Compositions and Methods for Reducing Endogenous Sulphide in Inflammatory Bowel Diseases

Field of the Invention

[001] The present invention relates to compositions for treating inflammatory bowel disease. The present invention also relates to dosage forms and methods of treating inflammatory bowel disease by administering the composition to a patient in need thereof.

Background

[002] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[003] The human intestinal microbiota consists of trillions of microorganisms, including at least 100 prevalent, and at least 1000 less common microbial species, harbouring over 100-fold more genes than those present in the human genome. The intestinal microbiota is composed predominantly of bacteria, yet also contains archaea, protozoa, fungi and viruses. The microbiota performs vital functions essential to health maintenance, including food processing, digestion of complex indigestible polysaccharides and synthesis of vitamins, and it secretes bioactive metabolites with diverse functions, ranging from inhibition of pathogens, metabolism of toxic compounds to modulation of host metabolism.

[004] Inflammatory bowel disease (IBD) is an increasingly prevalent, currently incurable condition believed to be caused by an abnormal immune response to the resident gut microbiome in genetically susceptible patients. The term IBD encompasses both ulcerative colitis (UC), Crohn’s disease (CD) and pouchitis. UC is characterised by chronic nongranulomatous inflammation that is limited to the colonic mucosa, typically involving the rectum and a variable proximal extent of the colon in continuity. CD is characterised by transmural, often granulomatous, inflammation that can involve any part of the gastrointestinal tract from the mouth to the anus.

[005] The clinical presentation of UC is characterised by bloody diarrhoea with associated urgency, tenesmus, and lower abdominal pain. CD most frequently involves the terminal ileum and perianal regions, and structuring and fistulising disease are common. The clinical presentation of CD varies widely according to disease distribution, but is typically one of abdominal pain, diarrhoea, and weight loss. Extra-intestinal manifestations (EIMs) of IBD affect up to 50% of patients, typically in parallel with disease activity, and are more common in CD than in UC. The range of EIM’s of IBD is broad, including arthropathy, venous thromboembolic disease, metabolic bone disease, uveitis and scleritis, and skin disease (pyoderma granulosum, erythema nodosum). Primary sclerosing cholangitis (PSC) is a hepatobiliary manifestation of IBD, affecting up to 5% of patients with IBD, which carries a high risk of progression to cholangiocarcinoma and liver transplantation. IBD patients are also at increased risk of intestinal dysplasia and malignancy, in particular, those with extensive colitis for whom colonoscopic surveillance is recommended.

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

[007] 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 the need for the identification of further bacteria for use in bacteriotherapy.

[008] UC is a relapsing and remitting inflammatory bowel disease characterised by superficial colonic mucosal inflammation that extends proximally from the rectum in a contiguous manner. Current treatments are hampered by incomplete efficacy and have the potential for unacceptable side-effects including allergy, intolerance, serious infection and malignancy due to long-term immunosuppression. The current immune targeted therapies for UC are inadequate because they do not address the primary drivers of the disease, only the secondary immune response.

[009] There is evidence that UC results from an energy deficiency state in colonocytes (colonic epithelial cells) induced by high levels of sulphide and nitric oxide (NO) depleting Coenzyme A (CoA) and inhibiting 0-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. The loss of the mucus and tight junction barrier then allows activation of the mucosa immune system in a natural killer T-cell driven IL-13- and IL-5-dependent, TH2-mediated, immune response.

[0010] The process is as follows: (1 ) high levels of NO are damaging to human tissue; (2) the source of colonic NO is from anaerobic bacterial respiration or activated immune cells; (3) nitrosation of CoA then produces S-nitroso CoA (inactive) and (a) prevents the acetylation required for lipogenesis and (b) diminishes 0-oxidation of butyrate in colonocytes; (4) colonocyte energy deficiency follows with the inability to synthesis mucus, lipids and proteins;

(5) barrier loss then leads to bacteria activating the immune system; (6) sulphide inhibits NO reductase leading to high intracellular levels of NO; and (7) the combination of NO and sulphide reproduces the lesion in colonocytes seen in ulcerative colitis. This relationship between NO and sulphide is further confirmed in experimental colonocyte cell culture, where NO alone does not replicate the lesion. Refer to Figure 1 for a diagrammatical representation of the inhibition of cellular respiration in colonocytes by luminal sulphide and NO in UC.

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

[0012] Butyrate is the primary energy source of colonocytes

[0013] Uniquely among human cells, colonocytes rely exclusively on bacterial metabolic products for their energy. This is principally in the form of butyrate, a short chain fatty acid that is produced by the fermentation of dietary fibre in the right colon by members of the colonic microbiota.

[0014] Butyrate oxidation is impaired in ulcerative colitis

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

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

[0017] In UC, measurements of anionic sulphide levels in the colon are elevated relative to healthy controls. In addition, the potential to produce sulphide in faecal samples was found to be 3-4-fold higher in UC than in control cases. This is likely due to the relatively high levels and active metabolisms of sulphate reducing, putrefying, protein fermenting, and amino acid utilising bacteria in patients with UC in both active and quiescent disease. Furthermore, NO is produced by both inflamed colonocytes and the colonic microbiota.

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

[0019] In UC, sulphidogenic microbiota have been demonstrated to be more abundant and active than in healthy individuals. In particular, sulphate-reducing bacteria (SRB) of the genus Desulfovibrio show greater diversity, abundance, and metabolic activity in faeces of patients with UC than in health. SRB such as Desulfovibrio reduce sulphur oxides (SO _X ) to produce sulphide. SRB are more abundant in active rather than quiescent UC and correlate significantly with disease severity, indicating a potential causative link. The increased abundance of sulphidogenic microbiota in parallel with increased luminal sulphide and disease severity is suggestive of anetiologyy involving microbial production of sulphide. Relevantly, sulphidogenic microbiota levels have also been observed to be increased in Crohn’s disease patients, suggesting an involvement of these metabolic pathways in this indication. [0020] Butyrate oxidation is impaired by nitric oxide and sulphide in combination

[0021] Neither sulphide alone nor NO alone reproduce the biochemical lesion of UC in human colonocytes. Normal human colonocytes in the presence of sulphide and NO demonstrate that the combination of sulphide and NO reduced butyrate oxidation, ketogenesis and lowered CoA levels as observed in the biochemical lesions in ulcerative colitis.

[0022] Impaired butyrate oxidation produces energy deficiency state in colonocytes

[0023] High levels of NO nitrosates the sulphydryl groups of CoA to S-nitroso-CoA thereby inhibiting the acyl transfer properties and energy forming function of acetyl-CoA. In addition, exposure of colonocytes to NO inactivates butyryl-CoA which is essential for 0- oxidation. Failure of 0-oxidation of butyrate leads to low levels of ATP and energy deficiency, diminishing the ability of the colonocyte to maintain the mucosal barrier. Attempts to treat UC with butyrate have not been successful and butyrate levels in UC patients have been noted to be either normal or elevated compared to healthy controls.

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

[0025] Both the quantity of glutathione and amount of reduced glutathione are altered in UC, suggesting either diminished synthesis or excessive consumption of glutathione. However, a significant increase in nitrosothiol groups in UC indicates that excessive consumption of glutathione is occurring. This consumption and depletion of glutathione occurs over time and induces a slow lesion identical to that seen in experimental models with human colonocytes exposed to sulphide and NO.

[0026] Failure of colonocyte tight junction assembly and mucus production

[0027] There are abnormalities of tight junctions and the mucous barrier in UC. These occur in active and quiescent disease indicating that they are not primarily due to active inflammation. Scanning electron microscopy of freeze-fractured colonocytes of UC shows diminished “meshing” of tight junctions between colonocytes. Another finding on electron microscopy in acute colitis is cell blebbing in colonocytes. Cell blebbing is characteristic of cellular energy deficiency via depletion of ATP and glutathione in many cell systems and is reversible. Colonocytes are the prime producers of mucus, the production of which is dependent on butyrate metabolism and which when inhibited by exogenous agents also diminishes mucus production.

[0028] Loss of colonic barrier leads to microbially triggered inflammation

[0029] The gastrointestinal tract contains a potent immune system that is separated from the luminal microorganisms by barriers of the mucous layer and epithelium. The innate immune system provides a further non-specific defense against invading organisms and is aided by the specific response to antigens provided by adaptive immune cells. Current evidence from human studies indicates 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] The management of UC involves both induction therapy (to induce remission) and maintenance therapy (to prevent further flares). The goal of treatment is maintenance of remission without steroids. Induction therapy is usually high dose oral 5-aminosalicylic acid compounds (5-ASAs) with or without topical 5-ASAs via enema or suppository. More severe flares require systemic corticosteroids (tapered over time and discontinued). Maintenance therapy choice in ulcerative colitis is determined by disease extent, severity, frequency of flares and past treatment history. The mainstays of maintenance therapy are the 5-ASAs used orally or topically. For patients with repeated flares, thiopurines should be used. In recent years, new biological agents have demonstrated efficacy for the maintenance of remission in UC. These are the anti-TNFa agents, infliximab, adalimumab and golimumab and the anti- integrin agent, vedolizumab. However, these agents are expensive and have incomplete efficacy.

[0032] Inadequacy of current therapies for ulcerative colitis

[0033] Current treatments are hampered by incomplete efficacy and have the potential for unacceptable side-effects including allergy, intolerance, serious infection, and malignancy due to long-term immunosuppression. The current treatments for UC are inadequate to maintain long-term remission in a significant proportion of patients. Many patients have chronic or relapsing inflammation of the colon leading to work and personal impairment and up to 30% require colectomy despite current therapies. Thiopurines and anti-TNF agents induce systemic immunosuppression, reducing the incidence and severity of flares but at the cost of increased risk of serious infections, malignancy, and particularly lymphoma. The major reasons for the poor therapeutic efficacy of current therapies are that most target the mucosal immune system, not the inciting cause of disease.

[0034] There is therefore a need in the art for effective treatments of inflammatory bowel disease. It is an objective of the invention to overcome one or more problems foreshadowed by the prior art.

Summary of the Invention

[0035] In a first aspect, the invention broadly resides in 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, wherein the microorganism is selected from the group consisting of: bacteria, archaea and yeast.

[0036] In a preferred embodiment, the microorganism comprises a phenotype and/or genes that are responsible for a phenotype selected from the group consisting of: reducing endogenous sulphide levels in the colon of a patient in need thereof; sulphide consumption; reducing sulphide and nitric oxide load on epithelial cells which affects cell respiration leading to a metabolic lesion; reducing relative abundance and or metabolic activity of sulphidogenic microbiota; reducing sulphide levels in the colon directly through consumption/assimilation/degradation; reducing sulphide levels, relative abundance and or metabolic activity of sulphidogenic microbiota in the colon by metabolic substrate competition; reducing sulphide levels, relative abundance and or metabolic activity of sulphidogenic microbiota in the colon by diverting metabolic substrates away from the production of sulphide; reducing sulphide levels, relative abundance and or metabolic activity of sulphidogenic microbiota in the colon by consuming H2; reducing sulphide levels, relative abundance and or metabolic activity of sulphidogenic microbiota by reducing release of metabolisable sulphur substrates; reducing sulphide levels, relative abundance and or metabolic activity of sulphidogenic microbiota by reducing sulphur 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 that are responsible for reducing endogenous sulphide levels in the colon of a patient in need thereof.

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

[0039] In a preferred embodiment, the microorganism comprises a phenotype and/or genes that are responsible for reducing sulphide and nitric oxide load on epithelial cells which affects cell respiration leading to a metabolic lesion.

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

[0041] In a preferred embodiment, the microorganism comprises a phenotype and/or genes that are responsible for reducing sulphide levels in the colon directly through consumption/assimilation/degradation. [0042] In a preferred embodiment, the microorganism comprises a phenotype and/or genes that are responsible for reducing sulphide levels, taxonomic diversity, number of species, relative abundance and or metabolic activity of sulphidogenic microbiota in the colon by metabolic substrate competition.

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

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

[0045] In a preferred embodiment, the microorganism comprises a phenotype and/or genes that are responsible for reducing sulphide levels, taxonomic diversity, number of species, relative abundance and or metabolic activity of sulphidogenic microbiota by reducing release of metabolizable sulphur substrates.

[0046] In a preferred embodiment, the microorganism comprises a phenotype and/or genes that are responsible for reducing sulphide levels, taxonomic diversity, number of species, relative abundance and or metabolic activity of sulphidogenic microbiota by reducing sulphur amino acid release (methionine, cysteine, homocysteine and/or taurine) into the colon.

[0047] In a preferred embodiment, the microorganism comprises a phenotype and/or genes that are 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 genes that are responsible for the phenotype of sulphide consumption.

[0049] In a further preferred embodiment, the sulphide is present in a form of sulphide selected from the group consisting of: a sulphide ion (S ² ); a hydrosulphide (HSj ion; a bisulphide ion (SH ), hydrogen sulphide (H2S); an organic compound containing the group R- SH (a thiol); sulphide metals (including but not limited to iron, copper, and molybdenum sulphides.

[0050] In a further preferred embodiment, the microorganism comprises a gene or several of the genes selected from the group consisting of: genes responsible for the use of sulphide in the biosynthesis of methionine and cysteine. [0051] In a further embodiment, the microorganism comprises a gene or several of the genes selected from the group consisting of: genes responsible for the anaerobic oxidation of sulphide.

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

[0053] In a further preferred embodiment, the microorganism comprises a gene or several of the genes selected from the group consisting of: genes coding 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 regarding sulphur and sulphide metabolism and handling in the colon; and genes responsible for pathways listed in Table 7 regarding homo-acetogenesis and hydrogen cycling within the colon.

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

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

[0056] In a further preferred embodiment, the microorganism comprises a 16S ribosomal RNA (rRNA) gene or contiguous whole genome sequence having a nucleotide sequence selected from the group consisting of: SEQ ID NOs 1 to 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 to 206.

[0058] In a further preferred embodiment, the microorganism comprises whole genome sequences having a nucleotide sequence selected from the group consisting of: SEQ ID NOs 207 to 412. [0059] In a further preferred embodiment, the microorganism comprises whole genome sequences 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 contigous whole genome sequence having a sequence identity to one or more SEQ ID NOs 1 to 9193, wherein 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 9 .5%; at least 91 %; at least 90.5% and at least 90%.

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

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

[0063] In a further preferred embodiment, the microorganism is cultured from a faecal or colonic biopsy sample.

[0064] In a further preferred embodiment, the microorganism comprises a community of microorganism cells derived from a stool or biopsy of one or more human donors.

[0065] In a further preferred embodiment, the community of microorganism cells comprises cultured microorganism cells.

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

[0067] In a further preferred embodiment, the community of microorganism cells comprises uncultured microorganism cells.

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

[0069] In a further preferred embodiment, the composition is a faecal transplant microbiota composition.

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

[0071] In a further preferred embodiment, the composition is a liquid. [0072] In a further preferred embodiment, after at least 4 weeks of storage at room temperature, said composition is capable of maintaining at least 50% cell viability relative to the initial cell viability immediately prior to storage.

[0073] In a further preferred embodiment, after at least 4 weeks of storage at room temperature said composition is capable of maintaining about 60% to about 80% cell viability relative to the initial cell viability immediately prior to the start of said 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 a further preferred embodiment, the composition comprises an insoluble fibre, a buffer, an antioxidant, an osmotic agent, an antifoaming 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 a resistant starch.

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

[0081] In a further preferred embodiment, the composition comprises a stabiliser and/or 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; saccharose; galactose-lactose; inulin; maltodextrin; glutathione; and any combination thereof.

[0083] In a further preferred embodiment, the said 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 further preferred embodiment, the said cryoprotectant is trehalose at a concentration of 2% to 15% in said lyophilized formulation.

[0085] In a further preferred embodiment, the said cryoprotectant is trehalose at a concentration of at least 5% in said lyophilized formulation. [0086] In a further preferred embodiment, the said cryoprotectant is trehalose at a concentration of at least 10% in said lyophilized formulation.

[0087] In a further preferred embodiment, the 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 said composition comprises one or more pharmaceutically acceptable carriers or excipients.

[0090] In a further preferred embodiment, the said composition is formulated as a geltab, pill, enema, microcapsule, capsule, or tablet.

[0091] In a further preferred embodiment, the capsule or tablet is enteric-coated, pH- dependent, slow-release, and/or gastro-resistant.

[0092] In a further preferred embodiment, the composition is adapted for administration orally or rectatly.

[0093] In a further preferred embodiment, every 200 mg of the composition comprises a pharmacologically active dose of microorganism cells or spores selected from the group consisting of: 10 ¹ to 10 ¹⁴ ; 10 ¹ to 10 ¹⁶ ; 10 ² to 10 ^14; 10 ³ to 10 ¹⁴ ; 10 ⁴ to 10 ¹⁴ ; 10 ⁵ to 10 ¹⁴ ;10 ⁶ to 10 ¹⁴ ; 10 ⁷ to 10 ¹⁴ ; 10® to 10 ¹⁴ ; 10 ⁴ to 10 ¹³ ; 10® to 10 ¹² ; 10® to 10 ¹¹ ; 10 ⁷ to 10 ¹ °; 10® to 10 ⁹ ; 10 ³ to 10 ¹³ ; 10 ³ to 10 ¹² ; 10 ³ to 10 ¹¹ ; 10 ³ to 10 ¹ °; 10 ³ to 10 ⁹ ; 10 ³ to 10®; 10 ³ to 10 ⁷ ; 10 ³ to 10 ⁵ ; 10 ³ to 10 ⁵ , and 10 ³ to 10 ⁴ colony forming units (cfu) or total cell count.

[0094] In a further preferred embodiment, the composition comprises a pharmacologically active dose of microorganism cells or spores selected from the group consisting of: from 1 cfu/mL to 10 cfu/mL, 100 cfu/mL to 1 thousand cfu/mL, 10 thousand cfu/mL to 100 thousand cfu/mL, from 10 cfu/mL to 10 ⁶ cfu/mL, from 100 cfu/mL to 10 ⁶ cfu/mL, from 1000 cfu/mL to 10 ⁶ cfu/mL, from 10000 cfu/mL to to 10 ⁶ cfu/mL, from 100000 cfu/mL to 10 ⁶ cfu/mL, from 10 ¹ cfu/mL to 10 ⁶ cfu/mL, 1 cfu/mL to 10 cfu/mL, 100 cfu/mL to 1 thousand cfu/mL, 10 thousand cfu/mL to 100 thousand cfu/mL, from 1 million cfu/mL to 10 million cfu/mL, from 10 million cfu/mL to 100 billion cfu/mL, from 10 million to 50 million cfu/mL, more preferably from 50 million to 100 million cfu/mL, from 100 million to 500 million cfu/mL, from 500 million to 1 billion cfu/mL, from 1 billion to 5 billion cfu/mL, from 5 billion to 10 billion cfu/mL, from 10 billion to 15 billion cfu/mL, from 1 5 billion to 20 billion cfu/mL, from 20 billion to 25 billion cfu/mL, from 25 billion to 30 billion cfu/mL, from 30 billion to 35 billion cfu/mL, from 35 billion to 40 billion cfu/mL from 40 billion to 45 billion cfu/mL, from 45 billion to 50 billion cfu/mL, from 50 billion to 55 billion cfu/mL, from 55 billion to 60 billion cfu/mL, from 60 billion to 65 billion cfu/mL, from 65 billion to 70 billion cfu/mL, from 70 billion to 75 billion cfu/mL, from 75 billion to 80 billion cfu/mL, from 80 billion to 85 billion cfu/mL, from 85 billion to 90 billion cfu/mL, from 90 billion to 95 billion cfu/mL, from 95 billion to 100 billion cfu/mL.

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

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

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

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

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

[00100] In a further preferred embodiment, the inflammatory bowel disease is selected from the group consisting of: ulcerative colitis; Crohn's disease; gastroenteritis; colitis; and pouchitis.

[00101] In a further preferred embodiment, the gastrointestinal disorder is selected from the group consisting of: irritable bowel syndrome; an ulcer of the gastrointestinal tract; a cancer of the gastrointestinal tract.

[00102] In a further preferred embodiment, the composition reduces endogenous sulphide levels in the colon of a patient in need thereof.

[00103] In a further preferred embodiment, the composition reduces sulphide and NO load on epithelial cells leading to a metabolic lesion via inhibition of cellular respiration.

[00104] In a further preferred embodiment, the composition reduces the taxonomic diversity, number of species, relative abundance, and/ or metabolic activity of sulphidogenic microbiota; reduces sulphide levels in the colon directly through consumption/assimilation/degradation; reduces sulphide levels in the colon via metabolic substrate competition; and or reduces sulphide levels in the colon by consuming hydrogen.

[00105] In a further preferred embodiment, the composition reduces the taxonomic diversity, number of species, relative abundance and/ or metabolic activity of sulphidogenic microbiota by reducing metabolizable sulphur substrates; reduce sulphur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

[00106] In a further preferred embodiment, the composition reduces the taxonomic diversity, number of species, relative abundance, and/or metabolic activity of sulphidogenic microbiota.

[00107] In a further preferred embodiment, the composition reduces sulphide levels in the colon directly through consumption/assimilation/degradation.

[00108] In a further preferred embodiment, the composition reduces sulphide levels in the colon by consuming hydrogen.

In a further preferred embodiment, the composition reduces the taxonomic diversity, number of species, relative abundance and/or metabolic activity of sulphidogenic microbiota by reducing metabolisable sulphur substrates.

[00109] In a further preferred embodiment, the composition reduces sulphur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

[00110] In a further preferred embodiment, the composition reduces sulphide levels in the colon via metabolic substrate competition. In a further preferred embodiment, the composition drives down undesired inflammation.

[00111] In a further preferred embodiment, the composition prevents or reduces activation of the mucosa immune system in a natural killer T-cell driven IL-13 and IL-5 dependent, TH2- mediated, immune response.

[00112] In a further preferred embodiment, the composition decreases inflammation in the subject when measured by a parameter selected from the group consisting of: TNFa signalling via NF-KB; IFNa signalling; IFNy signalling; IL6 JAK STAT3 signalling; activation of pro- apoptotic pathways; initiation of unfolded protein response.

[00113] In a further preferred embodiment, the composition down-regulates genes associated with pro-apoptotic pathways and the unfolded protein response, including genes selected from the group consisting of: CHAC1 , CEBPB, TRIB3, PPP1 R15A, DDIT3, ATF4 and XBP1.

[00114] 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 for treating or preventing ulcerative colitis. Preferably, the composition is used for treating or preventing IBD.

[00115] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Flavinofractor spp.

[00116] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Flavinofractor plautii.

[00117] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Christensenella spp.

[00118] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Christensenella minuta.

[00119] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Anaerobutyricum spp.

[00120] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Anaerobutyricum hallii.

[00121] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Escherichia-Shigella spp.

[00122] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Escherichia coli.

[00123] In a further preferred embodiment, the composition further comprises dietary fibre. Preferably, the dietary fibre is selected from the group consisting of: FOS, inulin, maltodextrin, and starch.

[00124] In a second aspect, the invention broadly resides in 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 fibre. Preferably, the dietary fibre is selected from the group consisting of: FOS, inulin, maltodextrin, and starch. [00125] In a third aspect, the invention broadly resides in a pharmaceutical composition comprising the composition of the first aspect of the invention, together with a pharmaceutically acceptable diluent or carrier. In one embodiment, the composition further comprises dietary fibre. Preferably, the dietary fibre is selected from the group consisting of: FOS, inulin, maltodextrin, ard starch.

[00126] In a fourth aspect, the invention broadly resides in an isolated non-inflammatory strain of microorganism comprising a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs 1 to 9193.

[00127] In a fifth aspec:, the invention broadly resides in an isolated non-inflammatory strain of microorganism comprising a contiguous whole genome sequence having a nucleotide sequence selected from the group consisting of: SEQ ID NOs 1 to 9193.

[00128] In a further preferred embodiment, the isolated non-inflammatory strain of microorganism according to the fourth aspect of the invention, wherein the at least one strain of microorganism comprises a 16S ribosomal RNA (rRNA) gene or conitgous whole genome sequence having a sequence identity to one or more SEQ ID NOs 1 to 9193, wherein the sequence identity is selec.ed from the group consisting of: at least 99.9%; at least 99%; at least 98%; at least 97%; a: least 96%; at least 95%; at least 94%; at least 93%; at least 92%; at least 91 %; and at least 90%.

[00129] In a fifth aspect, the invention broadly resides in a method of treating and/or preventing a gastrointestinal disorder in a patient in need thereof said method comprising the step of administering the composition of the invention to a patient in need thereof.

[00130] In a further preferred embodiment, the gastrointestinal disorder is gastrointestinal tract mucosal inflammation.

[00131] In a further preferred embodiment, the gastrointestinal disorder is dysbiosis.

[00132] 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), an enteric bacterial infection, a metabolic disease, a neuropsychiatric disorder, an autoimmune disease, an allergic disorder, hepatic encephalopathy, or a cancer.

[00133] In a further preferred embodiment, the gastrointestinal disorder is an inflammatory bowel disease. [00134] In a further preferred embodiment, the inflammatory bowel disease is selected from the group consisting of: ulcerative colitis; Crohn's disease; gastroenteritis; colitis; and pouchitis.

[00135] In a further preferred embodiment, the gastrointestinal disorder is selected from the group consisting of: irritable bowel syndrome; an ulcer of the gastrointestinal tract; a cancer of the gastrointestinal tract.

[00136] In a further preferred embodiment, the composition is administered orally or rectally.

[00137] In a further preferred embodiment, the said composition is administered to the patient using a dosing regimen selected from the group consisting of: once-off; 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; weekly; twice weekly; every 2 weeks; every 3 weeks; every 4 weeks; every 5 weeks; every 6 weeks; once monthly; twice monthly; every 2 months; every 3 months; every 4 months; every 5 months; every 6 months; yearly; twice yearly; every 2 years; every 3 years; every 4 years; and every 5 years.

[00138] In a further preferred embodiment, the composition reduces endogenous sulphide levels in the colon of a patient in need thereof.

[00139] In a further preferred embodiment, the composition reduces sulphide and nitric oxide load on epithelial cells leading to a metabolic lesion via inhibition of cellular respiration.

[00140] In a further preferred embodiment, the composition reduces nitric oxide production and/or reduces nitric oxide levels in the colon.

[00141] 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 sulphidogenic microbiota; reduces sulphide levels in the colon directly through consumption/assimilation/degradation; reduces sulphide levels, taxonomic diversity, number of, relative abundance and or metabolic activity of sulphidogenic microbiota via metabolic substrate competition; and/or reduces sulphide levels, taxonomic diversity, number of, relative abundance and or metabolic activity of sulphidogenic microbiota by consuming hydrogen.

[00142] 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 sulphidogenic microbiota. [00143] In a further preferred embodiment, the composition reduces sulphide levels in the colon directly through consumption/assimilation/degradation. by consuming hydrogen.

[00144] In a further preferred embodiment, the composition the taxonomic diversity, reduces the number of species, reduces the relative abundance and/or reduces the metabolic activity sulphidogenic microbiota via metabolic substrate competition.

[00145] In a further preferred embodiment, the composition reduces sulphide levels, reduces the taxonomic diversity, reduces the number of species, reduces the relative abundance and/or reduces the metabolic activity of sulphidogenic microbiota by consuming hydrogen.

[00146] 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 sulphidogenic microbiota by reducing metabolizable sulphur substrates in the colon; and/or reduces the taxonomic diversity, number of species, relative abundance and/or metabolic activity of sulphidogenic microbiota by reducing sulphur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

[00147] In a further preferred embodiment, the composition reduces the rate or concentration of sulphide produced in the colon by increasing the taxonomic diversity, the number of species, relative abundance and/or metabolic activity of sulphide consuming/assimilating/degrading isolates.

[00148] In a further preferred embodiment, the composition reduces the rate or concentration of sulphide produced in the colon by increasing the taxonomic diversity, number of species, relative abundance and/or metabolic activity of sulphide consuming/assimilating/degrading isolates relative to the number of species, the taxonomic diversity or relative abundance of sulphidogenic microbiota.

[00149] In a further preferred embodiment, the method drives down undesired inflammation.

[00150] 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.

[00151] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Flavinofractor spp. [00152] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Flavinofractor plautii.

[00153] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Christensenella spp.

[00154] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Christensenella minuta.

[00155] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Anaerobutyricum spp.

[00156] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Anaerobutyricum hallii.

[00157] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Escherichia-Shigella spp.

[00158] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Escherichia coli.

[00159] In a further preferred embodiment, the composition is administered with dietary fibre. Preferably, dietary fibre is selected from the group consisting of: FOS, inulin, maltodextrin, and starch. In one embodiment, the dietary fibre is administered concurrently or sequentially with the composition.

[00160] In a further preferred embodiment, the composition prevents or reduces activation of the mucosa immune system in a natural killer T-cell driven IL-13 and IL-5 dependent, TH2 mediated, immune response.

[00161] In a further preferred embodiment, the said method decreases inflammation in the subject when measured by a parameter selected from the group consisting of: TNFo signalling via NF-KB; IFNO signalling; IFNy signalling; IL6 JAK STAT3 signalling; activation of pro- apoptotic pathways; initiation of unfolded protein response.

[00162] In a further preferred embodiment, the said method down regulates genes associated with pro-apoptotic pathways and the unfolded protein response, including genes selected from the group consisting of: CHAC1 , CEBPB, TRIB3, PPP1 R15A, DDIT3, ATF4 and XBP1.

[00163] In a sixth aspect, the invention broadly resides in a method of reducing endogenous sulphide levels in the colon of a patient in need thereof. [00164] In a further preferred embodiment, the method reduces sulphide and nitric oxide load on epithelial cells leading to a metabolic lesion via inhibition of cellular respiration.

[00165] In a further prefe ^r red embodiment, the method reduces relative abundance and or metabolic activity of sulphidogenic microbiota.

[00166] In a further preferred embodiment, the method reduces sulphide levels in the colon directly through consumption/assimilation/degradation.

[00167] In a further preferred embodiment, the method reduces sulphide levels, taxonomic diversity, number of species, relative abundance and/or metabolic activity of sulphidogenic microbiota in the colon by metabolic substrate competition.

[00168] In a further preferred embodiment, the method reduces sulphide levels, taxonomic diversity, number of species, and relative abundance and/or metabolic activity of sulphidogenic microbiota ir the colon by consuming hydrogen.

[00169] In a further preferred embodiment, the method reduces sulphide levels, taxonomic diversity, number of species, and relative abundance and/or metabolic activity of sulphidogenic microbiota by reducing metabolisable sulphur substrates in the colon.

[00170] In a further preferred embodiment, the method reduces sulphide levels, the taxonomic diversity, numoer of species, relative abundance and/or metabolic activity of sulphidogenic microbiota by reducing sulphur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon.

[00171] In a further preferred embodiment, the method reduces sulphide levels, the taxonomic diversity, number of species, relative abundance and/or metabolic activity of sulphidogenic microbiota in the colon by reducing protein fermentation.

[00172] In a seventh aspect, the invention broadly resides in a method of preparing the biotherapeutic composition according to the second aspect of the invention, the method comprising mixing the composition according to the first aspect of the invention with an acceptable diluent or carrier.

[00173] In an eighth aspect, the invention broadly resides in a method of preparing the pharmaceutical composition according to the third aspect of the invention, the method comprising mixing the composition according to the first aspect of the invention with a pharmaceutically acceptable diluent or carrier. [00174] In a nineth aspect, the invention broadly resides in the use of the composition according to the first aspect of the invention in the manufacture of a medicament for reducing or preventing gastrointestinal disorder in a subject.

[00175] In a tenth aspect, the invention broadly resides in a dosage form comprising the composition of the first aspect of the invention.

[00176] In an eleventh aspect, the invention broadly resides in a kit comprising the dosage form according to the tenth aspect of the invention together with instructions for its use.

[00177] In a further aspect, the invention broadly resides in a nucleotide sequence selected from the group consisting of: SEQ ID NOs 1 to 9193.

[00178] In a further aspect, the invention is a nucleotide sequence having a sequence identity to one or more SEQ ID NOs 1 to 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%.

[00179] In one preferred embodiment, the nucleotide sequence is substantially purified or isolated.

[00180] In a further aspect, the invention is a micro-organism comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOs 1 to 9193.

[00181] In a further aspect, the invention is a micro-organism comprising a nucleotide sequence having a sequence identity to one or more SEQ ID NOs 1 to 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%.

[00182] In one preferred embodiment, the micro-organism is substantially purified or isolated.

[00183] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above.

Brief Description of the Drawings

[00184] Below is a brief description of each of the figures and drawings. [00185] Figure 1 shows tie diagrammatical representation of the processes that lead to ulcerative colitis induced by high levels of sulphide (either as a sulphide ion (S ^2- ), a bifsulphide ion (SHj, or hydrogen sulphide (H ₂ S)) and nitric oxide (NO).

[00186] Figure 2 shows a graphical representation of hydrogen competition between methanogens, acetogens and sulphate-reducing bacteria. Competition for hydrogen by methanogens and acetogens leads to a decrease in the activity and abundance of sulphate- reducing bacteria, resulting in decreased production of sulphide.

[00187] Figure 3 shows the BB265 drug discovery process detailing lead selection and BB265 mechanistic validation prior to clinical validation in a phase 1 trial.

[00188] Figure 4 shows the cysteine biosynthesis pathway outlined in red, a pathway by which sulphide can be directly consumed by colonic microbiota. This pathway was obtained from https://www.Qenome.ip/pathwav/map00270.

[00189] Figure 5 shows a table of taxa of all 143 isolates comprising the BB265 complex consortium along with all isolates outside of the consortium identified with a sulphide consumption phenotype. Complete taxonomic lineages and taxon identifications are displayed for each isolate.

[00190] Figure 6 shows a table of the V3-V4 hypervariable region of the 16S rRNA gene sequences taken from the isolates listed in Figure 5. These sequences are SEQ ID No 1-206 (a total of 206 sequences). These sequences are used for taxonomic classification of microbial species because the 16S rRNA gene, which is a small ribosomal subunit, is conserved amongst microbial species, whilst containing hypervariable regions, such as the V3-V4 region, which provides sufficient variation to determine species-level discrimination between 16 rRNA gene sequences. The sequences presented are DNA sequences encoding the ribosomal RNA.

[00191] Figure 7 shows a table of the full-length 16S rRNA gene sequences taken from the isolates listed in Figure 5. These sequences are SEQ ID No 207-412 (a total of 206 sequences). These sequences are used for taxonomic classification of microbial species because the 16S rRNA gene, which is a small ribosomal subunit, is conserved amongst microbial species whilst containing hypervariable regions, such as the V3-V4 region, which provides sufficient variation to determine species-level discrimination between 16 rRNA gene sequences. The sequences presented are DNA sequences encocing the ribosomal RNA.

[00192] Figure 8 shows a table of contiguous DNA sequences derived from whole genomes of the isolates listed in Table 5. These sequences are SEQ ID No 413 - 9193 (a total of 8781 sequences. These sequences taken together comprise the whole-genome sequence of the specific isolates listed in Table5. The sequences presented are DNA sequences encoding the ribosomal RNA.

[00193] Figure 9 shows the amount of sulphide, expressed in mM, consumed by microbial isolates identified to have a statistically significant sulphide consumption phenotype relative to the negative control when assayed for sulphide consumption using the in-house modified methylene blue assay.

[00194] Figure 10 shows a flow chart of the tri-tool consensus analysis used to identify taxa that discriminate between health and ulcerative colitis from large faecal metagenome datasets.

[00195] Figure 11 shows a phylogenetic tree displaying the sulphide phenotype of the 12,607 isolates from the inventors’ culture collection assayed for sulphide production. Tree construction was performed using FastTree v2.1 .11 to infer maximum likelihood trees with the generalised time-reversible (GTR) with CAT approximation model. The tree was visualised with R package ggtree v3.6.2 and R package ggtree Extra v1 .8.1 .

[00196] Figure 12a shows box and whisker plots representing the changes in the number of BB265 species in patients who achieved remission from FMT in a clinical trial. Week 0 indicates the number of species prior to remission when patient disease state was considered active; Week 12 is post FMT treatment during remission.

[00197] Figure 12b shows box and whisker plots representing the cumulative relative abundance of sulphide consumer species in patients who achieved remission from FMT in a clinical trial. Week 0 indicates the number or abundance of species prior to remission when patient disease state was considered active; Week 12 is post FMT Treatment during remission.

[00198] Figure 12c shows box and whisker plots representing the number of sulphide consumer species in patients who achieved remission from FMT in a clinical trial. Week 0 indicates the number or abundance of species prior to remission when patient disease state was considered active; Week 12 is post FMT treatment during remission.

[00199] Figure 13a shows box and whisker plots representing the average concentrations of aqueous sulphide (pM) in stool samples of Patient D post incubation in half strength YCFA with sodium sulphite and Solution I. Stool samples were collected during active disease and during remission following FMT.

[00200] Figure 13b shows box and whisker plots representing the average concentrations of aqueous sulphide (pM) in stool samples of Patient E post incubation in half strength YCFA with sodium sulphite and Solution I. Stool samples were collected during active disease and during remission following FMT.

[00201] Figure 13c shows bar graphs representing the number of BB265 species identified in stool samples from Patient D and E during active disease (Baseline) and remission following FMT.

[00202] Figure 13d shows bar graphs representing

[00203] the number of statistically significant sulphide consumer species identified in stool samples from Patient D and E during active disease (Baseline) and remission following FMT.

[00204] Figure 13e shows bar graphs representing the cumulative abundance of sulphidogenic species identified in stool samples from Patients D and E during active disease (Baseline) and remission following FMT.

[00205] Figure 14 shows box and whisker plots representing the average reduction in aqueous sulphide concentration (pM) in two ulcerative colitis patient stool samples (Patient A and B) co-incubated with a subset of the BB265 complex consortium comprising 127 of the 143 isolates. Reduction in aqueous sulphide is displayed as concentration (pM) change from negative control, which consists of stool incubated in media only. Phylogenetically diverse consumer mix (PDCM) refers to ulcerative colitis stool co-incubated with 19 isolates of BB265 phenotypically confirmed as sulphide consumers using the modified methylene blue assay. A double asterisk (**) indicates statistically significant difference between treatments (one-way Anova, P-value s 0.01 , n = 5).

[00206] Figure 15 shows box and whisker plots representing the average reduction in aqueous sulphide concentration in an ulcerative colitis patient stool sample (Patient A) co- incubated with 127 of the 143 isolates comprising the BB265 complex consortium. Reduction in aqueous sulphide is displayed as change in concentrations (pM) from the negative control, which consists of stool incubated in media only. Individual consumer refers to ulcerative colitis stool co-incubated with an individual sulphide consumer, bb0214, which displayed 670 pM sulphide consumption using the modified methylene blue assay. An asterisk (*) indicates statistically significant difference between treatments (one-way Anova, P-value 0.05, n = 5).

[00207] Figure 16 shows box and whisker plots representing the average reduction in aqueous sulphide concentration in ulcerative colitis patient stool samples (Patient A and C) co-incubated with the BB265 complex consortium. Reduction in aqueous sulphide is displayed as change in concentration (pM) from the negative control, which consists of stool incubated in media only. Individual consumer refers to ulcerative colitis stool co-incubated with the individual strong sulphide consumer, bb0214 which displayed 670 pM H2S consumption using the modified methylene blue assay. A double asterisk (**) indicates statistically significant difference between treatments (one-way Anova, P-value < 0.01 , n = 5).

[00208] Figure 17 shows a phylogenetic tree displaying the sulphide consumer and corresponding sulphidogenic phenotypes of the 275 purified microbial isolates assayed for sulphide consumption using the in-house modified methylene blue assay. Colours on tree tips represent phyla and the inner ring bar graph represents the average concentration of sulphide consumed by isolates (pM). Isolate identifiers are present for those isolates in the BB265 complex consortium and for isolates identified dith net average sulphide consumption. The inner-outer and outer-outer rings show sulphide production from cysteine and thiosulphate, respectively; ‘N’ refers to a negative phenotype whereas ‘Y’ refers to a positive phenotype. Tree construction was performed using FastTree v2.1 .11 to infer maximum likelihood trees with the generalised time-reversible (GTR) with CAT approximation model. The tree was visualised with R package ggtree v3.6.2 and R package ggtreeExtra v1 .8.1 .

[00209] Figure 18 shows a line graph representing the accumulation of H ₂ S overtime due to microbial production of hydrogen sulphide in an ulcerative colitis patient (Patient A) stool sample alone, healthy donor (Donor A) stool sample alone, and when the ulcerative colitis patient (Patient A) stool sample was co-incubated with the BB265 complex consortium, and a sulphide consumer ( bb0214). Healthy Donor A was used as a healthy comparison for hydrogen sulphide production. Concentration of H2S is displayed in pM.

[00210] Figure 19 shows a line graph representing the accumulation of H ₂ S overtime due to microbial production of hydrogen sulphide in an ulcerative colitis patient (Patient C) stool sample alone, healthy donor (Donor C) stool sample alone, and when the ulcerative colitis patient (Patient A) stool sample was co-incubated with the BB265 complex consortium, and a sulphide consumer ( bb0450). Healthy Donor C was used as a healthy comparison for sulphide production. Concentration of H ₂ S is displayed in pM.

[00211] Figure 20 shows a line graph representing the rate of H ₂ S production due to microbial production of hydrogen sulphide in an ulcerative colitis patient (Patient A) stool sample alone, healthy donor (Donor A) stool sample alone, and when the ulcerative colitis patient (Patient A) stool sample was co-incubated with the BB265 complex consortium, and a sulphide consumer ( bb0214). Healthy Donor A was used as a healthy comparison for sulphide production. Rate of H ₂ S production is displayed in pM hr ¹

[00212] Figure 21 shows a line graph representing the rate of H ₂ S production due to microbial production of hydrogen sulphide in an ulcerative colitis patient (Patient C) stool sample alone, healthy donor (Donor C) stool sample alone, and when the ulcerative colitis patient (Patient C) stool sample was co-incubated with the BB265 complex consortium, and a sulphide consumer (bb0450). Healthy Donor A was used as a healthy comparison for sulphide production. Rate of H ₂ S production is displayed in pM hr ¹

[00213] Figure 22 shows a dot plot representing the effect of fibre with and without with the 127-isolate subset of the BB265 complex consortium on the concentration of sulphide (pM) post fermentation of ulcerative colitis stool samples from Patients A , B and F. Fibres tested were: FOS, Inulin, Maltodextrin and Starch.

[00214] Figure 23a shows a bar graph representing the effect of co-culturing ulcerative colitis stool (Patient A and Patient C)with the 127-isolate subset of the BB265 complex consortium on the number and cumulative relative abundances of putative sulphidogenic species achieved from Shotgun metagenomics in the stool samples of Patient A and B.

[00215] Figure 23b shows a bar graph representing the effect of co-culturing ulcerative colitis stool (Patient and Patient C) with the 127-isolate subset of the BB265 complex consortium on the number and cumulative relative abundances of sulphide consuming species achieved from Shotgun metagenomics in the stool samples of Patient A and B.

[00216] Figure 23c shows a bar graph representing the effect of co-culturing ulcerative colitis stool (Patient and Patient C) with the 127-isolate subset of the BB265 complex consortium on the number and accumulate relative abundances of species comprising the BB265 complex consortium achieved from Shotgun metagenomics in the stool samples of Patient A and B

[00217] Figure 24a shows a bar graph representing the number and abundance of BB265, phenotypically identified consumers and producers achieved from shotgun metagenomics in the stool sample of Patient A when inoculated with the strong consumer bb0214 and the 127- isolate subset of BBB265 post incubation. Asterisks (***) indicate statistically significant difference between treatments (one-way Anova, P-value < 0.001 , n = 5).

[00218] Figure 24b shows a bar graph representing the cumulative relative abundance ofsulphidogens achieved from shotgun metagenomics in the stool sample of Patient A when inoculated with the strong consumer bb0214 and the 127-isolate subset of the BBB265 complex consortium post incubation. Asterisks (***) indicate statistically significant difference between treatments (one-way Anova, P-value s 0.001 , n = 5).

Detailed Description of the Invention [00219] For convenience, the following sections generally outline the various meanings of the terms used herein. Following this discussion, general aspects regarding compositions, use of medicaments and methods of the invention are discussed, followed by specific examples demonstrating the properties of various embodiments of the invention and how they can be employed.

[00220] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[00221] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent citec in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the information contained in that material should, however, be understood to be common general knowledge.

[00222] Manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and can be employed in the practice of the invention.

[00223] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

1. DEFINITIONS

[00224] The meaning of certain terms and phrases used in the specification, examples, and appended claims, a ^_ e provided below. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[00225] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%. [00226] The invention described herein may include one or more range of values (e.g. size, concentration etc.). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. For example, a person skilled in the field will understand that a 10% variation in upper or lower limits of a range can be totally appropriate and is encompassed by the invention. More particularly, the variation in upper or lower limits of a range will be 5% or as is commonly recognised in the art, whichever is greater.

[00227] In this application, the use of the singular also includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. 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 comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term “portion” can include part of a moiety or the entire moiety.

[00228] 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.

[00229] “Therapeutically effective amount” as used herein with respect to methods of treatment and in particular drug dosage, shall mean that dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment. It is emphasized that “therapeutically effective amount,” administered to a particular subject in a particular instance will not always be effective in treating the diseases described herein, even though such dosage is deemed a “therapeutically effective amount” by those skilled in the art. It is to be further understood that drug dosages are, in particular instances, measured as oral dosages, or with reference to drug levels as measured in blood. Amounts effective for such a use will depend on: the desired therapeutic effect; the potency of the biologically active material; the desired duration of treatment; the stage and severity of the disease being treated; the weight and general state of health of the patient; and the judgment of the prescribing physician. Treatment dosages need to be titrated to optimize safety and efficacy. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the indication for which the active agent is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titre the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage may range from about 0.1 mg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may 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.

[00230] The frequency of dosing will depend upon the pharmacokinetic parameters of the active agent and the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.

[00231] As used herein, a “carrier” can be any solvents, diluents, excipients or other vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.

[00232] As used herein, the term "pharmaceutically acceptable carrier" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a composition of the 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 of the formulation in which it is contained. The component has generally met the required standards of toxicological and manufacturing.

[00233] As used herein the term “subject” generally includes mammals such as: humans; farm animals such as sheep, goats, pigs, cows, horses, llamas; companion animals such as dogs and cats; primates; birds, such as chickens, geese and ducks; fish; and reptiles. The subject is preferably human.

[00234] As used herein, the “gastrointestinal tract” refers to the tract from the mouth to the anus which includes all the organs of the digestive system such as the esophagus, stomach, pancreas, liver, gallbladder, small intestine (including the ileum), caecum, large intestine, colon and rectum. Strains of the invention are at least useful for conditions of the terminal ileum, caecum or rectum. [00235] As used herein, a “non-inflammatory strain” refers to a strain of the invention which, when present in the gastrointestinal tract of a subject, preferably a human, is associated with a non-inflamed state. In one preferred embodiment, non-inflammatory strains of the invention have little or no cytotoxicity against mammalian cells in culture. In an embodiment, the strain results in less than 15%, less than 10% or less than 5% of cell death of the mammalian cells in culture. In an embodiment, a non-inflammatory strain of the invention causes less cell death when exposed to a given cell type than a strain which comprises a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence as shown 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, non-inflammatory strains of the invention have some level of cytotoxicity against mammalian cells in culture.

[00236] As used herein, an “inflammatory strain” refers to a strain of the invention which, when present in the gastrointestinal tract of a subject, preferably a human, is associated with an inflamed state. Inflammatory strains of the invention have cytotoxicity against mammalian cells in culture. In an embodiment, the strain results at least 40%, at least 45% or at least 50% of cell death of the mammalian cells in culture. In an embodiment, an inflammatory strain of the invention causes more cell death when exposed to a given cell type than a strain which comprises a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence as shown in any one of SEQ ID NOs presented in Figure 7 and Figure 8, or a nucleotide sequence at least 90% identical to one or more of SEQ ID NOs presented in Figure 7 and Figure 8. In an alterative embodiment, the inflammatory strain of the invention has little to no cytotoxicity against mammalian cells in culture.

[00237] As used herein, the term “bacteriotherapy” refers to the use of a bacterial isolate to treat or prevent a disease or a condition, or provide a health benefit, in a subject.

[00238] As used herein, the term “biotherapeutic” refers to a microorganism or combination thereof, such as bacterial isolate, that is useful for treating or preventing a disease or a condition, or provide a health benefit, in a subject.

[00239] The term "biotherapeutic composition" as used herein, refers to a formulation comprising a biotherapeutic preparation formulated together with one or more additional formulary ingredients to obtain a finished formulation suitable for delivery to a subject.

[00240] As used herein, the terms "treat,” "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject. Since every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population. Accordingly, a given subject or subject population may fail to respond or respond inadequately to treatment.

[00241] As used herein, the term “prevent”, “prevented”, or “preventing” when used with respect to the treatment of mucosal inflammation in the gastrointestinal refers to a prophylactic treatment which increases the resistance of a subject to mucosal inflammation in the gastrointestinal, in other words, decreases the likelihood that the subject will develop mucosal inflammation in the gastrointestinal as well as a treatment after mucosal inflammation in the gastrointestinal has begun in order to fight the inflammation, e.g., reduce or eliminate it altogether or prevent it from becoming worse.

[00242] As used hereini, the term “reducing”, or variations thereof refer to a reduction but not necessarily a complete abolition of gastrointestinal tract mucosal inflammation in a subject.

[00243] As used herein, the term "sample" refers to a collection of biological material obtained from a subject or a subject's surrounding environment, such as soil or water in the area that the subject inhabits. In some embodiments, the sample is obtained directly from the subject. For example, the sample can be a faecal sample or obtained during a colonoscopy. The sample may be in a form taken directly from the subject or surrounding environment, or it may be at least partially purified to remove at least some non-nucleic acid material. The purification may be slight, for instance amounting to no more than the concentration of the solids, or cells, of the samole into a smaller volume or the separation of cells from some or all of the remainder 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 the nucleic acids in the sample. In some embodiments, the predominant nucleic acid is DNA. The nucleic acid preparations can be pure or partially purified nucleic acid preparations. Techniques for the isolation of nucleic acid from samples, including complex samples, are numerous and well known in the art.

[00244] The unit “cfu” refers to "colony forming unit", which is the number of bacterial cells as revealed by microbiological counts on agar plates.

[00245] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[00246] Features of the invention will now be discussed with reference to the following non-limiting description and examples. 2. EMBODIMENTS

Composition

[00247] The present invention provides a composition for preventing or treating a gastrointestinal disorder in a subject in need thereof, said composition comprising at least one strain of bacteria.

[00248] 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.

[00249] Preferably the compositions are 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 solutions, for example phosphate- buffered saline. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. See, e.g., 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 herein incorporated by reference.

[00250] The composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulphite or sodium hydrogen-sulphite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetra acetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers; monosaccharides, disaccharides; and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); colouring, flavouring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants.

[00251] The optimal composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the biotherapeutic actives of the invention. The preferred form of the pharmaceutical composition depends on the intended mode of administration and therapeutic application.

[00252] The primary vehicle or carrier in a composition is aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, possibly supplemented with other materials. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. In one embodiment of the present invention, pharmaceutical compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of an aqueous solution.

[00253] The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

[00254] Additional compositions will be evident to those skilled in the art, including formulations of the invention in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Additional examples of sustained-sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, for example, films, or microcapsules. Sustained release matrices may 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 may also include liposomes, which can be prepared by any of several methods known in the art.

[00255] The composition to be used for in vivo administration can be filtered to remove undesirable components. This may be accomplished by filtration through filtration membranes. In addition, the compositions generally are placed into a sealed container to reduce exposure to oxygen. Once the pharmaceutical composition has been formulated, it may be stored in sealed containers.

[00256] The term "% sequence homology ", as used here, may for example be calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)). A comparison is made over the window corresponding to one of the aligned sequences, for example the shortest. The window may in some instances be defined by the target sequence. In other instances, the window may 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 correspondences in the target sequence is reported as % sequence homology, or percent identification.

[00257] In an embodiment, the % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Preferably, the GAP analysis aligns two sequences over their entire length.

[00258] The bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for instance, Handbook of Microbiological Media, Fourth Edition (2010) Ronald Atlas, CRC Press, Maintaining Cultures for Biotechnology and Industry (1996) Jennie C. Hunter-Cevera, Academic Press, as well as how detailed in the Examples using YCFA medium.

[00259] In yet a further preferred embodiment, the composition further comprises water.

[00260] In yet a further preferred embodiment, the composition is a liquid, such as an aqueous solution.

[00261] In yet a further preferred embodiment, further comprises a pharmaceutically acceptable carrier. [00262] In yet a further preferred embodiment, the composition 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. Preferably, the composition is stable for periods selected from the group consisting of: 6 months, 1 year and 2 years. In one example, the composition is stable at temperatures selected from the group consisting of: -80°C, -20 °C ,-4°C, 4°C, 18°C and 25°C.

[00263] Pharmaceutical and therapeutic compositions are within the scope of the invention.

[00264] The therapeutic composition of the invention may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the isolated bacteria present in the therapeutic composition. The precise nature of the pharmaceutically acceptable excipient or other material will depend on the route of administration, which may be, for example, oral or rectal. Many methods for the preparation of therapeutic compositions are known to those skilled in the art (see e.g. Robinson ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978).

[00265] The therapeutic composition of the invention may comprise a prebiotic, an antioxidant, a carrier, insoluble fibre, a buffer, an osmotic agent, an anti-foaming agent and/or a preservative.

[00266] The therapeutic composition may be made or provided in chemostat medium. Alternatively, the therapeutic composition may be made or provided in saline, e.g., 0.9% saline. It will be understood that any carrier or solution which does not impair viability of the bacteria present in the therapeutic composition and is compatible with administration to an individual may be used.

[00267] The therapeutic composition may be made or provided under reduced atmosphere, i.e., in the absence of oxygen. A synthetic stool preparation may be made or provided under N2, CO2, H2, or a mixture thereof, optionally with controlled levels of partial pressure of N2:CO2:H2.

[00268] The therapeutic composition may be for oral or rectal administration to the individual. Where the therapeutic composition is for oral administration, the therapeutic composition may be in the form of a capsule, or a tablet. Where the therapeutic composition is for rectal administration, the therapeutic composition may be in the form of an enema or through colonoscopic delivery. The preparation of suitable capsules, tablets and enema is well-known in the art. The capsule or tablet may comprise 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 but allow dissolution in the small intestine.

[00269] Orally dosed formulations, for example, can, in addition to the viable microorganisms comprise, inert compression aids, such as microcrystalline cellulose or oligosaccharide, flow aids, such as a silica gel, or a lubricant of, for example magnesium stearate (vegetable source) or stearic acid (vegetable source).

[00270] A composition disclosed herein can be used as, for example, a food supplement, an edible product or pharmaceutical product. When it is a food supplement, the composition can further comprise a conventional food supplement filler and/or an extender. The composition disclosed herein can also be included in any edible products, such as dairy products, including for example, a milk product, milk, yogurt, curd, ice-cream, dressing, and cheese, beverage products, meat products, and baked goods

[00271] Suppository formulations, for example, either for rectal use, can in addition to the compositions, comprise, for example, cocoa butter, polyethylene glycol, glycerin or gelatine.

[00272] The composition may comprise a disintegrant, a glidant, and/or a lubricant. Disintegrants aid in the breakup of the compacted mass when placed in a fluid environment. The disintegrant may be any suitable disintegrant such as for example, a disintegrant selected from the group consisting of sodium croscarmellose, crospovidone, gellan gum, hydroxypropyl cellulose, starch, and sodium starch glycolate. The glidant may be any suitable glidant such as for example, a glidant 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 in order to prevent the compacted powder mass from sticking to the equipment during the tableting or encapsulation process. The lubricant may be any suitable lubricant such as for example, a lubricant selected from the group consisting of calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumarate, and vegetable based fatty acids. In the composition and method 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; this weight percentage is a cumulative weight percentage taking into consideration all ingredients present in the carrier. [00273] Coatings can be used to control the solubility of the composition. Examples of coatings include carrageenan, cellulose acetate phthalate, ethylcellulose, gellan gum, maltodextrin, methacrylates, methylcellulose, microcrystalline cellulose, and shellac.

[00274] The composition may comprise one or more preservatives. Exemplary preservatives include antioxidants, chelating agents, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[00275] Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabi sulfite, propionic acid, glutathione, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabi sulfite, and sodium sulfite.

[00276] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. 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.

[00277] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxy benzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[00278] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[00279] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabi sulfite, potassium sulfite, potassium metabi sulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, NeoIone, Kathon, and Euxyl. [00280] The therapeutic composition may be lyophilized. The lyophilized therapeutic composition may comprise one or more stabilisers and/or cryoprotectants. The lyophilized therapeutic composition may be reconstituted using a suitable diluent prior to administration to the individual.

[00281 ] A therapeutic composition according to the present invention may be administered alone or in combination with other treatments, concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of dysbiosis, or a disease associated with dysbiosis as described herein. For example, a strain of the invention may be used in combination with an existing therapeutic agent for inflammatory bowel disease, irritable bowel syndrome, a metabolic disease, a neu'opsychiatric disorder, an autoimmune disease, an allergic disorder, a cancer, or hepatic encephalopathy.

[00282] For example, where the therapeutic compcsition is for the treatment of a dysbiosis associated with cancer, the therapeutic composition may optionally be administered in combination with a cancer immunotherapy, such as an immune check-point inhibitor, to the individual. Examples of check-point inhibitors which may be employed in this context include Programmed cell death protein 1 (PD-1 ) inhibitors, Programmed death-ligand 1 (PD-L1 ) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors. Manipulation of the gut microbiota in combination with immune check-point inhibitor treatment has been shown to improve efficacy of immune check-point inhibitors in treating cancer. In one preferred embodiment, the cancer in this context is colorectal cancer. In another embodiment, the cancer is renal cancer, lung cancer or melanoma.

[00283] In another embodiment, the composition of the invention further comprise immunomodulating compounds. In other embodiments, the immunomodulating compound is a cytokine, chemokine, or complement component that enhances expression of immune system accessory or adhesion molecules, their receptors, or combinations thereof. In some embodiments, the immunomodulating compound include interleukins, for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1a and MIP-1 b, complement components, or combinations thereof. In other embodiments, the immunomodulating compound stimulate expression, or enhanced expression of 0X40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1 , B7.2, TRAP, ICAM-1 , 2 or 3, cytokine receptors, or combination thereof. [00284] In another embodiment, the immunomodulatory compound induces or enhances expression of co-stimulatory molecules that participate in the immune response, which include, in some embodiments, CD40 or its ligand, CD28, CTLA-4 or a B7 molecule. In another embodiment, the immunomodulatory compound induces or enhances expression of a heat stable antigen (HSA), chondroitin sulfate-modified MHC invariant chain (li-CS), or an intracellular adhesion molecule 1 (ICAM-1).

[00285] The therapeutic compositions of the invention may be administered to an individual, preferably a human individual. Administration may be in a "therapeutically effective amount", this being sufficient to show benefit to the individual. Such benefit may be at least improvement or amelioration of at least one symptom. Thus "treatment" of a specified disease refers to amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular patient being treated, the clinical condition of the individual patient, the cause of the dysbiosis, the site of delivery of the composition, the type of therapeutic composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. A therapeutically effective amount or suitable dose of a therapeutic composition of the invention can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the therapeutic composition is for prevention or for treatment.

[00286] Formulary ingredients can be contacted with the preparation and mixed or prepared until a formulation is obtained. As will be clear to those of skill in the art, formulation conditions will generally be such that viable microorganisms are retained. In particular high temperatures, for example temperatures in excess of 40°C are avoided.

[00287] The amount of viable microorganisms included in a composition can vary and can be adjusted and optimized as will be appreciated by those of skill in the art. Such optimization may, for example, be achieved by preparing a series of different doses of a viable microorganism. The bacterial concentration in the composition can be, for example, from 10 ¹ cfu/mL to 10 ¹⁶ cfu/mL, 1 cfu/mL to 10 cfu/mL, 100 cfu/mL to 1 thousand cfu/mL, 10 thousand cfu/mL to 100 thousand cfu/mL, 1 million cfu/mL to 10 million cfu/mL, from 10 million cfu/mL to 100 billion cfu/mL, from 10 million to 50 million cfu/mL, more preferably from 50 million to 100 million cfu/mL, from 100 million to 500 million cfu/mL, from 500 million to 1 billion cfu/mL, from 1 billion to 5 billion cfu/mL, from 5 billion to 10 billion cfu/mL, from 10 billion to 15 billion cfu/mL, from 15 billion to 20 billion cfu/mL, from 20 billion to 25 billion cfu/mL, from 25 billion to 30 billion cfu/mL, from 30 billion to 35 billion cfu/mL, from 35 billion to 40 billion cfu/mL, from 40 billion to 45 billion cfu/mL, from 45 billion to 50 billion cfu/mL, from 50 billion to 55 billion cfu/mL, from 55 billion to 60 billion cfu/mL, from 60 billion to 65 billion cfu/mL, from 65 billion to 70 billion cfu/mL, from 70 billion to 75 billion cfu/mL, from 75 billion to 80 billion cfu/mL, from 80 billion to 85 billion cfu/mL, from 85 billion to 90 billion cfu/mL, from 90 billion to 95 billion cfu/mL, from 95 billion to 100 billion cfu/mL.

[00288] In an embodiment, the strain of the invention can be administered at, for example, a dosage of 0.01 to 100 x 10 ¹¹ cells/body, 0.1 to 10 x 10 ¹¹ cells/body or 0.3 to 5 x 10 ¹¹ cells/body. Furthermore, for example, the amount ingested per day as the bacteria can be 0.01 to 100 x 10 ¹¹ cells/60 kg body weight, 0.1 to 10 x 10 ¹¹ cells/60 kg body weight or 0.3 to 5 x 10 ¹¹ cells/60 kg body weight.

[00289] The content of at least one strain of bacteria contained in the orally ingested composition of the present invention may be determined as appropriate depending on its application form. As a dry microbial body it can be, for example, 5 to 50 w/w %, 1 to 75 w/w %, 0.1 to 100 w/w % or 1 to 100 w/w %.

[00290] In an embodiment, the composition is a controlled-release composition. As used herein, the term "controlled-release" refers to release or administration of a strain of the invention from a given dosage form in a controlled fashion in order to achieve the desired pharmacokinetic profile in vivo. An aspect of "controlled" delivery is the ability to manipulate the formulation and/or dosage form in order to establish the desired kinetics of release.

[00291] Procedures for preparing tablets, caplets, capsules and other forms of compositions of the invention are known to those of ordinary skill in the art and include without limitation wet granulation, dry granulation, and direct compression (for tablets and caplets).

[00292] Wet and dry granulation is used to manufacture tablets, caplets, or capsules. With granulation techniques, a chilsonation is used to manufacture the powder for the dosage forms. A chilsonator houses grooved, rotating rollers that are pressed tightly against one another by hydraulic pressure. Raw materials are placed into the hopper of the chilsonator and are fed by a system of horizontal and vertical screws into the rollers. As materials pass through the grooves in the rollers, it is compacted under very high pressure and emerges from the chilsonator as dense sheets. The sheets are milled into a fine granular powder using a Fitz mill and then passed through a screen to produce a uniform free flowing granule. The chilsonation process results in a finished powder that is two to four times denser than the starting material, a feature that permits the ingredients to be fashioned into the desired dosage form.

[00293] With dry granulation, the powder may be incorporated into a gelatin capsule or it may be mixed with gelatin to form a tablet or caplet. With wet granulation, the powder is moistened thus creating large "chunks" of material that are subsequently dried and milled to convert the chunks to particles of a desired size for the manufacturing process. Once the particles of a desired size are obtained, the particles are incorporated into a gelatin capsule or mixed with gelatin to form a tablet or caplet.

[00294] General considerations in formulation and/or manufacture can be found, 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

[00295] A composition of the invention can comprise a prebiotic. Because prebiotics have a chemical structure that resists digestion through the alimentary tract, they reach the colon as intact molecules where they are able to elicit systemic physiological functions and act as fermentable substrates for colonic microflora. Where a prebiotic is combined with a biotherapeutic, the resulting composition is sometimes referred to as a "synbiotic."

[00296] Examples of suitable prebiotics include, but are not limited to, oligosaccharide such as fructooligosaccharides, P95 Nutraflora®, for example, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, quercetin, human milk oligosaccharides, inulin oligosaccharides, mannan oligosaccharides, pyrodextrin, levan, maltotriose, pectic oligosaccharides, bimuno-galactooligosaccharides, arabinoxylan, fucoidan and resistant starches. Fructooligosaccharides can be extracted from, for example, chicory, artichokes, asparagus, dandelions, dahlias, endive, garlic, leeks, lettuce, and onions.

[00297] In an embodiment, the prebiotic comprises amino acids such as one or more or all of alanine, aspartic acid, glutamic acid, glycine, leucine, isoleucine, proline, serine, threonine and valine.

[00298] In an embodiment, the prebiotic comprises simple sugars which can be a monosaccharide (such as glucose, galactose or fructose) and/or a disaccharide (such as sucrose maltose or lactose).

[00299] In an embodiment, the prebiotic comprises from about 0.01 % (w/w) to about 75% (w/w), 5% (w/w) to about 50% (w/w), about 7.5% (w/w) to about 30% (w/w) or about 10% (w/w) to about 15% (w/w) of the composition. Other Microorganisms

[00300] In order to obtain the desired health benefit to the subject, it may be advantageous to include one or more additional biotherapeutic microorganisms in the composition. Thus, the composition may comprise more than one species/strain of microorganisms in addition to the strain of the invention, such as two, three, four, five or a higher plurality of species/strains of microorganisms. Non-limiting examples of biotherapeutics are suitable strains of the genera Absiella, Acetobacterium, Adlercreutzia, Aerococcus, Agathobaculum, Akkermansia, Alistipes, Allobaculum, Amedibacterium, Anaerobutyricum, Anaerofustis, Anaerostipes, Anaerotignum, Anaerotruncus, Bacillus, Bacteroides, Barnesiella, Bifidobacterium, Blautia, Butyricimonas, Carnobacterium, Christensenella, Ciostridium, Collinsella, Coprobacillus, Coprobacter, Coprococcus, Dorea, Enorma, Enterocloster, Enterococcus, Erysipelatoclostridium, Escherichia, 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, Peptoniphilus, Phocaeicola, Porphyromonas, Prevotella, Propionibacterium, Pusillimonas, Romboutsia, Roseburia, Ruminococcus, Ruthenibacterium, Sellimonas, Solibaculum, Sporolactobacillus, Sporomusa, Staphylococcus, Streptococcus, Subdoligranulum, Tetragenococcus, Thomasclavelia, Vagococcus, Vescimonas, Weisella. It is to be understood that the foregoing list is intended only to be illustrative and not a limiting representation of the biotherapeutics that may be included in the composition of the present invention. In this respect, any additional biotherapeutic species may also be used in the compositions of the present invention.

[00301] Some yeasts are also useful as biotherapeutics and are sometimes included in the compositions. One non-limiting example of a yeast used in biotherapeutics is Saccharomyces boulardii.

[00302] Some archaea are also useful as biotherapeutics and are sometimes included in the compositions. Non-limiting examples of an archaea used in biotherapeutics is Methanobrevibacter spp, including Methanobrevibacter smithii and Methanosphaera sp, including Methanobrevibacter stadtmanae.

Dosage Form [00303] Dosage forms are within the scope of the invention. In a preferred embodiment, the invention provides a dosage form comprising the composition as described in the first aspect of this invention. Preferably, the dosage form is stored in a sealed and sterile container.

Method for treating

[00304] Method for treating a gastrointestinal disorder are within the scope of the invention. In a preferred embodiment, the invention provides a method for treating a gastrointestinal disorder, wherein said method comprises the administration to a patient in need thereof a therapeutically effective amount of the composition as described in the first aspect of this invention.

[00305] In a further preferred embodiment, the dosage form is administered at an amount to at least partially treat a gastrointestinal disorder.

[00306] A subject that can be treated with the invention will include humans as well as other mammals and animals.

[00307] The effect of the administered therapeutic composition can be monitored by standard diagnostic procedures.

[00308] Methods of the invention can be used to treat or prevent a dysbiosis of the gastrointestinal tract in a subject. "Dysbiosis" in the context of the present invention refers to a state in which the normal diversity, relative proportions of species and/or function of the microbiota or microbiome, in particular the human gastrointestinal microbiota, is disrupted. Any disruption from the normal state of the microbiota in a healthy individual can be considered a dysbiosis, even if the dysbiosis does not result in a detectable decrease in health in the individual. In a preferred embodiment, the 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. In addition, or alternatively, "dysbiosis" may refer to an increase in the abundance of one or more bacteria, e.g. one or more pathogenic bacteria, in the microbiota of an individual relative to the abundance of said bacterium or bacteria in the microbiota of a healthy individual, i.e. an individual without a dysbiosis. The pathogenic bacteria present during dysbiosis are often Proteobacteria and resistant to one or more antibiotics. Examples of Proteobacteria include Escherichia, Salmonella, Campylobacter, Vibrio, Helicobacter, and Yersinia species.

[00309] The dysbiosis may be a dysbiosis associated with an enteric bacterial infection, such as an infection of the gastrointestinal tract with a pathogenic bacterium. Many bacteria capable of causing infections of the gastrointestinal tract in humans are known and include: gram positive bacteria, and gram negative bacteria. The pathogenic bacterium is preferably a pathogenic species of the genus 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 bacterium is a pathogenic species of the genus Clostridium or Escherichia. Most preferably, the pathogenic bacterium is Clostridium difficile or Escherichia coli.

[00310] Methods of the invention can be used to reduce or prevent gastrointestinal tract mucosal inflammation in a subject using compositions of the invention.

[00311] In an embodiment, the subject has, or is susceptible to having, an inflammatory bowel disease (IBD) such as Crohn's disease, ulcerative colitis and pouchitis. As used herein, the term "inflammatory bowel diseases (IBD)" has its general meaning in the art and refers to a group of inflammatory diseases of the colon and small intestine such as revised in the World Health Organisation Classification K20-K93 (ICD-10) such as Crohn disease (such as granulomatous enteritis; Crohn disease of small intestine; Crohn disease of large intestine; granulomatous and regional Colitis; Crohn disease of colon, large bowel and rectum; Crohn disease of both small and large intestine), Ulcerative colitis (such as Ulcerative (chronic) pancolitis; backwash ileitis; Ulcerative (chronic) proctitis; Ulcerative (chronic) rectosigmoiditis; Inflammatory polyps; Left sided colitis; left hemicolitis) and noninfective gastroenteritis and colitis (Gastroenteritis and colitis due to radiation; Toxic gastroenteritis and colitis; Allergic and dietetic gastroenteritis and colitis; Food hypersensitivity gastroenteritis or colitis; indeterminate colitis; specified noninfective gastroenteritis and colitis such as Collagenous colitis; Eosinophilic gastritis or gastroenteritis; Lymphocytic colitis Microscopic colitis (collagenous colitis or lymphocytic colitis); Noninfective gastroenteritis and colitis such as Diarrhoea; Enteritis; Ileitis; Jejunitis; Sigmoiditis) and postprocedural disorders of digestive system such as pouchitis. In an embodiment, the IBD is paediatric IBD.

[00312] In a further aspect, the present invention also relates to a faecal microbiota transplant composition comprising the strain of the invention. The term "faecal microbiota transplant composition" has its general meaning in the art and refers to any composition that can restore the faecal microbiota.

[00313] Administration to humans includes administration by a medical professional and self-administration. In general, in order to achieve a health benefit, single or multiple doses of the composition are administered, for example once-off, daily for a period of at least one week, at least two weeks, at least three weeks, at least six weeks, at least nine weeks, or at least twelve weeks. In one embodiment, the compositions can be administered for the remaining duration of a subject's life.

Device

[00314] Devices are within the scope of the invention. In a preferred embodiment, the invention provides a device, wherein the device comprises: (1 ) the composition as described in the first aspect of this invention; and (2) an applicator, container or material.

Use of a composition in the manufacture of a medicament

[00315] Uses are within the scope of this invention. In a preferred embodiment, the invention provides the use of a composition in the manufacture of a medicament for treating a gastrointestinal disorder.

Method for stabilising

[00316] Methods for stabilizing the compositions of the invention are within the scope of the invention.

[00317] In a further preferred embodiment, the said method protects the compositions of the invention against degradation.

[00318] In yet a further preferred embodiment, the composition of the invention retains its effective biological activity for a period selected from the group consisting of; greater than 24 hours; greater than 36 hours; greater than 48 hours.

[00319] The addition of approved pharmaceutical excipients to stabilise the compositions of the invention solutions is preferred from a safety standpoint, as the simpler methodology is likely to produce a less variable outcome and the choice of excipient can be limited to those with Generally Regarded as Safe (GRAS) status. Excipients for the stabilisation of protein solutions can be classified into four broad categories: salts, sugars, polymers or protein/amino acids, based on their chemical properties and mechanism of action. Salts (e.g. chlorides, nitrates) stabilise the tertiary structure of proteins by shielding charges through ionic interactions. Sugars (e.g. glycerol, sorbitol, fructose, trehalose) increase the surface tension and viscosity of the solution to prevent protein aggregation. Similarly, polymers (e.g. polyethylene glycol, cellulose derivatives) stabilise the protein tertiary structure by increasing the viscosity of the solution to prevent protein aggregation and intra- and inter-molecular electrostatic interactions between amino acids in the protein. Proteins (e.g. human serum albumin) are able to stabilise the structure of other proteins through ionic, electrostatic and hydrophobic interactions. Similarly, small amino acids with no net charge, such as alanine and glycine, stabilise proteins through the formation of weak electrostatic interactions.

[00320] As discussed above, the medicaments of the present invention may include one or more pharmaceutically acceptable carriers. The use of such media and agents for the manufacture of medicaments is well known in the art. Except insofar as any conventional media or agent is incompatible with the pharmaceutically acceptable material, use thereof in the manufacture of a pharmaceutical composition according to the invention is contemplated. Pharmaceutical acceptable carriers according to the invention may include one or more of the following examples: a. surfactants and polymers, including, however not limited to, polyethylene glycol (PEG), polyvinylpyrrolidone , polyvinylalcohol, crospovidone, polyvinylpyrrolidone-polyvinylacrylate copolymer, cellulose derivatives, HPMC, hydroxypropyl cellulose, carboxymethylethyl cellulose, hydroxypropylmethyl cellulose phthalate, polyacrylates and polymethacrylates, urea, sugars, polyols, and their polymers, emulsifiers, sugar gum, starch, organic acids and their salts, vinyl pyrrolidone and vinyl acetate; and/or b. binding agents such as various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose; and/or (3) filling agents such as lactose monohydrate, lactose anhydrous, microcrystalline cellulose and various starches; and/or c. filling agents such as lactose monohydrate, lactose anhydrous, mannitol, microcrystalline cellulose and various starches; and/or d. lubricating agents such as agents that act on the increased ability of the dosage form to be ejected from the packaging cavity, and/or e. sweeteners such as any natural or artificial sweetener including sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame K; and/or f. flavouring agents; and/or g. preservatives such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxy benzoic acid such as butyl paraben, alcohols such as ethyl or benzyl alcohol, phenolic chemicals such as phenol, or quarternary compounds such as benzalkonium chloride; and/or h. buffers; and/or i. diluents such as pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing; and/or j. absorption enhancer such as glyceryl trinitrate; and/or k. other pharmaceutically acceptable excipients.

[00321] Medicaments of the invention suitable for use in animals and in particular in human beings typically must be sterile and stable under the conditions of manufacture and storage.

Methods for Detection

[00322] A strain of the invention can be detected using a wide variety of known techniques. Conveniently the strain is detected using a nucleic acid-based detection system.

[00323] In an embodiment, nucleic acid sequencing is used. Illustrative non-limiting examples of 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 finds use in a Second Generation (a.k.a. Next Generation or Next- Gen), Third Generation (a.k.a. Next-Next-Gen), or Fourth Generation (a.k.a. N3-Gen) sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology.

[00324] In some embodiments, hybridization is employed in a detection method of the 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, a FISH assay is used. In other embodiments, nucleic acid amplification is used. Nucleic acids may be amplified prior to or simultaneous with detection. Conducting one or more amplification reactions may comprise 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, multiple 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 of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require that RNA be reversed transcribed to DNA prior to amplification (e.g., RT-PCR), whereas other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

[00325] Non-amplified or amplified nucleic acids can be detected by any conventional means. For example, the nucleic acids can be detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids. In another example, the nucleic acids are detected by sequencing. Illustrative non-limiting examples of detection methods are described herein.

[00326] Evaluation of an amplification process in "real-time" involves determining the amount of amplicon in the reaction mixture either continuously or periodically during the amplification reaction and using the determined values to calculate the amount of target sequence initially present in the sample. A variety of 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 methods disclosed in U.S. Pat. Nos. 6,303,305 and 6,541 ,205. Another method for determining the quantity of target sequence initially present in a sample, but which is not based on a real-time amplification, is disclosed in U.S. Pat. No. 5,710,029.

[00327] Amplification products may be detected n real time through a combination of primers specific to the region of interest and a non-specific fluorescent DNA binding dye. Such primers are designed so that they specifically target a region unique to the microbe of interest, leading to amplification of only the designed target. The primer design incorporates two individual primers that complementarily bind up and downstream of the targeted DNA site. During amplification the primers will bind up and downstream of the target of interest, generating an additional copy through polymerase chain reaction, with the DNA-binding dye being incorporated into the newly generated amplicon. Post amplification, the DNA-binding dye is excited via a specific wavelength of light and the emission at a different wavelength is measured. Binding of the fluorescent DNA-bindinc dye to the exponentially increasing amplicon of interest can be correlated with standards of known concentration through linear regression to determine the original copy number of the target region amplified. The methodology of using DNA-binding dyes is disclosed n U.S. Pat. No. 6,174,670. The specific use of the fluorescent DNA-binding dye SYBR green I is disclosed in U.S. Pat. No. 5,338,671 and 5,587,287.

[00328] Amplification products may 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 so that they emit differently detectable signals, depending on whether the probes are in a self-hybridized state or an altered state through hybridization to a target sequence. By way of non-limiting example, "molecular torches" are a type of self-hybridizing probe that includes distinct regions of self-complementarity (referred to as "the target binding domain" and "the target closing domain") which are connected by a joining region (e.g., nonnucleotide linker) and which hybridize to each other under predetermined hybridization assay conditions. In a preferred embodiment, molecular torches contain single-stranded base regions in the target binding domain that are from 1 to about 20 bases in length and are accessible for hybridization to a target sequence present in an amplification reaction under strand displacement conditions. Under strand displacement conditions, hybridization of the two complementary regions, which may be fully or partially complementary, of the molecular torch is favored, except in the presence of the target sequence, which will bind to the singlestranded region present in the target binding domain and displace all or a portion of the target closing domain. The target binding domain and the target closing domain of a molecular torch include a detectable label or a pair of interacting labels (e g., luminescent/quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized than when the molecular torch is hybridized to the target sequence, thereby permitting detection of probe- -target duplexes in a test sample in the presence of unhybridized molecular torches. Molecular torches and a variety of types of interacting label pairs are disclosed in U.S. Pat. No. 6,534,274, herein incorporated by reference in its entirety.

[00329] Another example of a detection probe having self-complementarity is a "molecular beacon." Molecular beacons include nucleic acid molecules having a target complementary sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target sequence present in an amplification reaction, and a label pair that interacts when the probe is in a closed conformation. Hybridization of the target sequence and the target complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation 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. Pat. Nos. 5,925,517 and 6,150,097.

[00330] In an embodiment, the method includes quantifying the amount of strain present in the sample

[00331] The present invention will now be described with reference to the following nonlimiting Examples. The description of the Examples is in no way limiting on the preceding paragraphs of this specification, however, is provided for exemplification of the methods and compositions of the invention.

Examples [00332] It will be apparent to persons skilled in the milling and pharmaceutical arts that numerous enhancements and modifications can be made to the above-described processes without departing from the basic inventive concepts. For example, in some applications the biologically active material may be pretreated and supplied to the process in the pretreated form. All such modifications and enhancements are considered to be within the scope of the present invention, the nature of which is to be 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 processes or compositions of the invention.

A EXAMPLE 1 - BB265 MECHANISM OF ACTION TO AMELIORATE THE METABOLIC LESION IN ULCERATIVE COLITIS

STUDY AIM

[00333] The aim of the therapy is to induce and maintain remission by reducing sulphide levels in the colon of patients with ulcerative colitis Materials, Methods and Results

THE COMPOSITIONS OF THIS INVENTION, INCLUDING THE BB265

[00334] The compositions of this invention, including the BB265 live biotherapeutic product, targets the proximal cause of colonocyte barrier disruption that results in immune activation.

REDUCING LUMINAL SULPHIDE BY 'TURNING OFF THE TAP’ FOR PRODUCTION

[00335] Reducing luminal sulphide can be accomplished by “turning off the tap” of sulphide production or by removing sulphide from the colon. Each of these mechanisms can be targeted using a live biotherapeutic product.

[00336] Microbial competition for metabolic substrates

[00337] Sulphidogenic microbiota use specific substrates for metabolism. As an example, sulphate-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, 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-sulphidogenic microbiota, which utilise these substrates, compete with sulphidogenic microbiota; such competition would limit the growth of sulphidogenic microbiota and lessen the production of luminal sulphide. Microbiota also compete with sulphidogenic organisms for electron acceptors. As an example, the two most dominant genera of SRB in the gut, Desulfovibrio and Desulfobulbus, both utilise molecular hydrogen (H2), derived from fermentation, as an electron acceptor in cellular respiration. Methanogenic archaea and/or acetogenic bacteria divert hydrogen (H ₂ ) away from sulphidogenic microbiota via competitive H ₂ uptake. Reducing luminal sulphide can be accomplished by competitive H ₂ uptake by methanogenic archaea, acetogenic bacteria or other microorganisms (Smith, N. W., Shorten, P. R., Altermann, E., Roy, N. C. & McNabb, W. C. Competition for Hydrogen Prevents Coexistence of Human Gastrointestinal Hydrogenotrophs in Continuous Culture. Front. Microbiol. 11 , 1073 (2020)). Methanogenic archaea, such as Methanobrevibacter smithii and acetogenic bacteria such as Alistipes spp., Blauti spp.and Roseburia spp. are diminished in ulcerative colitis and associated with clinical remission in FMT. Please refer to Figure 2 that shows a diagrammatic representation of hydrogen uptake by acetogens and methanogens that reduce sulphide production by SRB. Competition for metabolic substrates necessary for the growth and/or metabolism of sulphidogenic microbiota offers a therapeutic target to reduce endogenous sulphide in the colon.

[00338] Reduced sulphur substrate release in the colon

[00339] Sulphidogenic microbiota require sulphur substrates (such as inorganic sulphate, sulphite, thiosulphate, and tetrathionate, and organic cysteine, methionine, and taurine) to produce sulphide. In the colon, these substrates come from, but are not limited to, dietary sulphur sources, such as meat and cruciferous vegetables, host mucin, bile acids, and endogenous proteins (Wolf, P. G. et al. Diversity and distribution of sulphur 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). Though sulphur is ubiquitously present in the colon, much of it is bound in complex molecules inaccessible to sulphidogenic microbiota. In order for sulphidogenic microbiota to access sulphur to produce sulphide, other microbiota are often required to biotransform these compounds into accessible forms. As an example, SRB depend on saccharolytic bacteria, such as Bacteroides spp., to remove sulphate from host mucin (Tsai, H. H., Sunderland, D., Gibson, G. R., Hart, C. A. & Rhodes, J. M. A novel mucin sulphatase from human faeces: its identification, purification and characterization. Clin. Sci. 82, 447-454 (1992)). This reliance on the biotransformative properties of resident microbiota offers a therapeutic opportunity by which levels of colonic sulphide can be reduced. Specific microbiota that reduce the rate at which these substrates are released, biotransformed, or compete with biotransformers (such as via metabolic substrate or niche competition) reduce the amount of sulphur substrates available for the microbial production of sulphide. [00340] Colonic protein fermentation by bacteria is a major source of microbial sulphide and nitric oxide (NO) production. However, colonic bacteria ferment carbohydrates in preference to protein in the human colon and thus adequate amounts of dietary fibre reduce the amount of sulphur-containing amino acids released into the colon. In both human and animal studies, a high protein diet results in faecal microbiota changes that increase sulphide production and decrease SCFA production (Teigen, L. M. et al. Dietary Factors in Sulfur Metabolism and Pathogenesis of Ulcerative Colitis. Nutrients 11, (2019)). In addition, dietary fibres have been demonstrated to attenuate sulphide gas production in bioreactors containing stool from ulcerative colitis patients (Yao, C. K. et al. Modulation of colonic hydrogen sulphide production by diet and mesalazine utilizing a novel gas-profiling technology. Gut Microbes 9, 510-522 (2018)). Additionally, low protein, high resistant starch diets have been demonstrated to induce remission in ulcerative colitis..

[00341] Removing sulphide from the colon

[00342] Bacterial species can consume sulphide thereby reducing colonic sulphide levels. An example of sulphide consumption is through assimilation of sulphide to cysteine. Cysteine is synthesized from serine in a two-step process, commencing with transfer of the acetyl group from acetyl-CoA to serine, forming O-acetylserine (OAS), catalyzed by serine acetyltransferase (SAT). The second step, a condensation of OAS with sulphide to yield cysteine and acetate, catalyzed by O-acetylserine(thiol)lyase (OASTL) (Figure 4). Direct consumption, i.e., the biotransformation of sulphide, either metabolically or otherwise, can therefore reduce colonic levels of sulphide via direct removal.

B EXAMPLE 2 - THE DISCOVERY AND DEVELOPMENT OF BB265 AS A COMPLEX COMMUNITY LIVE BIOTHERAPEUTIC PRODUCT TO ADDRESS METABOLIC LESIONS IN ULCERATIVE COLITIS: AN OVERVIEW

STUDY AIM

[00343] To develop BB265 as a complex community live biotherapeutic product to address metabolic lesions in ulcerative colitis.

MATERIALS, METHODS AND RESULTS

APPROACH

[00344] BB265 is an encapsulated bioreactor-derived microbial consortium comprising specific microbial strains that reduce colonic sulphide which drives the onset of the chemical lesion seen in ulcerative colitis and can optionally include a collection of the globally most prevalent taxa or isolates (global core microbiome), taxa or isolates which best discriminate between health and ulcerative colitis, keystone taxa or isolates, and 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.

A COMPLEX COMMUNITY

[00345] Colonic microorganisms live in an ecosystem with many syntrophic relationships. Individual strains require many other species to sustain them via synergistic interactions. Keystone species are those that have a particularly critical role for the survival of many other species in the ecosystem, and in ecosystem stability and have outsized importance on the ecosystem often through uniquely held functions or functions that have limited redundancy within the system. (Banerjee, S., Schlaeppi, K. & van der Heijden, M. G. A. Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2018)). In addition, keystone species are also those linked with clinical efficacy, identified by their presence as a predictive factor for whole community recovery post-antibiotic treatment.

[00346] The inventors performed a global metagenomic meta-analysis to identify the most prevalent species shared across different geographical regions. This global core microbiome was then supplemented with the addition of previously identified keystone species and those identified informatically as keystones through multi-correlative network analysis. These bacterial isolates were drawn from the inventor’s culture collection and progressed to pilot process development to co-culture the defined community of species in bioreactors. This community supports the defined therapeutic candidate organisms, disrupts the existing community, and provides therapeutic potential themselves given the community contains many health associated organisms and has the emergent property of bolstering the effect of sulphide reduction by candidate microorganisms in ulcerative colitis.

IDENTIFICATION OF TARGETS AND LEADS

[00347] Bacteria or archaea with the following characteristics were identified as Leads considered for inclusion in the BB265 complex consortium: (1 ) globally prevalent taxa or isolates commonly found in healthy human faecal microbiomes, (2) taxa or isolates that best discriminate between the faecal microbiomes of health and ulcerative colitis (health associated), (3) keystone taxa or isolates, (4) phylogenetically diverse taxa or isolates, (5) sulphide consuming (or assimilating) taxa or isolates, (6) putative H2 consuming taxa or isolates such as methanogenic archaea and homoacetogenic bacteria, and (7) taxa or isolates that divert metabolic substrates away from sulphide production. [00348] Bacteria or archaea with the following characteristics were identified as Targets considered for exclusion from the BB265 complex consortium: (1) taxa or isolates that best discriminate between the faecal microbiomes of health and ulcerative colitis (disease- associated), (2) and taxa or isolates identified as sulphidogens.

IDENTIFICATION OF CANDIDATE THERAPEUTIC ORGANISMS

[00349] The inventors have developed unique phenotypic assays that identify sulphide producing microbial isolates; the inventor’s bacterial culture collection has been screened for sulphide production. The inventors have additionally developed unique phenotypic assays that identify sulphide consuming (or assimilating) microbial isolates; isolates identified as Leads with the ability to consume sulphide were considered candidate therapeutic organisms. Isolates identified as putative H2 consuming organisms via taxonomic and genomic means, such as methanogenic archaea and homoacetogenic bacteria, were also considered candidate therapeutic organisms.

VALIDATING MECHANISM OF ACTION OF BB265

[00350] The inventors validated the mechanism of action of the BB265 complex consortium in vitro by co-culturing faecal slurries from patients with active ulcerative colitis with the BB265 consortium. Unique phenotypic assays were developed to quantify sulphide production by the community, this was paired with measurements of real-time rates of production of H2S using H2S-specific commercial electrochemical sensors. The inventors demonstrated that the BB265 consortium significantly reduces sulphide concentration and rates of production when co-cultured with ulcerative colitis faecal communities. Additionally, the BB265 complex consortium was identified to have the emergent property of bolstering the effect of candidate microorganisms by reducing total concentration and rate of sulphide production more than the individual candidate organisms.

[00351] The 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 successfully went into remission due to receiving faecal microbiota transplantation in a randomized, controlled trial. This increase in BB265-associated taxa with remission was paired with a decrease in taxa identified as putative sulphidogens, supporting the hypothesis of sulphide reduction as a therapeutic target. Using unique phenotypic assays, the inventors assayed stool from patients prior to receiving faecal microbiota transplantation and then from stool post remission and identified a significant reduction in the sulphide-producing capacity of the community post-remission. [00352] The paired observations of (1 ) the BB265 complex consortium reducing sulphide concentration and rate of production in ulcerative colitis stool in vitro, (2) an increase in BB265- associated taxa and a decrease in putative sulphidogens in patients who successfully went into remission in a faecal microbiota transplantation trial, and (3) a significant reduction in sulphide-producing capacity of the ulcerative colitis stool community post-FMT in patients who went into clinical remission strongly supports the BB265 consortium as a therapy for ulcerative colitis, and further supports the mechanism of action of this therapy for reducing sulphide as a means to improve clinical outcomes.

PRE-BIOTIC FIBRE

[00353] The fermentation of fibre in the colon (particularly resistant starch and non-starch polysaccharides) occurs in preference to protein fermentation, resulting in reduced sulphur amino acid (cysteine) release into the colon and a decrease in sulphide production by resident microbiota (Teigen, L. M. et al. Dietary Factors in Sulfur Metabolism and Pathogenesis of Ulcerative Colitis. Nutrients 11, (2019). The inventors therefore tested fibre combinations that may potentiate our live biotherapeutic product in inflammatory bowel disease using those unique phenotypic assays and commercial HzS-specific electrochemical sensors as outlined above for validation of the BB265 complex consortium.

PHASE 1 HUMAN CLINICAL TRIAL

[00354] The final candidate consortium will be further tested in a phase 1 human trial. This final drug product will contain the enriched bacterial strains which target the underlying cause of the metabolic lesions in ulcerative colitis, further supported by a community comprising the most prevalent bacteria in healthy humans globally in addition to known keystone species that sustain important synergistic links in the colonic ecosystem. The phase 1 human trial will show that the inventor’s live biotherapeutic product acts early in the disease pathway to prevent barrier disruption by limiting the NO- and H2S-induced metabolic lesion. The phase 1 human trial will have the ability to assess important mechanistic endpoints e.g. therapeutic candidate’s ability to reduce sulphide in a patient with ulcerative colitis.

C EXAMPLE 3 -DEFINING ORGANISMS COMPRISING THE BB265 COMPLEX COMMUNITY LIVE BIOTHERAPEUTIC PRODUCT

STUDY AIM

[00355] To identify lead and candidate microorganisms and define the complex community of the BB265 live biotherapeutic product to address metabolic lesions in ulcerative colitis. MATERIALS, METHODS AND RESULTS

MOST PREVALENT COMMUNITY (MPC)

[00356] The widespread presence of microbial species commonly found in healthy human faecal microbiomes, regardless of differences in diet, lifestyle, and environmental conditions across various populations, indicates their importance as integral components of the human gut microbiota. Global prevalence of such taxa suggests they have co-evolved 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 these taxa fulfill fundamental roles by conserving core functions vital to human health. By interrogating healthy human faecal metagenomes across five geographies (Africa, South America, North America, Asia-Pacific, & Europe), we identified globally prevalent taxa to define a ‘core’ community structure, termed here as the 'Most Prevalent Community’ (MPC). A community structure determined by those species most commonly found within sequenced healthy human faecal microbiomes form a core community imperative to human gut ecology.

[00357] Microbial species found to be globally prevalent in healthy human faecal microbiomes were determined via publicly available Illumina paired-end shotgun metagenomes from faeces of individuals with a reported healthy phenotype downloaded from the European Nucleotide Archive (ENA). This analysis resulted in the generation of priority lists from 1 to 2, and 4 to 6 depending on global span and read coverage. Metagenomes with over five million reads were retained and the 882 remaining metagenomes were assigned to geographic regions based on their origin, including Africa, South America, North America, Europe and Asia-Pacific. Sequenced reads were trimmed using Trimmomatic version 0.39 and taxonomic classification was performed using Kraken version 2.1.2 through comparison to a custom database built from all available complete genomes in the National Center for Biotechnology Information (NCBI) Reference Sequence Database (RefSeq release 209). Reads mapping to Homo sapiens were removed and low abundance taxa with less than 0.001 % of total reads were removed to filter out spurious read classifications from each sample.

CO-OCCURRENCE INFORMED MOST PREVALENT COMMUNITY (COIMPC)

[00358] Microbial communities contain a plethora of complex interactions both between and within species and these synergistic relationships are integral to a functioning ecosystem (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 of considering these interactions is via co-occurrence analyses. Here, a co-occurrence informed; most prevalent community structure approach overlays co-occurrence data onto the prevalence data to maximise the likelihood of forming functional communities. Co-occurrence suggests a mutually beneficial relationship among bacteria of the MPC and more globally transient bacteria. The inclusion of bacteria outside of the MPC but positively correlated with bacteria forming the MPC will bolster and support the functioning of the core community through metabolic cooperation, cross-feeding, 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 Informed Most Prevalent Community (CoIMPC).

[00359] Bioinformatic analyses were performed to identify species co-occurring with globally prevalent species in the complex consortium. Specifically, network analysis was performed on the global metagenomic classification output with SpiecEasi v1.1.2 using Meinshausen-Buhlmann neighbourhood selection with 50 repetitions of the Stability Approach to Regularisation Selection (StARS), an nlambda value of 20 and a lambda. min.ratio of 0.01 . Species directly connected in the network to existing MPC taxa were selected for inclusion in the consortium based on this co-occurrence. These species have statistically inferred interactions with the complex consortium taxa, which implies a supportive role in the community.

IDENTIFICATION OF THE ‘HUB’ TAXA

[00360] Microbial taxa have varying levels of connectivity within their ecological community, with some taxa contributing more to community functioning and stability than others, referred to as hubs. Hubs have a high level of connectivity to other community members and are often essential for community structure and metabolic cycling (Banerjee, S., Schlaeppi, K. & van der Heijden, M. G. A. Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2018)). A method of identifying hub taxa in the gastrointestinal microbiome is through network analyses of sequencing data. Hubs are defined by their level of connectedness to other nodes in the network, inferring their importance in community functioning.

[00361] Bioinformatic network analyses were performed to identify statistically inferred hubs in the global metagenomic classification output. These analyses were performed using SpiecEasi with Meinshausen-Buhlmann neighbourhood selection and NetCOMI with SPRING association estimation, to identify taxa with the highest degree of centrality. These methods identify nodes with the highest connectivity, determining potential hubs within the network. Taxa identified as hubs, with high closeness, betweenness or eigenvector centrality were selected for inclusion. The species identified through these methods may have key roles in community structure determination and metabolic precesses.

IDENTIFICATION OF THE KEYSTONES

[00362] In addition to common and highly abundant microbial species, and those that cooccur with these species and act as ecological hubs, taxa that may be less abundant but functionally critical to the microbial ecosystem are considered. These keystone species have outsized importance on the ecosystem often through uniquely held functions or functions that have limited redundancy within the system (Banerjee, S., Schlaeppi, K. & van der Heijden, M. G. A. Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2018)). Here, keystone species are also those linked with clinical efficacy, identified by their presence as a predictive factor for whole community recovery post-antibiotic treatment.

[00363] Literature searches were performed to identify these important microbes. Within the researched literature, bioinformatic analyses were employed to identify organisms that carry critical functions that are not widely, if at all, carried by others. These 26 species are outlined in section 2e under the “Healthy gut environment” column. In addition, the researched literature detailed a meta-analysis of post-antibioiic recovery, revealing the presence of organisms that facilitate recovery of the gut microbial ecosystem following broad antibiotic loss thus identifying them as key players in recovery (Hajishengallis, G., Darveau, R. P. & Curtis, M. A. The keystone-pathogen hypothesis. Nat. Rev. Microbiol. 10, 717-725 (2012); Ze, X., Duncan, S. H., Louis, P. & Flint, H. J. 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, M. G. A. Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. M crobiol. 16, 567-576 (2018); Chng, K. R. et al. Metagenome-wide association analysis identifies microbial determinants of postantibiotic ecological recovery in the gut. Nature Ecology & Evolution 4, 1256-1267 (2020)). These 21 species are outlined in section 2e under ' Treat UC” and “Treat other IBD” columns. . These keystone species genomes were obtained from the National Center for Biotechnology Information (NCBI) Reference Sequence Database (RefSeq release 209).

IDENTIFICATION OF PHYLOGENETICALLY DIVERSE ISOLATES

[00364] Microbial species diversity within the gut microbiome is linked with healthy gut function (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)). Development of the complex consortium had a focus on diversity to ensure it mirrors that of a healthy gut microbiome.

[00365] 16S rRNA gene sequence-based similarity comparisons were used to identify culture collection isolates from BiomeBank that were phylogenetically distinct from the existing complex consortium. Isolates with a 16S sequence less than 98% identity, determined by NCBI-BLAST (blastn) (v2.13.0) comparison to any species or strain in the consortium, were iteratively included as they may fill ecological niches not covered by the initial community. Phylogenetic analyses were performed to evaluate phylogenetic coverage of the complex consortium with the addition of these diversity inclusion isolates.

IDENTIFICATION OF HEALTH AND DISEASE DISCRIMINATORY TAXA

[00366] Faecal microbiota transplant (FMT) has emerged as a potential treatment for ulcerative colitis, showing induction of remission in UC patients ). However, the clinical success of FMT is varied, possibly due to variation in preparation, delivery and donor . Since some donors have a greater observed success in recipient remission , it has been suggested that diversity or the presence of a specific combination of microbes may be responsible. Hence, there is a need for more defined microbiome therapeutics and the identification of specific microbial candidates.

[00367] Therefore, this analysis aims to define a consortium of taxa that discern between active UC, and healthy controls from a diverse array of publicly available faecal metagenomes, for the purpose of determining microbial targets associated with disease, or therapeutic bacteria and communities for disease treatment.

[00368] Data download

[00369] Publicly available Illumina paired shotgun metagenomes from feces of patients with ulcerative colitis, and controls were downloaded from the European Nucleotide Archive (ENA). Associated metadata was obtained from using SRA run selector, ENA toolkit and published metadata. Trimmomatic (v0.39) was used to remove low quality and adapter sequences. Kraken2 (v2.1.2) (Wood, D. E., Lu, J. & Langmead, B. Improved metagenomic analysis with Kraken 2. Genome Biol. 20, 257 (2019)) was used to determine taxonomic read classifications using a reference database built from all RefSeq complete genomes (release 209). The resulting kraken2 report files were input into Bracken for read count correction (Lu, J., Breitwieser, F. P., Thielen, P. & Salzberg, S. L. 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 by spurious read assignment during classification. A data table was created through merging individual sample bracken outputs.

[00370] Data curation

[00371] Two metadata stratifications were employed for data comparison, including: (1 ) Ulcerative colitis diagnosis vs non-IBD controls, (2) Active ulcerative colitis vs Healthy (different to non-IBD controls in that individuals that underwent endoscopy were removed).

[00372] Data analysis

[00373] Differentially abundant and discriminant 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, S. D. Analysis of compositions of microbiomes with bias correction. Nat. Commun. 11 , 3514 (2020)) and Selbal vO.1.0 Rivera-Pinto, J. Balances: a new perspective for microbiome analysis. Msystems (2018)) and discriminant taxa, between non-IBD controls and ulcerative colitis, and active ulcerative colitis vs reportedly healthy controls, were identified through determining consensus results. Filtered bracken outputs were passed to the ALDEx2 R package, which, for each sample, derived Monte-Carlo instances from the Dirichlet distribution and performed data normalisation using centered-log ratio transformation before statistically determining differentially abundant taxa through Welch’s test and Wilcoxon test. The resulting ^^Benjamini-Hochberg (BH) corrected P values were filtered to <0.05 to determine statistically significant differentially abundant taxa. Taxa were sorted by effect value for the highest and lowest effect scores.

[00374] A phyloseq object was created from the filtered bracken outputs and sample metadata, used for input into the ANCOM-BC R package. The ancombc function was run with default parameters, except with the inclusion of structural zeros and the neg_lb parameter set to true, and a conservative variance estimate (conserve set to true), as well as performing the global test. Resulting Holm-Bonferroni adjusted p values were used to identify statistically significant differentially abundant taxa (p<0.05)

[00375] Filtered bracken outputs were input into the R package selbal to determine taxa associated with metadata groups. The cross-validation function, selbal.cv, was run with ten iterations to identify components of the balance, or group of taxa, that form the inferred microbiome signature of each metadata group.

[00376] Analysis results Selbal identified the number of species needed to distinguish between non-IBD samples and UC samples was ten, with three associated with UC and seven associated with non-IBD. Using this group of species, this allowed for accurate classification of sample group, with a mean accuracy value of 0.7985. The number of species needed to distinguish between healthy samples and active UC samples was four, with one associated with UC and three associated with the healthy samples. Using this group of species, this allowed for accurate classification of sample group, with a mean accuracy value of 0.8420. Taxa identified by selbal, as well as statistically significant differentially abundant taxa, as determined by ALDEx2 and ANCOM- BC were combined to form consensus analysis, resulting in 52 therapeutic candidate taxa and 32 Targets.

PURIFICATION, BANKING, AND IDENTIFICATION OF BB265 CANDIDATES

[00377] When developing a microbial therapy, the purification, banking, and identification of microbial isolates within the consortium holds paramount importance. By isolating and characterising microbial strains of interest, this study also establishes a characterisation data package that can be incorporated into the regulatory filing for this targeted microbial therapy.

[00378] Microbial culture and Isolation

[00379] Fresh faecal 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 homogenised via vortex mixing for 5 min. Faecal homogenates were then serially diluted in sterile, pre-reduced PBS, spread plated onto multiple broad-range and selective bacteriological mediamedia and incubated anaerobically at 37 °C for 24-168 hours.

[00380] For isolating spore formers, 200 pL of stool homogenates were transferred into 1.5 mL microcentrifuge tubes and were either mixed with 600 pL of ethanol (undenatured), chloroform or heat shocked at 80 °C for varying amounts of time. Ethanol or chloroform samples were incubated at 25 °C at a range of time durations. After incubation or heat shock samples were centrifuged at 147,000 xg for 5 minu to pelletise. Supernatants were discarded and 600 pL of 1X PBS was added into each tube. Samples were vortexed for 5 min to resuspend. The above steps were repeated three times to remove residual ethanol. On the third wash, pellets were re-suspended in 200 pL 1X PBS and cultured on YCFA agar as described above.

[00381] Isolate purification

[00382] Post incubation, individual colonies were picked and re-streaked onto YCFA agar and incubated anaerobically at 37 °C for 24-72 hours. Individual colonies were then picked, re-streaked onto YCFA agar and incubated under the same conditions for purification. This was again repeated once more to complete purification.

[00383] Full-length 16S rRNA gene sequencing

Post purification, individual colonies were picked into 15 mL YCFA broth and incubated anaerobically at 37 °C overnight. Post incubation, 20 pL of overnight culture were diluted in 180 pL sterile PBS as 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.

[00384] Full-length 16S genes of each isolate were amplified by PCR using 7F (5'- AGAGTTTGATYMTGGCTCAG-3') and 151 OR (5'-ACGGYTACCTTGTTACGACTT-3') primers. Samples were run on a C1000 Touch™ thermal cycler (BioRad) with conditions: 95°C for 15 min, 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 2 min; PCR terminated with 8 min at 72°C and was held at 8°C. Resultant amplicons were stained with Gel Green Nucleic Acid Stain (Fisher Biotec) and run on a 1 % agarose gel at 100 V for -20 min. Amplicons were visualised by fluorescence under blue light using a MBE-200A BluViewTransiluminator (Major Science).

Samples were then purified using the EnzSAP™ PCR clean-up reagent following manufacturer's guidelines. Purified samples underwent capillary electrophoresis sequencing. Sequencing reactions were performed in a PTC-255 Peltier Thermal Cycler (MJ Research) using ABI PRISIM® BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems) following manufacturer’s guidelines. Singlepass sequencing was performed on each template using the 7F and 151 OR primers. The BigDye® Xterminator™ purification protocol was followed to purify fluorescently labelled fragments from unincorporated terminators. Samples were re-suspended in dd.fW and underwent electrophoresis using a ABI 3730x1 sequencer (Applied Biosystems).

[00385] Taxonomic determination

[00386] Raw sequence data output from capillary sequencing were quality filtered to remove reads containing < 600 bases total with a Phred quality score < 20. Quality filtered sequence chromatograms were then visually inspected to ensure purity of the sample. Forward and reverse paired-end reads were then merged using PEAR run by default settings. Merged reads were then trimmed left and right by 60 bp each to remove low quality bases. NCBI-BLAST (blastn) (v2.13.0) was then performed on merged sequences against the NCBI RefSeq 16S rRNA database (RefSeq release 209) and 16S databases constructed in-house comprising representatives of (1 ) globally prevalent taxa or isolates commonly found in healthy human faecal microbiomes, (2) taxa or isolates that best discriminate between the faecal microbiomes of health and ulcerative colitis (health associated), (3) keystone taxa or isolates, and (4) putative Hz consuming taxa or isolates such as methanogenic archaea and homoacetogenic bacteria with 97% sequence similarity set as the species-level cut off (Konstantinidis, K. T. & Tiedje, J. M. Genomic insights that advance the species definition for prokaryotes. Proc. Natl. Acad. Sci. U. S. A. 102, 2567-2572 (2005)). Isolates meeting these criteria with a query coverage of > 98% were then parsed for generation of V3-V4 16S rRNA sequences.

[00387] Generation of V3-V4 16S rRNA gene sequences

[00388] The 16S rRNA gene sequence V3-V4 hypervariable region was obtained for each sample by extraction from 16S reads obtained from capillary sequencing with the conserved forward (SEQ ID NO. p0001 : CCTACGGGNGGCWGCAG) and reverse (SEQ ID NO. p0002: GACTACHVGGGTATCTAATCC) regions as flanking markers using hyperex version 0.1.1 (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).

[00389] Generation of phylogenetic trees

[00390] 16S sequences were aligned with ssu-align v 0.1.1 with default parameters.

Resulting alignment files were input into FastTree V2.1.11 to infer maximum likelihood trees with the generalised time-reversible (GTR) with CAT approximation model. Trees were visualised with R package ggtree v3.6.2 and R package ggtreeExtra v1 .8.1 .

ISOLATE SCREENING FOR ANTIBIOTIC RESISTANCE, PHAGE ELEMENTS, AND VIRULENCE FACTORS

[00391] As the complex consortium is to be used as a microbial therapy, safety is paramount. Screening microbial isolates intended for use as a microbial therapy for antibiotic resistance, phage elements, and virulence factors is vital to ensure patient safety and effectiveness.

[00392] To ensure each isolate meets the regulatory safety profile, the following were considered:

[00393] a) only isolates sourced from healthy individuals with complete passage history were included.

[00394] b) all isolates were phenotypically screened for antibiotic-resistance and phage presence, genomically screened for anti-microbial resistance, virulence and phage elements, phenotypically screened for detrimental products, e.g., sulphide production, as per the inventor's defined mechanism of action for ulcerative colitis.

[00395] c) pathogen searches were performed via literature reviews to ensure known pathogens or species linked with disease were removed.

[00396] d) redundancy was considered, with at least 3 strains from the same species included to overcome knockout from above processes

[00397] e) redundant strains were removed, with preference given to isolates with lowest risk profile from above screening.

ISOLATE SCREENING FOR PRODUCTION OF SULPHIDE

[00398] Emerging evidence has pointed toward sulphide as a potentially deleterious agent contributing to the pathogenesis of ulcerative colitis (Gibson, G. R., Cummings, J. H. & Macfarlane, G. T. Growth and activities of sulphate-reducing bacteria in gut contents of healthy subjects and patients with ulcerative colitis. FEMS Microbiol. Lett. 86, 103-111 (1991 ); Pitcher, M. C. L., Beatty, E. R. & Cummings, J. H. The contribution of sulphate- reducing bacteria and 5-aminosalicylic acid to faecal sulphide in patients with ulcerative colitis. Gut 46, 64-72 (2000); Roediger, W. & Babidge, W. Nitric oxide effect on coloncyte metabolism: Co-action of sulphides and peroxide. An International Journal for Chemical Biology in Health and Disease 206, 159-167 (2000). In this context, the inventors embarked on a rigorous screening process of phenotyping microbial isolates for sulphide production. The primary focus was to identify isolates considered for inclusion in BB265 that did not produce sulphide, as these were considered ideal candidates for the therapeutic consortium.

[00399] A total of 12,607 microbial isolates, isolated from screened donors and present in the inventor's culture collection, were screened for a phenotype of sulphide production. Isolates were incubated anaerobically for 48 hours at 37 °C in a Whitley A55 HEPA Anaerovic Workstation (Don Whitley Scientific) (atmosphere: 10% H2, 10% CO2, 80% N2) in 200 pL YCFA broth. Post incubation, 20 pL culture were transferred to 180 pL YCFA broth supplemented with 0.1% each of sodium thiosulphate and ferric ammonium citrate. Cultures were then incubated for a further 48 hours and inspected for growth by turbidity and sulphide production by the visual presence of black precipitate. The addition of sodium thiosulphate provides isolates with an inorganic source of sulphur to assess reduction capacity to sulphide, whereas cysteine (1%) present in YCFA provides an organic source of sulphur, sulphide produced by microbiota from thiosulphate or cysteine reacts with ferric ammonium citrate to form iron sulphide (FeS), which can be seen visually as a black precipitate in the medium to confirm sulphide production. [00400] Of the 12,607 isolates screened, the inventors identified 2,694 isolates with a phenotype of sulphide production. Sulphide production was found to be phylogenetically diverse, with the phenotype identified in isolates across the major phyla Actinomycetota, Bacillota, Bacteroidota, Fusobacteria, Pseudomonadota, and Thermodesulfobacteriota (Figure 11 ). Interestingly, though sulphide has been identified as a deleterious agent in ulcerative colitis, the inventors identified that many taxa considered for inclusion in the BB265 complex consortium also showed a phenotype of sulphide production.

[00401] Where possible, the inventors selected isolates for inclusion in the BB265 consortium that showed a negative phenotype for sulphide production. However, where this could not occur, rather than dismissing these sulphide producing isolates outright, the inventors considered their potential role in ulcerative colitis management due to their prevalence in healthy individuals, keystone characteristics and anti-correlation with ulcerative colitis datasets. This decision was driven due to the relationship between sulphide and ulcerative colitis being nuanced and multifaceted. While the inventors posit sulphide as the deleterious agent in the context of ulcerative colitis, the human gut microbiome is a diverse and complex ecosystem, and microbial diversity is crucial for 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). Many sulphidogens identified in this study are linked to health, and likely engage in crucial roles that positively modulate the gut ecosystem, which, in the context of ulcerative colitis, may lead to an overall reduction in the rate and production of sulphide at the community level.

[00402] To further characterise lead isolates considered for inclusion in BB265, purified isolates were then assayed for production of sulphide from L-cysteine or thiosulphate independently by repeating the above process with media containing only L-cysteine or thiosulphate, respectively (Table 5).

[00403] In conclusion, sulphide production was identified as being a phylogenetically diverse phenotype, both present in taxa associated with health and ulcerative colitis. Isolates chosen for inclusion in the BB265 complex consortium were preferentially selected if they showed a negative phenotype for sulphide production; however, where this could not occur, isolates were still considered due to evidence for their association with health, importance as keystones, anticorrelation with ulcerative colitis, and hypothesised mechanisms of action, such as H2 consumption, which target sulphide production at the community level.

ISOLATE SCREENING FOR CONSUMPTION OF SULPHIDE [00404] This study aimed to phenotype candidate purified microbial isolates for sulphide consumption and which, therefore, may have the ability to reduce colonic concentrations of sulphide when introduced into a host when used as a treatment for ulcerative colitis.

[00405] Modified methylene blue assay for the determination of sulphide consuming microbial isolates

[00406] To identify sulphide consuming microbial isolates, we developed an in-house modified methylene blue assay based on chemistry in the American Public Health Association’s Standard Methods for the examination of sulphide in water and wastewater; referred to here 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 the determination of sulphide is a highly specific colorimetric test whereby N, N- dimethyl-p-phenylenediamine sulphate in the presence of an oxidising agent, such as acidified FeCh, reacts with sulphide to produce coloured methylene blue. As sulphide is the rate-limiting reactant, the amount of methylene blue produced is proportional to the original concentration of aqueous sulphide. The presence of methylene blue can be determined spectrophotometrically via absorption of light at 667 nm

[00407] Frozen isolates were thawed at 37 °C in the anaerobic cabinet, diluted 1 :10 in prereduced YCFA broth and left to incubate overnight. In triplicate, 100 pL 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. To this cuvette sodium sulphide (anhydrous) (NaS) was spiked as a source of aqueous sulphide at a concentration of 2 mM and the cuvette immediately capped to prevent the escape of volatile hydrogen sulphide (H2S). Negative controls consisting of modified YCFA (lacking L-cysteine) plus 2mM NaS without culture were prepared concurrently. Cuvettes were left to incubate anaerobically at 37 °C for 48 hr. Post incubation, growth of microbial isolates were identified visually by the presence of turbidity in cuvettes. Cuvettes were removed from the anaerobic cabinet, decapped and 200 pL culture broth added immediately to 3.8 mL dd.H ₂ O in a clean cuvette. To this cuvette, 345 pL of a solution containing FeCh (37.0 mM) and N, N-dimethyl-p-phenylenediamine sulphate salt (17.1 mM) dissolved in HCI (6 M) was added, the cuvette was immediately capped and inverted once to mix. Cuvettes were left for 10 min to allow for colour development and reaction to run to completion (4500-S2? SULPHIDE, in Standard Methods For the Examination of Water and Wastewater (American Public Health Association, (2017)) . OD 667 nm readings were then taken with a SPECTROstar Nano spectrophotometer (BMG Labtech) using MARS v4.01 for visualisation and data output.

[00408] Data and statistical analyses [00409] Data were exported from MARS to CSV format and raw OD 667 nm values transformed to sulphide concentration (mM) by comparison to a standard curve of known sulphide concentrations in Microsoft Excel v2208. Transformed data were imported into Prism (GraphPad) for statistical analysis and data visualisation. To determine whether microbial isolates exhibited a significant reduction in sulphide concentration post-incubation when compared to the negative control, a Welch’s t-test was performed with significance set to an alpha of 0.05 (Welch, B. L. The Generalization of 'Student’s' Problem when Several Different Population Variances are Involved. Biometrika 34, 28-35 (1947)).

[00410] Frozen isolates were thawed at 37 °C in the anaerobic cabinet, diluted 1 :10 in prereduced YCFA broth and left to incubate overnight. In triplicate, 100 pL 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.

[00411] Results

[00412] A total of 275 purified microbial isolates marked as candidates for inclusion in the BB265 complex consortium were phenotypically screened for consumption of sulphide (Figure 17). Of these, 154 isolates were identified that had an average net reduction in aqueous sulphide concentration in modified YCFA broth following incubation with 2 mM NaS anaerobically for 48 hours (Table 1 ). Of these, 87 isolates showed a statistically significant reduction in sulphide concentration compared to the negative control (Table 1 ; Figure 9). Taxa that differed significantly from the negative control comprised species in the genera Alisipes, Anaerobutyricum, Anaerofustis, Bacteroides, Blautia, Christensenella, Clostrdium, Collinsella, Coprobacillus, Coprococcus, Erysipelatoclostridium, Eubacterium, Flavonifractor, Intestinimonas, Longicatena, Massilimicrobiota, Parabacteroides, Roseburia, Ruminococcus, Streptococcus, and Thomasclavelia. Of these taxa, average net reduction in sulphide concentration ranged from 41 pM sulphide for Isolate bb0284: Bacteroides uniformis (± 4 SEM) to 697 pM sulphide for Isolate bb0627: Flavonifractor plautii (+ 55 SEM).

[00413] Table 1 - List of isolates identified with a sulphide consumption phenotype with average net reduction in sulphide compared to the negative control; corresponding sulphide production phenotype is also presented

00414] * indicates statistically significant sulphide consumption

[00415] Discussion

[00416] Sulphide is a cytotoxic metabolite produced in excess by resident colonic microbiota in patients with inflammatory bowel disease. Sulphide in this experiment is measured in the form of a sulphide ion (S ² ‘); a hydrosulphide (HS j ion; a bisulphide ion (SH' ), and hydrogen sulphide (H2S). Microbiota-derived sulphide in combination with nitric oxide plays a direct role in ulcerative colitis (UC) pathogenesis. These gases lead to sequestration of CoA, and depletion of glutathione and an inability of colonocytes (colonic epithelial cells) to produce energy via p-oxidation of butyrate. This biochemical lesion in ulcerative colitis results in an energy-deprived state for colonocytes and contributes to a loss of epithelial barrier function and ensuing mucosal inflammation.

[00417] Variation in fermentable dietary protein and carbohydrate reaching the colon influences production and detoxification of nitrogenous metabolites and sulphide by gut microbiota. Modern western dietary patterns observed in patients with ulcerative colitis create a colonic environment likely to be permissive of nitrogenous metabolites and excess sulphide production. Also contributing to colonic sulphide accumulation is intake of inorganic sulphur (sulphates/sulphites), nitrates/nitrites and, to a lesser extent, endogenous carbon substrate. In ulcerative colitis, measurements of anionic sulphide levels in the colon are elevated relative to healthy controls. In addition, the potential to produce sulphide in faecal samples has been found to be 3-4 fold higher in ulcerative colitis than in control cases. This is likely due to the relatively high levels of sulfidogenic microbiota, including SRB and cysteine degrading bacteria in patients with inflammatory bowel disease in both active and quiescent disease. Nitric oxide is produced by both inflamed colonocytes and the colonic microbiota.

[00418] Faecal microbiota transplantation (FMT) has demonstrated efficacy in the induction of remission of ulcerative colitis, indicating that the colonic microbiota are involved in disease pathogenesis and that modulation of the colonic microbiota can ameliorate disease. Given the evidence supporting sulphide as an etiological factor, modulation of the colonic microbiome in a way that reduces colonic levels of sulphide offers a promising therapeutic target.

[00419] In our study, we identify and then phenotype candidate microbial isolates considered for inclusion in BB265 with the capacity to consume sulphide and which may therefore reduce colonic levels of sulphide when introduced into a host. We identified 154 microbial isolates that, when incubated for 48 hours in the presence of 2 mM sulphide, decreased the concentration of aqueous sulphide when compared to the control (Table 1 ). While performed in vitro, our results show significant reduction in sulphide at concentrations within the physiologic range of luminal contents (0.3-3.4 mM), supporting the ability BB265 has to reduce sulphide in concentrations that are comparable to what is required in vivo. The majority of those isolates that differed significantly from the negative control comprised taxa which are commonly found diminished in individuals with ulcerative colitis, including but not limited to isolates of the genera: Bacteroides, Christensenella, Clostridium and Roseburia. This suggests that microbiota with the capacity to consume sulphide may be diminished in ulcerative colitis, and therefore methods to increase their abundance or metabolic activity in the colon may reduce colonic sulphide, allowing colonocytes to effectively respire, produce energy and maintain the mucosal barrier. In this way, these microbes can alleviate the sulphide and nitric oxide induced metabolic lesion that manifests with active inflammation in ulcerative colitis.

[00420] The isolates identified in this study offer therapeutic potential by consuming or biotransforming sulphide, either metabolically or otherwise, thereby reducing colonic levels of sulphide.

SELECTION OF THE FINAL CONSORTIUM COMPRISING BB265

[00421] The final list of isolates comprising the BB265 consortium consisted of 143 microbial isolates (Table 2). The BB265 complex consortium list is presented using the following taxonomic classifications. NB: Due to the ephemeral nature of taxonomic names, taxon IDs are also provided for each isolate in the BB265 consortium, including also those isolates not in the BB265 consortium but identified with a sulphide consumption phenotype (Figure 5). All past, present and future taxon names describing each species are to be covered here, including all homotypic, heterotypic, subjective, objective, nomenclatural and invalid synonyms, along with misapplied names.

[00422] Refer to the following results; * indicates taxa identified in the analyses considered for inclusion in BB265 but not present in the BB265 complex consortium

[00423] By domain: *Archaea; and Bacteria

[00424] Table 2 - List of taxa by Phylum

[00425] Table 3 - List of taxa by Genus

[00426] Table 4 - List of taxa by Species / 16S identification including 16S sequences for each species in the BB265 complex consortium or identified as sulphidogens

[00427] Table 5 - List of isolates comprising the 143 isolate BB265 complex consortium with corresponding sulphide consumption and production phenotype

D EXAMPLE 4 - IDENTIFICATION OF GENOMIC SIGNATURES OF THE BB265 COMPLEX COMMUNITY LIVE BIOTHERAPEUTIC PRODUCT PUTATIVELY INVOLED IN COLONIC SULPHIDE HANDLING

STUDY AIM [00428] To identify genes and gene clusters present in the genomes of isolates comprising the BB265 complex consortium putatively involved in colonic sulphide handling.

[00429] A list of genes implicated in sulphide metabolism was prepared with reference to the literature (Wolf, P. G. et al. Diversity and distribution of sulphur 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 for the members of the BB265 consortium were annotated using Prokka v1.14.6 to identify genes, and putative genes, that are present based on the DNA sequence (Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068-2069 (2014)). The lists of identified genes thus produced were then queried for those genes known, or inferred, to be involved with sulphide metabolism.

[00430] Table 6 - List of genes identified in the BB265 complex consortium putatively involved in sulphur and sulphide metabolism and handling within the colon

[00431] Table 7 - List of genes identified in the BB265 complex consortium putatively involved in homo-acetogenesis and hydrogen cycling within the colon

E EXAMPLE 5 - MECHANISTIC VALIDATION OF BB265 FOR THE TREATMENT OF ULCERATIVE COLITIS BY MODULATION OF COLONIC SULPHIDE

STUDY AIM

[00432] To mechanistically validate the BB265 complex consortium for the treatment of ulcerative colitis by modulating colonic sulphide

MATERIALS, METHODS AND RESULTS

F PREPARATION OF THE BB265 COMPLEX CONSORTIUM AS AN INOCULUM FOR MECHANISTIC VALIDATION EXPERIMENTS

[00433] The BB265 complex consortium was prepared as a mixed inoculum to be used going forward for validation experiments. Two mixed consortium inocula were prepared: (1 ) containing a subset of 127 isolates present in the final BB265 complex consortium (Table 8); this subset was prepared as validation experiments were undertaken before the remaining 16 isolates had completed screening as per section C. Example 3 - Defining organisms comprising the BB265 complex community live biotherapeutic product. The BB265 complex consortium was amended to additionally comprise these remaining 16 isolates post screening as they were considered as therapeutic candidates, and (2) the total BB265 complex consortium comprising the 143 isolates specified in section C. Example 3 - Defining organisms comprising the BB265 complex community live biotherapeutic product (Table 5).

[00434] Isolates comprising the inocula were grown anaerobically at 37 °C overnight in a Whitley A55 HEPA Anaerobic Workstation (Don Whitley Scientific) (atmosphere: 10% H2, 10% CO2, 80% N2) by inoculating 2.94 mL modified-YCFA broth with 30 pL thawed glycerol stocks of purified isolates. For slow growers, inoculation occurred the day prior and were incubated for 48 hours. Culture broth were then diluted 1 :1 in a Costar 48-well microplate to a final volume of 500 pL; diluted cultures were then read on a Spectrostar Nano spectrophotometer to determine OD 600 nm values. Cultures were then diluted individually with modified YCFA to achieve an OD 600 nm per isolate of 0.2. Equal volumes of diluted culture broth were then combined, diluted 1 :1 in 50% glycerol (final concentration 25%) and frozen at -80 °C. Total cell concentration of the mixed inocula were then determined by quantifying intact cell counts using a BactoBox® (SBT Instruments) as per manufacturer’s instructions. Using OD 600 nm as a proxy for cell number, and assuming each isolate present in the mixed inocula were present in equal abundance, cell concentration per isolate was determined by dividing the total cell number by the number of isolates in the inoculum. For slow growing isolates that did not reach an OD 600 nm value of 0.2, glycerol stocks were prepared as above and intact cell concentration per isolate determined using a BactoBox® (SBT Instruments) as per manufacturer’s instructions. When running validation experiments, slow growing isolates were combined with inocula at volumes adjusted to equalise cell concentration of all isolates in the inocula. These cell-number normalised mixes were then used as inocula for validation experiments moving forward.

[00435] Table 8 - List of isolates comprising the 127-isolate subset of the BB265 complex community inoculum

A PUTATIVE INVOLVEMENT OF TAXA COMPRISING THE BB265 COMPLEX

CONSORTIUM IN ULCERATIVE COLITS DISEASE RESOLUTION - AN FMT TRIAL

[00436] Background

[00437] Ulcerative colitis is a chronic inflammatory bowel disease derived from abnormal immune response, causing inflammation and ulceration of the colon and rectum. Recent advancement in the study of human gut microbiota demonstrated the association between gut dysbiosis and the development and progression of ulcerative colitis symptoms. One aspect of gut dysbiosis in ulcerative colitis patients is the imbalance of sulphide homeostasis, where excessive levels of sulphide can contribute to the pathogenesis of the disease. Targeting sulphide production in the gut can be a potential therapeutic strategy for ulcerative colitis. There is increased clinical evidence suggesting faecal microbiota transplantation (FMT) could restore gut microbiota and induce remission in ulcerative colitis patients. This study aims to determine the correlation between the production of sulphide in vitro and the abundance levels of the BB265 complex consortium isolates in ulcerative colitis patients who received FMT intervention. The result informs a putative involvement of the FMT-derived BB265 complex consortium in sulphide reduction and disease remission in ulcerative colitis patients.

[00438] Methods

[00439] Clinical trial

[00440] A randomised, controlled trial of FMT in adults with active, mild to moderate ulcerative colitis symptoms (Mayo scores 3 - 10) was performed between April 2019 and March 2020. All participants were 18 years old or older and provided written informed consent for participation. The trial was conducted in two phases: Phase 1 was a 12-week open label induction of ulcerative colitis remission, whereas Phase 2 was a 40-week open label maintenance of remission study. During Phase 1 , 35 eligible patients received a tapering dose of oral prednisolone, starting with 50 mg daily weaning to 0 mg over 8 weeks. At the beginning of week 5, patients received 3 L of polyethylene glycol bowel preparation and 4 mg of Loperamide was given orally 60 min prior to 200 mL of FMT via colonoscopy. This is followed by 2 faecal enemas of 50 mL via syringe into the rectum within 7 days (i.e., Day 3 or 4, and Day 6 or 7) of receiving the first FMT. 4 mg of Loperamide was given orally 30 min prior to administration of enema. The retention enemas were the same composition as the colonoscopic induction of remission FMT. Assessment was performed in week 12 and only 22 patients who showed clinical and endoscopic remission (Mayo score < 2 with no individual sub-score > 1) or clinical response (reduction in Total Mayo score > 30% and > 3 points, as well as a reduction in the rectal bleeding score of > 1 or a rectal bleeding score of 0 or 1 ) entered Phase 2 maintenance phase, where they received FMT blend with the same composition from the same donors used in Phase 1. These participants were openly randomized 1 :1 to receive either 4 weekly (i.e., 13 patients) or 8 weekly (i.e., 9 patients) of 50 mL FMT enemas for a total of 40 weeks. Randomisation was performed using an atmospheric noise randomisation algorithm (www.random.org). Maintenance of clinical remission or response over 40 weeks was assessed with four criteria: (i) time of flare (measured in weeks until SSCAI > 4), (ii) weeks in flare (weeks with SSCAI > 4), (iii) SSCAI < 3 at weeks 28 and 50, (iv) total Mayo score at week 50. Those patients from Phase I who did not achieve remission or response were invited to continue to provide faecal samples every 4 weeks for a further 40 weeks.

[00441 ] Metaaenomic sequencing and analysis

[00442] Genomic DNA was extracted from patient faecal samples using the FastDNA™ SPIN Kit for Soil DNA extraction kit (MPBio) as manufacturer's instructions. DNA quality was assessed using NanoDrop 2000c UV-vis spectrophotometer (Thermo Fisher, Waltham, MA, USA) and DNA subjected to metagenomic sequencing. DNA libraries were prepared using Celero EZTM DNA-Seq kits using protocol M01526 v1 and sequenced on an Illumina NextSeq2000 machine (109 bp PE), using Illumina protocol 1000000109376 v3. Metagenomes were quality trimmed and adapter sequences were removed with Trimmomatic v0.39, with leading and trailing bases below quality score 3 removed and reads below 50 base pairs removed. Potential contamination from human reads was removed by mapping to the human reference human genome (GRCh38) using bowtie v2.5.1 and removing reads that 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 (v mpa_vOct22_CHOCOPhlAnSGB_202212). MetaPhlAn4 outputs were merged and filtered for species-level results and the resulting data was used to identify taxonomic changes between samples of individuals who entered clinical and endoscopic remission, as defined above, and those who remained in active disease at the cessation of phase 1 .

[00443] The species MetaPhlAn4 output was filtered for baseline and remission samples for patient D (week 0 and week 52 samples) and E (week 0 and week 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 matching of taxa names, the number of matches, and the cumulative abundance of BB265 species in the metagenomes was calculated and compared between baseline and remission samples. This was repeated for species identified phenotypically as statistically significant consumers (Table 1 ).

[00444] A sulphide producing species list was generated by identifying species that had >70% of isolates in that species phenotypically identified as sulphide producers (Figure 11 ). Any BB265 species were removed from the list of sulphide producer species, and the resulting list of species was queried against the MetaPhlAn output for patients D and E, and the number of species matches, and cumulative abundance of species were compared between the baseline and remission samples.

[00445] To examine the abundance and species number of BB265, consumers and producers across all patients in the clinical trial, the species MetaPhlAn4 output was filtered for phase 1 samples, baseline (week 0) and end of phase 1 (week 12) for all patients. The number and cumulative abundance of BB265, phenotypically determined statistically significant consumer species, and sulphidogens, as defined above, were quantified at week 0 and week 12, comparing patients who entered clinical and endoscopic remission, and patients who did not enter clinical and endoscopic remission. Statistically significant differences between week 0 and week 12 for each group were determined using the Wilcoxon test with Bonferroni correction. Significance was determined as an adjusted p value of <0.05.

Determination of sulphide levels in baseline and remission stool samples

[00446] Faecal slurry preparation

[00447] Based on clinical trial criteria, stools from patients who showed clinical response and clinical remission were selected for sulphide determination. For each patient, both baseline sample (week 0, total Mayo score 3 - 10) and remission sample (week 12 or week 52, total Mayo score 0 - 2) were used in the experiment. Briefly, 10% faecal slurries were prepared by adding 1.25 g of stool in 12.5 mL of half strength YCFA and Solution I for all baseline and remission samples. The mixtures were mixed by vortexing for 5 min and centrifuged briefly. 2.5 mL of supernatant was transferred to gas chromatography (GC) vials containing either 2.5 mL of half strength YCFA supplemented with sodium sulfite as an inorganic sulphur source or Solution I, resulting in a final concentration of 5% faecal slurries. The GC vials were capped immediately and incubated anaerobically at 37 °C overnight. All experiments were performed in triplicate. Half-strength YCFA was used in this case as a complex medium, providing adequate nutrition for the growth of isolates in the BB265 complex consortium. Making the medium half-strength limits the total carbon and sulfur pool, ensuring sulphide was not produced in excess and allowing for accurate spectrophotometric quantification. Contrariwise, Solution I is a minimal medium, providing low-dose complex carbon sources as the only nutrient source to kickstart fermentation whilst minimising medium complexity to maintain the metabolic milieu as present in uclerative colitis stool. Solution I consists of the vitamin and mineral components of YCFA only with 5 g/L tryptone and 1 .25 g/L yeast extract.

[00448] Modified methylene blue assay for determination of sulphide

[00449] GC vials were removed from the anaerobic chamber and 200 pL of the mixture was transferred to cuvettes containing 3.8 mL of RO water. To this cuvette, 345 pL of a solution containing FeCI3 (37.0 mM) and N, N-dimethyl-p-phenylenediamine sulphate salt (17.1 mM) dissolved in HCI (6 M) was added, the cuvette was immediately capped and inverted once to mix. Cuvettes were left for 10 min to allow for colour development and reaction to run to completion. 1 mL of the samples were transferred to Eppendorf tubes and centrifuged at 15,000 x g for 5 min. Then, 750 pL of the supernatant were transferred to a 48-well microplate and OD 667 nm readings were then taken with a SPECTROstar Nano spectrophotometer (BMG Labtech) using MARS v4.01 for visualisation and data output.

[00450] Results [00451] Analysis of the metagenomes from phase 1 of the clinical trial dataset, identified that patients who went into remission (Clinical remission) had a significant increase in the number of BB265 species from week 0 to week 12. For patients who did not go into 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 sulphide consumer abundance from baseline to week 12 (Figure 12b).

[00452] Additionally, there was an increase in the number of consumer species from baseline to week 12 in the patients who entered clinical remission (Figure 12c). Reduction of aqueous sulphide after incubating faecal slurries from ulcerative colitis patients was observed in faecal samples taken during remission when compared to faecal samples from the same patients when in active disease. For Patient D, the concentration of aqueous sulphide in half strength YCFA with sodium sulfite and Solution I was reduced from 2355.1 pM (SD + 37) (week 0) to 1529.4 pM (SD + 124.4) (week 12), and 563.5 pM (SD + 23.2) (week 0) to 421 .6 pM (SD + 14.3) (week 12), respectively (Figure 13a). For Patient E, the concentration of aqueous sulphide in half strength YCFA with sodium sulfite and Solution I was reduced from 463.0 pM (SD + 19.8) (week 0) to 73.5 pM (SD + 1 .6) (week 12), and 332.3 pM (SD + 6.2) (week 0) to 272.2 pM (SD + 3.6) (week 12), respectively (Figure 13b).

[00453] To determine the correlation between phenotypically measured sulphide reduction in patients D and E from baseline to remission, metagenomic profiles of patients D and E were examined for BB265 species, sulphide consumer species and sulphidogenic species. Also identified was an increase in the number of BB265 species from the baseline (active) to remission samples in Patient E and to a lesser extent in Patient D (Figure 13c). Similarly, there was an increase in sulphide consumer species from baseline (active) to remission in both patients (Figure 13d).There was also a marked decrease in the cumulative relative abundance of sulphidogens, supporting the finding of decreased capacity of sulphide production in these samples (Figure 13e).

[00454] Discussion

[00455] Analysis of the metagenomes from patients in Phase I of the clinical trial supported the claims that BB265 species are important gut microbiota in the induction of remission for ulcerative colitis. The number of BB265 species and sulphide consumer species significantly increased from baseline to week 12 for patients that went into remission. Additionally, the statistically significant reduction in the relative abundance of consumer species in patients who did not enter remission also supports those isolates identified as sulphide consumers as a potential therapeutic for the treat ulcerative colitis. [00456] The results of the modified methylene blue assay correlated directly with the clinical data, where a reduction of aqueous sulphide in vitro corresponded to a decrease in total Mayo score in clinical observation. Specifically, for Patient D, the total Mayo score reduced from 3 in week 0 to 0 in week 52 and the modified methylene blue assay testing showed a significant decrease in the levels of aqueous sulphide in the clinical sample from week 0 to week 52. In addition, this correlation was also observed for Patient E, where the total Mayo score reduced from 8 in week 0 to 0 in week 12. This corresponded to elevated sulphide levels in the week 0 clinical sample and a significantly reduced level of hydrogen sulphide in the week 12 sample where the patient had achieved clinical remission.

[00457] The paired observations of: (1) an increase in the number of BB265-associated and sulphide consuming taxa, and a decrease in putative sulphidogen cumulativ relative abundance in patients who successfully went into remission due to FMT, and (2) a significant reduction in sulphide-producing capacity of the ulcerative colitis stool community post-FMT in patients who went into clinical remission strongly supports the BB265 consortium as a therapy for ulcerative colitis, and further supports the mechanism of action of this therapy for reducing sulphide as a means to improve clinical outcomes.

REDUCTION OF SULPHIDE BY THE BB265 COMPLEX CONSORTIUM WHEN COCULTURED WITH ULCERATIVE COLITIS FAECAL SLURRIES

[00458] Background

[00459] The BB265 complex consortium consists of a diverse array of metabolically active bacteria found in the healthy human gut microbiome. These bacteria are likely to play pivotal roles within the sulphur pathways of the human gut, contributing in diverse ways such as metabolising sulphur substrates, redirecting sulphide into various metabolites, and effectively consuming sulphide.

[00460] In our prior study, we have phenotypically identified specific healthy gut- associated microbes that can consume sulphide when present as individual organisms. However, these microbes exist as part of a complex microbial community within the gut microbiome and work symbiotically with other bacteria in the gut to perform essential functions. Hence, as a community it is possible these microbes potentiate the sulphide consuming capacity we have observed on an individual level, whilst providing the metabolic and ecological complexity necessary for health. We therefore sought to investigate whether the BB265 complex consortium can facilitate sulphide reduction as a co-culture. [00461] E.2.3. 1 Reduction of aqueous sulphide in ulcerative colitis faecal samples by coculture with a subset of the BB265 complex consortium compared to a phylogenetically diverse consumer community

[00462] Method

[00463] In-vitro assay

[00464] A bacterial consortium comprising 127 of the 143 isolates in the BB265 complex consortium was grown overnight anaerobically at 37 °C by inoculating 50 L of cell-number normalised glycerol stock into 20 mL of YCFA (Table 8).

[00465] A phylogenetically diverse consumer mix (PDCM) (Table 9), consisting of 19 phylogenetically diverse isolates, each identified to consume sulphide (range 20-670 pM) whilst being confirmed negative for su phide production was run in parallel to the 127-isolate subset of the BB265 complex consortium. The PDCM was run in parallel to assess community effects of sulphide consumption by a consumer community as a comparison to the 127-isolate subset of the BB265 complex consortium which itself contains many health-associated sulphidogens.

[00466] 50 pL glycerol stocks of cell-normalised PDCM and the 127-isolate subset of the

BB265 complex consortium were inoculated into 20 mL of pre-reduced YCFA tubes and incubated at 37°C for 24 hours anaerobically. After incubation, the cultures were pelletised by centrifugation at 12,000 x g for 10 min. The pellets were washed three times with 50 mL prereduced PBS to remove residual medium and resuspended in 20 mL pre-reduced Solution I.

[00467] Two ulcerative colitis stool samples were used in this study, referred to here as Patient A and Patient B. 16% (w/v) faecal slurries were prepared by adding 8 g of stool in a Falcon tube which was topped up with 50 mL of Solution I. Samples were vortexed for 5 min and pulsed briefly up to 100 rpm to pelletise large particles. 2.5 mL of supernatant was transferred to Hungate tubes containing 2.5 mL of Solution I, resulting in a final concentration of 8% (w/v) faecal slurry. Five replicates of faecal slurry per treatment were prepared and spiked with 10 pL of inoculum: the 127-isolate subset of BB265, the PDCM, and a negative control containing sterile media only. Hungate tubes were immediately capped to avoid loss of volatile H ₂ S and incubated anaerobically at 37 °C for 24 hours. Post incubation, 1 mL from each treatment was aliquoted into sterile 1 .5 mL microfuge tubes and stored at -80°C prior to DNA extraction. DNA extractions were performed using the FastDNA™ SPIN Kit for Soil (MPBio) DNA Extraction kit with DNA quality assessed using a NanoDrop 2000c UV-vis spectrophotometer (Thermo Fisher, Waltham, MA, USA). Once DNA was extracted, extracts were used for shotgun metagenomic sequencing. [00468] Metagenomic Sequencing

[00469] DNA libraries were prepared using Celero EZTM DNA-Seq kits using protocol M01526 v1 , and sequenced on an Illumina NextSeq2000 machine (109 bp PE), using Illumina protocol 1000000109376 v3. Reads were processed using a custom pipeline developed inhouse, which 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.

[00470] Table 9 - Isolates comprising the Phylogenetically Diverse Consumer Mix (PDCM)

[00471 ] Modified methylene blue assay

[00472] Hungate tubes were removed from the anaerobic chamber and 200 pL of the mixture was transferred to cuvettes containing 3.8 mL of RO water. To this cuvette, 345 pL of a solution containing FeCI3 (37.0 mM) and N, N-dimethyl-p-phenylenediamine sulphate salt (17.1 mM) dissolved in HCI (6 M) was added, the cuvette was immediately capped and inverted once to mix. Cuvettes were left for 10 min to allow for colour development and reaction to run to completion. Aliquots (1 mL) from color reacted suspensions were centrifuged at 12000 x g for 5 min and OD 667 nm readings of supernatants were then taken with a SPECTROstar Nano spectrophotometer (BMG Labtech) using MARS v4.01 for visualisation and data output.

[00473] Statistical analyses

[00474] To identify significant differences in sulphide consumption between treatment groups (stool only, stool plus the 127-isolate subset of the BB265 complex consortium, PDCM) within patients a one-way Anova was performed.

[00475] Results

[00476] For Patient A, when the faecal slurry was co-cultured with the 127-isolate subset of the BB265 complex consortium, an average reduction in aqueous sulphide of 44.5 pM (± 6.8 SD) was determined, a significant decrease in sulphide concentration when compared to the negative control (faecal slurry only). When co-cultured with the PDCM, a significant reduction compared to the negative control was also determined, with an average net reduction in sulphide of 24. pM (± 4.5 SD). Regarding sulphide reduction for Patient A, the 127-isolate subset of the BB265 complex consortium outperformed the PDCM significantly (Figure 14).

[00477] For Patient B, when the faecal slurry was co-cultured with the 127-isolate subset of the BB265 complex consortium, an average reduction in aqueous sulphide of 23.4 pM (± 4.7 SD) was determined, a significant decrease in sulphide concentration when compared to the negative control (faecal slurry only). When co-cultured with the PDCM, a significant reduction compared to the negative control was also determined, with an average net reduction in sulphide of 41.4 pM (± 8.0 SD). Contrariwise to Patient A, the PDCM significantly outperformed the 127-isolate subset of the BB265 complex consortium, consuming on average 18 pM more sulphide (Figure 14).

[00478] Metagenomic analysis of stool incubated with and without the 127-isolate subset of the BB265 complex consortium revealed distinct changes in microbial signatures due to the addition of the consortium. Regarding taxa comprising the BB265 complex consortium, Patient A had significantly fewer taxa autochthonous to the stool sample than Patient B (Figure 23c). Regardless, when stool was co-cultured with the 127-isolate subset of the BB265 complex consortium, post incubation there was increase in both samples of taxa comprising the BB265, both in number of taxa and relative abundances (Figure 23c). This increase in BB265 taxa paralleled with an increase in the number of sulphide consuming taxa post incubation (Figure 23b). In Patient A, a parallel increase in the relative abundances of sulphide consuming taxa was observed, whereas Patient B displayed an opposite trend, with a decrease in the total relative abundance of the community attributed to sulphide consuming taxa. In both Patients, the BB265 complex consortium had no effect on the number of sulphidogenic taxa identified, with the number of sulphidogenic taxa equal when comparing stool only with stool plus the 127-isolate subset of the BB265 complex consortium (Figure 23a). However, although the number of sulphidogens remained the same, addition of the 127-isolate subset of the BB265 complex consortium resulted in a decrease in the relative abundances of sulphidogenic taxa in both patients (Figure 23a).

[00479] Discussion

[00480] In both Patient A and B, when stool was co-cultured with the 127-isolate subset of the BB265 complex consortium, the number of sulphidogenic taxa remained unchanged when compared to the stool only sample; however, the cumulative relative abundance of these producers decreased (Figure 23a). This is analogous to the results observed in the clinical data for Patients D and E, which also showed that the cumulative relative abundance of sulphidogens decreased (Figure 13e). This supports the hypothesis that the BB265 consortium is working mechanistically akin to FMT-induction of remission in ulcerative colitis. Furthermore, this suggests that the 127-isolate subset of the BB265 consortium effectively reduced the prevalence of sulphidogens without diminishing their diversity. Though the total number of sulphide producing taxa remained unchanged, sulphide measurements indicated a significant reduction when compared to the control (Figure 14). This suggests that by modulating the abundances rather than decreasing diversity of sulphidogens, the deleterious effect of excess sulphide in ulcerative may be targeted. Modulation of abundances in this regard might enable for a treatment effect whilst maintaining the ecological diversity of the community which has been identified as a key component of a healthy microbiome.

[00481 ] These results elucidate that co-culturing ulcerative colitis stool with the 127-isolate subset of the BB265 complex consortium can significantly reduce sulphide. Notably, in the case of Patient A, there was an elevated sulphide reduction compared to the co-culture with the PDCM, which consists of 19 isolates displaying a higher net sulphide reduction in the consumer assay alone and consisting of no sulphidogens (Figure 14). These findings suggest that the additional species within the 127-isolate subset of the BB265 complex consortium support the phenotypic effect of sulphide consumers in the consortium by modulating complex community dynamics. However, in Patient B, the PDCM displayed higher sulphide reduction than the 127-isolate subset of the BB265 complex consortium, suggesting this bolstering effect may be patient specific (Figure 14).

[00482] Along with the potential for reducing colonic sulphide in ulcerative colitis by modulating the cumulative relative abundance of sulphidogens, a parallel increase in the number and cumulative relative abundance of sulphide consuming taxa was observed when stool was co-cultured with the 127-isolate subset of the BB265 complex consortium (Figure 23b). This suggests that the BB265 consortium may also act to reduce sulphide in ulcerative colitis by altering the ratio and abundances of sulphidogenic to sulphide consuming taxa, resulting in less production and greater consumption of sulphide by the community at any given time. These results give strong evidence that microbiota comprising the BB265 complex consortium have therapeutic potential in ulcerative colitis by modulating the dynamics of both sulphide production and consumption, resulting in a net reduction in sulphide levels when these taxa are present in the faecal samples of ulcerative colitis patients.

[00463] E.2.3.2 Reduction of aqueous sulphide in ulcerative colitis faecal sample by coculture with a subset of the BB265 complex consortium compared to an individual strong sulphide consumer

[00484] To further support the idea that community complexity may bolster the effect of sulphide reduction in ulcerative colitis, the inventors assessed how the 127-isolate subset of the BB265 complex consortium compares in sulphide reducing capacity to a single isolate identified as one of the strongest sulphide consumers individually.

[00485] Methods

[00486] In-vitro assay

[00487] In this study, bb0214 Ruminococcus bicirculans, which showed an average net reduction of 670 pM sulphide alone when assayed for consumption, was selected as a strong consumer. Glycerol stocks of the cell-number normalised 127-isolate subset of the BB265 complex community and bb0214 (50 pL) were inoculated into 20 ml_ of YCFA and incubated at 37 °C for 24 hours.

[00488] For the ulcerative colitis stool (Patient A), 32 g was dissolved in 50 mL of Solution I. The sample was vortexed for 5 min and pulsed briefly at 100 rpm to pelletise large particles and the supernatant transferred to a sterile 250 mL Shott bottle. The 24hr-grown 127-isolate subset of the BB265 complex consortium and bb0214 were centrifuged at 12000 x g for 10 min. After decanting the supernatant, pellets were washed with sterile 50 mL PBS three times. Final pellets were dissolved in 20 mL of sterile Solution I and vortexed until homogenised. Stool suspensions (2.5 mL) were transferred into sterile glass vials containing 2.5 mL of sterile Solution I, resulting in an 8% (w/v) final concentration of stool suspension. The 127-isolate subset of the BB265 complex consortium and bb0214 (100 pL) were inoculated into faecal slurries (100 pL) with 5 replicates; 100 pL sterile media was inoculated into 5 faecal slurry replicates as a negative control. Vials were incubated at 37 °C for 24 hours and the modified methylene blue assay was performed for measuring endpoint sulphide as described above. Post incubation, 1 mL from each treatment condition was aliquoted into sterile 1.5 mL microfuge tubes and stored at -80°C. DNA extractions were then performed using a FastDNA™ SPIN Kit for Soil (MPBio) DNA Extraction kit and DNA quality assessed using a NanoDrop 2000c UV-vis spectrophotometer (Thermo Fisher, Waltham, MA, USA). DNA extracts were then used to generate metagenomic sequencing datasets for each sample as per the methods described above.

[00489] Statistical analysis

[00490] To identify significant differences in sulphide consumption between treatment groups (stool only, stool plus the 127-isolate subset of BB265 complex consortium, stool plus bb0214 consumer) a one-way Anova was performed.

[00491] Results

[00492] When the faecal slurry from Patient A was incubated with the strong consumer bb0214 alone, an average net reduction in sulphide compared to the negative control (stool only) of 7.4 pM (± 8.4 SD) was observed. When incubated with the 127-isolate subset of the BB265 complex consortium a net average reduction in sulphide of 18.7 pM (± 3.2 SD) was observed, significantly greater than the single consumer alone (Figure 15).

[00493] The metagenomic analysis indicated that when the faecal slurry was incubated with the 127-isolate subset of the BB265 complex consortium there was a significant increase in the number of sulphide consuming taxa, both compared to the negative control (stool only) and stool incubated with bb0214. The count and cumulative abundance of sulphidogens exhibited a significant decrease in the faecal slurry treated with the 127-isolate subset, whereas no significant difference was observed when treated with bb0214. (Figure 24a and 24b).

[00494] Discussion

[00495] These results demonstrate that the 127-isolate subset of the BB265 complex consortium exhibits superior sulphide reduction in ulcerative colitis stool when compared to a strong sulphide consumer alone in the same context (Figure 15). The 127-isolate subset of the BB265 complex consortium concurrently reduces the number and cumulative relative abundance of sulphidogens while increasing the number of sulphide consumers in ulcerative colitis stool (Figure 24a and 24b), akin to the FMT-induction of remission observed in clinical samples, in both cases, leading to a reduction in sulphide (Figure 13eand 13d). This substantiates the notion that the microorganisms within the BB265 complex consortium engage in cooperative ecological interactions, effectively bolstering the reduction of sulphide in ulcerative colitis stool when contrasted with individual strong consumers in the same environment. Consequently, in this case, the utilisation of the 127-isolate subset of the BB265 complex consortium demonstrates a greater phenotype for sulphide reduction in ulcerative colitis than a strong consumer alone, whilst providing community complexity and diversity vital for health.

[00496] E.2.3.3 Reduction of aqueous sulphide in ulcerative colitis faecal samples by the

BB265 complex consortium compared to an individual strong sulphide consumer

[00497] Background

[00498] From previous experiments, we have determined that the 127-isolate subset of the BB265 complex consortium exhibits an elevated capacity for sulphide consumption when compared to individually phenotypically identified strong sulphide consumers. Consequently, it becomes imperative to expand our investigation to comprehend how the BB265 complex consortium, comprised of 143 genomically and phenotypically screened healthy gut bacteria, supports sulphide reduction in ulcerative colitis stool. This complex consortium displays extended taxonomic and metabolic diversity beyond that of the 127-isolate subset, potentially contributing to a wider range of beneficial functions within the human gut. Therefore, it becomes pivotal to study their collective contributions in the context of treating ulcerative colitis patients.

[00499] Methods

[00500] In-vitro assay

[00501] Cell-number normalised glycerol stocks (0.5% v/v inoculum) of the BB265 complex consortium were used to inoculate a 2 L bioreactor containing 1 L of sterile YCFA for 24 hours at 37 °C. The bioreactor harvest was withdrawn after 24 hours and prepared as 20 mL of 25% (v/v) glycerol stocks in 50 mL Falcon tubes by mixing 10 mL of harvest with 10 mL of 50% (v/v) glycerol. Glycerol stocks were frozen at -80 °C.

[00502] In this study, bb0214 Ruminococcus bicirculans, which showed 670 pM sulphide consumption in the consumer assay was selected as a strong consumer. Glycerol stock of bb0214 (50 pL) was inoculated into 20 mL of YCFA and incubated anaerobically at 37 °C for 24 hours.

[00503] The glycerol stock of the BB265 complex consortium bioreactor harvest (20 mL) was topped up to 50 mL with sterile pre-reduced PBS and immediately centrifuged at 12000 x g for 6 min. Concurrently, overnight grown bb0214 consumer was centrifuged at 12000 x g for 10 min. After decanting the supernatant, both pellets were washed with sterile 50 mL PBS three times. Pellets of the BB265 complex consortium and bb0214 were resuspended in 10 mL and 20 mL of Solution I, respectively, by vortexing.

[00504] Two ulcerative colitis stool samples (Patient A and C) were used in this study. A 5% (w/v) and 2.5% (w/v) faecal slurry of Patient A and C were prepared, respectively. Different concentrations of faecal slurry were prepared per donor to measure adequate sulphide; concentration was determined prior by assaying faecal slurries for sulphide production at various concentrations using the modified methylene blue assay. Herein, 7 g of stool sample from Patient A and 5 g of stool sample from Patient C were dissolved in 70 mL and 100 mL of pre-reduced Solution I, respectively. Each sample was vortexed for 5 min and pulsed briefly at 100 rpm to pelletise large particles. The supernatants were transferred to sterile 250 mL Schott bottles. Stool suspensions (2.5 mL) were transferred into sterile glass vials containing 2.5 mL of sterile Solution I preparing 5% and 2.5% (w/v) of final concentration of stool suspension of Patient A and C, respectively. The BB265 complex consortium, and bb0214 (100 pL) were inoculated into stool suspensions in 5 replicates. In addition, 100 pL of sterile medium was inoculated into 5 replicates for controls containing stool samples only. Samples were incubated anaerobically at 37 °C for 24 hours and the modified methylene blue assay performed for measuring endpoint sulphide as described above.

[00505] Statistical analysis

[00506] To identify significant differences in sulphide consumption between treatment groups (stool only, stool plus the BB265 complex consortium, stool plus bb0214 consumer) within patients a one-way Anova was performed.

[00507] Results

[00508] Post incubation, when incubated with the BB265 complex consortium, Patients A and C faecal slurries showed an average net reduction in sulphide of 10.5 pM (± 8.1 SD) and 12.7 pm (+ 3.6 SD), respectively. This was a significant reduction when compared to the negative control (stool only) and when co-cultured with bb0214 (Figure 16). No significant reduction in sulphide was observed for both patients when stool was co-cultured with the bb0214 consumer, with average net reduction of 1.0 pM (± 9.8 SD) and 1.7*1 O’ ¹⁴ pM (± 4.8 SD) sulphide for Patient A and C samples, respectively (Figure 16).

[00509] Discussion

[00510] These results have unveiled that BB265 exhibits a notably higher level of sulphide reduction compared to the strong individual consumer in both patient samples. This suggests that the microbial isolates within BB265 engage in cooperative interactions, enhancing sulphide-reducing pathways beyond what single sulphide consumers could achieve alone. [00511] E.2.3.4 Impact on the rate of hydrogen sulphide production in ulcerative colitis stool by the BB265 complex consortium and strongest consumers

[00512] Background

[00513] The modified methylene blue assay is a highly specific assay to detect total sulphides (H2S, HS and S ² ) in the aqueous phase of our bioreactor. This method allows endpoint data to be collected, when fermentation has run to completion and concentrations of sulphide between the aqueous phase and headspace have equilibrated. However, it does not allow real-time monitoring of the production of H2S in the bioreactor. This study aims to utilise commercial H ₂ S microsensors to monitor the production of H ₂ S in a closed bioreactor system in real-time whilst serving as a validation method for the results seen using the modified methylene blue assay. In addition, this study holds significance in elucidating the kinetics of H2S production. A comprehensive understanding of the rate of H2S production is crucial, as it allows us to examine the temporal release dynamics within the colon. Reducing the rate of H ₂ S production by this complex consortium emerges as a promising therapeutic approach for treating ulcerative colitis.

[00514] Methods

[00515] Calibration of Unisense Type II H ₂ S microsensor

[00516] The H ₂ S sensor(s) were connected to a fx-6 pA UniAmp amplifier and the SensorTrace Suite software was opened. The sensors were allowed a window of 2 hours minimum for pre-polarisation at 0 mV. This window of time was shortened in future uses based on the stability of the mV reading after 20 min. A solution of sterile, pure water and a standard pH buffer at pH 4 were purged with pure nitrogen gas for a minimum of 20 min to remove any oxygen dissolved in solution. A 200 mM solution of sodium sulphide was prepared in deoxygenated water by adding the required weight of sodium sulphide to the solution and very gently swirling with the lid on to prevent any oxygenation of the solution. Six 10 ml_ glass vials were purged with nitrogen to remove any oxygen and sealed with a butyl rubber stopper. Six 10 mL aliquots of the deoxygenated pH 4 solution were aliquoted into purged glass vials using a hypodermic needle through the rubber stopper to reduce mixing with air. 0, 1 and 2 mM sodium sulphide solutions were prepared in the deoxygenated pH 4 solution by adding the appropriate amount of dissolved 200 mM solution and gently inverting the vials. The hypodermic needle of the sensor was inserted through the butyl stopper and into the solution of the 0 mM calibration solution and a reading was taken when the signal became stable. This reading was inputted into the software with the corresponding H ₂ S concentration. This was repeated for the next highest concentration until all calibration points had been measured. The sensor was inserted into a sterile vial of pure water to remove any residual hydrogen sulphide. The millivolt (mV) signals corresponding to the respective concentrations of calibration points were then integrated into the curve, and the software was configured to create a standard curve, thereby preparing it for subsequent measurements.

[00517] Calibration of Unisense temperature sensor

[00518] The temperature sensor was connected to the fx-6 pA UniAmp amplifier and the SensorTrace Suite software was opened. The temperature sensor was placed in the known temperatures of 0°C, 22°C and 81 °C until the signal was stable. These temperatures were determined against a calibrated alcohol thermometer. The millivolt (mV) signals to the respective temperatures of calibration points were then integrated into the curve, and the software was configured to create a standard curve, thereby preparing it for subsequent measurements

[00519] Sensor Experiment

[00520] Individual consumers used in this study were bb0214 Ruminococcus bicirculans and bb0450 Clostridium paraputrificum. The glycerol stock of the BB265 complex consortium (20 mL) was immediately topped up to 50 mL with sterile pre-reduced PBS and then centrifuged for 12000 * g for 6 min. Concurrently, overnight grown sulphide consumers were centrifuged at 12000 ^x g for 10 min. After decanting the supernatants, all pellets were washed with sterile 50 mL PBS three times. The final consumer pellets and BB265 complex consortium were resuspended in 20 mL and 10 mL of sterile Solution I, respectively, and mixed via vortexing.

[00521] Stool samples from two ulcerative colitis patients (Patients A and C), were used in this experiment. Further, stool samples from screened healthy donors (Healthy Donor A and C) were used as negative controls in this study. Stool samples of 5% (w/v) for Patient A and Donor A and 2.5% (w/v) for Patient C and Donor C were prepared. Different concentrations of faecal slurry were prepared per donor to measure adequate sulphide; concentration was determined prior by assaying faecal slurries for sulphide production at various concentrations using the modified methylene blue assay. A 1 g and 0.5 g aliquot of Patient and Donor A and C stool were homogenised in 10 mL of Solution I, respectively. Each sample was vortexed for 5 min and briefly pulsed at 100 rpm to pelletise larger particles. Subsequently, 2.5 mL of supernatant from each sample was transferred into sterile glass vials containing 2.5 mL of Solution I, resulting in final stool suspension concentrations of 5% and 2.5% (w/v) for Patient and Donor A and C, respectively. Controls for H2S measurement were established by adding complex consortium and consumers in 100 pL aliquots. These prepared samples were subsequently used in the sensor experiments outlined below.

[00522] Using the sensors: The H2S needle-type sensors and temperature sensor were connected to the amperometry and temperature channels of the Fx-6 UniAmp, respectively. Subsequently, the sensors were initiated by scanning through the SensorTrace Logger software. Then, all sensors were securely placed inside a 37°C incubator and left for a few min until a stable signal was observed. Bottles containing samples were pierced with the H2S needle-type sensors until sensor tips were immersed in the stool suspension and incubated at 37°C. The recording time interval was adjusted to 300 seconds, and recording commenced while these samples incubated at 37°C for 24 hours. Following the incubation period, the recording was stopped, and the data was saved as an .xls file.

[00523] Results

[00524] In both cases, patient stool samples alone produced the most H2S. For the experiment run with Patient A and Donor A, the healthy donor produced the smallest amount of H ₂ S, with the bb0214 consumer outperforming the BB265 complex consortium in this regard (Figure 18 and 19). For the experiment with Patient C and Donor C, stool co-cultured with the bb0450 consumer produced the least amount of sulphide, followed by the healthy donor and then ulcerative colitis stool co-cultured with the BB265 complex consortium (Figure 18 and 19).

[00525] When comparing rate of production of H2S, both patient stool samples displayed a higher rate of H2S production compared to the ulcerative colitis samples incubated with the BB265 complex consortium (Figure 20 and 21 ). Patient A and C stools inoculated with BB265 had maximum H ₂ S production rate of - 30pM hr ¹ and -190 pM hr ¹ , which was lower than those stool samples alone, which had maximum H2S production rates of - 40pM hr ¹ and -250 pM hr ¹ , respectively. Both healthy donor samples had the lowest maximum H ₂ S production rates, indicating that healthy donors have lower rates of H ₂ S production than in ulcerative colitis patients. In terms of the H2S production rate with the consumer, bb0214 showed -43 pM hr ¹ with Patient A and bb0450 showed -200 pM hr ¹ with Patient C (Figure 20 and 21).

[00526] Discussion

[00527] These findings elucidate that stool samples obtained from individuals diagnosed with ulcerative colitis manifest a notably elevated rate of H ₂ S production. Importantly, the data demonstrates a concurrent reduction in both the rate and concentration of H2S production when the BB265 complex consortium is introduced, aligning with the outcomes of the modified methylene blue assay conducted on these specific patient samples alone and incubated with the BB265 complex consortium. The distinct behaviors observed in individual H ₂ S consumers within both patient stool samples confirm their varied phenotypic responses to H2S changes when these single isolates interact with the diverse bacterial populations present in ulcerative colitis patients. Consequently, the use of individual H ₂ S consumers may not consistently facilitate H ₂ S reduction in some ulcerative colitis patients, as their effectiveness appears to depend on the inter-variability between patient samples. In contrast, the BB265 complex consortium consistently demonstrates the same phenotypic response in both samples, underscoring its beneficial role in supporting H ₂ S reduction within the context of ulcerative colitis stool. These results highlight the pivotal role played by the BB265 complex consortium in the scope of ulcerative colitis treatment. The BB265 consortium exhibits the capacity to substantially mitigate the rate of H ₂ S production, a factor of paramount importance within the colon environment of ulcerative colitis patients. This reduction total and rate of H ₂ S holds significant therapeutic promise by effectively limiting the concentration of H ₂ S generated at any given moment, potentially offering substantial benefits to individuals affected with ulcerative colitis and also IBD.

G REDUCTION OF NITRITE PRODUCED BY ULCERATIVE COLITIS FAECAL COMMUNITIES WHEN CO-CULTURED WITH THE BB265 COMPLEX CONSORTIUM

[00528] Background

[00529] In conjunction with sulphide, nitric oxide (NO) has been implicated in the pathogenesis of ulcerative colitis. NO is a free radical produced endogenously by mammalian cells, such as colonocytes, that acts as a vasotransmitter with important physiological functions. In the context of ulcerative colitis, intraluminal NO has been observed to be increased, working alongside sulphide to inhibit P-oxidation to starve the colonocyte of cellular energy (Figure 1 ).

[00530] In the anaerobic environment of the colon, certain bacteria perform denitrification, a process that involves the stepwise reduction of nitrate (NO ₃ ‘) to nitrite (NO ₂ ~) and eventually to NO or other nitrogen oxides. This denitrification process is carried out by specific microbial populations, and the production of nitrite is a key intermediate step. It is plausible to assume that the majority of microbially-derived NO in stool samples originates from this anaerobic denitrification pathway.

[00531] Direct measurements of NO in stool are challenging due to its high reactivity and short half-life. As a result, measurements of NO ₂ ‘ are frequently used as a surrogate marker for NO. Nitrite is an intermediate product in the NO ₃ "-NO ₂ '-NO pathway, which is a common route for NO production in the gut. NO ₃ ~ is a stable and abundant molecule in the human diet, and it can be reduced to nitrite by certain commensal bacteria present in the gut. Subsequently, NO ₂ " can be further converted to NO under specific conditions, such as low oxygen levels, acidic pH, or in the presence of microbial enzymes like nitrite reductases. Moreover, it is important to note that NO ₂ ~ and NO exist in a dynamic equilibrium, with the conversion between the two influenced by environmental factors and enzymatic activities. Thus, higher levels of NO ₂ " in stool may indicate an increased potential for NO generation, further supporting the utility of nitrite measurement as a proxy for NO levels.

[00532] In this study, we aimed to investigate the role of NO in ulcerative colitis pathogenesis by assessing NO ₂ " levels in stool samples from patients with ulcerative colitis. Additionally, we sought to explore the impact of FMT on NO ₂ " levels in patients who achieved remission, hypothesising that modulation of the gut microbiota may alter NO ₂ ~ production. Furthermore, we conducted experiments where we co-cultured ulcerative colitis stool samples with the BB65 complex consortium to further validate the mechanism of action of BB265 on sulphide and NO modulation for the treatment of ulcerative colitis.

[00533] Methods

[00534] Sample selection

[00535] Patient samples were selected based on a positive sulphide phenotype observed in previous bioreactor experiments. Patient C was chosen due to the observation that sulphide concentration in the stool sample was reduced when incubated with BB265 complex consortium and an individual sulphide consumer. Patient D and E were selected as they showed clinical response and remission to FMT, and a significant reduction of sulphide concentrations in remission samples compared to baseline (active) samples.

[00536] Faecal slurry preparation

[00537] 10% (w/v) faecal slurries were prepared by adding 5 g of stool in 50 mL of Solution

I for Patient C, and 1 g of stool in 10 mL of half strength YCFA with sodium sulphite and Solution I for all baseline and remission samples of Patient D and E. The slurries were homogenised by vortexing for 5 min and centrifuged briefly up to 100 rpm to remove large particles. Subsequently, 2.5 mL of faecal slurry supernatant was transferred to gas chromatography (GC) vials containing either 2.5 mL of half strength YCFA with sodium sulfite or Solution I, resulting in a final concentration of 5% (w/v) faecal slurries.

[00538] Consumer isolate bb0214 Ruminococcus bicirculans was grown overnight in 20 mL YCFA broth. The glycerol stock of the BB265 complex consortium bioreactor harvest (20 mL) and bb0214 grown overnight were topped up to 50 mL with sterile pre-reduced PBS and immediately centrifuged at 12000 x g for 6 min. Samples were then washed three times with PBS as performed previously, and the BB265 complex consortium and bb0214 pellets resuspended in 10 and 20 mL Solution I by vortexing, respectively.

[00539] GC vials containing 5% faecal slurry of Patient C were spiked with 100 pL washed BB265 complex consortium and bb0214. Control samples were spiked with 100pL of Solution I only. Samples of Patient D and E were left unspiked to compare the difference in NO _Z ~ produced by the autochthonous mic’obial communities in baseline and remission samples.. Once inoculated, the GC vials were capped immediately and incubated anaerobically at 37 °C overnight. The experiment for Patient C was performed in triplicate whilst the experiment for Patient D and E was performed in duplicate due to limited sample weight.

[00540] Nitrite reduction test

[00541] A nitrite reduction test was performed using Microtest Nitrite 50 kit (Aquaspex). Post incubation, GC vials were removed from the anaerobic chamber and faecal slurries transferred from GC vials into Falcon tubes and centrifuged at 12,000 x g for 10 min. Then 1 mL of faecal slurry supernatant was transferred into a new Falcon tube containing 4 mL of RO water. To this Falcon tube, 5 drops of sulphuric acid were added, followed by 2 drops of indicator solution. The mixture was mixed by swirling each time 1 reagent was added. Finally, titration solution was added 5 pL at a time, swirled to mix until the colour of the mixture changed from red to pale blue. The volume of the titration stock solution used was recorded and the concentration of nitrite in the stool samples was calculated as follows:

[00542] 25 pL of Titration Stock Solution = 50 mg/L nitrite

[00543] Dilution factor = 5

[00544] Nitrite concentration (mg/L) = [(Volume of Titration Stock Solution x 50 mg/L)/25 pL] x 5

[00545] Results

[00546] Table 10 - Nitrite concentration (mg/L) in the stool samples of Patient C, D and E in different media types following incubation

[00547] Table 10 shows the concentrations of nitrite in the stool samples of Patient C, D and E after overnight incubation, with and without addition of the BB265 complex consortium and bb0214 consumer. A reduction of nitrite was observed in Patient C from 450 mg/L (+ 50 SD) to 366.67 mg/L (+ 28.87 SD) after incubation with the BB265 complex consortium. No reduction was observed for samples incubated with bb0214.

[00548] For patients who went into remission due to FMT treatment, stool samples incubated in half strength YCFA showed 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 the stool sample of Patient D incubated in Solution I.

[00549] Discussion

[00550] In this study, the inventors identified a significant reduction in NO ₂ ‘ concentration in stool samples from ulcerative colitis patients who had achieved remission following FMT (Table 10). This finding suggests a potential link between microbial alterations induced by FMT and the reduction of NO ₂ " production and subsequent production of NO. It is therefore plausible that FMT-induced shifts in the gut microbiota composition lead to changes in NO ₂ ‘- NO metabolism, thereby contributing to disease remission.

[00551] The inventors experiments involving co-culturing of ulcerative colitis stool samples with the BB265 complex consortium resulted in significantly lower NO ₂ “ levels in samples spiked with the BB265 complex consortium when compared to unspiked controls (Table 10). This outcome implies that the introduced microbial community had a suppressive effect on NO ₂ '-NO production during anaerobic fermentation. Moreover, the identification of decreased NO ₂ " levels in stool samples from ulcerative colitis patients parallels this trend, reinforcing the therapeutic potential of the BB265 complex consortium. This is further supported by the observation that taxa comprising the BB265 consortium were found to be in significantly higher number in stool metagenomes from patients that had achieved remission from FMT.

[00552] In conclusion, the inventors' findings highlight the therapeutic potential of the BB265 complex consortium for the treatment of ulcerative colitis by modulating colonic sulphide and NO. The parallel trends observed in the clinical FMT data and the BB265 complex consortium experiments provide compelling evidence for the validation of this mechanism of action specific to ulcerative colitis and also IBD.

BOLSTERING EFFECT OF FIBRE ON SULPHIDE MODULATION BY THE BB265 COMPLEX CONSORTIUM WHEN CO-CULTURED WITH ULCERATIVE COLITIS FAECAL SLURRIES

[00553] Background

[00554] The fermentation of fibre in the colon, particularly resistant starch and non-starch polysaccharides, takes preference over protein fermentation. This preference results in reduced release of sulphur amino acids, such as cysteine, into the colon, thereby diminishing sulphur amino acid fermentation and subsequently reducing sulphide production by the resident microbiota. Consequently, the inventors conducted tests on combinations of dietary fibres to enhance the efficacy of our live biotherapeutic product in treating Inflammatory Bowel Disease. These tests utilised unique phenotypic assays, as outlined above, to validate the effectiveness of the BB265 complex consortium. Furthermore, additional studies on the impact of these dietary fibres in terms of sulphide reduction could provide valuable insights for assessing dietary interventions aimed at rectifying the dysbiosis observed in ulcerative colitis.

[00555] Method

[00556] The cell-number normalised 127-isolate subset of the BB265 complex consortium was inoculated into 20 mL of YCFA and incubated anaerobically at 37 °C for 24 hours.

[00557] Three baseline (active) ulcerative colitis patient stool samples (Patient A, Patient B and Patient F) were used. Fibres used in this experiment were inulin, maltodextrin, starch, and FOS (fructo-oligosaccharides). Fecal samples (16% w/v) were prepared by dissolving 32 g stool into 100 mL of Solution I. Each sample was vortexed for 5 min and briefly pulsed at 100 rpm to pelletise larger particles. Subsequently, 2.5 mL of supernatant from each sample was transferred into sterile Hungate tubes containing 2.5 mL of Solution I with 2% of the respective fibre, resulting in an 8% (w/v) stool slurry, including 1 % (w/v) fibre.

[00558] The overnight grown 127-isolate subset of the BB265 complex consortium was centrifuged at 12000 x g for 10 min. After decanting the supernatant, pellets were washed with sterile 50 mL PBS three times. Final pellets were resuspended in 20 mL of sterile Solution I and vortexed to homogenise. The 127-isolate subset of the BB265 complex consortium (100 pL) was inoculated as samples into stool suspensions with and without fibres. Additionally, 100 pL of sterile medium was inoculated into tubes containing stool samples only, serving as controls. Subsequently, the tubes were incubated anaerobically at 37 °C for 24 hours, and an endpoint measurement of sulphide content was performed using the modified methylene blue assay, following the methodology employed in previous experiments.

[00559] Results

[00560] The data reveals that Patients A, B, and F had notably higher initial sulphide levels in their stools: 307.7 pM, 323.1 pM, and 350.8 pM, respectively. However, when the co-culture comprising the 127-isolate subset of the BB265 complex consortium was introduced, the sulphide levels in the stools of Patients A, B, and F decreased by 46.7 pM, 78.5 pM, and 1.1 pM, respectively. Remarkably, the dietary fibres exhibited substantial sulphide reduction capabilities. FOS, inulin, maltodextrin, and starch reduced sulphide levels in all three stool samples, ranging from 111.8 pM to 223.6 pM, 147.7 pM to 230.8 pM, 104.6 pM to 198.0 pM, and 143.6 pM to 213.9 pM, respectively. When these fibres were combined with the 127- isolate subset of BB265 complex consortium in ulcerative colitis stool, sulphide levels were altered to the ranges of 121.0 pM to 229.3 pM, 148.7 pM to 236.9 pM, 94.4 pM to 219.0 pM, and 142.6 pM to 213.9 pM, respectively (Figure 22).

[00561] Discussion

[00562] These results provide compelling evidence that dietary fibres, such as FOS, inulin, maltodextrin, and starch, make a significant contribution to the reduction of sulphide in ulcerative colitis stool samples. This confirms the role of dietary fibre in diminishing the fermentation of sulphur-containing proteins, subsequently leading to reduced sulphide production. Furthermore, the introduction of the 127-isolate subset of BB265 complex consortium in conjunction with these dietary fibres resulted in varying sulphide reduction, albeit to varying degrees depending on the specificity of stool sample and type of dietary fibre. This observation underscores how different dietary fibres can exert distinct influences on the microbiota, directing them toward diverse modes of carbohydrate fermentation.

[00563] Notably, the 127-isolate subset of the BB265 complex consortium, when supplemented with inulin, exhibited the ability to efficiently reduce high levels of sulphide in stool samples from all patients. This observation strongly suggests that the co-culture is proficient at promoting fibre fermentation over protein fermentation, particularly in the presence of inulin. In contrast, starch demonstrated lower sulphide consumption when combined with the 127-isolate subset, indicating its potentially limited fermentability by this consortium. Interestingly, FOS and maltodextrin exhibited varying interactions with the 127- isolate subset of BB265, resulting in fluctuations in the concentrations of reduced sulphide, depending on stool variations. This underscores the notion that dietary fibres have an evident impact on the complex consortium in terms of sulphide reduction in patients with ulcerative colitis and provides evidence for including fibre with the BB265 therapy to potentiate its ability to reduce sulphide levels.