microorganism selected from the group consisting of Christensenella spp.

[0154] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Christensenella minuta.

[0155] In a further preferred embodiment, the composition comprises at least one strain of microorganism selected from the group consisting of Anaerobutyricum spp.

[0156] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Anaerobutyricum hallii.

[0157] In a further preferred embodiment, the composition comprises at least one strain of microorganism selected from the group consisting of Escherichia-Shigella spp.

[0158] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of Escherichia coli.

[0159] In a more preferred embodiment, the composition is administered together with dietary fiber. Preferably, the dietary fiber is selected from the group consisting of FOS, inulin, maltodextrin, and starch. In one embodiment, the dietary fiber is administered simultaneously or sequentially with the composition.

[0160] In a further preferred embodiment, the composition prevents or reduces activation of the mucosal immune system in an IL-13 and IL-5 dependent, TH2-mediated immune response driven by natural killer T cells.

[0161] In a further preferred embodiment, the aforementioned method reduces inflammation in the subject as measured by a parameter selected from the group consisting of TNFα signaling via NF-κB, IFNα signaling, IFNγ signaling, IL6 JAK STAT3 signaling, activation of pro-apoptotic pathways, and initiation of the unfolded protein response.

[0162] In a further preferred embodiment, the aforementioned method downregulates genes associated with pro-apoptotic pathways and the unfolded protein response, including genes selected from the group consisting of CHAC1, CEBPB, TRIB3, PPP1R15A, DDIT3, ATF4, and XBP1.

[0163] In a sixth aspect, the present invention broadly relates to a method for reducing endogenous sulfide levels in the colon of a patient in need thereof.

[0164] In a further preferred embodiment, the method reduces the sulfide and nitric oxide load of epithelial cells, which cause metabolic pathology through inhibition of cellular respiration.

[0165] In a further preferred embodiment, the method reduces the relative abundance and / or metabolic activity of sulfidogenic microbiota.

[0166] In a more preferred embodiment, the method reduces sulfide levels in the colon directly through consumption / assimilation / degradation.

[0167] In a further preferred embodiment, the method reduces sulfide levels, taxonomic diversity, species number, relative abundance and / or metabolic activity of the sulfidogenic microbiota in the colon by metabolic substrate competition.

[0168] In a further preferred embodiment, the method reduces sulfide levels, taxonomic diversity, species number, and relative abundance and / or metabolic activity of sulfidogenic microbiota in the colon by consuming hydrogen.

[0169] In a further preferred embodiment, the method reduces sulfide levels, taxonomic diversity, species number, and relative abundance and / or metabolic activity of the sulfidogenic microbiota by reducing metabolizable sulfur substrates in the colon.

[0170] In a further preferred embodiment, the method reduces sulfide levels, taxonomic diversity, species numbers, relative abundance and / or metabolic activity of the sulfidogenic microbiota by reducing sulfur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon.

[0171] In a further preferred embodiment, the method reduces sulfide levels in the colon, the taxonomic diversity, number of species, relative abundance and / or metabolic activity of the sulfidogenic microbiota by reducing protein fermentation.

[0172] In a seventh aspect, the invention broadly relates to a method of preparing a biotherapeutic composition according to the second aspect of the invention, comprising the step of mixing a composition according to the first aspect of the invention with an acceptable diluent or carrier.

[0173] In an eighth aspect, the present invention broadly relates to a method of preparing a pharmaceutical composition according to the third aspect of the invention, comprising the step of mixing a composition according to the first aspect of the invention with a pharmaceutically acceptable diluent or carrier.

[0174] In a ninth aspect, the present invention broadly relates to the use of a composition according to the first aspect of the invention in the manufacture of a medicament for reducing or preventing gastrointestinal disorders in a subject.

[0175] In a tenth aspect, the present invention broadly relates to a dosage form comprising the composition of the first aspect of the invention.

[0176] In an eleventh aspect, the present invention broadly relates to a kit comprising a dosage form according to the tenth aspect of the invention together with instructions for its use.

[0177] In a further aspect, the present invention broadly relates to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-9193.

[0178] In a further aspect, the invention is a nucleotide sequence having sequence identity to one or more of SEQ ID NOs: 1-9193, wherein the sequence identity is selected from the group consisting of at least 99.9%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, and at least 90%.

[0179] In a preferred embodiment, the nucleotide sequence is substantially purified or isolated.

[0180] In a further aspect, the invention is a microorganism comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-9193.

[0181] In a further aspect, the present invention is a microorganism comprising a nucleotide sequence having sequence identity to one or more of SEQ ID NOs: 1-9193, wherein the sequence identity is selected from the group consisting of at least 99.9%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, and at least 90%.

[0182] In one preferred embodiment, the microorganism is substantially purified or isolated.

[0183] Further features of the present invention are described in more detail in the following description of several non-limiting embodiments thereof. This description is included merely for purposes of illustrating the present invention and is not to be understood as a limitation on the above summary, disclosure, or description of the invention. [Brief explanation of the drawings]

[0184] Below is a brief description of each of the figures and drawings.

[0185] [Figure 1] FIG. 1 shows a diagrammatic representation of the process leading to ulcerative colitis induced by elevated levels of sulfide (either as sulfide ion (S), bisulfide ion (SH), or hydrogen sulfide (HS)) and nitric oxide (NO).

[0186] [Figure 2] Figure 2 shows a diagrammatic representation of hydrogen competition between methanogens, acetogens, and sulfate-reducing bacteria. Competition for hydrogen by methanogens and acetogens causes a decrease in the activity and abundance of sulfate-reducing bacteria, resulting in a decrease in sulfide production.

[0187] [Figure 3] Figure 3 shows the BB265 drug discovery process detailing lead selection and mechanistic validation of BB265 prior to clinical validation in a Phase 1 trial.

[0188] [Figure 4] Figure 4 shows in red an overview of the cysteine ​​biosynthesis pathway, a pathway through which sulfide can be directly consumed by the colonic microbiota. This pathway was taken from https: / / www.genome.jp / pathway / map00270.

[0189] [Figure 5a] Figure 5 shows a table of taxa for all 143 isolates comprising the BB265 complex consortium, along with all isolates outside the consortium that were identified as having a sulfide-consuming phenotype. The complete taxonomic lineage and taxon identification is shown for each isolate. [Figure 5b] Figure 5 shows a table of taxa for all 143 isolates comprising the BB265 complex consortium, along with all isolates outside the consortium that were identified as having a sulfide-consuming phenotype. The complete taxonomic lineage and taxon identification is shown for each isolate. [Figure 5c] Figure 5 shows a table of taxa for all 143 isolates comprising the BB265 complex consortium, along with all isolates outside the consortium that were identified as having a sulfide-consuming phenotype. The complete taxonomic lineage and taxon identification is shown for each isolate. [Figure 5d] Figure 5 shows a table of taxa for all 143 isolates comprising the BB265 complex consortium, along with all isolates outside the consortium that were identified as having a sulfide-consuming phenotype. The complete taxonomic lineage and taxon identification is shown for each isolate. [Figure 5e] Figure 5 shows a table of taxa for all 143 isolates comprising the BB265 complex consortium, along with all isolates outside the consortium that were identified as having a sulfide-consuming phenotype. The complete taxonomic lineage and taxon identification is shown for each isolate. [Figure 5f] Figure 5 shows a table of taxa for all 143 isolates comprising the BB265 complex consortium, along with all isolates outside the consortium that were identified as having a sulfide-consuming phenotype. The complete taxonomic lineage and taxon identification is shown for each isolate. [Figure 5g]Figure 5 shows a table of taxa for all 143 isolates comprising the BB265 complex consortium, along with all isolates outside the consortium that were identified as having a sulfide-consuming phenotype. The complete taxonomic lineage and taxon identification is shown for each isolate.

[0190] [Figure 6] Figure 6 shows a table of the V3-V4 hypervariable regions of the 16S rRNA gene sequences obtained from the isolates listed in Figure 5. These sequences are SEQ ID NOS: 1-206 (206 sequences total). These sequences are used for taxonomic classification of microbial species because the 16S rRNA gene, a small ribosomal subunit, is conserved among microbial species while containing hypervariable regions, e.g., the V3-V4 regions, which provide sufficient variability to determine species-level discrimination among 16S rRNA gene sequences. The sequences presented are DNA sequences encoding ribosomal RNA.

[0191] [Figure 7] Figure 7 shows a table of full-length 16S rRNA gene sequences obtained from the isolates listed in Figure 5. These sequences are SEQ ID NOs: 207-412 (206 sequences total). These sequences are used for taxonomic classification of microbial species because the 16S rRNA gene, the small ribosomal subunit, is conserved among microbial species while containing hypervariable regions, e.g., the V3-V4 regions, which provide sufficient variability to determine species-level discrimination among 16S rRNA gene sequences. The sequences presented are DNA sequences encoding ribosomal RNA.

[0192] [Figure 8] Figure 8 shows a table of contiguous DNA sequences derived from the whole genomes of the isolates listed in Table 5. These sequences are SEQ ID NOS: 413-9193 (a total of 8781 sequences). Taken together, these sequences constitute the whole genome sequences of the particular isolates listed in Table 5. The sequences presented are DNA sequences encoding ribosomal RNA.

[0193] [Figure 9-1] FIG. 9 shows the amount of sulfide, expressed in mM, consumed by microbial isolates identified as having a statistically significant sulfide consumption phenotype compared to the negative control when assayed for sulfide consumption using an in-house modified methylene blue assay. [Figure 9-2] FIG. 9 shows the amount of sulfide, expressed in mM, consumed by microbial isolates identified as having a statistically significant sulfide consumption phenotype compared to the negative control when assayed for sulfide consumption using an in-house modified methylene blue assay.

[0194] [Figure 10] Figure 10 shows a flowchart of the consensus analysis using the three tools used to identify taxa discriminating between healthy and ulcerative colitis from a large fecal metagenomic database.

[0195] [Figure 11] Figure 11 shows a phylogenetic tree depicting the sulfide phenotypes of 12,607 isolates from our culture collection assayed for sulfide production. Tree construction was performed using FastTree v2.1.11, inferring a maximum likelihood tree using generalized time-reversible (GTR) with a CAT approximation model. Trees were visualized with the R package ggtree v3.6.2 and the R package ggtree Extra v1.8.1.

[0196] [Figure 12a] Figure 12a shows a boxplot depicting the change in BB265 species counts in patients who achieved remission with FMT in a clinical trial. Week 0 represents the species count before remission, when the patient's disease state was considered active, and week 12 represents the period during remission after FMT treatment.

[0197] [Figure 12b]Figure 12b shows a boxplot depicting the cumulative relative abundance of sulfide-consuming species in patients who achieved remission with FMT in a clinical trial. Week 0 represents the number or abundance of species before remission, when the patient's disease state was considered active, and week 12 represents the period during remission after FMT treatment.

[0198] [Figure 12c] Figure 12c shows a boxplot representing the number of sulfide-consuming species in patients who achieved remission with FMT in a clinical trial. Week 0 represents the number or abundance of species before remission, when the patient's disease state was considered active, and week 12 represents the period during remission after FMT treatment.

[0199] [Figure 13a] Figure 13a shows a boxplot depicting the mean concentrations (μM) of water-soluble sulfide in stool samples from patient D incubated in half-strength YCFA and Solution I containing sodium sulfite. Stool samples were collected during active disease and in remission after FMT.

[0200] [Figure 13b] Figure 13b shows a boxplot depicting the mean concentrations (μM) of water-soluble sulfide in stool samples from patient E incubated in half-strength YCFA and Solution I containing sodium sulfite. Stool samples were collected during active disease and in remission after FMT.

[0201] [Figure 13c] FIG. 13c shows a bar graph depicting the number of BB265 species identified in stool samples obtained from patients D and E during active disease (baseline) and remission after FMT.

[0202] [Figure 13d] Figure 13d shows a bar graph representing:

[0203] Number of statistically significant sulfide-consuming species identified in stool samples obtained from patients D and E during active disease (baseline) and remission after FMT.

[0204] [Figure 13e] FIG. 13e shows a bar graph depicting the cumulative abundance of sulfidogenic species identified in stool samples obtained from patients D and E during active disease (baseline) and remission after FMT.

[0205] [Figure 14] Figure 14 shows boxplots depicting the mean reduction in water-soluble sulfide concentrations (μM) in stool samples from two ulcerative colitis patients (patients A and B) co-incubated with a subset of the BB265 complex consortium, including 127 of the 143 isolates. Water-soluble sulfide reduction is shown as the change in concentration (μM) from the negative control, consisting of stool incubated in medium alone. The phylogenetically diverse consumer mix (PDCM) refers to ulcerative colitis stool co-incubated with 19 isolates of BB265 that were phenotypically confirmed as sulfide consumers using a modified methylene blue assay. Double asterisks (**) indicate statistically significant differences between treatments (one-way Anova, P value ≦0.01, n=5).

[0206] [Figure 15] Figure 15 shows a boxplot depicting the mean reduction in water-soluble sulfide concentration in stool samples from an ulcerative colitis patient (Patient A) co-incubated with 127 of the 143 isolates comprising the BB265 complex consortium. Water-soluble sulfide reduction is shown as the change in concentration (μM) from the negative control, which consisted of stool incubated in medium alone. Individual consumers refer to ulcerative colitis stool co-incubated with the individual sulfide consumer bb0214, which showed 670 μM sulfide consumption using a modified methylene blue assay. An asterisk (*) indicates a statistically significant difference between treatments (one-way Anova, P value ≦0.05, n=5).

[0207] [Figure 16]Figure 16 shows boxplots depicting the mean reduction in water-soluble sulfide concentration in stool samples from ulcerative colitis patients (patients A and C) co-incubated with the BB265 complex consortium. Reduction in water-soluble sulfide is shown as the change in concentration (μM) from the negative control consisting of stool incubated in medium alone. Individual consumers refer to ulcerative colitis stool co-incubated with the individual strong sulfide consumer bb0214, which showed 670 μM HS consumption using a modified methylene blue assay. Double asterisks (**) indicate statistically significant differences between treatments (one-way Anova, P value ≦0.01, n=5).

[0208] [Figure 17] Figure 17 shows a phylogenetic tree depicting the sulfide-consuming and corresponding sulfide-producing phenotypes of 275 purified microbial isolates assayed for sulfide consumption using an in-house modified methylene blue assay. The color at the top of the tree represents the phylum, and the bars in the inner ring represent the average concentration (μM) of sulfide consumed by the isolates. Isolate identifiers are shown for isolates in the BB265 complex consortium and for isolates identified as having a net average sulfide consumption. The inner and outer rings indicate sulfide production from cysteine ​​and thiosulfate, respectively, with "N" indicating a negative phenotype and "Y" indicating a positive phenotype. Tree construction was performed using FastTree v2.1.11, inferring a maximum likelihood tree using generalized time-reversible (GTR) with a CAT approximation model. Trees were visualized with the R package ggtree v3.6.2 and the R package ggtreeExtra v1.8.1.

[0209] [Figure 18]Figure 18 shows a line graph depicting the accumulation of HS over time due to microbial hydrogen sulfide production in a stool sample from a patient with ulcerative colitis (Patient A) alone, a healthy donor (Donor A) alone, and a stool sample from a patient with ulcerative colitis (Patient A) co-incubated with the BB265 complex consortium and a sulfide consumer (bb0214). Healthy Donor A was used as a healthy control for hydrogen sulfide production. The concentration of HS is shown in μM.

[0210] [Figure 19] Figure 19 shows a line graph depicting the accumulation of HS over time due to microbial hydrogen sulfide production in stool samples from an ulcerative colitis patient (Patient C), a healthy donor (Donor C), and an ulcerative colitis patient (Patient A) co-incubated with the BB265 complex consortium and sulfide consumer (bb0450). Healthy Donor C was used as a healthy control for sulfide production. HS concentrations are shown in μM.

[0211] [Figure 20] Figure 20 shows a line graph depicting the rate of H2S production resulting from microbial hydrogen sulfide production in a stool sample from an ulcerative colitis patient (Patient A) alone, a stool sample from a healthy donor (Donor A) alone, and a stool sample from an ulcerative colitis patient (Patient A) co-incubated with the BB265 complex consortium and a sulfide consumer (bb0214). Healthy Donor A was used as a healthy control for sulfide production. The rate of H2S production is shown in μM hr-1.

[0212] [Figure 21] Figure 21 shows a line graph depicting the rate of H2S production resulting from microbial hydrogen sulfide production in a stool sample from an ulcerative colitis patient (Patient C) alone, a stool sample from a healthy donor (Donor C) alone, and a stool sample from an ulcerative colitis patient (Patient C) co-incubated with the BB265 complex consortium and a sulfide consumer (bb0450). Healthy Donor A was used as a healthy control for sulfide production. The rate of H2S production is shown in μM hr-1.

[0213] [Figure 22] Figure 22 shows a dot plot depicting the effect of fiber on sulfide (μM) concentrations after fermentation of ulcerative colitis stool samples obtained from patients A, B, and F with and without a subset of 127 isolates of the BB265 complex consortium. The fibers tested were FOS, inulin, maltodextrin, and starch.

[0214] [Figure 23a] Figure 23a shows a bar graph depicting the effect of co-cultivating ulcerative colitis stool (patients A and C) with a subset of 127 isolates of the BB265 complex consortium on the number and cumulative relative abundance of putative sulfidogenic species, achieved by shotgun metagenomics in stool samples from patients A and B.

[0215] [Figure 23b] Figure 23b shows a bar graph depicting the effect of co-culturing ulcerative colitis stool (Patient A and Patient C) with a subset of 127 isolates of the BB265 complex consortium on the number and cumulative relative abundance of sulfide-consuming species, achieved by shotgun metagenomics in stool samples from Patients A and B.

[0216] [Figure 23c] Figure 23c shows a bar graph depicting the effect of co-culturing ulcerative colitis stool (Patient A and Patient C) with a subset of 127 isolates of the BB265 complex consortium on the number and cumulative relative abundance of species comprising the BB265 complex consortium, achieved by shotgun metagenomics on stool samples from Patients A and B.

[0217] [Figure 24a]Figure 24a shows a bar graph depicting the number and abundance of phenotypically identified consumers and producers of BB265 achieved by shotgun metagenomics in stool samples from patient A after incubation with a subset of 127 isolates of the potent consumers bb0214 and BBB265. Asterisks (***) indicate statistically significant differences between treatments (one-way Anova, P value ≦0.001, n=5).

[0218] [Figure 24b] Figure 24b shows a bar graph depicting the cumulative relative abundance of sulfidogens achieved by shotgun metagenomics in stool samples from patient A after incubation with a subset of 127 isolates of the potent consumer bb0214 and BBB265 complex consortium. Asterisks (***) indicate statistically significant differences between treatments (one-way Anova, P value ≦0.001, n=5). DETAILED DESCRIPTION OF THE INVENTION

[0219] Detailed Description of the Invention For convenience, the following section will generally outline various meanings of terms used herein. This discussion is followed by general aspects relating to the compositions, pharmaceutical uses, and methods of the present invention, followed by specific examples illustrating the properties of various embodiments of the present invention and how they may be utilized.

[0220] Those skilled in the art will recognize that the invention described herein may be subject to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes, individually or collectively, all of the steps, properties, formulations, and compounds referred to or indicated in this specification, as well as any and all combinations of steps or properties, or any two or more of them.

[0221] Each document, reference, patent application, or patent cited in this document is expressly incorporated herein by reference in its entirety and shall be read and considered by the reader as part of this document. It is for the sake of brevity only that documents, references, patent applications, or patents cited in this document are not repeated in this document. None of the cited materials or the information contained therein, however, is to be understood as common general knowledge.

[0222] Manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned in this specification or any document incorporated herein by reference are incorporated herein by reference and may be used in the practice of this invention.

[0223] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for illustrative purposes only. Functionally equivalent products, formulations, and methods are clearly within the scope of the invention described herein.

[0224] 1.Definition The meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. In the event of an apparent discrepancy between a term's usage in the art and its definition provided herein, the definition provided herein shall control.

[0225] Except as in the working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein shall be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" may mean ±1%.

[0226] The invention described herein may include one or more ranges of values ​​(e.g., size, concentration, etc.). A range of values ​​is understood to include all values ​​within the range, for example, the values ​​defining the range and values ​​adjacent to the range that produce the same or substantially the same results as the values ​​immediately adjacent to the values ​​defining the boundaries of the range. For example, a person skilled in the art would understand that a 10% variation in the upper or lower limit of a range may be fully appropriate and be encompassed by the present invention. More specifically, the variation in the upper or lower limit of a range may be 5% or, whichever is greater, as generally recognized in the art.

[0227] In this application, the use of the singular also includes the plural unless otherwise indicated. In this application, the use of "or" means "and / or" unless otherwise indicated. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" encompass both elements and components that contain one unit and elements and components that contain more than one subunit, unless otherwise indicated. Also, the use of the term "moiety" can include part of a moiety or the entire moiety.

[0228] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0229] "Therapeutically effective amount," as used herein in relation to methods of treatment and specifically drug dosage, refers to a dosage that provides a specific pharmacological response, wherein the drug is administered to a significant number of subjects in need of such treatment. It is emphasized that a "therapeutically effective amount" administered to a particular subject in a particular case does not necessarily mean that such a dosage is effective for treating the diseases described herein, even if such a dosage is considered to be a "therapeutically effective amount" by those skilled in the art. It should be further understood that the drug dosage, in a particular case, refers to the drug level measured as an oral dosage or measured in blood. The amount effective for such use will depend on the desired therapeutic effect, the potency of the biologically active material, the desired treatment duration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician. Treatment dosages may need to be titrated to optimize safety and effectiveness. Those skilled in the art will therefore understand that dosage levels appropriate for treatment will vary depending, in part, on the indication for which the active agent is used, the route of administration, and the patient's size (weight, body surface area, or organ size) and condition (age and general health). Thus, clinicians can titrate dosages and modify the route of administration to obtain optimal therapeutic effect. Typical dosages can range from about 0.1 mg / kg up to about 100 mg / kg or more, depending on the factors discussed above. In other embodiments, dosages can range from 0.1 mg / kg up to about 100 mg / kg, or 1 mg / kg up to about 100 mg / kg, or 5 mg / kg up to about 100 mg / kg.

[0230] The frequency of administration will depend on the pharmacokinetic parameters of the active agent and formulation used. Typically, the clinician will administer the composition until the dosage achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more administrations over time (which may or may not contain the same amount of the desired molecule), or as continuous infusion via an implanted device or catheter. Further investigation of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of routine work performed by those skilled in the art. The appropriate dosage can be confirmed through the use of appropriate dose-response data.

[0231] As used herein, a "carrier" can be any solvent, diluent, excipient, or other vehicle, dispersing or suspending aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, solid binder, lubricant, and the like, suitable for the particular dosage form desired.

[0232] As used herein, the term "pharmaceutically acceptable carrier" component may refer to a component that is not biologically or otherwise undesirable, i.e., the component can be incorporated into the compositions of the present invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components in the formulation in which it is included. The component generally meets necessary toxicological and manufacturing standards.

[0233] As used herein, the term "subject" generally includes mammals, such as humans, livestock animals, such as sheep, goats, pigs, cows, horses, and llamas, pets, such as dogs and cats, primates, birds, such as chickens, geese, and ducks, fish, and reptiles. The subject is preferably a human.

[0234] As used herein, "gastrointestinal tract" refers to the tract from the mouth to the anus, including all organs of the digestive system, such as the esophagus, stomach, pancreas, liver, gallbladder, small intestine (including the ileum), cecum, large intestine, colon, and rectum. The strains of the present invention are useful for conditions in at least the terminal ileum, cecum, or rectum.

[0235] As used herein, "non-inflammatory strain" refers to a strain of the present invention that, when present in the gastrointestinal tract of a subject, preferably a human, is associated with a non-inflammatory condition. In one preferred embodiment, the non-inflammatory strain of the present invention has little or no cytotoxicity against mammalian cells in culture. In some embodiments, the strain causes cell death in less than 15%, less than 10%, or less than 5% of mammalian cells in culture. In some embodiments, the non-inflammatory strain of the present invention, when exposed to a given cell type, causes less cell death than a strain containing a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence set forth in any one of SEQ ID NOS: 1-9193, or a nucleotide sequence at least 90% identical to one or more of SEQ ID NOS: 1-9193. In an alternative embodiment, the non-inflammatory strain of the present invention has some level of cytotoxicity against mammalian cells in culture.

[0236] As used herein, "inflammatory strain" refers to a strain of the present invention that, when present in the gastrointestinal tract of a subject, preferably a human, is associated with an inflammatory condition. The inflammatory strain of the present invention is cytotoxic to mammalian cells in culture. In some embodiments, the strain causes cell death of at least 40%, at least 45%, or at least 50% of mammalian cells in culture. In some embodiments, the inflammatory strain of the present invention, when exposed to a given cell type, causes more cell death than a strain comprising a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence set forth in any one of the SEQ ID NOs: 7 and 8, or a nucleotide sequence at least 90% identical to one or more of the SEQ ID NOs: 7 and 8. In alternative embodiments, the inflammatory strain of the present invention has little or no cytotoxicity to mammalian cells in culture.

[0237] As used herein, the term "bacterial therapy" refers to the use of a bacterial isolate to treat or prevent a disease or condition or to provide a health benefit in a subject.

[0238] As used herein, the term "biotherapeutic agent" refers to a microorganism or combination thereof, e.g., a bacterial isolate, that is useful to treat or prevent a disease or condition or provide a health benefit in a subject.

[0239] The term "biotherapeutic composition," as used herein, refers to a formulation comprising a biotherapeutic preparation formulated with one or more additional formulation components to obtain a final formulation suitable for delivery to a subject.

[0240] As used herein, the terms "treat," "treating," "treatment," and grammatical variations thereof refer to subjecting an individual subject to a desired protocol, regimen, process, or remedy to obtain a physiological response or outcome in the subject. Because not all treated subjects may respond to a particular treatment protocol, regimen, process, or remedy, treatment does not require that the desired physiological response or outcome be achieved in all subjects or subject populations. Thus, a given subject or subject population may fail to respond or may respond inadequately to treatment.

[0241] As used herein, the terms "prevent," "prevented," or "preventing," when used in reference to the treatment of mucosal inflammation in the digestive tract, refer to prophylactic treatment that increases a subject's resistance to mucosal inflammation in the digestive tract, in other words, reduces the likelihood that a subject will develop mucosal inflammation in the digestive tract, as well as treatment to combat inflammation after digestive tract mucosal inflammation has begun, for example, to reduce or eliminate it all together or prevent it from getting worse.

[0242] As used herein, the term "reduce" or variations thereof refers to a reduction, but not necessarily a complete elimination, of gastrointestinal mucosal inflammation in a subject.

[0243] As used herein, the term "sample" refers to a collection of biological material obtained from a subject or the subject's environment, such as soil or water from the area where the subject lives. In some embodiments, the sample is obtained directly from the subject. For example, the sample may be a fecal sample or may be obtained during a colonoscopy. The sample may be in a form obtained directly from the subject or the environment, or may be at least partially purified to remove at least some non-nucleic acid material. The degree of purification may be minor, for example, merely concentrating the sample's solids or cells into a smaller volume or separating cells from some or all of the rest of the sample. In some embodiments, nucleic acids are isolated from the sample. Such isolated preparations include reverse transcription products and / or PCR amplification products of nucleic acids in the sample. In some embodiments, the predominant nucleic acid is DNA. The nucleic acid preparation may be pure or partially purified. Techniques for isolating nucleic acids from samples, including complex samples, are numerous and well known in the art.

[0244] The unit "cfu" refers to "colony forming units," which is the number of bacterial cells revealed by counting the number of organisms on an agar plate.

[0245] Other definitions of optional terms used herein can be found in the detailed description of the present invention and are applied throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

[0246] The properties of the present invention will now be described with reference to the following non-limiting descriptions and examples.

[0247] 2. Embodiment composition The present invention provides a composition for preventing or treating a gastrointestinal disorder in a subject in need thereof, the composition comprising at least one bacterial strain.

[0248] In a further preferred embodiment, the composition is selected from the group consisting of a therapeutic composition, a pharmaceutical composition, a cosmetic composition, and a veterinary composition.

[0249] Preferably, the composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline, such as phosphate-buffered saline. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is contemplated for use in therapeutic compositions, except insofar as it is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the composition. For example, see Remington's Pharmaceutical Sciences, 19th Ed. (1995, Mack Publishing Co., Easton, Pa.) and Remington's The Science and Practice of Pharmacy, 23 rd Edition. (2020, Mack Publishing Co., Easton, Pa.), which are incorporated herein by reference.

[0250] The compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or penetration of the composition. Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffering agents (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, These include, but are not limited to, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapol), stability enhancers (sucrose or sorbitol), tonicity enhancers (e.g., alkyl metal halides, preferably sodium chloride or potassium chloride), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants.

[0251] The optimal composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the biotherapeutic active agent of the present invention. The preferred form of the pharmaceutical composition depends on the intended mode of administration and therapeutic application.

[0252] The primary vehicle or carrier in the composition is water-soluble. For example, suitable vehicles or carriers can be water for injection or saline, possibly supplemented with other materials. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer with a pH of about 7.0-8.5 or acetate buffer with a pH of about 4.0-5.5, which may further contain sorbitol or a suitable substitute thereof. In one embodiment of the present invention, pharmaceutical compositions can be prepared for storage by mixing a selected composition having the desired purity in the form of an aqueous solution with optional formulating agents.

[0253] Formulation components are present in concentrations that are acceptable to the site of administration. For example, buffering agents are used to maintain the composition at physiological pH or slightly lower, typically within the pH range of about 5 to about 8.

[0254] Additional compositions, including the formulations of the present invention in sustained or controlled delivery formulations, will be apparent to those skilled in the art.Technologies for formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art.Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules.Sustained-release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

[0255] The composition to be used for in vivo administration can be filtered to remove undesirable components.This can be achieved by filtering through a filtration membrane.In addition, the composition is generally placed in a sealed container to reduce exposure to oxygen.After pharmaceutical composition is formulated, it can be stored in a sealed container.

[0256] The term "sequence homology %" as used herein can be calculated, for example, as follows: A query sequence is aligned to a target sequence using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)). A comparison is made over a window corresponding to one of the aligned sequences, e.g., the shorter one. In some cases, the window can be defined by the target sequence. In other cases, the window can be defined by the query sequence. The nucleic acid residues (nucleotides) at each position are compared, and the percentage of positions in the query sequence that have identical counterparts in the target sequence is reported as sequence homology % or identity percent.

[0257] In one embodiment, the percent identity of polynucleotides is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) using a gap creation penalty of 5 and a gap extension penalty of 0.3. Preferably, GAP analysis aligns two sequences over their entire length.

[0258] Bacterial strains for use in the present invention can be cultured using standard microbiological techniques as detailed, for example, in Handbook of Microbiological Media, Fourth Edition (2010) Ronald Atlas, CRC Press, Maintaining Cultures for Biotechnology and Industry (1996) Jennie C. Hunter-Cevera, Academic Press, and using YCFA medium as detailed in the Examples.

[0259] In an even more preferred embodiment, the composition further comprises water.

[0260] In an even more preferred embodiment, the composition is a liquid, for example an aqueous solution.

[0261] In an even more preferred embodiment, it further comprises a pharmaceutically acceptable carrier.

[0262] In an even more preferred embodiment, the composition retains its effective biological activity for a period selected from the group consisting of more than 24 hours, more than 36 hours, and more than 48 hours. Preferably, the composition is stable for a period selected from the group consisting of 6 months, 1 year, and 2 years. In one example, the composition is stable at a temperature selected from the group consisting of -80°C, -20°C, -4°C, 4°C, 18°C, and 25°C.

[0263] Pharmaceutical and therapeutic compositions are within the scope of the present invention.

[0264] The therapeutic compositions of the present invention may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials shall be non-toxic and shall not interfere with the effectiveness of the isolated bacteria present in the therapeutic composition. The exact nature of the pharmaceutically acceptable excipients or other materials will depend on the route of administration, which may be, for example, oral or rectal. Numerous methods for preparing therapeutic compositions are known to those skilled in the art (see, for example, Robinson ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978).

[0265] Therapeutic compositions of the present invention may include prebiotics, antioxidants, carriers, insoluble fiber, buffering agents, osmolality adjusting agents, anti-foaming agents, and / or preservatives.

[0266] The therapeutic composition may be prepared or provided in a chemostat medium. Alternatively, the therapeutic composition may be prepared or provided in saline, e.g., 0.9% saline. It will be understood that any carrier or solution may be used that does not impair the viability of the bacteria present in the therapeutic composition and is compatible with administration to an individual.

[0267] Therapeutic compositions may be made or provided under a reducing atmosphere, i.e., in the absence of oxygen. Synthetic stool preparations may be made or provided under N, CO, H, or mixtures thereof, optionally with controlled levels of N:CO:H partial pressure.

[0268] The therapeutic composition may be for oral or rectal administration to an individual. When the therapeutic composition is for oral administration, it may be in the form of a capsule or tablet. When the therapeutic composition is for rectal administration, it may be in the form of an enema or delivered via colonoscopy. The preparation of suitable capsules, tablets, and enemas is well known in the art. The capsule or tablet may include a coating to protect the capsule or tablet from stomach acid. For example, the capsule or tablet may be enteric-coated, pH-dependent, slow-release, and / or gastro-resistant. Such capsules and tablets are used, for example, to minimize dissolution of the capsule or tablet in the stomach while allowing dissolution in the small intestine.

[0269] Orally administered formulations may contain, for example, in addition to viable microorganisms, inert compression aids such as microcrystalline cellulose or oligosaccharides, flow aids such as silica gel, or lubricants made from, for example, magnesium stearate (vegetable source) or stearic acid (vegetable source).

[0270] The compositions disclosed herein can be used as, for example, dietary supplements, food products or pharmaceutical products.When being a dietary supplement, the compositions can further comprise conventional dietary supplement fillers and / or bulking agents.The compositions disclosed herein can also be included in any food product, for example, dairy products, including dairy products, milk, yogurt, curd, ice cream, dressing and cheese, beverage products, meat products and baked goods.

[0271] Suppository formulations, e.g., for rectal use, may contain, in addition to the composition, for example, cocoa butter, polyethylene glycol, glycerin, or gelatin.

[0272] The composition may contain a disintegrant, a glidant, and / or a lubricant. The disintegrant can aid in the disintegration of the compressed material when placed in a fluid environment. The disintegrant can be any suitable disintegrant, such as a disintegrant selected from the group consisting of croscarmellose sodium, crospovidone, gellan gum, hydroxypropyl cellulose, starch, and sodium starch glycolate. The lubricant can be any suitable lubricant, such as a lubricant selected from the group consisting of silicon dioxide, colloidal silicon dioxide, and talc. Lubricants are generally always used in the manufacture of dosage forms by direct compression to prevent the compressed powder material from adhering to the equipment during the tableting or encapsulation process. The lubricant can be any suitable lubricant, such as a lubricant selected from the group consisting of calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumarate, and vegetable-based fatty acids. In the compositions and methods of the present invention, the carrier may be present in the composition in a range of approximately 30% w / w to approximately 98% w / w, where this weight percentage is a cumulative weight percentage taking into account all components present in the carrier.

[0273] Coating agents can be used to control the solubility of the composition, and examples of coating agents include carrageenan, cellulose acetate phthalate, ethyl cellulose, gellan gum, maltodextrin, methacrylates, methyl cellulose, microcrystalline cellulose, and shellac.

[0274] The compositions may include one or more preservatives. Exemplary preservatives include antioxidants, chelating agents, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0275] Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, glutathione, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0276] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., edetate sodium, edetate disodium, edetate trisodium, edetate calcium disodium, edetate dipotassium, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0277] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid, and phenylethyl alcohol.

[0278] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0279] Other preservatives include tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.

[0280] The therapeutic composition may be lyophilized. The lyophilized therapeutic composition may contain one or more stabilizers and / or cryoprotectants. The lyophilized therapeutic composition can be reconstituted using a suitable diluent prior to administration to an individual.

[0281] Therapeutic compositions according to the invention can be administered alone or in combination with other treatments, simultaneously or sequentially with one or more other therapeutic agents, or as a combined preparation, for the treatment of dysbiosis or dysbiosis-associated diseases described herein. For example, the strains of the invention can be used in combination with existing therapeutic agents for inflammatory bowel disease, irritable bowel syndrome, metabolic disorders, neuropsychiatric disorders, autoimmune diseases, allergic disorders, cancer, or hepatic encephalopathy.

[0282] For example, when the therapeutic composition is for the treatment of cancer-associated dysbiosis, the therapeutic composition may be administered to an individual, optionally in combination with cancer immunotherapy, such as an immune checkpoint inhibitor. Examples of checkpoint inhibitors that can be used in this context include programmed cell death protein 1 (PD-1) inhibitors, programmed death-ligand 1 (PD-L1) inhibitors, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors. Manipulation of the gut microbiota in combination with immune checkpoint inhibitor treatment has been shown to improve the effectiveness of immune checkpoint inhibitors in cancer treatment. In one preferred embodiment, the cancer in this context is colorectal cancer. In another embodiment, the cancer is kidney cancer, lung cancer, or melanoma.

[0283] In another embodiment, the compositions of the present invention further comprise an immunomodulatory compound. In other embodiments, the immunomodulatory compound is a cytokine, chemokine, or complement component, their receptor, or combination thereof that enhances the expression of immune system accessory or adhesion molecules. In some embodiments, the immunomodulatory compound includes an interleukin, e.g., interleukin 1-15, interferon alpha, beta, or gamma, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), a chemokine, e.g., neutrophil-activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1a and MIP-1b, a complement component, or a combination thereof. In other embodiments, the immunomodulatory compound stimulates expression or enhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2, or 3, a cytokine receptor, or a combination thereof.

[0284] In another embodiment, the immunomodulatory compound induces or enhances the expression of costimulatory molecules involved in the immune response, which in some embodiments include CD40 or its ligand, CD28, CTLA-4, or B7 molecules. In another embodiment, the immunomodulatory compound induces or enhances the expression of heat-stable antigen (HSA), chondroitin sulfate-modified MHC invariant chain (Ii-CS), or intracellular adhesion molecule 1 (ICAM-1).

[0285] Therapeutic compositions of the present invention can be administered to an individual, preferably a human individual. Administration can be in a "therapeutically effective amount," sufficient to show benefit to the individual. Such benefit can be, at a minimum, the improvement or alleviation of at least one symptom. Thus, "treatment" of a specified disease refers to the alleviation of at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated, the particular patient being treated, the individual patient's clinical condition, the cause of the dysbiosis, the site of delivery of the composition, the type of therapeutic composition, the method of administration, the schedule of administration, and other factors known to medical professionals. Prescribing treatment, e.g., determining dosage, is within the responsibility of general practitioners and other physicians and may depend on the severity and / or progression of the symptoms of the disease being treated. The therapeutically effective amount or suitable dose of a therapeutic composition of the present invention can be determined by comparing its in vitro and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other test animals to humans are known. The exact dose will depend on several factors, including whether the therapeutic composition is for prophylaxis or treatment.

[0286] The ingredients can be mixed or prepared until the preparation is contacted with the preparation. As will be apparent to those skilled in the art, the formulation conditions will generally be such that viable microorganisms are maintained. In particular, high temperatures, e.g., temperatures above 40°C, are avoided.

[0287] The amount of viable microorganisms included in the composition can vary and can be adjusted and optimized, as will be understood by those skilled in the art. Such optimization can be achieved, for example, by preparing a series of different doses of viable microorganisms. The bacterial concentration in the composition can be, for example, 10 1 cfu / mL ~10 16 cfu / mL, 1 cfu / mL to 10 cfu / mL, 100 cfu / mL to 1,000 cfu / mL, 10,000 cfu / mL to 100,000 cfu / mL, 1 million cfu / mL to 10 million cfu / mL, 10 million cfu / mL to 100 billion cfu / mL, 10 million to 50 million cfu / mL, more preferably 50 million to 100 million cfu / mL, 100 million to 500 million cfu / mL, 500 million to 1 billion cfu / mL, 1 billion to 5 billion cfu / mL, 5 billion to 10 billion cfu / mL, 10 billion to 15 billion cfu / mL, 15 billion to 20 billion cfu / mL, 20 billion to 25 billion cfu / mL, 25 to 30 billion cfu / mL, 30 to 35 billion cfu / mL, 35 to 40 billion cfu / mL, 40 to 45 billion cfu / mL, 45 to 50 billion cfu / mL, 50 to 55 billion cfu / mL, 55 to 60 billion cfu / mL, 60 to 65 billion cfu / mL, 65 to 70 billion cfu / mL, 70 to 75 billion cfu / mL, 75 to 80 billion cfu / mL, 80 to 85 billion cfu / mL, 85 to 90 billion cfu / mL, 90 to 95 billion cfu / mL, or 95 to 100 billion cfu / mL.

[0288] In one embodiment, the strains of the present invention are, for example, 0.01 to 100 × 10 11 cells / body, 0.1-10×10 11 cells / body, or 0.3–5×10 11 The dosage can be 0.01 to 100 × 10 cells / body. 11 cells / 60 kg body weight, 0.1-10 x 10 11 cells / 60 kg body weight, or 0.3–5 × 10 11 cells / 60 kg body weight.

[0289] The content of at least one bacterial strain in the orally ingested composition of the present invention can be determined appropriately depending on the application form, and as a dry microbial organism, it can be, for example, 5 to 50 wt.%, 1 to 75 wt.%, 0.1 to 100 wt.%, or 1 to 100 wt.%.

[0290] In some embodiments, the composition is a controlled release composition. As used herein, the term "controlled release" refers to the release or administration of the strain of the present invention from a given dosage form in a controlled manner to achieve a desired pharmacokinetic profile in vivo. One aspect of "controlled" delivery is the ability to manipulate the formulation and / or dosage form to establish the desired release kinetics.

[0291] Procedures for preparing tablets, caplets, capsules, and other forms of the compositions of the present invention are known to those skilled in the art and include, without limitation, wet granulation, dry granulation, and direct compression (for tablets and caplets).

[0292] Wet and dry granulation are used to manufacture tablets, caplets, or capsules. A granulation technique, chilsonation, is used to produce powders for dosage forms. A chilsonator has rotating grooved rollers that are hydraulically pressed tightly together. Raw material is placed in the chilsonator's hopper and fed to the rollers by a horizontal and vertical screw system. As the material passes through the roller grooves, it is compressed under very high pressure and exits the chilsonator as a dense sheet. The sheet is milled into a fine powder using a Fitz mill and then passed through a screen to produce uniform, free-flowing granules. The chilsonation process produces a final powder that is two to four times denser than the starting material, a property that allows the ingredients to be formed into the desired dosage form.

[0293] In dry granulation, the powder can be incorporated into a gelatin capsule or it may be mixed with gelatin to form a tablet or caplet. In wet granulation, the powder is wetted, thereby creating large "chunks" of material, which are then dried and milled to convert the chunks into particles of the size desired for the manufacturing process. Once the desired size particles are obtained, they are incorporated into a gelatin capsule or mixed with gelatin to form a tablet or caplet.

[0294] General formulation and / or manufacturing considerations are discussed, for example, in Remington's The Science and Practice of Pharmacy, 23 rd Edition. (2020, Mack Publishing Co., Easton, Pa.), which is incorporated by reference. Prebiotics

[0295] The compositions of the present invention may contain prebiotics. Prebiotics have a chemical structure that resists digestion through the digestive tract, allowing them to reach the colon as intact molecules, where they can induce systemic physiological functions and act as a fermentable substrate for the colonic microflora. When prebiotics are combined with biological therapeutic agents, the resulting compositions may be referred to as "synbiotics."

[0296] Examples of suitable prebiotics include, but are not limited to, oligosaccharides such as fructooligosaccharides, P95 Nutraflora®, such as galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, quercetin, human milk oligosaccharides, inulin oligosaccharides, mannan oligosaccharides, pyrodextrin, levan, maltotriose, pectic oligosaccharides, bimno-galactooligosaccharides, arabinoxylan, fucoidan, and resistant starch. Fructooligosaccharides can be extracted from, for example, chicory, artichoke, asparagus, dandelion, dahlia, endive, garlic, leek, lettuce, and onion.

[0297] In certain embodiments, the prebiotic comprises one or more or all of the amino acids, for example, alanine, aspartic acid, glutamic acid, glycine, leucine, isoleucine, proline, serine, threonine, and valine.

[0298] In certain embodiments, the prebiotic comprises a simple sugar, which can be a monosaccharide (eg, glucose, galactose, or fructose) and / or a disaccharide (eg, sucrose, maltose, or lactose).

[0299] In certain embodiments, the prebiotics comprise from about 0.01% (wt / wt) to about 75% (wt / wt), 5% (wt / wt) to about 50% (wt / wt), about 7.5% (wt / wt) to about 30% (wt / wt), or about 10% (wt / wt) to about 15% (wt / wt) of the composition. Other microorganisms

[0300] It may be beneficial to include one or more additional biotherapeutic microorganisms in the composition to obtain a desired health benefit in a subject. Thus, the composition may include more than one microbial species / strain in addition to the strains of the present invention, for example, two, three, four, five, or more microbial species / strains. Non-pharmaceutical examples of biotherapeutic agents include Absiella, Acetobacterium, Adlercreutzia, Aerococcus, Agathobaculum, Akkermansia, Alistipes, Allobaculum, Amedibacterium, Anaerobutyricum, Anaerofustis, Anaerostipes, Anaerotignum, Anaerotruncus, Bacillus, Bacteroides , Barnesiella, Bifidobacterium, Blautia, Butyricimonas, Carnobacterium, Christensenella, Clostridium, Collinsella, Coprobacillus, Coprobacter, Coprococcus, Dorea, Enorma, Enterocloster, Enterococcus, Erysipelatoclostridium, Escherichi a, Eubacterium, Faecalibacillus, Faecalibacterium, Finegoldia, Flavonifractor, Flintibacter, Gemmiger, Holdemanella, Hungatella, Intestinimonas, Lachnoanaerobaculum, Lachnospira, Lacrimispora, Lactobacillus, Lactococcus, Leuconostoc , Ligilactobacillus, Longicatena, Massilimicrobiota, Megamonas, Merdibacter, Methanobrevibacter, Negativibacillus, Odoribacter, Oenococcus, Oscillospira, Oscillospiraceae, Parabacteroides, Paraclostridium, Parolsenella, Pediococcus,Suitable strains are those of the genera Peptoniphilus, Phocaeicola, Porphyromonas, Prevotella, Propionibacterium, Pusillimonas, Romboutsia, Roseburia, Ruminococcus, Ruthenibacterium, Sellimonas, Solibaculum, Sporolactobacillus, Sporomusa, Staphylococcus, Streptococcus, Subdoligranulum, Tetragenococcus, Thomasclavelia, Vagococcus, Vescimonas, and Weisella. It should be understood that the foregoing list is intended to be merely exemplary of the biotherapeutic agents that may be included in the compositions of the present invention and does not represent a limitation. In this regard, any additional biotherapeutic species may also be used in the compositions of the present invention.

[0301] Some yeasts are also useful as biotherapeutics and may be included in the compositions. One non-limiting example of a yeast used in a biotherapeutic is Saccharomyces boulardii.

[0302] Some archaea are also useful as biotherapeutics and may be included in the compositions. Non-limiting examples of archaea used in biotherapeutics are Methanobrevibacter spp., including Methanobrevibacter smithii, and Methanosphaera sp., including Methanobrevibacter stadtmanae. Dosage form

[0303] Dosage forms are within the scope of the present invention. In a preferred embodiment, the present invention provides a dosage form comprising the composition described in the first aspect of the present invention. Preferably, the dosage form is stored in a sealed and sterile container. Methods for Treatment

[0304] Methods for treating gastrointestinal disorders are within the scope of the present invention. In a preferred embodiment, the present invention provides a method for treating gastrointestinal disorders, comprising administering to a patient in need thereof a therapeutically effective amount of a composition described in the first aspect of the present invention.

[0305] In a more preferred embodiment, the dosage form is administered in an amount that at least partially treats the gastrointestinal disorder.

[0306] Subjects that can be treated according to the present invention include humans, as well as other mammals and animals.

[0307] The effects of the administered therapeutic compositions can be monitored by standard diagnostic procedures.

[0308] The methods of the present invention can be used to treat or prevent gastrointestinal dysbiosis in a subject. In the context of the present invention, "dysbiosis" refers to a state in which the normal diversity, relative species ratio, and / or function of the microbiota or microbiome, particularly the human gastrointestinal microbiota, is disrupted. Any disruption from the normal state of the microbiota in a healthy individual can be considered dysbiosis, even if it does not result in a detectable decrease in the individual's health status. In a preferred embodiment, dysbiosis may be associated with one or more pathological symptoms. For example, "dysbiosis" may refer to a decrease in the microbial diversity of the microbiota. Additionally or alternatively, "dysbiosis" may refer to an increase in the abundance of one or more bacteria, e.g., one or more pathogenic bacteria, in an individual's microbiota compared to the abundance of said one or more bacteria in the microbiota of a healthy individual, i.e., an individual without dysbiosis. Pathogenic bacteria present in dysbiosis are often Proteobacteria and are resistant to one or more antibiotics. Examples of Proteobacteria include Escherichia, Salmonella, Campylobacter, Vibrio, Helicobacter, and Yersinia species.

[0309] The dysbiosis may be a dysbiosis associated with enterobacterial infection, for example, a gastrointestinal infection caused by pathogenic bacteria.Many bacteria are known to cause gastrointestinal infection in humans, including gram-positive and gram-negative bacteria.The pathogenic bacteria are preferably pathogenic species of the genera Clostridium, Escherichia, Enterococcus, Klebsiella, Enterobacter, Proteus, Salmonella, Shigella, Staphylococcus, Vibrio, Aeromonas, Campylobacter, Plesiomonas, Bacillus, Helicobacter, Listeria, or Yersinia. Preferred examples of such pathogenic bacteria include Clostridium difficile, Clostridium perfringens, Clostridium botulinum, Escherichia coli, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Campylobacter fetus, Campylobacter jejuni, Aeromonas hydrophila, Plesiomonas shigelloides, Bacillus cereus, Helicobacter pylori, Listeria monocytogenes, and Yersinia enterocolitica. More preferably, the pathogenic bacteria are pathogenic species of the genus Clostridium or Escherichia. Most preferably, the pathogenic bacteria are Clostridium difficile or Escherichia coli.

[0310] The methods of the present invention can be used to reduce or prevent gastrointestinal mucosal inflammation in a subject using the compositions of the present invention.

[0311] In some embodiments, the subject has or is predisposed to have inflammatory bowel disease (IBD), such as Crohn's disease, ulcerative colitis, and pouchitis. As used herein, the term "inflammatory bowel disease (IBD)" has its general meaning in the art and refers to inflammatory diseases of the colon and small intestine, such as those as revised in World Health Organization Classification K20-K93 (ICD-10), including Crohn's disease (e.g., granulomatous enteritis, Crohn's disease of the small intestine, Crohn's disease of the large intestine, granulomatous and regional colitis, Crohn's disease of the colon, large intestine, and rectum, Crohn's disease of both the small intestine and large intestine), ulcerative colitis (e.g., ulcerative (chronic) pancolitis, reflux ileitis, and ulcerative colitis). IBD refers to the group consisting of ulcerative (chronic) proctitis, ulcerative (chronic) rectosigmoiditis, inflammatory polyps, left-sided colitis, left hemicolitis), and non-infectious gastrointestinal inflammation and colitis (radiation-induced gastrointestinal inflammation and colitis, toxic gastrointestinal inflammation and colitis, allergic and diet-induced gastrointestinal inflammation and colitis, food hypersensitivity gastrointestinal inflammation or colitis, unclassifiable colitis, designated non-infectious gastrointestinal inflammation and colitis, e.g., collagenous colitis, eosinophilic gastritis or gastrointestinal inflammation, lymphocytic colitis, microscopic colitis (collagenous colitis or lymphocytic colitis), non-infectious gastrointestinal inflammation and colitis, e.g., diarrhea, enteritis, ileitis, jejunitis, sigmoid colitis), and post-procedural disorders of the digestive system, e.g., pouchitis. In one embodiment, the IBD is pediatric IBD.

[0312] In a further aspect, the present invention also relates to fecal microbiota transplant compositions comprising the strains of the present invention. The term "fecal microbiota transplant composition" has its general meaning in the art and refers to any composition capable of restoring fecal microbiota.

[0313] Administration to humans includes administration by medical professionals and self-administration.Generally, to achieve health benefits, single or multiple doses of composition are administered, for example, once and every day for at least 1 week, at least 2 weeks, at least 3 weeks, at least 6 weeks, at least 9 weeks or at least 12 weeks.In one embodiment, composition can be administered for the remaining period of the life of the subject. device

[0314] Devices are within the scope of the present invention. In a preferred embodiment, the present invention provides a device comprising: (1) a composition described in the first aspect of the present invention; and (2) an applicator, container, or material. Use of the composition in the manufacture of a medicament

[0315] The uses are within the scope of the present invention. In a preferred embodiment, the present invention provides the use of the composition in the manufacture of a medicament for treating a gastrointestinal disorder. Methods for stabilization

[0316] Methods for stabilizing the compositions of the present invention are within the scope of the present invention.

[0317] In a further preferred embodiment, the method protects the compositions of the present invention from degradation.

[0318] In an even more preferred embodiment, the composition of the present invention retains its effective biological activity for a period selected from the group consisting of greater than 24 hours, greater than 36 hours, and greater than 48 hours.

[0319] The addition of approved pharmaceutical excipients to stabilize solutions of the compositions of the present invention is preferred from a safety standpoint, as simpler approaches are more likely to produce less variable results and limit excipient choices to those with Generally Recognized as Safe (GRAS) status. Excipients for stabilizing protein solutions can be classified into four broad categories based on their chemical properties and mechanism of action: salts, sugars, polymers, or protein / amino acids. Salts (e.g., chlorides, nitrates) stabilize the tertiary structure of proteins by sealing charges through ionic interactions. Sugars (e.g., glycerol, sorbitol, fructose, trehalose) increase the surface tension and viscosity of the solution, preventing protein aggregation. Similarly, polymers (e.g., polyethylene glycol, cellulose derivatives) stabilize the tertiary structure of proteins by increasing the viscosity of the solution, preventing protein aggregation and intramolecular and intermolecular electrostatic interactions between the amino acids of proteins. Proteins (e.g., human serum albumin) can stabilize the structure of other proteins through ionic, electrostatic, and hydrophobic interactions. Similarly, small amino acids that have no net charge, such as alanine and glycine, stabilize proteins through the formation of weak electrostatic interactions.

[0320] As mentioned above, the medicament of the present invention can comprise one or more pharmaceutically acceptable carriers.The use of such media and agents for the preparation of medicaments is well known in the art.Any conventional media or agent is contemplated for use in the preparation of pharmaceutical compositions according to the present invention, except insofar as it is incompatible with pharmaceutically acceptable materials.The pharmaceutically acceptable carrier according to the present invention can include one or more of the following examples: a. surfactants and polymers, including but not limited to polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, crospovidone, polyvinylpyrrolidone-polyvinyl acrylate copolymer, cellulose derivatives, HPMC, hydroxypropyl cellulose, carboxymethyl ethyl cellulose, hydroxypropyl methylcellulose phthalate, polyacrylates and polymethacrylates, urea, sugars, polyols and their polymers, emulsifiers, sugar gums, starches, organic acids and their salts, vinylpyrrolidone, and vinyl acetate; and / or b. binders, such as various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, and / or (3) fillers, such as lactose monohydrate, lactose anhydrous, microcrystalline cellulose, and various starches, and / or c. fillers, such as lactose monohydrate, lactose anhydrous, mannitol, microcrystalline cellulose, and various starches, and / or d. lubricants, e.g., agents that act to increase the ability of the dosage form to be released from the packaging gap, and / or e. sweeteners, e.g., any natural or artificial sweetener, e.g., sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame K, and / or f. flavoring agents, and / or g. preservatives, such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid, such as butylparaben, alcohols, such as ethyl or benzyl alcohol, phenolic chemicals, such as phenol, or quaternary compounds, such as benzalkonium chloride, and / or h. buffering agents, and / or i. a diluent, e.g., a pharmaceutically acceptable inert filler, e.g., microcrystalline cellulose, lactose, dicalcium phosphate, sugar, and / or mixtures of any of the foregoing, and / or j. absorption enhancers, e.g., glyceryl trinitrate, and / or k. Other pharmaceutically acceptable excipients.

[0321] Medicaments of the invention suitable for use in animals, and particularly humans, typically must be sterile and stable under the conditions of manufacture and storage.

[0322] Methods for detection The strains of the invention can be detected using a wide range of known techniques. Conveniently, the strains are detected using nucleic acid-based detection systems.

[0323] In some embodiments, nucleic acid sequencing is used.Nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing.In some embodiments, the technology provided herein is used in second-generation (also known as next generation or next-generation), third-generation (also known as next-generation) or fourth-generation (also known as N3 generation) sequencing technology, including but not limited to pyrosequencing, sequencing by ligation, single molecule sequencing, sequencing by synthesis (SBS), massively parallel clone, massively parallel single molecule SBS, massively parallel single molecule real-time, massively parallel single molecule real-time nanopore technology.

[0324] In some embodiments, hybridization is utilized in the detection method of the present invention. Illustrative, non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blot. In one embodiment, FISH assay is used. In other embodiments, nucleic acid amplification is used. Nucleic acids can be amplified before or simultaneously with detection. Performing one or more amplification reactions can include one or more PCR-based amplifications, non-PCR-based amplifications, or a combination thereof. Illustrative, non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), nested PCR, linear amplification, multiplex displacement amplification (MDA), real-time SDA, rolling circle amplification, circle-to-circle amplification transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Those skilled in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

[0325] Non-amplified or amplified nucleic acid can be detected by any conventional means.For example, nucleic acid can be detected by hybridization with detectably labeled probe and measuring the resulting hybrid.In another example, nucleic acid is detected by sequencing.The exemplary and non-limiting example of detection method is described herein.

[0326] "Real-time" evaluation of the amplification process involves continuously or periodically during the amplification reaction, calculating the amount of target sequence initially present in the sample using a predetermined value, and determining the amount of amplicon in the reaction mixture.Various methods for determining the amount of initial target sequence present in a sample based on real-time amplification are well known in the art.These include the methods disclosed in U.S. Patent No. 6,303,305 and U.S. Patent No. 6,541,205.Another method for determining the amount of target sequence initially present in a sample that is not based on real-time amplification is disclosed in U.S. Patent No. 5,710,029.

[0327] The amplification product can be detected in real time through a combination of primers specific to the target region and a nonspecific fluorescent DNA-binding dye. Such primers are designed to specifically target a region unique to the target microorganism, resulting in amplification of only the designed target. The primer design incorporates two separate primers that complementarily bind upstream and downstream of the targeted DNA site. During amplification, the primers bind upstream and downstream of the target, generating additional copies through the polymerase chain reaction, and the DNA-binding dye is incorporated into the newly generated amplicon. After amplification, the DNA-binding dye is excited with specific wavelengths of light, and the emission of different wavelengths is measured. The binding of the fluorescent DNA-binding dye to the exponentially increasing target amplicon can be correlated with a standard of known concentration by determining the original copy number of the amplified target region through linear regression. A technique using DNA-binding dyes is disclosed in U.S. Patent No. 6,174,670. The specific use of the fluorescent DNA binding dye SYBR green I is disclosed in US Pat. Nos. 5,338,671 and 5,587,287.

[0328] Amplification products can be detected in real time through the use of various self-hybridizing probes, most of which have a stem-loop structure. Such self-hybridizing probes are labeled to emit detectable signals differently depending on whether the probe is self-hybridized or altered through hybridization to a target sequence. As a non-limiting example, a "molecular torch" is a type of self-hybridizing probe that contains distinct self-complementary regions (referred to as a "target binding domain" and a "target closing domain") connected by a binding region (e.g., a non-nucleotide linker), which hybridize to each other under predetermined hybridization assay conditions. In a preferred embodiment, the molecular torch is 1 to about 20 bases long and contains a single-stranded base region within the target binding domain that is accessible for hybridization to a target sequence present in an amplification reaction under strand-displacing conditions. Under strand displacement conditions, hybridization of the two complementary regions of the molecular torch, which can be fully complementary or partially complementary, is preferred, except in the presence of a target sequence that binds to the single-stranded region present in the target binding domain and replaces all or part of the target closing domain. The target binding domain and target closing domain of the molecular torch contain a detectable label or an interacting label pair (e.g., luminescent / quencher) that is arranged so that when the molecular torch self-hybridizes, a different signal is generated than when the molecular torch hybridizes to the target sequence, thereby enabling the detection of probe-target duplexes in test samples in the presence of unhybridized molecular torches. Molecular torches and various types of interacting label pairs are disclosed in U.S. Patent No. 6,534,274, which is incorporated herein by reference in its entirety.

[0329] Another example of a detection probe with self-complementarity is a "molecular beacon." A molecular beacon comprises a nucleic acid molecule having a target-complementary sequence, an affinity pair (or nucleic acid arm) that holds the probe in a closed configuration in the absence of a target sequence present in the amplification reaction, and a label pair that interacts when the probe is in a closed configuration. Hybridization of the target sequence and the target-complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open configuration. The shift to the open configuration is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and 25 EDANS). Molecular beacons are disclosed in U.S. Patent Nos. 5,925,517 and 6,150,097.

[0330] In some embodiments, the method includes quantifying the amount of the strain present in the sample.

[0331] The present invention will now be described with reference to the following non-limiting examples, which are in no way a limitation on the preceding paragraphs of this specification, but are provided for illustration of the methods and compositions of the present invention. [Example]

[0332] It will be apparent to those skilled in the art of grinding and pharmaceuticals that numerous enhancements and modifications can be made to the above-described process without departing from the basic inventive concept. For example, in some applications, the biologically active material may be pretreated and fed to the process in a pretreated form. All such modifications and enhancements are considered to be within the scope of the present invention, the nature of which is determined from the foregoing description and the appended claims. Furthermore, the following examples are provided for illustrative purposes only and are not intended to limit the scope of the process and compositions of the present invention.

[0333] A Example 1 Mechanism of action of BB265 in alleviating metabolic lesions in ulcerative colitis Research objectives The goal of this treatment is to induce and maintain remission by reducing sulfide levels in the colon of patients with ulcerative colitis.

[0334] Materials, methods, and results Compositions of the Invention Comprising BB265 The compositions of the present invention, including the BB265 live biotherapeutic product, target proximal causes of colonic cell barrier disruption that lead to immune activation.

[0335] Reduces intestinal sulfide by "turning off" production. Reducing intraluminal sulfide can be achieved by "turning the plug" on sulfide production or by removing sulfide from the colon. Each of these mechanisms can be targeted using live bacterial biotherapeutic preparations.

[0336] Microbial competition for metabolic substrates

[0337] Sulfidogenic microbiota use specific substrates for metabolism. For example, sulfate-reducing bacteria (SRB) preferentially use volatile fatty acids (acetate, propionate, butyrate), organic acids (lactate, valerate, succinate, pyruvate), amino acids (alanine, glutamate, serine), and ethanol as electron donors in cellular respiration (Plugge, C.M., Zhang, W., Scholten, J.C.M. & Stams, A.J.M. Metabolic flexibility of sulfate-reducing bacteria. Front. Microbiol. 2, 81 (2011). Non-sulfidogenic microbiota that utilize these substrates will compete with sulfidogenic microbiota, and such competition will limit the growth of the sulfidogenic microbiota and reduce intraluminal sulfide production. Microbiota also compete with sulfidogenic organisms for electron acceptors. For example, Desulfovibrio and Desulfobulbus, the two most dominant SRB genera in the gut, both utilize fermentation-derived molecular hydrogen (H2) as an electron acceptor in cellular respiration. Methanogenic archaea and / or acetogenic bacteria divert hydrogen (H2) from sulfidogenic microbiota through competitive H2 uptake. Reducing intraluminal sulfide can be achieved through competitive H2 uptake by methanogenic archaea, acetogenic bacteria, or other microorganisms (Smith, NW, Shorten, PR, Altermann, E., Roy, NC & McNabb, WC). Competition for Hydrogen Prevents Coexistence of Humans. Gastrointestinal Hydrogenotrophs in Continuous Culture. Front. Microbiol. 11, 1073 (2020)).Methanogenic archaea, e.g., Methanobrevibacter smithii, and acetogenic bacteria, e.g., Alistipes spp., Blauti spp., and Roseburia spp., are reduced in ulcerative colitis and are associated with clinical remission in FMT. See Figure 2, which shows a diagrammatic representation of hydrogen uptake by acetogens and methanogens, which reduces sulfide production by SRB. Competition for metabolic substrates required for the growth and / or metabolism of sulfide-producing microbiota provides a therapeutic target for reducing endogenous sulfide in the colon.

[0338] Reduced release of sulfur substrates in the colon

[0339] Sulfidogenic microbiota require sulfur substrates (e.g., inorganic sulfate, sulfite, thiosulfate, and tetrathione, as well as organic cysteine, methionine, and taurine) to produce sulfide. In the colon, these substrates are derived from, but not limited to, dietary sulfur sources, such as meat and cruciferous plants, host mucin, bile acids, and endogenous proteins (Wolf, PG et al. Diversity and distribution of sulfur metabolism in the human gut microbiome and its association with colorectal cancer. bioRxiv 2021.07.01.450790 (2021) doi:10.1101 / 2021.07.01.450790). Sulfur is ubiquitously present in the colon, but most of it is bound in complex molecules that are inaccessible to sulfidogenic microbiota. For sulfidogenic microbiota to access sulfur and produce sulfide, other microbiota often need to biotransform these compounds into accessible forms. For example, SRB rely on saccharolytic bacteria, such as Bacteroides spp., to remove sulfate from host mucin (Tsai, HH, Sunderland, D., Gibson, GR, Hart, CA & Rhodes, JM. A novel mucin sulfatase from human faeces: its identification, purification, and characterization. Clin. Sci. 82, 447-454 (1992)). This reliance on the biotransformation properties of resident microbiota provides therapeutic opportunities to reduce colonic sulfide levels. Certain microbiota that reduce the rate at which these substrates are released, biotransformed, or compete with biotransformers (e.g., via metabolic substrate or niche competition) reduce the amount of sulfur substrate available for microbial sulfide production.

[0340] Bacterial colonic protein fermentation is the primary source of microbial sulfide and nitric oxide (NO) production. However, colonic bacteria preferentially ferment carbohydrates over protein in the human colon, and therefore, adequate dietary fiber reduces the amount of sulfur-containing amino acids released into the colon. In both human and animal studies, a high-protein diet leads to changes in the fecal microbiota that increase sulfide production and decrease SCFA production (Teigen, LM et al. Dietary Factors in Sulfur Metabolism and Pathogenesis of Ulcerative Colitis. Nutrients 11, (2019)). Additionally, dietary fiber has been shown to attenuate sulfide gas production in bioreactors containing stool from ulcerative colitis patients (Yao, CK et al. Modulation of colonic hydrogen sulfide production by diet and mesalazine utilizing a novel gas-profiling technology. Gut Microbes 9, 510-522 (2018)). In addition, a low protein, high resistant starch diet has been shown to induce remission in ulcerative colitis.

[0341] Removes sulfide from the colon

[0342] Bacterial species can consume sulfide, thereby reducing colonic sulfide levels. An example of sulfide consumption is through the assimilation of sulfide into cysteine. Cysteine ​​is synthesized from serine in a two-step process, beginning with the transfer of an acetyl group from acetyl-CoA to serine, catalyzed by serine acetyltransferase (SAT), to form O-acetylserine (OAS). The second step is the condensation of OAS with sulfide, catalyzed by O-acetylserine (thiol) lyase (OASTL), to produce cysteine ​​and acetate (Figure 4). Direct consumption, i.e., the biotransformation of sulfide, metabolically or otherwise, can therefore reduce colonic sulfide levels by direct removal.

[0343] B Example 2 Discovery and development of BB265 as a multicommunity probiotic biotherapeutic to address metabolic pathology in ulcerative colitis: An overview Research objectives To develop BB265 as a multicommunity live bacterial biotherapeutic formulation to address metabolic pathology in ulcerative colitis. Materials, methods, and results approach

[0344] BB265 is an enclosed bioreactor-derived microbial consortium that includes specific microbial strains that reduce colonic sulfide, which drives the development of chemical lesions seen in ulcerative colitis, and may optionally include the most globally predominant taxa or isolates (global core microbiome), taxa or isolates that best discriminate between healthy and ulcerative colitis, keystone taxa or isolates, and a collection of phylogenetically diverse taxa or isolates. The BB265 drug discovery process, detailing lead selection and validation prior to consortium validation in a Phase 1 study, is presented in Figure 3.

[0345] Complex Community Colonic microorganisms survive in ecosystems through numerous mutualistic nutritional relationships. Individual strains require numerous other species for their persistence through synergistic interactions. Keystone species play particularly important roles for the survival of numerous other species in an ecosystem and for ecosystem stability, often exerting significant ecological importance through uniquely conserved functions or functions with limited redundancy within the system (Banerjee, S., Schlaeppi, K. & van der Heijden, MGA Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2018)). Additionally, keystone species have also been associated with clinical efficacy, and their presence has been identified as a predictor of community-wide recovery following antibiotic treatment.

[0346] We performed a global metagenomic meta-analysis to identify the most common species shared across different regions. This global core microbiome was then supplemented with previously identified keystone species and those informatically identified as keystones through multiple correlation network analysis. These bacterial isolates were drawn from our culture collection and advanced to pilot process development for co-cultivation with a defined species community in a bioreactor. Given that this community contains numerous health-related organisms and possesses emergent properties that enhance the effects of candidate microbial sulfide reduction in ulcerative colitis, this community may complement a defined therapeutic candidate organism, disrupt existing communities, and provide therapeutic potential in its own right.

[0347] Target and lead identification Bacteria or archaea with the following characteristics were identified as leads for consideration for inclusion in the BB265 complex consortium: (1) globally predominant taxa or isolates commonly found in healthy human fecal microbiomes, (2) taxa or isolates that best discriminate between healthy and ulcerative colitis fecal microbiomes (health-associated), (3) keystone taxa or isolates, (4) phylogenetically diverse taxa or isolates, (5) sulfide-consuming (or assimilating) taxa or isolates, (6) putative H2-consuming taxa or isolates, e.g., methanogenic archaea and homoacetogenic bacteria, and (7) taxa or isolates that divert metabolic substrates away from sulfide production.

[0348] Bacteria or archaea with the following characteristics were identified as targets to consider for removal from the BB265 complex consortium: (1) taxa or isolates that best discriminate between healthy and ulcerative colitis fecal microbiomes (disease-associated), and (2) taxa or isolates identified as sulfidogens.

[0349] Identification of candidate therapeutic organisms The inventors have developed a unique phenotypic assay to identify sulfide-producing microbial isolates, and their bacterial culture collection has been screened for sulfide production. The inventors have further developed a unique phenotypic assay to identify sulfide-consuming (or assimilating) microbial isolates, and isolates identified as leads with the ability to consume sulfide are considered candidate therapeutic organisms. Isolates identified by taxonomic and genomic means as putative H2-consuming organisms, such as methanogenic archaea and homoacetogenic bacteria, are also considered candidate therapeutic organisms.

[0350] Verify the mechanism of action of BB265 We validated the mechanism of action of the BB265 complex consortium in vitro by co-culturing fecal slurries obtained from patients with active ulcerative colitis with the BB265 consortium. We developed a unique phenotypic assay to quantify sulfide production by the community and paired it with real-time measurements of H2S production rate using a commercially available H2S-specific electrochemical sensor. We demonstrated that the BB265 consortium significantly reduced sulfide concentration and production rate when co-cultured with ulcerative colitis fecal communities. In addition, we identified emergent properties of the BB265 complex consortium that enhanced the action of candidate microorganisms over individual candidate organisms by reducing total sulfide concentration and production rate.

[0351] The present inventors further supported the mechanism of action of BB265 by identifying an increase in taxa comprising the BB265 consortium in patients with active ulcerative colitis who underwent fecal microbiota transplantation and successfully achieved remission in a randomized controlled trial. This increase in BB265-associated taxa with remission was paired with a decrease in taxa identified as putative sulfidogens, supporting the hypothesis of sulfide reduction as a therapeutic target. Using a unique phenotypic assay, the inventors assayed stool samples obtained from patients before fecal microbiota transplantation and then after remission and identified a significant decrease in the sulfide-producing capacity of the community after remission.

[0352] The paired observations of (1) the BB265 complex consortium reducing sulfide concentrations and production rates in ulcerative colitis stool in vitro, (2) an increase in BB265-associated taxa and a decrease in putative sulfidogens in patients who successfully achieved remission in fecal microbiota transplant studies, and (3) a significant reduction in the sulfide-producing capacity of ulcerative colitis stool communities after FMT in patients who achieved clinical remission strongly support the BB265 consortium as a treatment for ulcerative colitis and further support the mechanism of action of this treatment to reduce sulfide as a means to improve clinical prognosis.

[0353] Prebiotic Fiber Fermentation of fiber (especially resistant starch and non-starch polysaccharides) in the colon occurs in preference to protein fermentation, resulting in reduced release of sulfur amino acids (cysteine) into the colon and reduced sulfide production by the resident microbiota (Teigen, LM et al. Dietary Factors in Sulfur Metabolism and Pathogenesis of Ulcerative Colitis. Nutrients 11, (2019)). We therefore tested fiber combinations that could enhance our live bacterial biotherapeutic formulation in inflammatory bowel disease using intrinsic phenotypic assays and a commercially available H2S-specific electrochemical sensor, as outlined above for validation of the BB265 complex consortium.

[0354] Phase 1 human clinical trial The final candidate consortium will be further tested in a Phase 1 human trial. This final drug product will contain enriched bacterial strains that target the underlying causes of metabolic lesions in ulcerative colitis, further supported by a community containing the most prevalent bacteria in healthy humans worldwide, in addition to known keystone species that maintain important synergistic relationships in the colonic ecosystem. Phase 1 human trials will demonstrate that our live bacterial biotherapeutic formulation acts to prevent barrier disruption by limiting NO- and H2S-induced metabolic lesions early in the disease pathway. Phase 1 human trials will have the ability to evaluate important mechanistic endpoints, such as the ability of therapeutic candidates to reduce sulfide in patients with ulcerative colitis.

[0355] C Example 3 Definition of the organisms that make up the BB265 multicommunity live bacterial biotherapeutic formulation Research objectives Identifying lead candidate microorganisms and defining a complex community for the BB265 live bacterial biotherapeutic formulation to address metabolic pathology in ulcerative colitis.

[0356] Materials, methods, and results Most Dominant Community (MPC) The widespread presence of microbial species commonly found in healthy human fecal microbiomes across various populations, regardless of differences in diet, lifestyle, and environmental conditions, indicates their importance as an essential component of the human gut microbiota. The global prevalence of such taxa suggests that they have coevolved with humans, forming a mutualistic relationship necessary for human health (Sharon, I. et al. The Core Human Microbiome: Does It Exist and How Can We Find It? A Critical Review of the Concept. Nutrients 14, (2022)). Furthermore, despite differences in community structure across global populations, the core functions of the gut microbiota remain relatively stable, indicating that these taxa play a fundamental role by preserving core functions crucial to human health. By examining healthy human fecal metagenomes across five regions (Africa, South America, North America, Asia-Pacific, and Europe), we identified globally dominant taxa that define the "core" community structure, referred to herein as the "most predominant community" (MPC). The community structure defined by the most commonly found species within the sequenced healthy human fecal microbiome forms a core community essential to the human gut ecosystem.

[0357] The globally dominant microbial species in healthy human fecal microbiomes were determined by publicly available Illumina paired-end shotgun metagenomics from feces of individuals with documented healthy phenotypes downloaded from the European Nucleotide Archive (ENA). This analysis resulted in the generation of priority lists ranked 1-2 and 4-6 according to global span and read coverage. Metagenomes with over 5 million reads were retained, and the remaining 882 metagenomes were assigned to regions based on their origin, including Africa, South America, North America, Europe, and Asia-Pacific. Sequencing reads were trimmed using Trimmomatic version 0.39, and taxon classification was performed using Kraken version 2.1.2 through comparison with a custom database constructed from all available complete genomes in the National Center for Biotechnology Information (NCBI) Reference Sequence Database (RefSeq release 209). Misclassified reads were filtered from each sample by removing reads that mapped to Homo sapiens and removing low abundance taxa that accounted for less than 0.001% of total reads.

[0358] Most Dominant Community Based on Co-occurrence (COIMPC) Microbial communities involve numerous complex interactions, both interspecific and intraspecific, and these synergistic relationships are essential for ecosystem functioning (Sharon, I. et al. The Core Human Microbiome: Does It Exist and How Can We Find It? A Critical Review of the Concept. Nutrients 14, (2022)). One way to examine these interactions is through co-occurrence analysis. Herein, a co-occurrence-based most-dominant community structure approach maximizes the likelihood of forming functional communities by overlaying co-occurrence data with dominance data. Co-occurrence suggests mutually beneficial relationships between bacteria in the MPC and more widespread, transient bacteria. Including bacteria outside the MPC but positively correlated with bacteria that form the MPC strengthens and supports core community function through metabolic cooperation, cross-feeding, and ecological network stability and resilience, among other means. After defining the MPC, this core community was supplemented with supporting taxa identified through co-occurrence analysis to form the "co-occurrence-based most-dominant community" (CoIMPC).

[0359] Bioinformatics analysis was performed to identify species that co-occur with globally dominant species in the composite consortium. Specifically, network analysis was performed on the output of the global metagenomic classification using SpiecEasi v1.1.2 with Meinshausen-Buhlmann neighborhood selection, with the Stability Approach to Regularization Selection (StARS) repeated 50 times with an nlambda value of 20 and a lambda.min. ratio of 0.01. Species directly connected to existing MPC taxa in the network were selected for inclusion in the consortium based on this co-occurrence. These species have statistically inferred interactions with the composite consortium taxa, indicating a supporting role in the community.

[0360] Identifying "hub" taxa Microbial taxa have varying levels of connectivity within their ecological communities, with some taxa contributing more than others to the functionality and stability of the community and referred to as hubs. Hubs have high levels of connectivity to other community members and are often essential for community structure and metabolic cycling (Banerjee, S., Schlaeppi, K. & van der Heijden, MGA Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2018)). A method for identifying hub taxa in the gut microbiome is through network analysis of sequencing data. Hubs are defined by the level of connectivity to other nodes in the network, which infers their importance in community functionality.

[0361] Bioinformatics network analyses were performed to identify statistically inferred hubs in the global metagenomic classification output. These analyses were performed using SpiecEasi with Meinshausen-Buhlmann neighbor selection and NetCOMI with SPRING association estimation to identify taxa with the highest degree of centrality. These methods identify nodes with the highest connectivity and determine potential hubs within the network. Taxa identified as hubs with high nearness centrality, betweenness centrality, or eigenvector centrality were selected for inclusion. Species identified through these methods may have important roles in determining community structure and metabolic processes.

[0362] Identifying the keystone In addition to common and high-abundance microbial species and those that co-occur with these species and act as ecological hubs, we consider taxa that are low in abundance but may be functionally important to microbial ecosystems. These keystone species often have significant ecological importance through unique functions or functions with limited redundancy within the system (Banerjee, S., Schlaeppi, K. & van der Heijden, MGA Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2018)). Herein, keystone species are also identified as those associated with clinical efficacy, and their presence is a predictor of community-wide recovery after antibiotic treatment.

[0363] A literature search was conducted to identify these key microorganisms. Within the literature reviewed, bioinformatics analysis was used to identify organisms with important functions not widely, if at all, possessed by others. These 26 species are outlined in Section 2e under "Healthy Gut Environment." Additionally, the reviewed literature details meta-analyses of post-antibiotic recovery, revealing the presence of organisms that promote the recovery of the gut microbial ecosystem after widespread antibiotic eradication, thus identifying them as key factors in recovery (Hajishengallis, G., Darveau, RP & Curtis, MA The keystone-pathogen hypothesis. Nat. Rev. Microbiol. 10, 717-725 (2012); Ze, X., Duncan, SH, Louis, P. & Flint, HJ Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon. ISME J. 6, 1535-1543 (2012); Rottjers, L. & Faust, K. Can we predict keystones? Nature reviews. Microbiology vol. 17 193 (2019); Banerjee, S., Schlaeppi, K. & van der Heijden, MGA Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2018), Chng, KR et al. Metagenome-wide association analysis identifies microbial determinants of post-antibiotic ecological recovery in the gut. Nature Ecology & Evolution 4, 1256-1267 (2020)).These 21 species are outlined in Section 2e under the headings "Treatments for UC" and "Treatments for other IBDs." The genomes of these keystone species were obtained from the National Center for Biotechnology Information (NCBI) Reference Sequence Database (RefSeq release 209).

[0364] Identification of phylogenetically diverse isolates The diversity of microbial species within the gut microbiome is associated with healthy gut function (Mosca, A., Leclerc, M. & Hugot, JP. Gut Microbiota Diversity and Human Diseases: Should We Reintroduce Key Predators in Our Ecosystem? Front. Microbiol. 7, 455 (2016)). The development of the multidisciplinary consortium focused on diversity to ensure it reflected that of a healthy gut microbiome.

[0365] Culture collection isolates that were phylogenetically distinct from the existing composite consortium were identified from BiomeBank using similarity comparisons based on 16S rRNA gene sequences. Isolates with 16S sequences that were less than 98% identical to any species or strain in the consortium, as determined by NCBI-BLAST (blastn) (v2.13.0) comparison, were included iteratively because they may fill ecological niches not covered by the initial community. Phylogenetic analysis was performed to assess the phylogenetic coverage of the composite consortium with these diversity-encompassing isolates.

[0366] Identifying taxa that discriminate between health and disease Fecal microbiota transplantation (FMT) has emerged as a potential treatment for ulcerative colitis and has been shown to induce remission in UC patients. However, the clinical success of FMT is variable, likely due to variability in preparation, delivery, and donors. Some donors have been observed to have greater success in achieving remission in recipients, suggesting that the presence of microbial diversity or specific combinations may be responsible. Therefore, there is a need for more defined microbiome therapeutics and the identification of specific microbial candidates.

[0367] Therefore, this analysis aims to define a consortium of taxa that discriminate between active UC and healthy controls from a diverse array of publicly available fecal metagenomes, with the goal of determining microbial targets associated with disease or therapeutic bacteria and communities for disease treatment.

[0368] Download data

[0369] Publicly available Illumina paired shotgun metagenomics derived from feces of patients with ulcerative colitis and controls were downloaded from the European Nucleotide Archive (ENA). Associated metadata was obtained using the SRA run selector, the ENA toolkit, and publicly available metadata. Trimmomatic (v0.39) was used to remove low-quality sequences and adapter sequences. Kraken2 (v2.1.2) (Wood, DE, Lu, J. & Langmead, B. Improved metagenomic analysis with Kraken 2. Genome Biol. 20, 257 (2019)) was used to determine taxonomic read classification using a reference database (release 209) constructed from all RefSeq complete genomes. The resulting kraken2 report files were input into Bracken for read count correction (Lu, J., Breitwieser, FP, Thielen, P. & Salzberg, SL Bracken: Estimating species abundance in metagenomics data. PeerJ Comput. Sci. 3, e104 (2017)). Any read counts below 0.001% per sample were removed to reduce bias due to mis-assignment of reads during classification. Data tables were created by integrating the braken outputs for individual samples.

[0370] Data curation

[0371] Two metadata stratifications were utilized for data comparison: (1) ulcerative colitis diagnosis versus non-IBD controls, and (2) active ulcerative colitis versus healthy (which differs from non-IBD controls in that individuals who underwent endoscopy were excluded).

[0372] Data analysis

[0373] Differentially present and discriminatory taxa between metadata groups were identified using ALDEx2 v1.29.1 (Gloor, G. ALDEx2: ANOVA-Like Differential Expression tool for compositional data. ALDEx manual modular 20, 1-11 (2015)), ANCOM-BC v1.6.4 (Lin, H. & Peddada, SD Analysis of compositions of microbiomes with bias correction. Nat. Commun. 11, 3514 (2020)), and Selbal v0.1.0 Rivera-Pinto, J. Balances: a new perspective for microbiome analysis. Msystems (2018)). Discriminatory taxa between non-IBD controls and ulcerative colitis, and between active ulcerative colitis and reportedly healthy controls, were identified through adjudication of consensus results. The filtered bracken output was sent to the ALDEx2 R package, and for each sample, a Monte Carlo instance was derived from the Dirichlet distribution. After data normalization using a centered log-ratio transformation, differentially represented taxa were statistically determined via Welch's test and Wilcoxon's test. The resulting Benjamin-Hochberg (BH)-corrected P values ​​were filtered for <0.05 to determine statistically significant differentially represented taxa. Taxa were sorted by effect value for highest and lowest effect scores.

[0374] A phyloseq object was created from the filtered bracken output and specimen metadata and used as input to the ANCOM-BC R package. The ancombc function was run with default parameters, except for the inclusion of structural zeros and the neg_lb parameter set to true, as well as conservative variance estimation (conservative set to true) and global testing. The resulting Holm-Bonferroni adjusted p-values ​​were used to identify statistically significantly differentially abundant taxa (p<0.05).

[0375] The filtered bracken output was input into the R package selbal to determine the taxa associated with the metadata sets. The cross-validation function selbal.cv was run with 10 iterations to identify the balance components, or groups of taxa, that form the inferred microbiome signature for each metadata set.

[0376] Analysis results Selbal identified 10 species required to distinguish between non-IBD and UC samples, with three associated with UC and seven associated with non-IBD. This grouping allowed for accurate classification of the sample groups, with a mean accuracy of 0.7985. The number of species required to distinguish between healthy and active UC samples was four, with one associated with UC and three associated with healthy samples. This grouping allowed for accurate classification of the sample groups, with a mean accuracy of 0.8420. The taxa identified by Selbal and the statistically significantly differentially abundant taxa determined by ALDEx2 and ANCOM-BC were combined to form a consensus analysis, resulting in 52 therapeutic candidate taxa and 32 targets.

[0377] Purification, storage, and identification of BB265 candidates When developing a microbial therapy, the purification, storage, and identification of microbial isolates within a consortium are of high importance. By isolating and characterizing the targeted microbial strains, this study also builds a characterization data package that can be incorporated into regulatory filings for this targeted microbial therapy.

[0378] Microbial culture and isolation

[0379] Fresh fecal samples were transferred to a Whitley A55 HEPA Anaerobic Workstation (Don Whitley Scientific) (atmosphere: 10% H2, 10% CO2, 80% N2) within 1 hour of donation, mixed with pre-reduced phosphate-buffered saline (PBS) to a concentration of 100 mg / mL, and homogenized by vortex mixing for 5 minutes. The fecal homogenates were then serially diluted in sterile pre-reduced PBS and plated onto multiple broad-spectrum and selective bacterial media and incubated anaerobically at 37°C for 24–168 hours.

[0380] To isolate sporogens, 200 μL of stool homogenate was transferred to a 1.5 mL microcentrifuge tube and mixed with 600 μL of ethanol (non-denatured), chloroform, or heat-shocked at 80°C for various times. The ethanol or chloroform samples were incubated at 25°C for a range of time periods. After incubation or heat shock, the samples were pelleted by centrifugation at 147,000 x g for 5 minutes. The supernatant was discarded, and 600 μL of 1x PBS was added to each tube. The samples were resuspended by vortexing for 5 minutes. This step was repeated three times to remove residual ethanol. After the third wash, the pellet was resuspended in 200 μL of 1x PBS and plated on YCFA agar as described above.

[0381] Isolate purification

[0382] After incubation, individual colonies were picked and restreaked onto YCFA agar and incubated anaerobically at 37°C for 24-72 hours. Individual colonies were then picked and restreaked onto YCFA agar and incubated under the same conditions for purification. This process was repeated once more to complete the purification.

[0383] Full-length 16S rRNA gene sequencing After purification, individual colonies were picked into 15 mL of YCFA broth and incubated anaerobically overnight at 37°C. After incubation, 20 μL of the overnight culture was diluted into 180 μL of sterile PBS as a template for PCR amplification and 16S rRNA gene capillary sequencing. The remaining overnight culture was diluted with sterile pre-reduced glycerol (25% final concentration) and immediately frozen at -80°C.

[0384] The full-length 16S gene of each isolate was amplified by PCR using the 7F (5'-AGAGTTTGATYMTGGCTCAG-3') and 1510R (5'-ACGGYTACCTTGTTACGACTT-3') primers. Samples were run in a C1000 Touch™ thermal cycler (BioRad) using the following conditions: 95°C for 15 minutes, 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 2 minutes, followed by 35 cycles. PCR was terminated at 72°C for 8 minutes and held at 8°C. The resulting amplicons were stained with Gel Green nucleic acid dye (Fisher Biotec) and run on a 1% agarose gel at 100V for approximately 20 minutes. Amplicons were visualized by fluorescence under blue light using an MBE-200A BluView Transiluminator (Major Science). The samples were then purified using EnzSAP™ PCR clean-up reagents according to the manufacturer's guidelines. The purified samples were subjected to capillary electrophoresis sequencing. Sequencing reactions were performed in a PTC-255 Peltier thermal cycler (MJ Research) using the ABI PRISIM® BigDye™ Terminator Cycle Sequencing Kits with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems) according to the manufacturer's guidelines. Single-pass sequencing was performed on each template using 7F and 1510R primers. Fluorescently labeled fragments were purified from unincorporated terminators according to the BigDye® Xterminator™ purification protocol. The samples were resuspended in dd.H2O and electrophoresed using an ABI 3730xl sequencer (Applied Biosystems).

[0385] Taxonomic judgment

[0386] The raw sequence data output from capillary sequencing was quality filtered to remove reads with a Phred quality score below 20 and less than 600 total bases. The quality-filtered sequence chromatograms were then visually inspected to ensure sample purity. Forward and reverse paired-end reads were then merged using PEAR running at default settings. The merged reads were then trimmed by 60 bp on each side to remove low-quality bases. NCBI-BLAST (blastn) (v2.13.0) was then run against the merged sequences, setting a species-level cutoff of 97% sequence similarity, against the NCBI RefSeq 16S rRNA database (RefSeq release 209) and an in-house constructed 16S database containing (1) globally dominant taxa or isolates widely found in healthy human fecal microbiomes, (2) taxa or isolates that best discriminate between healthy and ulcerative colitis fecal microbiomes (health-associated), (3) keystone taxa or isolates, and (4) putative H-consuming taxa or isolates, e.g., representatives of methanogenic archaea and homoacetogenic bacteria (Konstantinidis, KT & Tiedje, JM Genomic insights that advance the species definition for prokaryotes. Proc. Natl. Acad. Sci. USA 102, 2567-2572). (2005)). Isolates meeting these criteria with query coverage of 98% or greater were then analyzed for generation of V3-V4 16S rRNA sequences.

[0387] Generation of V3-V4 16S rRNA gene sequences

[0388] The 16S rRNA gene sequence V3-V4 hypervariable regions were obtained for each sample by extraction from 16S reads obtained from capillary sequencing using hyperex version 0.1.1, using the conserved forward (sequence number p0001: CCTACGGGNGGCWGCAG) and reverse (sequence number p0002: GACTACHVGGGTATCTAATCC) regions as flanking markers (Ebou, A., Koua, D. & Zeze, A. HyperEx: A Tool to Extract Hypervariable Regions from 16S rRNA Sequencing Data. bioRxiv 2021.09.03.455391 (2021) doi:10.1101 / 2021.09.03.455391).

[0389] Genealogy tree generation

[0390] The 16S sequences were aligned using ssu-align v 0.1.1 with default parameters. The resulting alignment file was input into FastTree v2.1.11 to infer a maximum likelihood tree using generalized time reversible (GTR) with a CAT approximation model. The tree was visualized with the R package ggtree v3.6.2 and the R package ggtreeExtra v1.8.1.

[0391] Screening isolates for antibiotic resistance, phage elements, and virulence factors As complex consortia are being used as microbial therapies, safety is of paramount importance. Screening microbial isolates intended for use as microbial therapies for antibiotic resistance, phage elements, and virulence factors is important to ensure patient safety and efficacy.

[0392] To ensure that each isolate met the regulatory safety profile, the following were considered:

[0393] a) Only isolates sourced from healthy individuals with complete history were included.

[0394] b) All isolates were phenotypically screened for antibiotic resistance and phage presence, genomically screened for antimicrobial resistance, virulence, and phage elements, and phenotypically screened for harmful products, e.g., sulfide production, according to our defined mechanism of action for ulcerative colitis.

[0395] c) A pathogen search was performed by literature review to ensure removal of known pathogens or species associated with disease.

[0396] d) At least three strains from the same species were included to allow for redundancy and overcome knockouts from the processes mentioned above.

[0397] e) Redundant strains were removed in favor of isolates with the lowest risk profile according to the screening described above.

[0398] Screening of isolates for sulfide production Recent evidence suggests that sulfides may be a harmful substance that contributes to the pathogenesis of ulcerative colitis (Gibson, GR, Cummings, JH & Macfarlane, GT Growth and activities of sulfate-reducing bacteria in gut contents of healthy subjects and patients with ulcerative colitis. FEMS Microbiol. Lett. 86, 103-111 (1991); Pitcher, MCL, Beatty, ER & Cummings, JH The contribution of sulfate-reducing bacteria and 5-aminosalicylic acid to faecal sulfide in patients with ulcerative colitis. Gut 46, 64-72 (2000); Roediger, W. & Babidge, W Nitric oxide effect on coloncyte metabolism: Co-action of sulfides and peroxide. An International Journal for Chemical Biology in Health and Disease 206, 159-167 (2000). In this context, we undertook a rigorous screening process to phenotype microbial isolates for sulfide production. The primary focus was to identify isolates for consideration for inclusion in BB265 that did not produce sulfide, as these would be considered ideal candidates for the therapeutic consortium.

[0399] A total of 12,607 microbial isolates isolated from screened donors and present in our culture collection were screened for sulfide-producing phenotypes. Isolates were anaerobically incubated in 200 μL of YCFA broth at 37°C for 48 hours in a Whitley A55 HEPA Anaerobic Workstation (Don Whitley Scientific) (atmosphere: 10% H2, 10% CO2, 80% N2). After incubation, 20 μL of culture was transferred to 180 μL of YCFA broth supplemented with 0.1% sodium thiosulfate and ferric ammonium citrate. The cultures were then incubated for an additional 48 hours and examined for growth by turbidity and for sulfide production by visual inspection of the presence of a black precipitate. The addition of sodium thiosulfate provided the isolates with an inorganic sulfur source for assessing their sulfide-reducing potential, while the presence of cysteine ​​(1%) in YCFA provided an organic sulfur source. Sulfide produced by the microbiota from thiosulfate or cysteine ​​reacts with ferric ammonium citrate to form iron sulfide (FeS), which can be seen as a black precipitate in the medium, confirming sulfide production.

[0400] Of the 12,607 isolates screened, we identified 2,694 isolates with a sulfide-producing phenotype. Sulfide production was found to be phylogenetically diverse, with the phenotype identified in isolates across the major phyla Actinomycetota, Bacillota, Bacteroidota, Fusobacteria, Pseudomonadota, and Thermodesulfobacteriota (FIG. 11). Interestingly, although sulfide has been identified as a deleterious substance in ulcerative colitis, we determined that many taxa under consideration for inclusion in the BB265 complex consortium also exhibited the sulfide-producing phenotype.

[0401] When possible, we selected isolates that showed a negative phenotype for sulfide production for inclusion in the BB265 consortium. However, when this was not possible, rather than completely dismissing these sulfide-producing isolates, we considered their potential role in the management of ulcerative colitis due to their prevalence in healthy individuals, keystone features, and anti-correlation with the ulcerative colitis dataset. This decision was made due to the subtle and multifaceted nature of the relationship between sulfide and ulcerative colitis. Although we affirm sulfide as a harmful substance in the context of ulcerative colitis, the human gut microbiome is a diverse and complex ecosystem, and microbial diversity is crucial to health (Mosca, A., Leclerc, M. & Hugot, J.P. Gut Microbiota Diversity and Human Diseases: Should We Reintroduce Key Predators in Our Ecosystem? Front. Microbiol. 7, 455 (2016)). Numerous sulfidogens identified in this study are associated with health and likely play an important role in positively modulating the gut ecosystem, which may result in an overall reduction in sulfide rates and production at the community level in the context of ulcerative colitis.

[0402] To further characterize lead isolates for consideration for inclusion in BB265, purified isolates were then independently assayed for sulfide production from L-cysteine ​​or thiosulfate by repeating the process described above using media containing only L-cysteine ​​or thiosulfate, respectively (Table 5).

[0403] In conclusion, sulfide production was identified as a phylogenetically diverse phenotype, present in both healthy and ulcerative colitis-associated taxa. Isolates selected for inclusion in the BB265 complex consortium were prioritized if they exhibited a negative phenotype for sulfide production; however, if this was not possible, isolates were still considered due to their association with health, keystone importance, inverse correlation with ulcerative colitis, and hypothesized mechanisms of action targeting sulfide production at the community level, e.g., H consumption.

[0404] Screening isolates for sulfide consumption This study aimed to phenotype candidate purified microbial isolates with respect to sulfide consumption, and therefore, may have the ability to reduce colonic concentrations of sulfide when introduced into a host when used as a treatment for ulcerative colitis.

[0405] A modified methylene blue assay for the determination of sulfide-consuming microbial isolates.

[0406] To identify sulfide-consuming microbial isolates, we developed an in-house modified methylene blue assay based on the chemical reaction in the American Public Health Association's Standard Methods for the Examination of Sulfide in Water and Wastewater, referred to herein as the "MB method" (4500-S2? SULPHIDE. in Standard Methods For the Examination of Water and Wastewater (American Public Health Association, 2017)). The MB method for determining sulfide is a highly specific colorimetric test in which N,N-dimethyl-p-phenylenediamine sulfate reacts with sulfide in the presence of an oxidizing agent, e.g., acidified FeCl, to produce methylene blue coloration. Because sulfide is the rate-limiting reactant, the amount of methylene blue produced is proportional to the original concentration of aqueous sulfide. The presence of methylene blue can be determined spectrophotometrically by light absorption at 667 nm.

[0407] Frozen isolates were thawed at 37°C in an anaerobic cabinet, diluted 1:10 in pre-reduced YCFA broth, and incubated overnight. In triplicate, 100 μL of the overnight culture was used to inoculate 3.85 mL of modified YCFA broth (lacking L-cysteine) in a sterile cuvette spiked with 0.1% L-serine. Sodium sulfide (anhydrous) (NaS) was spiked into the cuvette at a concentration of 2 mM as a source of water-soluble sulfide, and the cuvette was immediately capped to prevent the escape of volatile hydrogen sulfide (HS). A negative control consisting of modified YCFA (lacking L-cysteine) plus 2 mM NaS, but without culture, was prepared simultaneously. The cuvette was incubated anaerobically at 37°C for 48 hours. After incubation, growth of the microbial isolate was visually confirmed by the presence of turbidity in the cuvette. The cuvette was removed from the anaerobic cabinet, uncapped, and 200 μL of culture broth was immediately added to 3.8 mL of dd.H2O in a new cuvette. 345 μL of a solution containing 37.0 mM FeCl3 and 17.1 mM N,N-dimethyl-p-phenylenediamine sulfate dissolved in 6 M HCl was added to the cuvette. The cuvette was immediately capped and mixed by inversion once. The cuvette was left for 10 minutes to allow color development and the reaction to proceed to completion (4500-S2? SULPHIDE. in Standard Methods For the Examination of Water and Wastewater (American Public Health Association, (2017))). OD readings at 667 nm were then taken on a SPECTROstar Nano spectrophotometer (BMG Labtech) using MARS v4.01 for visualization and data output.

[0408] Data and statistical analysis

[0409] Data were exported from MARS in CSV format, and raw OD 667 nm values ​​were converted to sulfide concentrations (mM) by comparison to a standard curve of known sulfide concentrations in Microsoft Excel v2208. Transformed data were imported into Prism (GraphPad) for statistical analysis and data visualization. To determine whether microbial isolates showed a significant reduction in sulfide concentration after incubation compared to the negative control, a Welch t-test was performed with significance set at alpha 0.05 (Welch, BL The Generalization of 'Student's' Problem when Several Different Population Variances are Involved. Biometrika 34, 28-35 (1947)).

[0410] Frozen isolates were thawed at 37°C in an anaerobic cabinet, diluted 1:10 in pre-reduced YCFA broth, and incubated overnight. In triplicate, 100 μL of the overnight culture was used to inoculate 3.85 mL of modified YCFA broth (lacking L-cysteine) in a sterile cuvette spiked with 0.1% L-serine.

[0411] result

[0412] A total of 275 purified microbial isolates marked as candidates for inclusion in the BB265 complex consortium were phenotypically screened for sulfide consumption (Figure 17). From these, 154 isolates were identified that had an average net reduction in soluble sulfide concentration in modified YCFA broth after 48 hours of anaerobic incubation with 2 mM NaS (Table 1). Of these, 87 isolates showed a statistically significant reduction in sulfide concentration compared to the negative control (Table 1, Figure 9). Taxa that were significantly different from the negative control included species of the genera Alisipes, Anaerobutyricum, Anaerofustis, Bacteroides, Blautia, Christensenella, Clostridium, Collinsella, Coprobacillus, Coprococcus, Erysipelatoclostridium, Eubacterium, Flavonifractor, Intestinimonas, Longicatena, Massilimicrobiota, Parabacteroides, Roseburia, Ruminococcus, Streptococcus, and Thomasclavelia. Among these taxa, the mean net reduction in sulfide concentration ranged from 41 μM sulfide (± 4 SEM) in isolate bb0284: Bacteroides uniformis to 697 μM sulfide (± 55 SEM) in isolate bb0627: Flavonifractor plautii.

[0413] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0414] *Indicates statistically significant sulfide consumption

[0415] Consideration

[0416] Sulfide is a cytotoxic metabolite overproduced by the resident colonic microbiota in patients with inflammatory bowel disease. In this experiment, sulfide was measured as sulfide ions (S 2- ), hydrogen sulfide (HS - ) ion, bisulfide ion (SH - Microbiota-derived sulfides, in combination with nitric oxide, play a direct role in the pathogenesis of ulcerative colitis (UC). These gases cause CoA sequestration, glutathione depletion, and impaired energy production by colonocytes (colonic epithelial cells) via butyrate β-oxidation. This biochemical lesion in UC leads to an energy-depleted state of colonocytes, contributing to the loss of epithelial barrier function and subsequent mucosal inflammation.

[0417] Variations in the fermentable dietary proteins and carbohydrates reaching the colon affect the production and detoxification of nitrogen-containing metabolites and sulfide by the gut microbiota. The modern Western dietary pattern observed in patients with ulcerative colitis creates a colonic environment likely permissive for the production of nitrogen-containing metabolites and excess sulfide. Intake of inorganic sulfur (sulfate / sulfite), nitrate / nitrite, and, to a lesser extent, endogenous carbon substrates also contribute to colonic sulfide accumulation. Measured anionic sulfide levels in the colon are elevated in ulcerative colitis compared with healthy controls. Additionally, sulfide production capacity in fecal samples has been found to be 3-4 times higher in ulcerative colitis patients than in control cases. This is likely due to the relatively high levels of sulfide-producing microbiota, including SRBs and cysteine-degrading bacteria, in patients with inflammatory bowel disease in both active and dormant disease states. Nitric oxide is produced by both colonocytes and the colonic microbiota under inflamed conditions.

[0418] Fecal microbiota transplantation (FMT) has shown efficacy in inducing remission of ulcerative colitis, indicating that the colonic microbiota is involved in the pathogenesis of the disease and that modulation of the colonic microbiota may alleviate the disease. Given the evidence supporting sulfide as a pathological factor, modulation of the colonic microbiome in a manner that reduces colonic sulfide levels provides a promising therapeutic target.

[0419] In this study, we identified and then phenotyped candidate microbial isolates for inclusion in BB265 that possess the ability to consume sulfide and, therefore, reduce colonic sulfide levels when introduced into a host. We identified 154 microbial isolates that, when incubated for 48 hours in the presence of 2 mM sulfide, exhibited reduced concentrations of soluble sulfide compared to the control (Table 1). Although performed in vitro, our results demonstrated significant reductions in sulfide at concentrations within the physiological range of intestinal luminal contents (0.3-3.4 mM), supporting the ability of BB265 to reduce sulfide to concentrations equivalent to those required in vivo. The majority of these isolates that were significantly different from the negative control included taxa commonly found to be reduced in individuals with ulcerative colitis, including, but not limited to, isolates from the genera Bacteroides, Christensenella, Clostridium, and Roseburia. This suggests that microbiota capable of consuming sulfide may be reduced in ulcerative colitis, and therefore, methods that increase their abundance or metabolic activity in the colon may reduce colonic sulfide, allowing colonocytes to effectively respire, produce energy, and maintain the mucosal barrier. In this way, these microorganisms may alleviate the sulfide- and nitric oxide-induced metabolic lesions that accompany active inflammation in ulcerative colitis.

[0420] The isolates identified in this study offer therapeutic potential by consuming or biotransforming sulfide, either metabolically or otherwise, thereby reducing sulfide levels in the colon.

[0421] Selection of the final consortium that will comprise BB265 The final list of isolates comprising the BB265 consortium consisted of 143 microbial isolates (Table 2). The BB265 composite consortium list is presented using the following taxonomic classification: NB: Due to the ephemeral nature of taxonomic names, taxon identifiers are also provided for each isolate in the BB265 consortium, including isolates not included in the BB265 consortium but identified as having a sulfide-consuming phenotype (Figure 5). All past, present, and future taxon names describing each species are covered here, including all homotypic, heterotypic, subjective, objective, nomenclatural, and invalid synonyms, as well as misused names.

[0422] With reference to the results below, * denotes taxa identified in the analysis considered for inclusion in BB265 but not present in the BB265 combined consortium

[0423] By domain: *Archaea and Bacteria

[0424] [Table 2]

[0425] [Table 3-1] [Table 3-2]

[0426] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0427] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0428] D Example 4 Identification of the genomic signature of the BB265 complex community probiotic biotherapeutic formulation putatively involved in the regulation of colonic sulfide Research objectives To identify genes and gene clusters present in the genomes of isolates comprising the BB265 complex consortium that are putatively involved in the regulation of colonic sulfide.

[0429] A list of genes involved in sulfide metabolism was prepared based on the literature (Wolf, PG et al. Diversity and distribution of sulfur metabolism in the human gut microbiome and its association with colorectal cancer. bioRxiv 2021.07.01.450790 (2021) doi:10.1101 / 2021.07.01.450790). Genome assemblies of BB265 consortium members were annotated using Prokka v1.14.6 to identify existing and putative genes based on DNA sequence (Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068-2069 (2014)). The resulting list of identified genes was then queried for genes known or suspected to be involved in sulfide metabolism.

[0430] [Table 6-1] [Table 6-2]

[0431] [Table 7]

[0432] E Example 5 Mechanistic validation of BB265 for the treatment of ulcerative colitis by modulating colonic sulfide Research objectives Mechanistic validation of the BB265 complex consortium for the treatment of ulcerative colitis by modulating colonic sulfide Materials, methods, and results F. Preparation of BB265 complex consortium as inoculum for mechanistic validation experiments

[0433] A BB265 complex consortium was prepared as a mixed inoculum to be used for validation experiments. Two mixed consortium inocula were prepared: (1) one containing a subset of the 127 isolates present in the final BB265 complex consortium (Table 8). This subset was prepared as a validation experiment and was obtained before completing screening of the remaining 16 isolates according to Section C. Example 3—Definition of Organisms Constituting the BB265 Complex Community-Live Biotherapeutic Formulation. The BB265 complex consortium was modified to include these 16 isolates, as they were considered therapeutic candidates after screening. (2) The entire BB265 complex consortium (Table 5) contained the 143 isolates specified in Section C. Example 3—Definition of Organisms Constituting the BB265 Complex Community-Live Biotherapeutic Formulation.

[0434] Isolates containing the inoculum were grown anaerobically overnight at 37°C in a Whitley A55 HEPA Anaerobic Workstation (Don Whitley Scientific) (atmosphere: 10% H2, 10% CO2, 80% N2) by inoculating 2.94 mL of modified YCFA broth with 30 μL of a thawed glycerol stock of the purified isolate. For slow growers, inoculation was performed the day before and incubated for 48 hours. The culture broth was then diluted 1:1 in a Costar 48-well microplate to a final volume of 500 μL, and the diluted cultures were then read in a Spectrostar Nano spectrophotometer to determine the OD600nm value. The cultures were then individually diluted with modified YCFA to achieve an OD600nm of 0.2 for each isolate. Equal volumes of the diluted culture broth were then combined, diluted 1:1 in 50% glycerol (final concentration 25%), and frozen at -80°C. The total cell concentration of the mixed inoculum was then determined by quantifying the intact cell count using a BactoBox® (SBT Instruments) according to the manufacturer's instructions. Using OD 600 nm as an approximation of cell number, the cell concentration per isolate was determined by dividing the total cell number by the number of isolates in the inoculum, assuming equal abundance of each isolate present in the mixed inoculum. For slow-growing isolates that did not reach an OD 600 nm value of 0.2, glycerol stocks were prepared as described above, and the intact cell concentration per isolate was determined using a BactoBox® (SBT Instruments) according to the manufacturer's instructions. When performing validation experiments, the slow-growing isolates were combined with the inoculum in a volume adjusted to ensure equal cell concentrations of all isolates in the inoculum. These cell number-normalized mixtures were then used as inoculum for proceeding with validation experiments.

[0435] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0436] Putative involvement of taxa comprising the BB265 complex consortium in the resolution of ulcerative colitis disease - the FMT study

[0437] background Ulcerative colitis is a chronic inflammatory bowel disease (IBD) resulting from an abnormal immune response that leads to inflammation and ulceration of the colon and rectum. Recent advances in the study of the human gut microbiota have demonstrated a relationship between intestinal dysbiosis and the onset and progression of ulcerative colitis symptoms. One aspect of intestinal dysbiosis in ulcerative colitis patients is an imbalance in sulfide homeostasis, and excessive levels of sulfide may contribute to the pathogenesis of the disease. Targeting sulfide production in the intestine may be a potential therapeutic strategy for ulcerative colitis. Increasing clinical evidence suggests that fecal microbiota transplantation (FMT) can restore the gut microbiota and induce remission in ulcerative colitis patients. This study aimed to determine the correlation between in vitro sulfide production and the abundance levels of the BB265 complex consortium isolate in ulcerative colitis patients undergoing FMT intervention. The results indicate the putative involvement of the FMT-derived BB265 complex consortium in sulfide reduction and disease remission in ulcerative colitis patients.

[0438] method

[0439] Clinical trials

[0440] A randomized, controlled trial of FMT in adults with active, mild-to-moderate ulcerative colitis symptoms (Mayo score 3-10) was conducted from April 2019 to March 2020. All participants were 18 years of age or older and provided written informed consent to participate. The trial consisted of two phases: Phase 1 was a 12-week, open-label induction of ulcerative colitis remission, while Phase 2 was a 40-week, open-label remission maintenance study. During Phase 1, 35 eligible patients received tapered doses of oral prednisolone, starting at 50 mg daily and tapering to 0 mg over 8 weeks. At the beginning of week 5, patients received a 3-liter polyethylene glycol bowel preparation and 4 mg of loperamide orally over 60 minutes before 200 mL of FMT via colonoscopy. This was followed by two 50 mL fecal enemas administered intrarectally via syringe within seven days of receiving the first FMT (i.e., on days 3 or 4 and 6 or 7). Four mg of loperamide was given orally 30 minutes before administration of the enema. The retention enema was of the same composition as the colonoscopic remission-induction FMT. At week 12, evaluation was performed. Only 22 patients who demonstrated clinical and endoscopic remission (Mayo score of 2 or less, with no individual subscores greater than 1) or clinical response (a total Mayo score reduction of 30% or more, a reduction of 3 or more points, and a rectal bleeding score reduction of 1 or more, or a rectal bleeding score of 0 or 1) entered the maintenance phase of phase 2, where patients received the FMT blend, using the same composition derived from the same donor used in phase 1. These participants were randomized 1:1 in an open-label fashion to receive 50 mL FMT enemas either 4 times per week (i.e., 13 patients) or 8 times per week (i.e., 9 patients) for a total of 40 weeks. Randomization was performed using an atmospheric noise randomization algorithm (www.random.org).Maintenance of clinical remission or response over 40 weeks was assessed by four criteria: (i) time to flare (measured in weeks to SSCAI > 4), (ii) number of weeks in flare (number of weeks SSCAI > 4), (iii) SSCAI < 3 at weeks 28 and 50, and (iv) total Mayo score at week 50. Patients from phase I who did not achieve remission or response continued to provide stool samples every 4 weeks for an additional 40 weeks.

[0441] Metagenomic sequencing and analysis

[0442] Genomic DNA was extracted from patient fecal samples using the FastDNA™ SPIN Kit for Soil DNA Extraction Kit (MPBio) according to the manufacturer's instructions. DNA quality was assessed using a NanoDrop 2000c UV-vis spectrophotometer (Thermo Fisher, Waltham, MA, USA), and the DNA was subjected to metagenomic sequencing. DNA libraries were prepared using the Celero EZ™ DNA-Seq kit, protocol M01526 v1, and sequenced on an Illumina NextSeq2000 machine (109 bp PE) using Illumina protocol 1000000109376 v3. Metagenomes were quality trimmed, adapter sequences were removed with Trimmomatic v0.39, leading and trailing bases with a quality score less than 3 were removed, and reads shorter than 50 base pairs were removed. Potential contaminants from human reads were removed by mapping them to the human reference genome (GRCh38) using bowtie v2.5.1 and removing reads aligned to the human genome. Taxonomic assignment was performed for each metagenome using MetaPhlAn4 (v4.0.6) with default parameters and the default MetaPhlAn4 database (vmpa_vOct22_CHOCOPhlAnSGB_202212). MetaPhlAn4 output was integrated and filtered for species-level results, and the resulting data were used to identify taxonomic changes between samples from individuals who entered clinical and endoscopic remission, as defined above, and those who remained in active disease at the end of Phase 1.

[0443] The MetaPhlAn4 output for species was filtered for baseline and remission samples from patients D (week 0 and 52 samples) and E (week 0 and 12 samples). BB265 species names identified by BLAST comparison to the NCBI RefSeq 16S rRNA database were queried by species name against the species taxa in the MetaPhlAn4 output. Through taxon name matches and the number of matches, the cumulative abundance of BB265 species in the metagenome was calculated and compared between baseline and remission samples. This was repeated for species identified as phenotypically statistically significant consumers (Table 1).

[0444] A list of sulfide-producing species was generated by identifying species within which greater than 70% of isolates were phenotyped as sulfide producers (Figure 11). Any BB265 species were removed from the list of sulfide-producing species, and the resulting species list was queried against the MetaPhlAn output for patients D and E, and the number of species matches and cumulative species abundance were compared between baseline and remission samples.

[0445] To examine the abundance and number of species of BB265, consumers, and producers across all patients in the clinical trial, the species MetaPhlAn4 output was filtered for all patient Phase 1 samples, baseline (Week 0) and end of Phase 1 (Week 12). The number and cumulative abundance of BB265, statistically significant consumer species determined by phenotype, and sulfidogens, as defined above, were quantified at Weeks 0 and 12, and patients who entered clinical and endoscopic remission were compared with those who did not. For each group, statistically significant differences between Weeks 0 and 12 were determined using the Wilcoxon test with Bonferroni correction. Significance was determined as an adjusted p-value of less than 0.05.

[0446] Determination of sulfide levels in stool samples at baseline and at the time of remission Preparation of fecal slurry

[0447] Stool samples obtained from patients who demonstrated clinical response and clinical remission based on clinical trial criteria were selected for sulfide determination. For each patient, both a baseline sample (week 0, total Mayo score 3–10) and a remission sample (week 12 or 52, total Mayo score 0–2) were used in the experiment. Briefly, for all baseline and remission samples, a 10% fecal slurry was prepared by adding 1.25 g of stool to 12.5 mL of half-strength YCFA and Solution I. The mixture was mixed by vortexing for 5 minutes and briefly centrifuged. 2.5 mL of the supernatant was transferred to a gas chromatography (GC) vial containing 2.5 mL of either half-strength YCFA or Solution I supplemented with sodium sulfite as an inorganic sulfur source, resulting in a final fecal slurry concentration of 5%. The GC vial was immediately capped and anaerobically incubated overnight at 37°C. All experiments were performed in triplicate. Half-strength YCFA was used in this case as a complex medium providing adequate nutrients for the growth of isolates in the BB265 complex consortium. Making the medium half-strength limited the total carbon and sulfur pools, ensuring that sulfide was not overproduced and allowing accurate spectrophotometric quantification. In contrast, Solution I is a minimal medium that provides a low dose of a complex carbon source as the sole nutrient source to initiate fermentation while minimizing medium complexity to maintain the metabolic environment present in ulcerative colitis stool. Solution I consists of the vitamin and mineral components of YCFA with only 5 g / L tryptone and 1.25 g / L yeast extract.

[0448] Modified methylene blue assay for the determination of sulfide

[0449] The GC vial was removed from the anaerobic chamber, and 200 μL of the mixture was transferred to a cuvette containing 3.8 mL of RO water. 345 μL of a solution containing 37.0 mM FeCl3 and 17.1 mM N,N-dimethyl-p-phenylenediamine sulfate dissolved in 6 M HCl was added to the cuvette, which was immediately capped and mixed by inversion once. The cuvette was left for 10 minutes to allow color development and the reaction to proceed to completion. 1 mL of the sample was transferred to an Eppendorf tube and centrifuged at 15,000 × g for 5 minutes. 750 μL of the supernatant was then transferred to a 48-well microplate, and OD readings at 667 nm were then obtained using a SPECTROstar Nano spectrophotometer (BMG Labtech), with MARS v4.01 used for visualization and data output.

[0450] result

[0451] Metagenomic analysis from a phase 1 clinical trial dataset identified that patients who entered remission (clinical remission) had a significant increase in the number of BB265 species from week 0 to week 12. For patients who did not enter remission, the number of BB265 species did not change significantly between baseline and remission samples (Figure 12a). Patients who did not enter clinical remission also had a significant decrease in the abundance of sulfide consumers from baseline to week 12 (Figure 12b).

[0452] Additionally, there was an increase in the number of consuming species in patients who entered clinical remission from baseline to week 12 (Figure 12c). A reduction in water-soluble sulfide after incubation of fecal slurries obtained from ulcerative colitis patients was observed in fecal samples collected during remission compared with fecal samples obtained from the same patients with active disease. For patient D, the concentration of water-soluble sulfide in half-strength YCFA and Solution I containing sodium sulfite was reduced from 2355.1 μM (SD ± 37) at week 0 to 1529.4 μM (SD ± 124.4) at week 12 and from 563.5 μM (SD ± 23.2) at week 0 to 421.6 μM (SD ± 14.3) at week 12, respectively (Figure 13a). For patient E, the concentration of water-soluble sulfide in half-strength YCFA and Solution I containing sodium sulfite was reduced from 463.0 μM (SD ± 19.8) (week 0) to 73.5 μM (SD ± 1.6) (week 12) and from 332.3 μM (SD ± 6.2) (week 0) to 272.2 μM (SD ± 3.6) (week 12), respectively (Figure 13b).

[0453] To determine the correlation of phenotypically measured sulfide reduction in patients D and E from baseline to remission, the metagenomic profiles of patients D and E were examined for BB265, sulfide-consuming, and sulfide-producing species. An increase in the number of BB265 species was also identified in patient E from baseline (active) to remission samples, to a lesser extent than in patient D (Figure 13c). Similarly, there was an increase in sulfide-consuming species from baseline (active) to remission in both patients (Figure 13d). There was also a significant decrease in the cumulative relative abundance of sulfidogens, supporting the finding of a decrease in sulfide-producing capacity in these samples (Figure 13e).

[0454] Consideration

[0455] Analysis of metagenomes obtained from patients in Phase I of clinical trials supported the assertion that the BB265 species is an important gut microbiota in inducing remission in ulcerative colitis. The numbers of BB265 and sulfide-consuming species significantly increased from baseline to week 12 in patients who entered remission. Additionally, the statistically significant decrease in the relative abundance of consuming species in patients who did not enter remission also supports isolates identified as sulfide-consuming species as potential therapeutic agents for the treatment of ulcerative colitis.

[0456] The results of the modified methylene blue assay directly correlated with clinical data, with a reduction in water-soluble sulfide in vitro corresponding to a decrease in the total Mayo score in clinical observations. Specifically, for patient D, the total Mayo score was reduced from 3 at week 0 to 0 at week 52, and the modified methylene blue assay test demonstrated a significant decrease in water-soluble sulfide levels in clinical samples from week 0 to week 52. In addition, this correlation was also observed for patient E, whose total Mayo score was reduced from 8 at week 0 to 0 at week 12. This corresponded to high sulfide levels in the clinical samples at week 0 and significantly reduced hydrogen sulfide levels in the samples at week 12, when the patient achieved clinical remission.

[0457] The paired observations of (1) an increase in the number of BB265-associated and sulfide-consuming taxa and a decrease in the cumulative relative abundance of putative sulfidogens in patients who successfully entered remission due to FMT, and (2) a significant reduction in the sulfide-producing capacity of the ulcerative colitis stool community after FMT in patients who entered clinical remission, strongly support the BB265 consortium as a treatment for ulcerative colitis and further support the mechanism of action of this treatment to reduce sulfide as a means to improve clinical prognosis.

[0458] Sulfide reduction by the BB265 complex consortium when co-cultured with ulcerative colitis fecal slurry background

[0459] The BB265 complex consortium is composed of a diverse array of metabolically active bacteria found in the healthy human gut microbiome. These bacteria likely play a pivotal role in the human intestinal sulfur pathway, contributing in diverse ways, for example, to metabolizing sulfur substrates, redirecting sulfide to various metabolites, and effectively consuming sulfide.

[0460] In previous studies, we phenotypically identified specific healthy gut-associated microorganisms capable of consuming sulfide when present as individual organisms. However, these microorganisms exist as part of complex microbial communities within the gut microbiome, functioning symbiotically with other bacteria in the gut to perform essential functions. Thus, as a community, these microorganisms may enhance the sulfide-consuming capabilities we observed at the individual level and provide the metabolic and ecological complexity necessary for a healthy state. Therefore, we sought to investigate whether the BB265 complex consortium could promote sulfide reduction as a co-culture.

[0461] E.2.3.1 Co-culture with a subset of the BB265 complex consortium reduces water-soluble sulfide in ulcerative colitis fecal samples compared to a phylogenetically diverse consumer community

[0462] method

[0463] In vitro assays

[0464] A bacterial consortium containing 127 of the 143 isolates in the BB265 complex consortium was grown anaerobically overnight at 37 °C by inoculating 50 μL of cell number-normalized glycerol stock into 20 mL of YCFA (Table 8).

[0465] A phylogenetically diverse consumer mix (PDCM) (Table 9), consisting of 19 phylogenetically diverse isolates each identified as consuming sulfide (ranging from 20 to 670 μM) and confirmed negative for sulfide production, was run in parallel with a subset of 127 isolates from the BB265 complex consortium. The PDCM was run in parallel with a subset of 127 isolates from the BB265 complex consortium, which itself contains numerous healthy-associated sulfidogens, to assess the community effect of sulfide consumption by the consumer community.

[0466] A 50 μL glycerol stock of a subset of 127 isolates from the cell number-normalized PDCM and BB265 complex consortium was inoculated into a 20 mL pre-reduced YCFA tube and incubated anaerobically at 37°C for 24 hours. After incubation, the culture was pelleted by centrifugation at 12,000 × g for 10 minutes. The pellet was washed three times with 50 mL of pre-reduced PBS to remove residual medium and resuspended in 20 mL of pre-reduced Solution I.

[0467] Stool samples from two patients with ulcerative colitis, referred to herein as Patient A and Patient B, were used in this study. A 16% (wt / vol) fecal slurry was prepared by placing 8 g of stool into a Falcon tube and adding 50 mL of Solution I. The sample was vortexed for 5 minutes and gently pulsed at a maximum of 100 rpm to pellet large particles. 2.5 mL of the supernatant was transferred to a Hungate tube containing 2.5 mL of Solution I, resulting in a final fecal slurry concentration of 8% (wt / vol). Five fecal slurry replicates per treatment were prepared and spiked with 10 μL of inoculum: a subset of 127 isolates of BB265, PDCM, and a negative control containing sterile medium alone. The Hungate tubes were immediately capped to avoid loss of volatile HS and incubated anaerobically at 37°C for 24 hours. After incubation, 1 mL of each treatment was aliquoted into a sterile 1.5 mL microcentrifuge tube and stored at -80°C prior to DNA extraction. DNA extraction was performed using the FastDNA™ SPIN Kit for Soil (MPBio) DNA extraction kit, and DNA quality was assessed using a NanoDrop 2000c UV-vis spectrophotometer (Thermo Fisher, Waltham, MA, USA). After DNA extraction, the extracts were used for shotgun metagenomic sequencing.

[0468] Metagenomic sequencing

[0469] DNA libraries were prepared using the Celero EZ™ DNA-Seq kit, protocol M01526 v1, and sequenced on an Illumina NextSeq2000 machine (109 bp PE) using Illumina protocol 1000000109376 v3. Reads were processed using an in-house developed custom pipeline that uses Trimmomatic v0.39 to remove adapters and low-quality bases, Bowtie2 v2.5.1 to align reads to the human genome for removal, and Metaphlan v4.0.6 to quantify the abundance of taxa present in each sample.

[0470] [Table 9]

[0471] Modified methylene blue assay

[0472] The Hungate tube was removed from the anaerobic chamber, and 200 μL of the mixture was transferred to a cuvette containing 3.8 mL of RO water. 345 μL of a solution containing 37.0 mM FeCl3 and 17.1 mM N,N-dimethyl-p-phenylenediamine sulfate dissolved in 6 M HCl was added to the cuvette. The cuvette was immediately capped and mixed by inversion once. The cuvette was left for 10 minutes to allow color development and the reaction to proceed to completion. An aliquot (1 mL) from the color-reacted suspension was centrifuged at 12,000 × g for 5 minutes, and an OD reading of the supernatant at 667 nm was then obtained on a SPECTROstar Nano spectrophotometer (BMG Labtech), using MARS v4.01 for visualization and data output.

[0473] statistical analysis

[0474] One-way anovas were performed to identify significant differences in sulfide consumption between treatment groups (stool only, stool plus a subset of 127 isolates of the BB265 complex consortium, PDCM) within patients.

[0475] result

[0476] For patient A, when fecal slurry was co-cultured with a subset of 127 isolates from the BB265 complex consortium, a mean reduction in water-soluble sulfide of 44.5 μM (±6.8 SD) was determined, a significant reduction in sulfide concentration compared to the negative control (fecal slurry only). When co-cultured with PDCM, a significant reduction was also determined compared to the negative control, with a mean net reduction in sulfide of 24 μM (±4.5 SD). The subset of 127 isolates from the BB265 complex consortium performed significantly better than PDCM for sulfide reduction in patient A (FIG. 14).

[0477] For patient B, when fecal slurry was co-cultured with a subset of 127 isolates from the BB265 complex consortium, a mean reduction in water-soluble sulfide of 23.4 μM (±4.7 SD) was determined, a significant decrease in sulfide concentration compared to the negative control (fecal slurry only). When co-cultured with PDCM, a significant reduction was also determined compared to the negative control, with a mean net reduction of sulfide of 41.4 μM (±8.0 SD). In contrast to patient A, PDCM performed significantly better than the subset of 127 isolates from the BB265 complex consortium, consuming an average of 18 μM more sulfide (Figure 14).

[0478] Metagenomic analysis of stool samples incubated with and without a subset of the 127 isolates from the BB265 complex consortium revealed clear changes in microbial signatures due to the addition of the consortium. Patient A had significantly fewer taxa unique to their stool samples than patient B (Figure 23c). Regardless, when stool was cocultured with a subset of the 127 isolates from the BB265 complex consortium, there was an increase in both the number and relative abundance of taxa comprising BB265 in both samples after incubation (Figure 23c). This increase in BB265 taxa was paralleled by an increase in the number of sulfide-consuming taxa after incubation (Figure 23b). A parallel increase in the relative abundance of sulfide-consuming taxa was observed in patient A, while patient B showed the opposite trend, with a decrease in the total relative abundance of the community attributed to sulfide-consuming taxa. In neither patient did the BB265 complex consortium have an effect on the number of sulfidogenic taxa identified; the number of sulfidogenic taxa was comparable when stool alone was compared to stool plus the subset of 127 isolates from the BB265 complex consortium (Fig. 23a). However, although the number of sulfidogens remained the same, the addition of the subset of 127 isolates from the BB265 complex consortium resulted in a decrease in the relative abundance of sulfidogenic taxa in both patients (Fig. 23a).

[0479] Consideration

[0480] In both patients A and B, when stool samples were cocultured with a subset of the 127 isolates from the BB265 complex consortium, the number of sulfidogenic taxa remained unchanged compared to stool-only samples; however, the cumulative relative abundance of these producers decreased (Fig. 23a). This was similar to the results observed in the clinical data of patients D and E, who also showed a decrease in the cumulative relative abundance of sulfidogens (Fig. 13e). This supports the hypothesis that the BB265 consortium acts mechanistically similarly to induce remission by FMT in ulcerative colitis. Furthermore, this suggests that the subset of 127 isolates from the BB265 consortium effectively reduced the dominance of sulfidogens without reducing their diversity. Although the total number of sulfidogenic taxa remained unchanged, sulfide measurements showed a significant reduction compared to controls (Fig. 14). This suggests that the deleterious effects of excess sulfide in ulcerative colitis can be targeted by modulating the abundance of sulfidogens rather than reducing their diversity. In this regard, modulation of abundance may allow for treatment while maintaining the biodiversity of communities identified as important components of the healthy microbiome.

[0481] These results demonstrate that co-culturing ulcerative colitis stool with a subset of the 127 isolates of the BB265 complex consortium significantly reduced sulfide. Notably, in the case of patient A, there was a higher sulfide reduction compared to co-culture with PDCM, which consisted of 19 isolates that showed high net sulfide reduction in the consumer assay alone and were not composed of sulfidogens (Figure 14). These findings suggest that additional species within the subset of 127 isolates of the BB265 complex consortium may enhance the phenotypic effects of sulfide consumers in the consortium by modulating the complex community dynamics. However, in patient B, PDCM demonstrated a higher sulfide reduction than the subset of 127 isolates of the BB265 complex consortium, suggesting that this enhancement may be patient-specific (Figure 14).

[0482] Along with the ability to reduce colonic sulfide in ulcerative colitis by modulating the cumulative relative abundance of sulfidogens, a parallel increase in the number and cumulative relative abundance of sulfide-consuming taxa was observed when stool was cocultured with a subset of the 127 isolates of the BB265 complex consortium (Figure 23b). This suggests that the BB265 consortium may also act to reduce sulfide in ulcerative colitis by altering the ratio and abundance of sulfide-producing taxa relative to sulfide-consuming taxa, resulting in lower production and higher consumption of sulfide by the community at any given time point. These results provide strong evidence that the microbiota, including the BB265 complex consortium, has therapeutic potential in ulcerative colitis by modulating the dynamics of both sulfide production and consumption, resulting in a net reduction in sulfide levels when these taxa are present in fecal samples from ulcerative colitis patients.

[0483] E.2.3.2 Co-culture with a subset of the BB265 complex consortium reduces water-soluble sulfide in ulcerative colitis fecal samples compared to individual potent sulfide consumers

[0484] To further support the idea that community complexity may potentiate the effects of sulfide reduction in ulcerative colitis, we evaluated how a subset of 127 isolates from the BB265 complex consortium compares their sulfide-reducing ability to a single isolate individually identified as one of the most potent sulfide consumers.

[0485] method

[0486] In vitro assays

[0487] In this study, bb0214 Ruminococcus bicirculans, which alone showed an average net reduction of 670 μM sulfide when assayed for consumption, was selected as a potent consumer. A subset of 127 isolates from the cell-number-normalized BB265 complex community and a glycerol stock (50 μL) of bb0214 were inoculated into 20 mL of YCFA and incubated at 37°C for 24 h.

[0488] For ulcerative colitis stool (Patient A), 32 g was dissolved in 50 mL of Solution I. The sample was vortexed for 5 minutes and briefly pulsed at 100 rpm to pellet large particles, and the supernatant was transferred to a sterile 250 mL Schott bottle. A subset of 127 isolates of the BB265 complex consortium and bb0214, grown for 24 hours, was centrifuged at 12,000 × g for 10 minutes. After decanting the supernatant, the pellet was washed three times with 50 mL of sterile PBS. The final pellet was dissolved in 20 mL of sterile Solution I and vortexed until homogenized. The stool suspension (2.5 mL) was transferred to a sterile glass vial containing 2.5 mL of sterile Solution I to obtain a final concentration of 8% (weight / volume) stool suspension. A subset of 127 isolates from the BB265 complex consortium and bb0214 (100 μL) were inoculated into quintuplicate fecal slurry (100 μL), and 100 μL of sterile medium was inoculated into five replicate fecal slurry samples as a negative control. Vials were incubated at 37°C for 24 hours, and a modified methylene blue assay was performed to measure endpoint sulfide as described above. After incubation, 1 mL from each treatment condition was aliquoted into sterile 1.5 mL microcentrifuge tubes and stored at -80°C. DNA extraction was then performed using the FastDNA™ SPIN Kit for Soil (MPBio) DNA extraction kit, and DNA quality was assessed using a NanoDrop 2000c UV-vis spectrophotometer (Thermo Fisher, Waltham, MA, USA). The DNA extracts were then used to generate metagenomic sequencing datasets for each sample according to the methods described above.

[0489] statistical analysis

[0490] A one-way anova was performed to identify significant differences in sulfide consumption between treatment groups (stool only, stool plus a subset of 127 isolates of the BB265 complex consortium, and stool plus bb0214 consumers).

[0491] result

[0492] When fecal slurry obtained from patient A was incubated with the potent consumer bb0214 alone, a mean net reduction in sulfide of 7.4 μM (±8.4 SD) was observed compared to the negative control (stool only). When incubated with a subset of 127 isolates of the BB265 complex consortium, a mean net reduction in sulfide of 18.7 μM (±3.2 SD) was observed, which was significantly higher than the single consumer alone (Figure 15).

[0493] Metagenomic analysis showed that when fecal slurries were incubated with a subset of 127 isolates from the BB265 complex consortium, there was a significant increase in the number of sulfide-consuming taxa compared to both the negative control (stool only) and stool incubated with bb0214. The number and cumulative abundance of sulfidogens showed a significant decrease in fecal slurries treated with the subset of 127 isolates, while no significant differences were observed when treated with bb0214 (Figures 24a and 24b).

[0494] Consideration

[0495] These results demonstrate that a subset of 127 isolates from the BB265 complex consortium exhibited superior sulfide reduction in ulcerative colitis stool compared to strong sulfide consumers alone in the same setting (Figure 15). Similar to the induction of remission by FMT observed in clinical samples, the subset of 127 isolates from the BB265 complex consortium simultaneously reduced the number and cumulative relative abundance of sulfidogens and increased the number of sulfide consumers in ulcerative colitis stool (Figures 24a and 24b), resulting in sulfide reduction in both cases (Figures 13e and 13d). This supports the idea that microorganisms within the BB265 complex consortium engage in cooperative ecological interactions that effectively enhance sulfide reduction in ulcerative colitis stool compared to individual strong consumers in the same environment. As a result, in this case, utilization of a subset of 127 isolates from the BB265 complex consortium provides a stronger phenotype for sulfide reduction in ulcerative colitis than potent consumers alone, while simultaneously providing the community complexity and diversity important for healthy conditions.

[0496] E.2.3.3 Reduction of water-soluble sulfide in ulcerative colitis fecal samples by the BB265 complex consortium compared with individual potent sulfide consumers

[0497] background

[0498] From previous experiments, we have determined that a subset of 127 isolates from the BB265 complex consortium exhibits a higher sulfide consumption capacity than the potent sulfide consumers identified by individual phenotypes. As a result, this requires us to expand our investigation to understand how the BB265 complex consortium, composed of 143 genomically and phenotypically screened healthy gut bacteria, helps reduce sulfide in ulcerative colitis stool. This complex consortium exhibits expanded taxonomic and metabolic diversity beyond that of the subset of 127 isolates, potentially contributing to a broader range of beneficial functions in the human intestine. Therefore, it is crucial to study their collective contribution in the context of treating ulcerative colitis patients.

[0499] method

[0500] In vitro assays

[0501] A cell number-normalized glycerol stock (0.5% v / v inoculum) of the BB265 complex consortium was used to inoculate a 2 L bioreactor containing 1 L of sterile YCFA for 24 h at 37°C. The bioreactor harvest was removed after 24 h and 20 mL of 25% (v / v) glycerol stock was prepared in a 50 mL Falcon tube by mixing 10 mL of the harvest with 10 mL of 50% (v / v) glycerol. The glycerol stock was frozen at -80°C.

[0502] In this study, bb0214 Ruminococcus bicirculans, which showed consumption of 670 μM sulfide in the consumer assay, was selected as a potent consumer. A glycerol stock of bb0214 (50 μL) was inoculated into 20 mL of YCFA and incubated anaerobically at 37°C for 24 hours.

[0503] A 20 mL glycerol stock of the BB265 complex consortium bioreactor harvest was brought to 50 mL with sterile pre-reduced PBS and immediately centrifuged at 12,000 × g for 6 minutes. Concurrently, overnight-grown bb0214 consumers were centrifuged at 12,000 × g for 10 minutes. After decanting the supernatant, both pellets were washed three times with 50 mL of sterile PBS. The BB265 complex consortium and bb0214 pellets were resuspended in 10 mL and 20 mL of Solution I, respectively, by vortexing.

[0504] Two ulcerative colitis stool samples (Patients A and C) were used in this study. 5% (wt / vol) and 2.5% (wt / vol) fecal slurries were prepared for Patients A and C, respectively. Different concentrations of fecal slurries were prepared for each donor to measure the appropriate sulfide, which was previously determined by assaying the fecal slurries for sulfide production at various concentrations using a modified methylene blue assay. Herein, 7 g of stool sample obtained from Patient A and 5 g of stool sample obtained from Patient C were dissolved in 70 mL and 100 mL of Pre-Reduced Solution I, respectively. Each sample was vortexed for 5 minutes and briefly pulsed at 100 rpm to pellet large particles. The supernatant was transferred to a sterile 250 mL Schott bottle. Stool suspensions (2.5 mL) were transferred to sterile glass vials containing 2.5 mL of sterile Solution I to prepare stool suspensions for patients A and C at final concentrations of 5% and 2.5% (weight / volume), respectively. The BB265 complex consortium and bb0214 (100 μL) were inoculated into the stool suspensions in quintuplicate. In addition, 100 μL of sterile medium was inoculated into five replicates as a control containing stool sample alone. Samples were incubated anaerobically at 37°C for 24 hours, and a modified methylene blue assay was performed to measure endpoint sulfide as described above.

[0505] statistical analysis

[0506] A one-way anova was performed to identify significant differences in sulfide consumption between treatment groups (stool only, stool plus BB265 combined consortium, stool plus bb0214 consumers) within patients.

[0507] result

[0508] After incubation, fecal slurries from patients A and C showed a mean net reduction in sulfide of 10.5 μM (±8.1 SD) and 12.7 μM (±3.6 SD) when incubated with the BB265 complex consortium, respectively. This was a significant reduction compared to the negative control (stool only) and when co-cultured with bb0214 (FIG. 16). When stool was co-cultured with bb0214 consumers, no significant reduction in sulfide was observed for either patient, with a reduction of 1.0 μM (±9.8 SD) and 1.7×10 for samples from patients A and C, respectively. -14 There was a mean net reduction in sulfide of 1 μM (±4.8 SD) (FIG. 16).

[0509] Consideration

[0510] These results revealed that BB265 exhibited significantly higher levels of sulfide reduction compared to the potent individual consumers in both patient samples, suggesting that the microbial isolates within BB265 engage in cooperative interactions to enhance sulfide-reducing pathways beyond what a single sulfide consumer could achieve alone.

[0511] E.2.3.4 Effect of the BB265 complex consortium and its most potent consumer on hydrogen sulfide production rate in ulcerative colitis stool

[0512] background

[0513] The modified methylene blue assay was used to measure total sulfide (HS, HS) in the aqueous phase of our bioreactor. - , and S 2-This method is a highly specific assay for detecting HS. This method allows for the collection of endpoint data when fermentation is complete and the sulfide concentration between the aqueous phase and headspace has equilibrated. However, it does not allow for real-time monitoring of HS production in bioreactors. This study aims to utilize a commercially available HS microsensor to monitor HS production in real time in a sealed bioreactor system, while simultaneously serving as a validation method for results confirmed using a modified methylene blue assay. Additionally, this study is important in elucidating the kinetics of HS production. A comprehensive understanding of HS production rate is crucial, as it allows for the examination of temporal release kinetics within the colon. Reducing HS production rate by this complex consortium emerges as a promising therapeutic approach for treating ulcerative colitis.

[0514] method

[0515] Calibration of Unisense Type II H2S Microsensor

[0516] The H2S sensor was connected to an fx-6 pA UniAmp amplifier and the SensorTrace Suite software was opened. A minimum 2-hour window was allowed for the sensor to prepolarize to 0 mV. This time window was shortened for future use based on the stability of the mV reading after 20 minutes. A solution of sterile, pure water and a standard pH buffer solution at pH 4 was purged with pure nitrogen gas for a minimum of 20 minutes to remove any dissolved oxygen in the solution. A 200 mM sodium sulfide solution was prepared in deoxygenated water by adding the required weight of sodium sulfide to the solution and stirring very slowly with the cap on to prevent any oxygenation of the solution. Six 10 mL glass vials were purged with nitrogen to remove any oxygen and sealed with butyl rubber stoppers. Six 10 mL aliquots of the deoxygenated pH 4 solution were aliquoted into the purged glass vials using a hypodermic needle pierced through the rubber stopper to reduce air contamination. 0, 1, and 2 mM sodium sulfide solutions were prepared in deoxygenated pH 4 solution by adding the appropriate amount of dissolved 200 mM solution and gently inverting the vial. The hypodermic needle of the sensor was inserted through the butyl stopper into the 0 mM calibration solution, and a reading was taken when the signal stabilized. This reading, along with the corresponding HS concentration, was entered into the software. This was repeated for the next higher concentration until all calibration points had been measured. The sensor was inserted into a sterile vial of pure water to remove any residual hydrogen sulfide. The millivolt (mV) signals corresponding to each concentration of the calibration points were then inserted into the curve, and the software was configured to generate a standard curve, thereby preparing for subsequent measurements.

[0517] Unisense Temperature Sensor Calibration

[0518] The temperature sensor was connected to an fx-6 pA UniAmp amplifier and the SensorTrace Suite software was opened. The temperature sensor was placed at known temperatures of 0°C, 22°C, and 81°C until the signal stabilized. These temperatures were determined against a calibrated alcohol thermometer. The millivolt (mV) signal for each temperature of the calibration points was then fitted into a curve and the software was configured to generate a standard curve, thereby preparing for subsequent measurements.

[0519] Sensor Experiment

[0520] The individual consumers used in this study were bb0214 Ruminococcus bicirculans and bb0450 Clostridium paraputrificum. A 20 mL glycerol stock of the BB265 complex consortium was immediately brought to 50 mL with sterile pre-reduced PBS and then centrifuged at 12,000 × g for 6 minutes. Concurrently, overnight-grown sulfide consumers were centrifuged at 12,000 × g for 10 minutes. After decanting the supernatant, all pellets were washed three times with 50 mL of sterile PBS. The final consumer pellet and the BB265 complex consortium were resuspended in 20 mL and 10 mL of sterile Solution I, respectively, and mixed by vortexing.

[0521] Stool samples obtained from two ulcerative colitis patients (Patients A and C) were used in this experiment. Additionally, stool samples obtained from screened healthy donors (Healthy Donors A and C) were used as negative controls in this study. 5% (weight / volume) stool samples were prepared for Patient A and Donor A, and 2.5% (weight / volume) stool samples were prepared for Patient C and Donor C. Different concentrations of fecal slurry were prepared for each donor to measure the appropriate sulfide, which was previously determined by assaying the fecal slurry for sulfide production at various concentrations using a modified methylene blue assay. Aliquots of 1 g and 0.5 g of stool from Patient A and Donor C, respectively, were homogenized in 10 mL of Solution I. Each sample was vortexed for 5 minutes and gently pulsed at 100 rpm to pellet large particles. Subsequently, 2.5 mL of supernatant from each sample was transferred to a sterile glass vial containing 2.5 mL of Solution I, resulting in final stool suspension concentrations of 5% and 2.5% (weight / volume) for patient A and donors C, respectively. Controls for HS measurements were established by adding 100 μL of sterile Solution I to each vial. Each vial was inoculated with the BB265 complex consortium and consumer in 100 μL aliquots. These prepared samples were subsequently used in the sensor experiments outlined below.

[0522] Sensor use: The H2S needle sensor and temperature sensor were connected to the amperometry and temperature channels of the Fx-6 UniAmp, respectively. The sensors were then initiated by scanning through the SensorTrace Logger software. All sensors were then fixedly placed in a 37°C incubator and left for several minutes until a stable signal was observed. The bottle containing the sample was pierced with the H2S needle sensor until the tip of the sensor was immersed in the fecal suspension, and incubated at 37°C. The recording time interval was adjusted to 300 seconds, and recordings were made while the samples were incubated at 37°C for 24 hours. After the incubation period, recording was stopped, and the data was saved as an .xls file.

[0523] result

[0524] In both cases, the patient stool sample alone produced the most HS. For experiments performed with patient A and donor A, the healthy donor produced the least amount of HS, with the bb0214 consumer performing better in this regard than the BB265 complex consortium (Figures 18 and 19). For experiments with patient C and donor C, stool co-cultured with the bb0450 consumer produced the least amount of sulfide, followed by the healthy donor and the ulcerative colitis stool co-cultured with the BB265 complex consortium (Figures 18 and 19).

[0525] When comparing the H2S production rates, stool samples from both patients showed higher H2S production rates compared to ulcerative colitis samples incubated with the BB265 complex consortium (Figures 20 and 21). The stools of patients A and C inoculated with BB265 produced approximately 30 μM H2S. -1 and at approximately 190 μM -1 and the maximum H2S production rate is approximately 40 μM / h, respectively. -1 and at approximately 250 μM -1This was lower than the stool sample alone, which had a maximum H2S production rate of 100 μM / s. All healthy donor samples had the lowest maximum H2S production rates, indicating that healthy donors have lower H2S production rates than ulcerative colitis patients. Regarding the H2S production rate in the human body, bb0214 produced approximately 43 μM / s in patient A. -1 bb0450 showed a significant improvement in the efficacy of BB0450 at approximately 200 μM in patient C. -1 (Figures 20 and 21).

[0526] Consideration

[0527] These findings reveal that stool samples obtained from individuals diagnosed with ulcerative colitis exhibit significantly elevated H2S production rates. Importantly, the data demonstrate a simultaneous reduction in H2S production rate and concentration upon introduction of the BB265 complex consortium, consistent with the results of a modified methylene blue assay performed on these specific patient samples incubated alone and with the BB265 complex consortium. The distinct behavior observed for individual H2S consumers within both patient stool samples confirms the various phenotypic responses to H2S changes when these single isolates interact with the diverse bacterial populations present in ulcerative colitis patients. As a result, the use of individual H2S consumers may not be able to consistently promote H2S reduction in some ulcerative colitis patients, as their efficacy appears to depend on inter-patient variability. In contrast, the BB265 complex consortium consistently exhibited the same phenotypic response in both samples, highlighting its beneficial role in supporting H2S reduction within the context of ulcerative colitis stool. These results highlight the pivotal role played by the BB265 complex consortium in the spectrum of ulcerative colitis treatment. The BB265 consortium demonstrates its ability to substantially reduce the rate of H2S production, a crucial factor within the colonic environment of ulcerative colitis patients. This reduction in the total amount and rate of H2S, by effectively limiting the concentration of H2S produced at any given time, holds significant therapeutic promise and is likely to provide substantial benefit to individuals suffering from both ulcerative colitis and IBD.

[0528] Reduction of nitrite produced by ulcerative colitis fecal communities when co-cultured with G BB265 complex consortium

[0529] background Nitric oxide (NO), along with sulfide, has been implicated in the pathogenesis of ulcerative colitis. NO is a free radical endogenously produced by mammalian cells, e.g., colonocytes, that acts as a vasotransmitter with important physiological functions. In the context of ulcerative colitis, intraluminal NO is increased and, together with sulfide, has been observed to act to inhibit β-oxidation and deplete cellular energy in colonocytes (Figure 1).

[0530] In the anaerobic environment of the colon, certain bacteria can convert nitrates (NO3 - ) to nitrite (NO2 - ), ultimately undergoing denitrification, a process involving the stepwise reduction of NO or other nitrogen oxides. This denitrification process is carried out by specific microbial populations, with nitrite production being a critical intermediate step. It is reasonable to assume that the majority of microbially derived NO in fecal samples originates from this anaerobic denitrification pathway.

[0531] Direct measurement of NO in feces is difficult due to its high reactivity and short half-life. - Measurements of NO are often used as surrogate markers for NO. Nitrite is a common pathway for NO production in the intestine. - -NO2 - -NO pathway intermediate. NO3 - is a stable and abundant molecule in the human diet and can be reduced to nitrite by certain commensal bacteria present in the gut. - can be further converted to NO under certain conditions, such as low oxygen levels, acidic pH, or in the presence of microbial enzymes such as nitrite reductase. -It is important to note that NO and NO exist in a dynamic equilibrium, and the conversion between them is influenced by environmental factors and enzymatic activity. Therefore, high NO levels in feces - Levels may indicate increased likelihood of NO production, further supporting the usefulness of nitrite measurements as an approximation of NO levels.

[0532] In this study, we investigated NO2 in stool samples obtained from patients with ulcerative colitis. - We aimed to investigate the role of NO in the pathogenesis of ulcerative colitis by assessing its levels. In addition, we aimed to investigate the role of NO in the pathogenesis of ulcerative colitis by modulating the gut microbiota. - We hypothesized that NO2 production may be altered in patients who achieved remission. - We aimed to investigate the effect of FMT on sulfide and NO levels. Furthermore, we performed experiments in which ulcerative colitis stool samples were co-cultured with the BB65 complex consortium to further validate the mechanism of action of BB265 on sulfide and NO modulation in the treatment of ulcerative colitis.

[0533] method

[0534] Sample Selection

[0535] Patient samples were selected based on the positive sulfide phenotype observed in previous bioreactor experiments. Patient C was selected because sulfide concentrations in stool samples were observed to be reduced when incubated with the BB265 complex consortium and individual sulfide consumers. Patients D and E were selected because they demonstrated clinical response and remission to FMT, with significant reductions in sulfide concentrations in remission samples compared to baseline (active) samples.

[0536] Preparation of fecal slurry

[0537] A 10% (wt / vol) fecal slurry was prepared for Patient C by adding 5 g of stool to 50 mL of Solution I, and for all baseline and remission samples from Patients D and E by adding 1 g of stool to 10 mL of half-strength YCFA containing sodium sulfite and Solution I. The slurry was homogenized by vortexing for 5 min and briefly centrifuged at a maximum of 100 rpm to remove large particles. Subsequently, 2.5 mL of the fecal slurry supernatant was transferred to a gas chromatography (GC) vial containing 2.5 mL of half-strength YCFA containing sodium sulfite or Solution I to obtain a final concentration of 5% (wt / vol) fecal slurry.

[0538] The consumer isolate bb0214, Ruminococcus bicirculans, was grown overnight in 20 mL of YCFA broth. A glycerol stock (20 mL) of the BB265 complex consortium bioreactor harvest and the overnight-grown bb0214 was brought to 50 mL with sterile pre-reduced PBS and immediately centrifuged at 12,000 × g for 6 minutes. The sample was then washed three times with PBS as before, and the BB265 complex consortium and bb0214 pellets were resuspended in 10 and 20 mL of Solution I, respectively, by vortexing.

[0539] A GC vial containing 5% fecal slurry from patient C was spiked with 100 μL of the washed BB265 complex consortium and bb0214. A control sample was spiked with 100 μL of Solution I only. Samples from patients D and E were left unspiked to measure NO2 produced by the indigenous microbial community. - Differences in β-glucan were compared between baseline and remission samples. After inoculation, GC vials were immediately capped and incubated anaerobically at 37°C overnight. Patient C's experiments were performed in triplicate, while patients D and E's experiments were performed in duplicate due to limited sample weight.

[0540] Nitrite reduction test

[0541] Nitrite reduction tests were performed using a Microtest Nitrite 50 kit (Aquaspex). After incubation, the GC vial was removed from the anaerobic chamber, and the fecal slurry was transferred from the GC vial to a Falcon tube and centrifuged at 12,000 × g for 10 minutes. Then, 1 mL of the fecal slurry supernatant was transferred to a new Falcon tube containing 4 mL of RO water. Five drops of sulfuric acid were added to the Falcon tube, followed by two drops of indicator solution. The mixture was mixed by swirling once after each reagent addition. Finally, 5 μL of titration solution was added at a time and swirled until the color of the mixture changed from red to light blue. The volume of titration stock solution used was recorded, and the nitrite concentration in the stool sample was calculated as follows:

[0542] 25 μL titration stock solution = 50 mg / L nitrite

[0543] Dilution factor = 5

[0544] Nitrite concentration (mg / L) = [(volume of titration stock solution x 50 mg / L) / 25 μL] x 5

[0545] result

[0546] [Table 10-1] [Table 10-2]

[0547] Table 10 shows the concentrations of nitrite in stool samples from patients C, D, and E after overnight incubation with and without the addition of the BB265 complex consortium and bb0214 consumers. A reduction in nitrite from 450 mg / L (±50 SD) to 366.67 mg / L (±28.87 SD) was observed in patient C after incubation with the BB265 complex consortium. No reduction was observed for samples incubated with bb0214.

[0548] For patients who entered remission due to FMT treatment, stool samples incubated in half-strength YCFA showed a significant reduction in nitrite concentrations from 2016.67 mg / L (±28.87 SD) to 816.67 mg / L (±57.74 SD) for patient D and from 1550 mg / L (±70.71 SD) to 750 mg / L (±0 SD) for patient E. No reduction was observed for patient D's stool sample incubated in Solution I.

[0549] Consideration

[0550] In this study, we investigated NO2 in stool samples obtained from ulcerative colitis patients who achieved remission after FMT. - We identified a significant decrease in NO2 concentrations (Table 10). This finding supports the FMT-induced microbial changes and NO2 - This suggests a potential link between the FMT-induced shift in gut microbiota composition and the subsequent reduction in NO production. - It is plausible that this may result in alterations in NO metabolism, thereby contributing to disease remission.

[0551] Our experiments involving co-culturing ulcerative colitis stool samples with the BB265 complex consortium demonstrated significantly lower NO2 levels in samples spiked with the BB265 complex consortium compared to non-spiked controls. - The results showed that the introduced microbial community significantly increased NO2 production during anaerobic fermentation. -Furthermore, the results showed that the stool samples obtained from patients with ulcerative colitis had an inhibitory effect on NO production. - The identification of decreased levels parallels this trend and strengthens the therapeutic potential of the BB265 complex consortium, which is further supported by the observation that taxa comprising the BB265 consortium were found to be significantly more abundant in stool metagenomes obtained from patients who achieved remission with FMT.

[0552] In conclusion, our findings highlight the therapeutic potential of the BB265 complex consortium for the treatment of ulcerative colitis by modulating colonic sulfide and NO. The parallel trends observed in the clinical FMT data and the BB265 complex consortium experiments provide a compelling rationale for validating this mechanism of action specific to ulcerative colitis and IBD.

[0553] Enhanced effect of fiber on sulfide modulation by the BB265 complex consortium when co-cultured with ulcerative colitis fecal slurry

[0554] background The fermentation of fiber in the colon, particularly resistant starch and non-starch polysaccharides, occurs preferentially over protein fermentation. This preference reduces the release of sulfur amino acids, such as cysteine, into the colon, thereby reducing sulfur amino acid fermentation and subsequently sulfide production by the resident microbiota. As a result, we tested dietary fiber combinations that enhance the efficacy of our probiotic biotherapeutic formulation in the treatment of inflammatory bowel disease. These tests utilize the unique phenotypic assays outlined above to verify the efficacy of the BB265 complex consortium. Furthermore, further research into the effects of these dietary fibers on sulfide reduction could provide valuable insights for evaluating dietary interventions aimed at correcting the dysbiosis observed in ulcerative colitis.

[0555] method

[0556] A subset of 127 cell number-normalized isolates of the BB265 complex consortium was inoculated into 20 mL of YCFA and incubated anaerobically at 37°C for 24 h.

[0557] Stool samples from three baseline (active) ulcerative colitis patients (Patient A, Patient B, and Patient F) were used. The fibers used in this experiment were inulin, maltodextrin, starch, and FOS (fructo-oligosaccharides). Fecal samples (16% wt / vol) were prepared by dissolving 32 g of feces in 100 mL of Solution I. Each sample was vortexed for 5 minutes and gently pulsed at 100 rpm to pellet large particles. Subsequently, 2.5 mL of supernatant from each sample was transferred to a sterile Hungate tube containing 2.5 mL of Solution I with 2% of the respective fiber, resulting in an 8% (wt / vol) stool slurry with 1% (wt / vol) fiber.

[0558] A subset of 127 isolates from the BB265 complex consortium grown overnight was centrifuged at 12,000 × g for 10 minutes. After decanting the supernatant, the pellet was washed three times with 50 mL of sterile PBS. The final pellet was resuspended in 20 mL of sterile Solution I and vortexed to homogenize. A subset of 127 isolates from the BB265 complex consortium (100 μL) was inoculated into fecal suspensions with and without fiber as samples. In addition, 100 μL of sterile medium was inoculated into tubes containing only the fecal sample to serve as controls. The tubes were then anaerobically incubated at 37°C for 24 hours, and endpoint measurements of sulfide content were performed using a modified methylene blue assay, following the procedure used in the previous experiment.

[0559] result

[0560] The data show that patients A, B, and F had significantly elevated initial sulfide levels in their stools (307.7 μM, 323.1 μM, and 350.8 μM, respectively). However, when cocultures containing a subset of 127 isolates from the BB265 complex consortium were introduced, the sulfide levels in the stools of patients A, B, and F decreased by 46.7 μM, 78.5 μM, and 1.1 μM, respectively. Notably, dietary fiber demonstrated substantial sulfide-reducing potential. FOS, inulin, maltodextrin, and starch reduced sulfide levels in all three stool samples by 111.8 μM to 223.6 μM, 147.7 μM to 230.8 μM, 104.6 μM to 198.0 μM, and 143.6 μM to 213.9 μM, respectively. When these fibers were combined with a subset of 127 isolates of the BB265 complex consortium in ulcerative colitis stool, sulfide levels varied between 121.0 μM and 229.3 μM, 148.7 μM and 236.9 μM, 94.4 μM and 219.0 μM, and 142.6 μM and 213.9 μM, respectively (Figure 22).

[0561] Consideration

[0562] These results provide compelling evidence that dietary fibers, such as FOS, inulin, maltodextrin, and starch, significantly contribute to sulfide reduction in ulcerative colitis stool samples. This confirms the role of dietary fiber in reducing the fermentation of sulfur-containing proteins and subsequently reducing sulfide production. Furthermore, the introduction of a subset of the 127 isolates of the BB265 complex consortium in combination with these dietary fibers resulted in variable sulfide reduction, although the extent varied depending on the specificity of the stool sample and the type of dietary fiber. This observation highlights how different dietary fibers can exert different effects on the microbiota, directing them toward diverse forms of carbohydrate fermentation.

[0563] Notably, a subset of the 127 isolates from the BB265 complex consortium, when supplemented with inulin, demonstrated the ability to efficiently reduce high sulfide levels in stool samples from all patients. This observation strongly suggests that the coculture, especially in the presence of inulin, is better at promoting fiber fermentation than protein fermentation. In contrast, starch, when combined with the subset of 127 isolates, exhibited low sulfide consumption, indicating potentially limited fermentability by this consortium. Interestingly, FOS and maltodextrin exhibited various interactions with the subset of 127 isolates from BB265, resulting in variability in sulfide concentrations that were reduced depending on stool variation. This reinforces the idea that dietary fiber has a clear impact on the complex consortium in terms of sulfide reduction in patients with ulcerative colitis and provides a rationale for including fiber in BB265 therapy to enhance its ability to reduce sulfide levels.

Claims

1. 1. A composition for preventing or treating a gastrointestinal disorder in a subject in need thereof, said composition comprising at least one strain of a microorganism; the microorganism is selected from the group consisting of bacteria and archaea; The microorganisms reduce endogenous sulfide levels in the colon of a patient in need thereof; sulfide consumption; reducing the sulfide and nitric oxide load of epithelial cells affecting cellular respiration, causing metabolic pathology; reducing the relative abundance and / or metabolic activity of sulfidogenic microbiota; reducing sulfide levels in the colon directly through consumption / assimilation; reducing sulfide levels, the relative abundance and / or metabolic activity of sulfidogenic microbiota in the colon by metabolic substrate competition; reducing sulfide levels, the relative abundance and / or metabolic activity of sulfidogenic microbiota in the colon by diverting metabolic substrates away from sulfide production; H ２ reducing sulfide levels, the relative abundance of sulfidogenic microbiota, and / or metabolic activity in the colon by consuming a diet rich in sulfur; reducing sulfide levels, the relative abundance of sulfidogenic microbiota, and / or metabolic activity in the colon by reducing the release of metabolizable sulfur substrates; reducing sulfide levels, the relative abundance of sulfidogenic microbiota, and / or metabolic activity in the colon by reducing sulfur amino acid release (methionine, cysteine, homocysteine, and / or taurine) into the colon; reducing colonic protein fermentation; reducing nitric oxide production in the colon; reducing nitric oxide levels in the colon; and inducing apoptosis of colonocytes in the lesions to resolve the induced stable inflammatory state. composition.

2. 10. The composition of claim 1, wherein the microorganism comprises a gene responsible for reducing endogenous sulfide levels in the colon of a patient in need thereof.

3. The composition of claim 1 , wherein the microorganism comprises a gene responsible for sulfide consumption.

4. The composition of claim 1, wherein the microorganism contains genes responsible for reducing sulfide and nitric oxide load in epithelial cells, which affects cellular respiration and leads to metabolic lesions.

5. 2. The composition of claim 1, wherein the microorganism comprises a gene responsible for reducing the relative abundance and / or metabolic activity of sulfidogenic microbiota.

6. 10. The composition of claim 1, wherein the microorganism comprises a gene responsible for reducing sulfide levels in the colon directly through consumption / assimilation.

7. 2. The composition of claim 1, wherein the microorganism comprises a gene responsible for reducing sulfide levels, the relative abundance and / or metabolic activity of sulfidogenic microbiota in the colon through metabolic substrate competition.

8. 2. The composition of claim 1, wherein the microorganism comprises a gene responsible for reducing sulfide levels, the relative abundance of sulfidogenic microbiota, and / or metabolic activity in the colon by diverting metabolic substrates away from sulfide production.

9. The microorganism is H ２ 2. The composition of claim 1, comprising a gene responsible for reducing sulfide levels, the relative abundance of sulfide-producing microbiota, and / or metabolic activity in the colon upon consumption of said composition.

10. 2. The composition of claim 1, wherein the microorganism comprises a gene responsible for reducing sulfide levels, the relative abundance of sulfidogenic microbiota, and / or metabolic activity by reducing the release of metabolizable sulfur substrates.

11. 2. The composition of claim 1, wherein the microorganism comprises a gene responsible for reducing sulfide levels, the relative abundance of sulfidogenic microbiota, and / or metabolic activity by reducing sulfur amino acid release (methionine, cysteine, homocysteine, and / or taurine) into the colon.

12. The composition of claim 1 , wherein the microorganism comprises a gene responsible for reducing colonic protein fermentation.

13. The composition of claim 1 , wherein the microorganism comprises a gene responsible for reducing nitric oxide production in the colon.

14. The composition of claim 1 , wherein the microorganism comprises a gene responsible for reducing nitric oxide levels in the colon.

15. 2. The composition of claim 1, wherein the microorganism contains a gene responsible for inducing apoptosis of colonocytes in the lesion and resolving the induced stable inflammatory state.

16. The sulfide is a sulfide ion (S ２－ ), bisulfide ion (SH － ), organic compounds containing R-SH groups (thiols), and hydrogen sulfide (H ２ 16. The composition of claim 1, wherein the sulfide is present in the form of a sulfide selected from the group consisting of:

17. 17. The composition of any one of claims 1 to 16, wherein the microorganism comprises a gene selected from the group consisting of genes responsible for sulfide utilization in the biosynthesis of methionine and cysteine.

18. 18. The composition of any one of claims 1 to 17, wherein the microorganism comprises genes selected from the group consisting of genes responsible for pathways 2.3.1.30, 2.5.1.47, and / or 2.5.1.65 of Figure 4, genes responsible for pathways listed in Table 6 for sulfur and sulfide metabolism and regulation in the colon, and genes responsible for pathways listed in Table 7 for homoacetogenesis and hydrogen cycling in the colon.

19. 19. The composition of any one of claims 1 to 18, wherein the microorganism is selected from a phylum, genus, species, or isolate selected from the group consisting of: a species selected from the group listed in Figure 5 and any combination of these species; an isolate selected from the group listed in Table 1 and any combination of these isolates; a phylum selected from the group listed in Table 2 and any combination of these phyla; a genera selected from the group listed in Table 3 and any combination of these genera; a species selected from the group listed in Table 4 and any combination of these species; an isolate selected from the group listed in Table 5 and any combination of these isolates; an isolate selected from the group listed in Table 8 and any combination of these isolates; and an isolate selected from the group listed in Table 9 and any combination of these isolates.

20. 20. The composition of any one of claims 1 to 19, wherein the microorganism comprises a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 9193.

21. 21. The composition of any one of claims 1 to 20, wherein the microorganism comprises a 16S ribosomal RNA (rRNA) gene having sequence identity to one or more of SEQ ID NOs: 1-9193, and the sequence identity is selected from the group consisting of at least 99.9%, at least 99.5%, at least 99%, at least 98.5%, at least 98%, at least 97.5%, at least 97%, at least 96.5%, at least 96%, at least 95.5%, at least 95%, at least 94.5%, at least 94%, at least 93.5%, at least 93%, at least 92.5%, at least 92%, at least 91.5%, at least 91%, at least 90.5%, and at least 90%.

22. 22. The composition of any one of claims 1 to 21, wherein the microorganism is a fecal or colonic microorganism.

23. 23. The composition of any one of claims 1 to 22, wherein the microorganism is non-inflammatory.

24. 24. The composition of any one of claims 1 to 23, wherein the microorganism is cultured from a fecal or colon biopsy sample.

25. 25. The composition of any one of claims 1 to 24, wherein the microorganisms comprise a microbial cell community derived from stool or biopsies of one or more human donors.

26. 26. The composition of any one of claims 1 to 25, wherein the microbial cell community comprises cultured microbial cells.

27. 27. The composition of any one of claims 1 to 26, wherein the cultured microbial cells are derived from multiple human donors.

28. 28. The composition of any one of claims 1 to 27, wherein the microbial cell community comprises uncultured microbial cells.

29. 29. The composition of any one of claims 1 to 28, wherein the uncultured microbial cells are derived from a single human donor.

30. 30. The composition of any one of claims 1 to 29, which is a fecal transplant microbiota composition.

31. 31. The composition of any one of claims 1 to 30, which is lyophilized.

32. 32. The composition of any one of claims 1 to 31, which is a liquid.

33. 33. The composition of any one of claims 1 to 32, which is capable of maintaining at least 50% cell viability after storage at room temperature for at least 4 weeks compared to the initial cell viability immediately prior to storage.

34. 34. The composition of any one of claims 1 to 33, which is capable of maintaining about 60% to about 80% cell viability after storage at room temperature for at least 4 weeks compared to the initial cell viability immediately before the start of said storage.

35. 35. The composition of any one of claims 1 to 34, comprising a prebiotic.

36. 36. The composition of any one of claims 1 to 35, comprising a carrier.

37. 37. The composition of any one of claims 1 to 36, comprising insoluble fiber, an antioxidant, a buffering agent, an osmolality adjusting agent, an anti-foaming agent, and / or a preservative.

38. 38. The composition of any one of claims 1 to 37, comprising a chemostat medium.

39. 39. The composition of any one of claims 1 to 38, comprising a saline composition.

40. 40. The composition of any one of claims 1 to 39, comprising resistant starch.

41. 41. The composition of any one of claims 1 to 40, which is lyophilized together with a pharmaceutically acceptable excipient.

42. 42. The composition of any one of claims 1 to 41, comprising a stabilizing agent and / or a cryoprotectant.

43. 43. The composition of any one of claims 1 to 42, wherein the cryoprotectant is selected from the group consisting of trehalose, mannitol, sucrose, glycerol, sorbitol, DMSO, propylene glycol, ethylene glycol, sucrose, galactose-lactose, and any combination thereof.

44. 44. The composition of any one of claims 1 to 43, wherein the cryoprotectant further comprises a compound selected from the group consisting of glycerol, polyethylene glycol (PEG), glycerin, erythritol, arabitol, xylitol, sorbitol, glucose, lactose, ribose, and any combination thereof.

45. 45. The composition of any one of claims 1 to 44, wherein the cryoprotectant is trehalose at a concentration of 2% to 15% in the lyophilized formulation.

46. 46. ​​The composition of any one of claims 1 to 45, wherein the cryoprotectant is trehalose at a concentration of at least 5% in the lyophilized formulation.

47. 47. The composition of any one of claims 1 to 46, wherein the cryoprotectant is trehalose at a concentration of at least 10% in the lyophilized formulation.

48. 48. The composition of any one of claims 1 to 47, which is a pharmaceutical composition.

49. 49. The composition of any one of claims 1 to 48, wherein the at least one strain of microorganism is diluted with an inert powdered diluent.

50. 50. The composition of any one of claims 1 to 49, comprising one or more pharmaceutically acceptable carriers or excipients.

51. 51. The composition of any one of claims 1 to 50, formulated as a gel tablet, pill, enema, microcapsule, capsule, or tablet.

52. 52. The composition of any one of claims 1 to 51, wherein the capsule or tablet is enteric coated, pH dependent, slow release, and / or gastric acid resistant.

53. 53. The composition of any one of claims 1 to 52, adapted for oral or rectal administration.

54. For every 200 mg of the composition, １ ~10 １６ , 10 ３ ~10 １４ , 10 ４ ~10 １４ , 10 ５ ~10 １４ , 10 ６ ~10 １４ , 10 ７ ~10 １４ , 10 ８ ~10 １４ , 10 ４ ~10 １３ , 10 ５ ~10 １２ , 10 ６ ~10 １１ , 10 ７ ~10 １０ , 10 ８ ~10 ９ , 10 ３ ~10 １３ , 10 ３ ~10 １２ , 10 ３ ~10 １１ , 10 ３ ~10 １０ , 10 ３ ~10 ９ , 10 ３ ~10 ８ , 10 ３ ~10 ７ , 10 ３ ~10 ６ , 10 ３ ~10 ５ , and 10 ３ ~10 ４ 54. The composition of any one of claims 1 to 53, comprising a pharmacologically active dose of microbial cells or spores selected from the group consisting of colony forming units (cfu) or total cell count.

55. 10 １ cfu / mL to 10 ６ cfu / ml, 1 cfu / mL to 10 cfu / mL, 100 cfu / mL to 1,000 cfu / mL, 10,000 cfu / mL to 100,000 cfu / mL, 10 million cfu / mL to 100 billion cfu / mL, 10 million to 50 million cfu / mL, more preferably 50 million to 100 million cfu / mL, 100 million to 500 million cfu / mL, 500 million to 1 billion cfu / mL, 1 billion to 5 billion cfu / mL, 5 billion to 10 billion cfu / mL, 10 billion to 15 billion cfu / mL, 15 billion to 20 billion cfu / mL, 20 billion to 25 billion cfu / mL, 25 billion to 30 billion cfu / mL, 30 billion to 35 billion cfu / mL 55. The composition of any one of claims 1 to 54, comprising a pharmacologically active dose of microbial cells or spores selected from the group consisting of: 1 L, 35-40 billion cfu / mL, 40-45 billion cfu / mL, 45-50 billion cfu / mL, 50-55 billion cfu / mL, 55-60 billion cfu / mL, 60-65 billion cfu / mL, 65-70 billion cfu / mL, 70-75 billion cfu / mL, 75-80 billion cfu / mL, 80-85 billion cfu / mL, 85-90 billion cfu / mL, 90-95 billion cfu / mL, 95-100 billion cfu / mL.

56. 56. The composition of any one of claims 1 to 55, wherein the composition comprises a pharmacologically active dose of microbial cells or spores, and the concentration of the microbial cells or spores as dry microbial matter is selected from the group consisting of 5-50% w / w, 1-75% w / w, 0.01-100% w / w, 0.1-100% w / w, and 1-100% w / w.

57. 57. The composition of any one of claims 1 to 56, wherein the gastrointestinal disorder is gastrointestinal mucosal inflammation.

58. 58. The composition of any one of claims 1 to 57, wherein the gastrointestinal disorder is a dysbiosis.

59. 59. The composition of any one of claims 1 to 58, wherein the inflammation is associated with one or more disorders selected from the group consisting of inflammatory bowel disease (IBD), pouchitis, irritable bowel syndrome (IBS), enterobacterial infection, metabolic disease, neuropsychiatric disorder, autoimmune disease, allergic disorder, hepatic encephalopathy, or cancer.

60. 60. The composition of any one of claims 1 to 59, wherein the gastrointestinal disorder is inflammatory bowel disease.

61. 61. The composition of any one of claims 1 to 60, wherein the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, gastroenteritis, enteritis, and pouchitis.

62. 62. The composition of any one of claims 1 to 61, wherein the gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, gastrointestinal ulcers, and gastrointestinal cancer.

63. 63. The composition of any one of claims 1 to 62, which reduces endogenous sulfide levels in the colon of a patient in need thereof.

64. 64. A composition according to any one of claims 1 to 63, which reduces sulfide and NO load in epithelial cells, which cause metabolic lesions through inhibition of cellular respiration.

65. 65. The composition of any one of claims 1 to 64, which reduces the relative abundance, number of species, and / or metabolic activity of sulfidogenic microbiota, reduces sulfide levels in the colon directly through consumption / assimilation, reduces sulfide levels in the colon through metabolic substrate competition, and / or reduces sulfide levels in the colon by consuming hydrogen.

66. 66. The composition of any one of claims 1 to 65, wherein the composition reduces the relative abundance, species number, and / or metabolic activity of sulfidogenic microbiota by reducing metabolizable sulfur substrates, and reduces sulfur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

67. 67. A composition according to any one of claims 1 to 66, which induces apoptosis of colonocytes in the lesion and resolves the operation of the induced stable inflammatory state.

68. 68. The composition of any one of claims 1 to 67, which reduces unwanted inflammation.

69. 69. The composition of any one of claims 1 to 68, which reduces unwanted inflammation.

70. 70. The composition of any one of claims 1 to 69, which prevents or reduces activation of the mucosal immune system in an IL-13 and IL-5 dependent TH2-mediated immune response driven by natural killer T cells.

71. 71. The composition of any one of claims 1 to 70, which reduces inflammation in the subject as measured by a parameter selected from the group consisting of TNFα signaling via NF-κB, IFNα signaling, IFNγ signaling, IL6 JAK STAT3 signaling, activation of pro-apoptotic pathways, and initiation of the unfolded protein response.

72. 72. The composition of any one of claims 1 to 71, which downregulates genes associated with pro-apoptotic pathways and the unfolded protein response, including genes selected from the group consisting of CHAC1, CEBPB, TRIB3, PPP1R15A, DDIT3, ATF4, and XBP1.

73. 73. The composition of any one of claims 1 to 72, wherein the at least one strain of microorganism is a strain selected from the group consisting of Flavinofractor spp., Flavinofractor plati, Christensenella spp., Christensenella minuta, Anaerobutyricum spp., Anaerobutyricum hallii, Escherichia-Shigella spp., and Escherichia coli, or a combination of two or more strains thereof.

74. 74. A composition according to any one of claims 1 to 73 for use in treating or preventing ulcerative colitis.

75. 77. A biotherapeutic composition comprising the composition of claims 1-76 together with an acceptable diluent or carrier.

76. 75. A pharmaceutical composition comprising the composition of claims 1 to 74 together with a pharmaceutically acceptable diluent or carrier.

77. An isolated, non-inflammatory strain of a microorganism comprising a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-9193.

78. 78. The isolated non-inflammatory strain of a microorganism of claim 77, wherein the at least one strain of a microorganism comprises a 16S ribosomal RNA (rRNA) gene having sequence identity to one or more of SEQ ID NOs: 1-9193, wherein the sequence identity is selected from the group consisting of at least 99.9%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, and at least 90%.

79. 75. A method for treating and / or preventing a gastrointestinal disorder in a patient in need thereof, comprising administering to a patient in need thereof a composition according to any one of claims 1 to 74.

80. 80. The method of claim 79, wherein the gastrointestinal disorder is gastrointestinal mucosal inflammation.

81. 81. The method of any one of claims 79 to 80, wherein the gastrointestinal disorder is a dysbiosis.

82. 82. The method of any one of claims 79 to 81, wherein the inflammation is associated with one or more disorders selected from the group consisting of inflammatory bowel disease (IBD), pouchitis, irritable bowel syndrome (IBS), enterobacterial infection, metabolic disease, neuropsychiatric disorder, autoimmune disease, allergic disorder, hepatic encephalopathy, or cancer.

83. 83. The method of any one of claims 79 to 82, wherein the gastrointestinal disorder is inflammatory bowel disease.

84. 84. The method of any one of claims 79 to 83, wherein the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, gastroenteritis, enteritis, and pouchitis.

85. 85. The method of any one of claims 79 to 84, wherein the gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, gastrointestinal ulcers, and gastrointestinal cancer.

86. 86. The method of any one of claims 79 to 85, wherein the composition is administered orally or rectally.

87. 87. The method of any one of claims 79-86, wherein the composition is administered to the patient using a dosing regimen selected from the group consisting of once, hourly, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 12 hours, once daily, twice daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, once per week, twice per week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, once per month, twice per month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, once per year, twice per year, every 2 years, every 3 years, every 4 years, and every 5 years.

88. 88. The method of any one of claims 79 to 87, wherein the composition reduces endogenous sulfide levels in the colon of a patient in need thereof.

89. 89. The method of any one of claims 79 to 88, wherein the composition reduces sulfide and nitric oxide load in epithelial cells that cause metabolic lesions through inhibition of cellular respiration.

90. 90. The method of any one of claims 79 to 89, wherein the composition reduces nitric oxide production and / or reduces nitric oxide levels in the colon.

91. 91. The method of any one of claims 79 to 90, wherein the composition reduces the relative abundance of sulfidogenic microbiota, reduces the number of species and / or metabolic activity, reduces sulfide levels in the colon directly through consumption / assimilation, reduces sulfide levels, the relative abundance, number of species and / or metabolic activity of sulfidogenic microbiota through metabolic substrate competition, and / or reduces sulfide levels, the relative abundance, number of species and / or metabolic activity of sulfidogenic microbiota by consuming hydrogen.

92. 92. The method of any one of claims 79 to 91, wherein the composition reduces the relative abundance, number of species, and / or metabolic activity of sulfidogenic microbiota by reducing metabolizable sulfur substrates in the colon, and / or reduces the relative abundance, number of species, and / or metabolic activity of sulfidogenic microbiota by reducing protein fermentation and thereby reducing sulfur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon.

93. 93. The method of any one of claims 79 to 92, wherein unwanted inflammation is reduced.

94. 94. The composition of any one of claims 79 to 93, wherein the composition prevents or reduces activation of the mucosal immune system in an IL-13 and IL-5 dependent TH2-mediated immune response driven by natural killer T cells.

95. 95. The method of any one of claims 79 to 94, wherein the method reduces inflammation in the subject as measured by a parameter selected from the group consisting of TNFα signaling via NF-κB, IFNα signaling, IFNγ signaling, IL6 JAK STAT3 signaling, activation of pro-apoptotic pathways, and initiation of the unfolded protein response.

96. 96. The method of any one of claims 79 to 95, wherein the method downregulates genes associated with pro-apoptotic pathways and the unfolded protein response, including genes selected from the group consisting of CHAC1, CEBPB, TRIB3, PPP1R15A, DDIT3, ATF4, and XBP1.

97. A method for reducing endogenous sulfide levels in the colon of a patient in need thereof.

98. 98. The method of claim 97, which reduces sulfide and NO load in epithelial cells that cause metabolic lesions through inhibition of cellular respiration.

99. 98. The method of claim 97, wherein the relative abundance, species number, and / or metabolic activity of sulfidogenic microbiota is reduced.

100. 98. The method of claim 97, wherein sulfide levels are reduced in the colon directly through consumption / assimilation.

101. 98. The method of claim 97, wherein the sulfide levels, relative abundance, species number, and / or metabolic activity of sulfidogenic microbiota in the colon are reduced by metabolic substrate competition.

102. 98. The method of claim 97, wherein consuming hydrogen reduces sulfide levels, the relative abundance, species number, and / or metabolic activity of sulfidogenic microbiota in the colon.

103. 98. The method of claim 97, wherein the sulfide levels, relative abundance, species number, and / or metabolic activity of sulfidogenic microbiota are reduced by reducing metabolizable sulfur substrates in the colon.

104. 98. The method of claim 97, wherein sulfide levels, relative abundance, species number, and / or metabolic activity of sulfidogenic microbiota are reduced by reducing sulfur amino acid release into the colon (methionine, cysteine, homocysteine, taurine).

105. 98. The method of claim 97, wherein the sulfide levels, relative abundance, species number, and / or metabolic activity of sulfidogenic microbiota in the colon are reduced by reducing protein fermentation.

106. 106. The method of any one of claims 79 to 105, wherein the inflammatory bowel disease is ulcerative colitis.

107. 107. The method of any one of claims 79-106, wherein the composition comprises a strain selected from the group consisting of Flavinofractor spp., Flavinofractor plati, Christensenella spp., Christensenella minuta, Anaerobutyricum spp., Anaerobutyricum hallii, Escherichia-Shigella spp., and Escherichia coli.

108. 76. A method for preparing the biotherapeutic composition of claim 75, comprising the step of mixing the composition of any one of claims 1 to 76 with an acceptable diluent or carrier.

109. 77. A method for preparing the pharmaceutical composition of claim 76, comprising the step of mixing the composition of any one of claims 1 to 74 with a pharmaceutically acceptable diluent or carrier.

110. 75. Use of a composition according to any one of claims 1 to 74 in the manufacture of a medicament for reducing or preventing gastrointestinal disorders in a subject.

111. 75. A dosage form comprising the composition of any one of claims 1 to 74.

112. 112. A kit comprising the dosage form of claim 111 together with instructions for its use.

113. 112. A kit comprising the dosage form of claim 111 together with instructions for its use.

114. A nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-9193.

115. A nucleotide sequence having sequence identity to one or more of SEQ ID NOs: 1-9193, wherein the sequence identity is selected from the group consisting of at least 99.9%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, and at least 90%.

116. 116. The nucleotide sequence of claim 114 or 115, which is substantially purified or isolated.

117. A microorganism comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 9193.

118. 1. A microorganism comprising a nucleotide sequence having sequence identity to one or more of SEQ ID NOs: 1-9193, wherein the sequence identity is selected from the group consisting of at least 99.9%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, and at least 90%.

119. 119. A microorganism according to claim 117 or 118, which is substantially purified or isolated.

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