Compositions Comprising Bacterial Species and Related Methods

Abstract

The present disclosure generally relates to bacterial strains of the genus Christensenella, such as the Christensenella sp. P152-H6d bacterial strain, and compositions comprising such bacterial strains. The disclosure further relates to methods of using such bacterial strains and compositions to prevent or treat disorders, such as inflammatory disorders, gastrointestinal disorders, metabolic disorders, and / or dysbiosis.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 893,142, filed August 28, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated by reference in its entirety. The ASCII copy was created on August 28, 2020, is named ASP-058WO_SL.txt, and is 3,693,554 bytes in size. [Background technology]

[0003] The gastrointestinal (GI) tract, as well as other organ systems, is a complex biological system that contains a community of many different organisms, including diverse bacterial strains. Hundreds of different species can form symbiotic communities in the GI tract and other organs of healthy individuals. Furthermore, the microorganisms present in the gut not only play a crucial role in digestive health but also affect the immune system. Disorders or imbalances in biological systems, such as the GI tract, can include changes in the types and numbers of bacteria in the gut, which can lead to or be indicators of ill health and / or the development of disease.

[0004] Members of the Christensenellaceae family have been characterized as highly heritable gut commensal bacteria that are associated with several beneficial health aspects. For example, Goodrich et al. reported a microbiome study across >1,000 fecal samples from 416 twin pairs, and observed that Christensenellaceae was the most highly heritable taxon and was significantly more abundant in subjects with a lean body mass index (BMI) (<25) compared with subjects with an obese BMI (>30). Goodrich et al., Cell 159:789-799 (2014). Goodrich et al. further demonstrated that Christensenellaceae is associated with reduced weight gain in germ-free mice inoculated with fecal samples from lean and obese humans, and that adding Christensenella minuta, a member of the Christensenellaceae family, to donor feces reduced increased adiposity in recipient mice, suggesting that Christensenellaceae promotes a lean host phenotype. Goodrich et al., 2014. Zhou et al. demonstrated that fecal microbiota transplantation (FMT) is effective in attenuating high-fat diet (HED)-induced steatohepatitis in mice and that attenuation is associated with increased abundance of Christensenellaceae. Zhou et al., Sci Rep. 7(1):1529 (2017). Enrichment of members of the Christensenellaceae family has also been observed in fecal and urine samples from healthy versus pediatric and young adult inflammatory bowel disease (IBD) patients (Papa et al., PLoS ONE 7, e39242 (2012)).

[0005] Morotomi et al. first reported the Christensenellaceae as a distinct division within the Cloistridiales, along with the first genus Christensenella and the first isolated species, Christensenella minuta. Morotomi et al., Int J Syst Evol Microbiol. 62(Pt 1):144-149 (2012). Other reported isolated members of the Christensenella genus include C. massiliensis (Ndongo et al., New Microbes New Infect. 12:69-70 (2016)) and C. timonensis (Ndongo et al., New Microbes New Infect. 13:32-33 (2016)). Summary of the Invention [Problem to be solved by the invention]

[0006] Given the growing evidence for the role of the Christenseneraceae family in maintaining beneficial health conditions, such as lean body mass, low inflammation, and a balanced microbiota, there is a need to identify additional members of the Christenseneraceae family and the Christensenera genus, particularly those that exhibit potentially beneficial anti-inflammatory properties, such as short-chain fatty acid production and / or anti-inflammatory cytokine production, and have the ability to treat disorders, such as inflammatory disorders (e.g., IBD) and metabolic disorders (e.g., obesity, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH)). [Means for solving the problem]

[0007] Provided herein are compositions, e.g., pharmaceutical compositions, containing species or strains of the genus Christensenella, such as the species or strain referred to herein as Christensenella sp. P152-H6d. The terms Christensenella sp. P152-H6d, Christensenella P152-H6d, P152-H6d, Christensenella ASMB, and Christensenella ASMB P152-H6d are used interchangeably herein. Unless otherwise indicated, it is understood that these terms can refer not only to species but also to strains of the species. For example, Christensenella sp. P152-H6d can refer not only to the species Christensenella sp. P152-H6d, but also to the strain type of the species, Christensenella sp. P152-H6d (e.g., the strain deposited under accession number DSM33237). The species Christensenella sp. P152-H6d may alternatively refer to Christensenalla californii.

[0008] In one aspect, provided herein is a composition comprising a bacterial strain of the genus Christensenella, wherein the bacterial strain comprises a 16s rRNA gene sequence having at least about 98% sequence identity to the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the composition further comprises an excipient, diluent, and / or carrier. In some embodiments, the composition or the bacterial strain in the composition is lyophilized, freeze dried, or spray dried.

[0009] In some embodiments, the Christensenella bacterial strain can increase the secretion of CCL-18 and / or IL-10 by human cells, such as THP-1 macrophages, monocyte-derived dendritic cells (moDCs), or peripheral blood mononuclear cells (PBMCs) in vitro; for example, the Christensenella bacterial strain increases the secretion of CCL-18 by human THP-1 macrophages when co-cultured with the human THP-1 macrophages. In some embodiments, the Christensenella bacterial strain comprises a 16s rRNA gene sequence having at least about 98.5%, 98.65%, 99%, or 99.5% sequence identity to the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the Christensenella bacterial strain comprises the 16s rRNA gene sequence of SEQ ID NO: 1. In some embodiments, the Christensenella bacterial strain shares at least 70% DNA-DNA hybridization with Christensenella sp. P152-H6d deposited under accession number DSM 33237. In some embodiments, the Christensenella bacterial strain comprises a nucleotide sequence having at least about 70% identity to any one of SEQ ID NOs: 2-28. In some embodiments, the Christensenella bacterial strain comprises a genome having at least 95% average nucleotide identity (ANI) with the genome of Christensenella sp. P152-H6d deposited under accession number DSM 33237. In some embodiments, the Christensenella bacterial strain comprises a genome having at least 96.5% average nucleotide identity (ANI) and at least 60% alignment fraction (AF) with the genome of Christensenella sp. P152-H6d deposited under accession number DSM 33237. In some embodiments, the Christensenella bacterial strain is Christensenella sp. P152-H6d, deposited under accession number DSM33237.

[0010] In some embodiments, the Christensenella bacterial strain does not sporulate. In some embodiments, the Christensenella bacterial strain of the composition is viable. In some embodiments, the bacterial strain is capable of at least partially colonizing the intestine of a human subject. In some embodiments, the composition is suitable for oral delivery to a subject. In some embodiments, the composition comprising the Christensenella bacterial strain is formulated as an enteric formulation. In some embodiments, the enteric formulation is formulated as a capsule, tablet, caplet, pill, troche, lozenge, powder, or granule. In some embodiments, the composition is formulated as a suppository, suspension, emulsion, or gel. In some embodiments, the composition comprises at least 1 x 10 3 The composition comprises a bacterial strain of up to 100 CFUs. In some embodiments, the composition comprises a therapeutically effective amount of the bacterial strain sufficient to prevent or treat the disorder when administered to a subject in need thereof. In some embodiments, the disorder is selected from the group consisting of inflammatory disorders, gastrointestinal disorders, inflammatory bowel disease, cancer, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, insulin deficiency, insulin resistance-related disorders, insulin sensitivity, impaired glucose tolerance, prediabetes, diabetes, high body mass index (BMI), excess fat, obesity, overweight, cardiovascular disease, atherosclerosis, hyperlipidemia, hyperglycemia, dyslipidemia, and hypertension. In some embodiments, the gastrointestinal disorder is selected from the group consisting of ulcerative colitis, Crohn's disease, and irritable bowel syndrome.

[0011] In some embodiments, the composition comprises an excipient selected from the group consisting of a filler, a binder, a disintegrant, and any combination thereof. In some embodiments, the excipient is selected from the group consisting of cellulose, polyvinylpyrrolidone, silicon dioxide, stearyl fumarate or a pharmaceutically acceptable salt thereof, and any combination thereof. In some embodiments, the composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of fructooligosaccharides, trehalose, and combinations thereof. In some embodiments, the fructooligosaccharide is Raftilose® (a fructooligosaccharide derived from inulin). In some embodiments, the composition is suitable for bolus administration or bolus release. In some embodiments, the composition comprises a Christensenella bacterial strain and at least one or more additional bacterial strains.

[0012] In another aspect, provided herein is a bacterial strain of the genus Christensenella, wherein the bacterial strain comprises a 16s rRNA gene sequence having at least about 98% sequence identity to the polynucleotide sequence of SEQ ID NO:1.

[0013] In some embodiments, the Christensenella bacterial strain is capable of increasing the secretion of CCL-18 and / or IL-10 by human cells, such as THP-1 macrophages, monocyte-derived dendritic cells (moDCs), or peripheral blood mononuclear cells (PBMCs) in vitro; for example, the Christensenella bacterial strain increases the secretion of CCL-18 by human THP-1 macrophages when co-cultured with the human THP-1 macrophages. In some embodiments, the Christensenella bacterial strain comprises a 16s rRNA gene sequence having at least about 98.5%, 98.65%, 99%, or 99.5% sequence identity to the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the Christensenella bacterial strain comprises the 16s rRNA gene sequence of SEQ ID NO: 1. In some embodiments, the Christensenella bacterial strain shares at least 70% DNA-DNA hybridization with Christensenella sp. P152-H6d deposited under accession number DSM 33237. In some embodiments, the Christensenella bacterial strain comprises a nucleotide sequence having at least about 70% identity to any one of SEQ ID NOs: 2-28. In some embodiments, the Christensenella bacterial strain comprises a genome having at least 95% average nucleotide identity (ANI) with the genome of Christensenella sp. P152-H6d deposited under accession number DSM 33237. In some embodiments, the Christensenella bacterial strain comprises a genome having at least 96.5% average nucleotide identity (ANI) and at least 60% alignment fraction (AF) with the genome of Christensenella sp. P152-H6d deposited under accession number DSM 33237. In some embodiments, the Christensenella bacterial strain is Christensenella sp. P152-H6d, deposited under accession number DSM33237. In some embodiments, the Christensenella bacterial strain is viable. In some embodiments, the bacterial strain is capable of at least partially colonizing the intestine of a human subject.

[0014] In another aspect, provided herein is a food product comprising a Christensenella bacterial strain described herein.

[0015] In another aspect, provided herein are methods for preventing or treating a disorder in a subject in need thereof, such as an inflammatory disorder, a gastrointestinal disorder, an inflammatory bowel disease, cancer, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, insulin deficiency, insulin resistance-related disorders, insulin sensitivity, impaired glucose tolerance, prediabetes, diabetes, high body mass index (BMI), excess fat, obesity, overweight, cardiovascular disease, atherosclerosis, hyperlipidemia, hyperglycemia, dyslipidemia, and hypertension, comprising administering to the subject a therapeutically effective amount of a Kristensenella bacterial strain described herein or a composition comprising a Kristensenella bacterial strain described herein. In some embodiments, the gastrointestinal disorder is ulcerative colitis, Crohn's disease, or irritable bowel syndrome. Also provided herein are methods for treating a dysbiosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Kristensenella bacterial strain described herein or a composition comprising a Kristensenella bacterial strain described herein. Also provided herein are methods of modifying the gut microbiome in a subject (e.g., a subject in need thereof), comprising administering to the subject a therapeutically effective amount of a Christensenerella bacterial strain described herein or a composition comprising a Christensenerella bacterial strain described herein. Also provided herein are methods of treating a skin disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Christensenerella bacterial strain described herein or a composition comprising a Christensenerella bacterial strain described herein. In some embodiments, the skin disorder is selected from the group consisting of psoriasis, eczema, dermatitis (e.g., eczematous dermatitis, atopic and seborrheic dermatitis, allergic or irritant contact dermatitis, fissure eczema, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis, radiation dermatitis, and stasis dermatitis), and acne. In some embodiments of the methods provided herein, the method further comprises administering a prebiotic to the subject. In some embodiments, the subject is selected from the group consisting of a human, a companion animal, and a livestock animal.

[0016] The present disclosure can be more fully understood with reference to the following figures. The present invention also relates to the following: [Item 1] 1. A composition comprising: A bacterial strain of the genus Christensenella, comprising a 16s rRNA gene sequence having at least about 98% sequence identity to the polynucleotide sequence of SEQ ID NO: 1; and Excipients, diluents, and / or carriers wherein the bacterial strain is freeze-dried, lyophilized, or spray-dried. [Item 2] 2. The composition of item 1, wherein the bacterial strain comprises a 16s rRNA gene sequence having at least about 98.5%, 99%, or 99.5% sequence identity to the polynucleotide sequence of SEQ ID NO:1. [Item 3] 3. The composition of claim 1 or 2, wherein the bacterial strain comprises a 16s rRNA gene sequence of SEQ ID NO: 1. [Item 4] 4. The composition of any one of items 1 to 3, wherein the bacterial strain shares at least 70% DNA-DNA hybridization with Christensenella sp. P152-H6d deposited under accession number DSM33237. [Item 5] 5. The composition of any one of items 1 to 4, wherein the bacterial strain comprises a nucleotide sequence having at least about 70% identity to any one of SEQ ID NOs: 2 to 28. [Item 6] 6. The composition of any one of items 1 to 5, wherein the bacterial strain comprises a genome having at least 95% average nucleotide identity (ANI) with the genome of Christensenella sp. P152-H6d deposited under accession number DSM33237. [Item 7] 7. The composition of any one of items 1 to 6, wherein the bacterial strain comprises a genome having an average nucleotide identity (ANI) of at least 96.5% and an alignment fraction (AF) of at least 60% with the genome of Christensenella sp. P152-H6d deposited under accession number DSM33237. [Item 8] 8. The composition of any one of items 1 to 7, wherein the bacterial strain is Christensenella sp. P152-H6d deposited under accession number DSM33237. [Item 9] 9. The composition of any one of items 1 to 8, wherein the bacterial strain is capable of increasing the production of an anti-inflammatory gene product by human cells. [Item 10] 10. The composition of item 9, wherein the anti-inflammatory gene product is selected from the group consisting of CCL-18, IL-10, IL-1RA, and MCP-1. [Item 11] 11. The composition of any one of items 1 to 10, wherein the bacterial strain is capable of reducing or attenuating the production of inflammatory gene products by human cells. [Item 12] 12. The composition of item 11, wherein the inflammatory gene product is selected from the group consisting of IL12-p40, IL-1β, IL-17A, IL-21, IFN-γ and TNF-α. [Item 13] 13. The composition of any one of items 9 to 12, wherein the human cells are selected from the group consisting of THP-1 macrophages, moDCs and PBMCs. [Item 14] 14. The composition of any one of items 1 to 13, wherein the bacterial strain is capable of reducing or attenuating the production of one or more serum biomarkers selected from the group consisting of lipocalin-2 / NGAL, serum amyloid A (SAA), and granulocyte colony-stimulating factor (G-CSF) in a cell, tissue, or subject. [Item 15] 15. The composition of any one of items 1 to 14, further comprising one or more additional bacterial strains. [Item 16] 16. The composition of item 15, wherein the one or more additional bacterial strains comprise a strain of Anaerostipes caccae. [Item 17] 17. The composition according to item 16, wherein the Anaerostipes cacae strain is the Anaerostipes cacae strain P127-A10a deposited under accession number DSM 33531. [Item 18] A pharmaceutical unit comprising: A bacterial strain mixture comprising a bacterial strain of the genus Christensenella, wherein the bacterial strain comprises a 16s rRNA gene sequence having at least about 98% sequence identity to the polynucleotide sequence of SEQ ID NO:1, wherein each bacterial strain in the bacterial strain mixture is in lyophilized form and the pharmaceutical unit is at least 1 x 10 8 a bacterial strain mixture having viable bacterial organisms; and pharmaceutically acceptable excipients A pharmaceutical unit comprising: [Item 19] 19. The pharmaceutical unit according to item 18, wherein the mixture of bacterial strains further comprises a bacterial strain of the species Anaerostipes cacae. [Item 20] 20. The composition or unit according to any one of items 1 to 19, formulated as an enteric formulation. [Item 21] 21. The composition or unit according to item 20, wherein the enteric formulation is formulated as a capsule, tablet, caplet, pill, troche, lozenge, powder, or granule. [Item 22] 22. The composition or unit according to any one of items 1 to 21, formulated as a suppository, suspension, emulsion or gel. [Item 23] At least 1 x 10 3 23. The composition or unit according to any one of items 1 to 22, comprising a bacterial strain of CFU. [Item 24] 24. The composition or unit according to any one of items 1 to 23, comprising a therapeutically effective amount of the bacterial strain sufficient to prevent or treat the disorder when administered to a subject in need thereof. [Item 25] 25. The composition or unit according to item 24, wherein the disorder is selected from the group consisting of inflammatory disorders, gastrointestinal disorders, inflammatory bowel disease, cancer, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, insulin deficiency, insulin resistance-related disorders, insulin sensitivity, impaired glucose tolerance, prediabetes, diabetes, high body mass index (BMI), excess fat, obesity, overweight, cardiovascular disease, atherosclerosis, hyperlipidemia, hyperglycemia, dyslipidemia and hypertension. [Item 26] 26. The composition or unit according to item 25, wherein the gastrointestinal disorder is selected from the group consisting of ulcerative colitis, Crohn's disease, and irritable bowel syndrome. [Item 27] 27. The composition or unit of any one of the preceding items, wherein the excipient is selected from the group consisting of a filler, a binder, a disintegrant, and any combination thereof. [Item 28] 27. The composition or unit according to any one of the preceding items, wherein the excipient is selected from the group consisting of cellulose, polyvinylpyrrolidone, silicon dioxide, stearyl fumarate or a pharmaceutically acceptable salt thereof, and any combination thereof. [Item 29] 29. The composition or unit according to any one of items 1 to 28, wherein the composition further comprises a cryoprotectant. [Item 30] 30. The composition or unit of item 29, wherein the cryoprotectant is selected from the group consisting of fructooligosaccharides, trehalose, and combinations thereof. [Item 31] 31. The composition or unit according to item 30, wherein the fructooligosaccharide is Raftilose®. [Item 32] 32. A composition or unit according to any one of items 1 to 31, which is suitable for bolus administration or bolus release. [Item 33] 33. The composition or unit according to any one of items 1 to 32, wherein the bacterial strain is capable of at least partially colonizing the intestine of a human subject. [Item 34] 34. The composition or unit according to any one of items 1 to 33, which is suitable for oral delivery to a subject. [Item 35] 35. The composition or unit according to any one of items 1 to 34, wherein the bacterial strain is viable. [Item 36] 36. The composition or unit according to any one of items 1 to 35, comprising at least one or more additional bacterial strains. [Item 37] 37. The composition or unit of any one of items 1 to 36, wherein the composition loses up to 3 logs of colony forming units (cfu) upon storage at 4°C for 6 months. [Item 38] 38. A food product comprising a composition or unit according to any one of items 1 to 37. [Item 39] 38. A method of treating dysbiosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition or unit according to any one of items 1 to 37. [Item 40] 38. A method of modifying the gut microbiome in a subject, comprising administering to the subject a therapeutically effective amount of the composition or unit of any one of items 1 to 37. [Item 41] 38. A method for treating a gastrointestinal disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition or unit according to any one of items 1 to 37. [Item 42] 42. The method of item 41, wherein the gastrointestinal disorder is ulcerative colitis (UC), Crohn's disease, or irritable bowel syndrome. [Item 43] 38. A method of treating an inflammatory disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition or unit of any one of items 1 to 37. [Item 44] 38. A method of treating a skin disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition or unit of any one of items 1 to 37. [Item 45] 45. The method of claim 44, wherein the skin disorder is selected from the group consisting of psoriasis, eczema, dermatitis (e.g., eczematous dermatitis, atopic and seborrheic dermatitis, allergic or irritant contact dermatitis, fissure eczema, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis, radiation dermatitis, and stasis dermatitis), and acne. [Item 46] 38. A method for treating non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, insulin deficiency, insulin resistance-related disorders, insulin sensitivity, impaired glucose tolerance, prediabetes, diabetes, high body mass index (BMI), excess fat, obesity, overweight, cardiovascular disease, atherosclerosis, hyperlipidemia, hyperglycemia, dyslipidemia, or hypertension in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition or unit of any one of items 1 to 37. [Item 47] 47. The method of any one of items 39 to 46, further comprising administering a prebiotic to the subject. [Item 48] 48. The method of any one of items 39 to 47, wherein the subject is selected from the group consisting of humans, companion animals, and livestock animals. [Brief explanation of the drawings]

[0017] [Figure 1]Figure 1 shows (A) a maximum likelihood (ML) phylogenetic tree constructed using the 16S rDNA sequences of the nearest neighbors of Christensenella sp. P152-H6d (P152-H6d) along with other genera. This phylogenetic tree demonstrates that Christensenella is a monophyletic genus with clear taxonomic delineation among its member species, with each individual Christensenella species clearly clustering in a single clade. (B) BLASTn search results for the Christensenella P152-H6d (C. sp. P152-H6d) 16S rDNA gene sequence (SEQ ID NO: 1). The closest match across the entire P152-H6d 16S rRNA sequence length of 1442 bp is Christensenella timonensis, with 97.85% sequence identity. [Figure 2] Figure 2 depicts an ANI-based phylogenetic tree characterizing Christensenella sp. P152-H6d, other Christensenella species, and other genera. FastANI was used to calculate paired ANI values ​​between the genomes of the depicted strains. The paired ANI values ​​were used to create a distance matrix in Phylip format. Phylogenetic relationships between these genomes were inferred using the neighbor-joining method using the R package BionNJ. Branch lengths are proportional to ANI distances. [Figure 3] Figure 3 depicts a scanning electron micrograph (18.64k magnification) of Christensenella sp. P152-H6d bacterial cells. [Figure 4] Figure 4 depicts the short-chain fatty acid (SCFA) production profile of Christensenella sp. P152-H6d (Christensenella ASMB P152-H6d) cultured for 72 hours. The levels of butyrate, propionate, and acetate in the batch culture supernatant were analyzed by HPLC (ABPDU, Berkeley, CA). Uninoculated YCFAC medium was used as a negative control. [Figure 5]Figure 5 depicts the effect of Christensenella sp. P152-H6d on CCL-18 production in human THP-1 macrophages. (A) THP-1 macrophages were co-cultured with PBS alone, PBS plus E. coli LPS, Christensenella sp. P152-H6d, and an immunomodulatory bacterial strain control (known to induce proinflammatory cytokines), and supernatants were collected and assayed for CCL-18 production. Each test substance was evaluated in quadruplicate, and results represent at least two independent experiments. *p value ≤ 0.05 (one-way ANOVA). (B) CCL-18 production in THP-1 macrophages is increased in a dose-dependent manner by Christensenella sp. P152-H6d. *p value ≤ 0.05 (individual Student's t-test). [Figure 6] Figure 6 depicts the effect of Christensenella sp. P152-H6d on (A) IL12-p40 and (B) TNF-α production in human THP-1 macrophages. THP-1 macrophages were co-cultured with PBS alone, PBS plus E. coli LPS, Christensenella sp. P152-H6d, and an immunomodulatory bacterial strain control (known to induce proinflammatory cytokines), and supernatants were collected and assayed for IL12-p40 and TNF-α production. Each test article was evaluated in quadruplicate, and results represent at least two independent experiments. p-value ≤ 0.05 (one-way ANOVA). [Figure 7] Figure 7 depicts the effect of Christensenella sp. P152-H6d on (A) & (C) IL-10, (B) IL-1RA, and (D) MCP1 production in (A) & (B) human monocyte-derived dendritic cells (MoDCs) and (C) & (D) human peripheral blood mononuclear cells (PBMCs). Human cells were cultured with PBS alone and co-cultured with Christensenella sp. P152-H6d, respectively, and supernatants were collected and assayed for cytokine production. Each test article was evaluated in quadruplicate, and results are representative of at least two independent experiments. [Figure 8]Figure 8 depicts the effect of Christensenella sp. P152-H6d (C.P152-H6d) in a human THP-1 macrophage in vitro cytokine assay in the presence of Crohn's disease (CD) fecal flora. THP-1 macrophage supernatants were collected at the end of the assay, and IL-12p40 concentrations in the culture supernatants were plotted for PBS control, CD fecal flora, Christensenella sp. P152-H6d alone, and CD fecal flora supplemented with the indicated v / v amounts of Christensenella sp. P152-H6d. Each test article was evaluated in quadruplicate, and results are representative of at least two independent experiments. [Figure 9] Figure 9 depicts the effect of Christensenella sp. P152-H6d on dorsal skin thickness in an imiquimod (IMQ)-induced psoriasis-like skin inflammation mouse model. *<0.05 by two-way ANOVA with Dunnett's post-hoc analysis. [Figure 10] Figure 10 depicts the effect of Christensenella sp. P152-H6d on skin redness (erythema) in an oxazolone-induced atopic dermatitis mouse model. Erythema clinical scores provided: (A) over time, and (B) as AUC. 2-way ANOVA with Dunnett's post-hoc analysis yielded **<0.005, ****<0.0001. [Figure 11] Figure 11 depicts the effect of Christensenella sp. P152-H6d on dorsal skin desquamation in an oxazolone-induced atopic dermatitis mouse model. Clinical scores of dorsal skin desquamation provided: (A) over time, and (B) as AUC. ****<0.0001 by two-way ANOVA with Dunnett's post-hoc analysis. [Figure 12] FIG. 12 depicts the effect of Christensenella sp. P152-H6d on body weight in a DSS-induced colitis mouse model. [Figure 13]Figure 13 depicts the effect of administration of Christensenella sp. P152-H6d (C.P152-H6d) and anti-IL12p40 antibody (anti-IL12-p40) on the production of colonic cytokines: (A) IL-1β, (B) IL-17α, and (C) TNF-α, respectively, in a mouse model of DSS-induced colitis. The right panel depicts the correlation between the levels of colonic cytokines produced and the % weight loss induced by DSS administration. [Figure 14] Figure 14 depicts the effect of Christensenella sp. P152-H6d on body weight in a DSS-induced colitis mouse model. *p<0.05, two-way ANOVA with Benjamini, Krieger, and Yekutieli method for multiple comparisons to limit false discovery rate, analyzed by GraphPad Prism. [Figure 15] Figure 15 depicts the levels of IBD disease activity biomarkers in plasma on day 14 of DSS-induced colitis following administration of vehicle, Christensenella sp. P152-H6d (C.P152-H6d), and anti-IL12-p40 antibody, respectively. (A) Granulocyte colony-stimulating factor (G-CSF), (B) Lipocalin-2 / NGAL, and (C) Serum amyloid A (SAA) were assessed by ELISA, and levels were normalized to ml of plasma analyzed. (D) Histology scores for the distal colon following administration of vehicle, Christensenella sp. P152-H6d, and anti-IL12-p40 antibody, respectively. A scoring system of 1 to 5 was assigned to each sample in the categories of subacute inflammation, colonic gland damage / loss, erosion, thickening, and submucosal edema. The histology score shown here is the sum of the scores for all five categories per mouse. (A)-(C): *p<.05, multiple comparisons to control for false discovery rate, one-way ANOVA with Benjamini, Krieger, and Yekutieli method, analyzed with GraphPad Prism. (D): *p<.05, multiple comparisons to control for false discovery rate, Kruskal-Wallis test with Benjamini, Krieger, and Yekutieli method, analyzed with GraphPad Prism. [Figure 16]Figure 16 depicts the effect of Christensenella sp. P152-H6d on body weight in a Citrobacter rodentium-induced colitis mouse model. % body weight change is presented: (A) over time, and (B) as AUC. [Figure 17] Figure 17 depicts the effect of Christensenella sp. P152-H6d on (A) colon length and (B) colon weight in a Citrobacter rodentium-induced colitis mouse model. (C) Colon weight / length ratio. LLOD = lower limit of detection. [Figure 18] Figure 18 depicts the effect of Christensenella sp. P152-H6d on the production of (A) IFN-γ, (B) IL-1β, (C) IL-21, and (D) TNF-α in colon tissue in a Citrobacter rodentium-induced colitis mouse model. LLOD = lower limit of detection. [Figure 19] Figure 19 depicts the levels of lipocalin-2 / NGAL in plasma on day 14 of Citrobacter-induced colitis following administration of vehicle, Christensenella sp. P152-H6d, and a control bacterial strain (bacterium Y). Levels were assessed by ELISA and normalized to ml of plasma analyzed. [Figure 20] Figure 20 depicts the effects of Christensenella sp. P152-H6d, Anaerostipes caccae, and the combination of Christensenella sp. P152-H6d and Anaerostipes caccae on (A) body weight and (B) histology scores for the distal colon in a TNBS-induced colitis mouse model. A scoring system of 1 to 5 was assigned to each specimen in the categories of subacute inflammation, colonic gland damage / loss, erosion, thickening, and submucosal edema. The histology score shown here is the sum of the scores for all five categories per mouse. *p<0.05, multiple comparisons to reduce false discovery rate, Kruskal-Wallis test with Benjamini, Krieger, and Yekutieli methods, analyzed by GraphPad Prism. [Figure 21]Figure 21 depicts the effects of Christensenella sp. P152-H6d (C.P152-H6d), Anaerostipes cacae (A. cacae), and the combination of Christensenella sp. P152-H6d and Anaerostipes cacae on the production of (A) IL-1β, (B) IL-12p40, and (C) TNF-α in human THP-1 macrophages. THP-1 macrophage supernatants were collected at the end of the assay, and cytokine concentrations in the culture supernatants were assessed by ELISA. Each test article was evaluated in quadruplicate, and results are representative of at least two independent experiments. [Figure 22] Figure 22 depicts PCR amplicons from a PCR-based screen for additional strains of Christensenella sp. P152-H6d from healthy human fecal samples. (A) Amplicons generated from Christensenella sp. P152-H6d-specific primers (Ch_relA_AA_2) and primers spanning homologous regions of three different members of the Christensenella genus (Ch_Fred). Ch = Christensenella sp. P152-H6d, Cmi = C. minuta, Cma = C. massiliensis, and Ct = C. timonensis. (B) PCR amplicons confirming the isolation of four additional strains of Christensenella sp. P152-H6d (strains P235-A1a (lane A1), P235-A3a (lane A3), P237-A7a (lane A7), and P237-B12a (lane B12). ChA = Christensenella sp. P152-H6d. DETAILED DESCRIPTION OF THE INVENTION

[0018] I. Bacterial strains In one aspect, provided herein is a species or strain of the genus Christensenella, referred to herein as Christensenella sp. P152-H6d, as well as compositions, e.g., pharmaceutical compositions, comprising Christensenella sp. P152-H6d. The terms Christensenella sp. P152-H6d, Christensenella P152-H6d, P152-H6d, Christensenella ASMB, and Christensenella ASMB P152-H6d are used interchangeably herein. Unless otherwise indicated, it is understood that these terms can refer to not only species but also strains of the species. For example, Christensenella sp. P152-H6d can refer not only to the species Christensenella sp. P152-H6d, but also to the strain type of the species, Christensenella sp. P152-H6d (e.g., the strain deposited under accession number DSM33237). The species Christensenella sp. P152-H6d may alternatively refer to Christensenella californii.

[0019] As used herein, the term "species" refers to a taxonomic entity conventionally defined by genomic sequence and phenotypic characteristics. A "strain" is a specific instance of a species that has been isolated and purified according to conventional microbiological techniques. The bacterial species and / or strains described herein include living and / or viable species and / or strains. In some embodiments, the bacterial species and / or strains described herein include vegetative and non-sporulating forms of bacteria. One of skill in the art will recognize that the genus Christensenera may undergo taxonomic reorganization. Thus, contemplated Christensenera species are intended to include Christensenera species that have been renamed and / or reclassified as well as species that may subsequently be renamed and / or reclassified.

[0020] In some embodiments, the bacterial strain Christensenella sp. P152-H6d contains a 16S rRNA gene sequence with a certain percent identity to a reference sequence. rRNA, 16S rDNA, 16S rRNA, 16S, 18S, 18S rRNA, and 18S rDNA refer to nucleic acids that are components of or encode ribosomal components. Ribosomes have two subunits, called the small subunit (SSU) and the large subunit (LSU). Ribosomal RNA genes (rDNAs) and their complementary RNA sequences are variable but sufficiently conserved to allow molecular comparisons between organisms, and are therefore widely used to determine evolutionary relationships between organisms. In embodiments, the 16S rDNA sequence of the 30S SSU may be used for molecular-based taxonomic assignment of prokaryotes. For example, 16S sequences are sometimes used for phylogenetic reconstruction because, although generally highly conserved, they contain specific hypervariable regions with sufficient nucleotide diversity to differentiate most bacterial genera and species. 16S rDNA sequence data are used to provide taxonomic classification, but closely related bacterial strains classified within the same genus and species may exhibit different biological phenotypes.

[0021] Thus, bacterial strains of the Christensenella sp. P152-H6d species provided herein include strains that comprise a 16s rRNA gene sequence having a certain percent identity to SEQ ID NO: 1. In some embodiments, the bacterial strain is a strain of the genus Christensenella that comprises a 16s rRNA gene sequence that has at least 97.90% sequence identity to the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the bacterial strain comprises a 16S rRNA gene sequence having at least about 97.95%, about 98.00%, about 98.05%, about 98.1%, about 98.15%, about 98.2%, about 98.25%, about 98.3%, about 98.35%, about 98.4%, about 98.45%, about 98.5%, about 98.55%, about 98.6%, about 98.65%, about 98.7%, about 98.75%, about 98.80%, about 98.85%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9% identity to the polynucleotide sequence of SEQ ID NO:1. In certain embodiments, the bacterial strain comprises a 16s rRNA gene sequence identical to SEQ ID NO: 1. In some embodiments, the above-referenced sequence identity is over at least about 70% of SEQ ID NO: 1. In other embodiments, the above-referenced sequence identity is over at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO: 1.

[0022] In some embodiments, the bacterial strain of Christensenella sp. P152-H6d comprises a genomic sequence (e.g., the entire genomic sequence or a fragment or contig thereof) having a certain percent identity to one or more of SEQ ID NOs: 2-28. In some embodiments, the Christensenella sp. P152-H6d strain comprises the polynucleotide sequence of any one of SEQ ID NOs: 2-28, or a nucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the polynucleotide sequence of any one of SEQ ID NOs: 2-28. In some embodiments, the genome of Christensenella sp. strain P152-H6d comprises each of the polynucleotide sequences set forth in SEQ ID NOs: 2-28, or each polynucleotide sequence having at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to each of the polynucleotide sequences set forth in SEQ ID NOs: 2-28.

[0023] In some embodiments, the bacterial strain Christensenella sp. P152-H6d comprises a whole genome sequence having at least about 70% identity across at least 70% of its genome to the sum of all genomic contigs represented by SEQ ID NOs:2-28. In some embodiments, the whole genome sequence has at least about 75%, 80%, 85%, 90%, 95%, or more than 95% identity to the sum of all genomic contigs represented by SEQ ID NOs:2-28. In some embodiments, the above-referenced sequence identity spans at least 75%, 80%, 85%, 90%, 95%, or more than 95% of the whole genome sequence of the bacterial strain. In some embodiments, the bacterial strain Christensenella sp. P152-H6d comprises a whole genome sequence comprising coding regions having at least about 70% identity across at least 70% of the whole coding region of its genome to coding regions within the sum of all genomic contigs represented by SEQ ID NOs:2-28. In some embodiments, the coding regions within the entire genome sequence have at least about 75%, 80%, 85%, 90%, 95%, or more than 95% identity to the coding regions within the sum of all genome contigs represented by SEQ ID NOs: 2-28. In some embodiments, the above-referenced sequence identity spans at least 75%, 80%, 85%, 90%, 95%, or more than 95% of the coding regions within the entire genome sequence of the bacterial strain.

[0024] In some embodiments, the bacterial strains of the Christensenella sp. P152-H6d species provided herein comprise a relA gene sequence having a particular percent identity to SEQ ID NO: 33. In some embodiments, the bacterial strain is a strain of the genus Christensenella that comprises a relA gene sequence having at least about 85% sequence identity to the polynucleotide sequence of SEQ ID NO: 33. In some embodiments, the bacterial strain comprises a relA gene sequence having at least about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9% identity to the polynucleotide sequence of SEQ ID NO: 33. In certain embodiments, the bacterial strain comprises a relA gene sequence identical to SEQ ID NO: 33. In some embodiments, the above-referenced sequence identity is over at least about 70% of SEQ ID NO: 33. In other embodiments, the above-referenced sequence identity is over at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO: 33.

[0025] The identity of a bacterial strain of the species Christensenella sp. P152-H6d can be determined, for example, by sequencing the 16s rRNA gene sequence or genome sequence (e.g., the entire genome sequence or a fragment or contig thereof) of the bacterial strain using any sequencing method known in the art, including, for example, Sanger sequencing. One example of a sequencing technology useful for identifying Christensenella sp. P152-H6d strains is the Illumina platform. The Illumina platform is based on fold-back PCR and amplification of DNA on a solid surface (e.g., a flow cell) using anchored primers (e.g., capture oligonucleotides). For sequencing by the Illumina platform, bacterial DNA is fragmented and adapters are added to the ends of the fragments. DNA fragments are attached to the surface of the flow cell channel by capturing oligonucleotides capable of hybridizing to the adapter ends of the fragments. The DNA fragments are then extended and bridge-amplified. After multiple cycles of solid-phase amplification and subsequent denaturation, an array of millions of spatially immobilized nucleic acid clusters or colonies of single-stranded nucleic acids is generated. Each cluster may contain approximately hundreds to thousands of copies of the same template single-stranded DNA molecule. The Illumina platform uses a sequencing-by-synthesis method in which sequencing nucleotides containing a detectable label (e.g., a fluorophore) are sequentially added to the free 3' hydroxyl group. After incorporation of the nucleotide, the label can be excited using laser light of a wavelength specific to the labeled nucleotide. An image is captured, and the identity of the nucleotide base is recorded. These steps can be repeated to sequence the remaining bases. Sequencing by this technique is described, for example, in U.S. Patent Application Publication Nos. 2011 / 0009278, 2007 / 0014362, 2006 / 0024681, and 2006 / 0292611, and U.S. Patent Nos. 7,960,120, 7,835,871, 7,232,656, and 7,115,200.Another example of a sequencing technology useful for identifying strains of Christensenella sp. P152-H6d is the SOLiD technology by Applied Biosystems, Life Technologies Corporation (Carlsbad, Calif.). In SOLiD sequencing, bacterial DNA may be cleaved into fragments, and adapters may be attached to the ends of the fragments to generate a library. A clonal bead population may be prepared in a microreactor containing templates, PCR reaction components, beads, and primers. After PCR, the templates may be denatured, and bead enrichment may be performed to isolate beads with extended primers. Templates on selected beads undergo 3' modification to enable covalent attachment to a slide. Sequences may be determined by sequential hybridization and ligation with several primers. A set of four fluorescently labeled dinucleotide probes competes for ligation to the sequencing primers. Multiple cycles of ligation, detection, and cleavage are performed, with the number of cycles determining the final read length. Another example of a sequencing technique useful for identifying strains of Christensenella sp. P152-H6d is Ion Torrent sequencing. In this technique, bacterial DNA is cut into fragments, and then oligonucleotide adapters are ligated to the ends of the fragments. The fragments are then attached to a surface, and each base in the fragment is released during base incorporation. + This can be resolved by measuring ions, a technique described, for example, in U.S. Published Patent Application Nos. 2009 / 0026082, 2009 / 0127589, 2010 / 0035252, 2010 / 0137143, and 2010 / 0188073.

[0026] Once the polynucleotide sequence of a bacterial strain (e.g., 16s rRNA gene sequence or genome sequence) is obtained, sequence identity with the polynucleotide sequence of Christensenella sp. P152-H6d can be determined in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, BLAT (BLAST-like alignment tool), ALIGN, or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis, using the algorithm employed by the Blastp, blastn, blastx, tblastn, and tblastx programs (Karlin et al., PROC. NATL. ACAD. SCI. USA 87:2264-2268 (1990); Altschul, J. MOL. EVOL. 36, 290-300 (1993); Altschul et al., NUCLEIC ACIDS RES. 25:3389-3402 (1997), which are incorporated herein by reference), is adapted for sequence similarity searching. For a discussion of basic issues in searching sequence databases, see Altschul et al., NATURE, GENETICS 6:119-129 (1994), which is incorporated herein by reference in its entirety. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Search parameters for histograms, classification descriptions, alignments, expectation (i.e., the statistical significance threshold for reporting matches against database sequences), cutoffs, matrices, and filters are at default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al. (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, incorporated herein by reference in its entirety).Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty), R=10 (gap extension penalty), wink=1 (generate word hits every wink positions along the query), and gapw=16 (sets the window width within which gapped alignments are generated). Equivalent Blastp parameter settings would be Q=9, R=2, wink=1, and gapw=32. Searches may be performed using NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g., -G, cost for opening a gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost for extending a gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for nucleotide mismatch [integer]: default = -3; -r, reward for nucleotide mismatch [integer]: default = 1; -e, expectation value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, dropoff (X) for BLAST extension in bits: default = 20 for blastn / 7 otherwise; -X, X dropoff value for gapped alignments (in bits): default = 15 for all programs except for blastn; and -Z, final X dropoff value for gapped alignments (in bits): 50 for blastn, 25 otherwise). Sequence-to-sequence best-match comparisons, available in the GCG package version 10.0, use DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty); equivalent settings for protein comparisons are GAP=8 and LEN=2.

[0027] In certain embodiments, the bacterial strain Christensenella sp. P152-H6d provided herein is Christensenella sp. P152-H6d, strain P152-H6d. A deposit of Christensenella sp. P152-H6d, strain P152-H6d, was made on August 12, 2019, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Brunswick, Germany). This deposit was assigned the accession number DSM33237. The 16s rRNA gene sequence of Christensenella sp. P152-H6d, strain P152-H6d, is provided herein as SEQ ID NO: 1, and the genome sequence of Christensenella sp. P152-H6d, strain P152-H6d, is provided herein as SEQ ID NOs: 2-28. In other specific embodiments, the bacterial strain of Christensenella sp. P152-H6d provided herein is a Christensenella sp. strain selected from the group consisting of P235-A1a, P235-A3a, P237-A7a, and P237-B12a. Isolation of these specific strains is described in Example 7, below.

[0028] Additional bacterial strains of the Christensenella sp. P152-H6d species provided herein include Christensenella strains having a DNA-DNA hybridization (DDH) value of about 70% or greater with Christensenella sp. P152-H6d, strain P152-H6d. In certain embodiments, Christensenella sp. P152-H6d strain is a strain having a DNA-DNA hybridization value of greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99%, or any range between any of these values, with Christensenella sp. P152-H6d, strain P152-H6d. Any method for determining DNA-DNA hybridization values ​​known in the art may be used to assess the degree of DNA-DNA hybridization, including, but not limited to, the spectrophotometric method for determining percent renaturation described by De Ley et al. (J Biochem 12 133-142 (1970)), slight modification of hybridization temperature (Gavini et al., Ecology in Health and Disease 12 40-45 (2001)), and the method described by Grimont et al., Curr Microbiol 4, 325-330 (1980) and Rossello-Mora, Molecular Identification, Systematics and Population Structure of Prokaryotes pp.23-50 (2006). In some embodiments, the degree of DNA-DNA hybridization is determined by digital DNA-DNA hybridization (dDDH) analysis, for example, using a genome-to-genome distance calculation online tool (see Meier-Kolthoff et al., BMC Bioinformatics 14:60 (2013)). In certain embodiments, Christensenella sp. P152-H6d is a strain having a DDH or dDDH value of about 70% or greater than that of Christensenella sp. P152-H6d, strain P152-H6d.In some embodiments, the DDH or dDDH value is greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of Christensenella sp. P152-H6d, strain P152-H6d, or any range between any of these values.

[0029] Additional bacterial strains of the species Christensenella sp. P152-H6d provided herein include Christensenella strains having an average nucleotide identity (ANI) of 95% or greater with Christensenella sp. P152-H6d, strain P152-H6d. In some embodiments, the ANI is about 95% or greater, about 95.5% or greater, about 96% or greater, about 96.5% or greater, about 97% or greater, about 97.5% or greater, about 98% or greater, about 98.5% or greater, about 99% or greater, about 99.5% or greater, or 100% with Christensenella sp. P152-H6d, strain P152-H6d, or any range between any of these values. The average nucleotide identity (ANI) of shared genes between two strains is a robust means for comparing the genetic relatedness between strains, and it is known that an ANI value of about 95% corresponds to the 70% DNA-DNA hybridization criterion for defining species. See, for example, Konstantinidis and Tiedje, Proc Natl Acad Sci USA, 102(7):2567-72 (2005), Goris et al., Int J Syst Evol Microbiol. 57(Pt 1):81-91 (2007), and Jain et al., Nat Commun. 9(1):5114 (2018). In some embodiments, the ANI between two bacterial genomes is calculated from a pairwise comparison of all sequences shared between any two strains and can be determined, for example, using any of several publicly available ANI tools, including, but not limited to, OrthoANI using usearch (Yoon et al., Antonie van Leeuwenhoek 110:1281-1286 (2017)), ANI Calculator, JSpecies (Richter and Rossello-Mora, Proc Natl Acad Sci USA 106:19126-19131 (2009)), and JSpeciesWS (Richter et al., Bioinformatics 32:929-931 (2016)). Other methods for determining the ANI of two genomes are known in the art.See, e.g., Konstantinidis, KT and Tiedje, JM, Proc. Natl. Acad. Sci. USA, 102:2567-2572 (2005); Varghese et al., Nucleic Acids Research, 43(14):6761-6771 (2015); and Jain et al., Nat Commun. 9(1):5114 (2018). In certain embodiments, the ANI between two bacterial genomes can be determined using alignment-based methods, e.g., by averaging the nucleotide identities of orthologous genes identified as bidirectional best hits (BBHs). Protein-coding genes of a first genome (genome A) and a second genome (genome B) are compared at the nucleotide level using a similarity search tool, such as NSimScan (Novichkov et al., Bioinformatics 32(15):2380-23811 (2016). The results are then filtered to retain only BBHs that show at least 70% sequence identity over at least 70% of the length of the shorter sequence in each BBH pair. The ANI of genome A to genome B is defined as the sum of the alignment length multiplied by the percent identity for all BBHs divided by the sum of the BBH gene lengths. In another specific embodiment, the ANI between two bacterial genomes may be determined using alignment-free methods, such as FastANI, which uses alignment-free approximate sequence mapping to assess genomic relatedness. Jain et al., Nat Commun. 9(1):5114 (2018). FastANI has been demonstrated to reveal clear genetic discontinuities between species, with 99.8% of all 8 billion genome pairs analyzed conforming to intraspecific ANI values ​​of >95% and interspecific ANI values ​​of <83%. Thus, in some embodiments, bacterial strains with an average nucleotide identity (ANI) of 95% or greater with the genome of Christensenella sp. P152-H6d are identified as bacterial strains of the species Christensenella sp. P152-H6d.

[0030] Additional bacterial strains of the Christensenella sp. P152-H6d species provided herein include Christensenella strains having an alignment fraction (AF) with Christensenella sp. P152-H6d, strain P152-H6d, of 60% or greater. In some embodiments, the AF is about 65% or greater, about 70% or greater, about 75% or greater, about 80% or greater, about 85% or greater, about 90% or greater, about 95% or greater, or 100% with Christensenella sp. P152-H6d, strain P152-H6d, or any range between any of these values. In some embodiments, the AF is calculated by dividing the sum of the lengths of all BBH genes by the sum of the lengths of all genes in genome A. This calculation is performed separately from genome A to genome B and from genome B to genome A.

[0031] In certain embodiments, Christensenella sp. strain P152-H6d comprises a genome that has about 95% or more ANI and 60% or more AF with the genome of Christensenella sp. P152-H6d, strain P152-H6d. In another specific embodiment, Christensenella sp. strain P152-H6d comprises a genome that has about 96.5% or more ANI and 60% or more AF with the genome of Christensenella sp. P152-H6d, strain P152-H6d.

[0032] Additional bacterial strains of the Christensenella sp. P152-H6d species provided herein include Christensenella strains having genomic characteristics identical or nearly identical to Christensenella sp. P152-H6d, strain P152-H6d. Such genomic characteristics include, for example, genome size, G+C content, number of coding sequences, and number of tRNAs. In some embodiments, Christensenella sp. P152-H6d strains comprise genomes between about 2.75 and about 2.85 megabases (Mb) in size. In some embodiments, Christensenella sp. P152-H6d strains comprise genomes between about 2.80 and about 2.85 megabases (Mb). In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome approximately 2.75, 2.76, 2.77, 2.78, 2.79, 2.80, 2.81, 2.82, 2.83, 2.84, or approximately 2.85 Mb in size. In certain embodiments, the Christensenella sp. P152-H6d strain comprises a genome approximately 2.82 Mb in size. In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome having a G+C content of between about 48% and about 50%. In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome having a G+C content of between about 48.5% and about 49.5%. In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome with a G+C content of about 48.6%, 48.7%, 48.8%, 48.9%, 49.0%, 49.1%, 49.2%, 49.3%, 49.4%, or about 49.5%. In certain embodiments, the Christensenella sp. P152-H6d strain comprises a genome with a G+C content of about 48.91%. In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome comprising about 2600-2800 coding sequences. In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome comprising about 2650-2750 coding sequences. In some embodiments, the Christensenella sp. strain P152-H6d comprises a genome comprising coding sequences of about 2650, 2655, 2660, 2665, 2670, 2675, 2680, 2685, 2690, 2695, 2700, 2705, 2710, 2715, 2720, 2725, 2730, 2735, 2740, 2745 or about 2750.In certain embodiments, the Christensenella sp. P152-H6d strain comprises a genome comprising about 2671 coding sequences. In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome comprising about 35-50 tRNA sequences. In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome comprising about 37-45 tRNA sequences. In some embodiments, the Christensenella sp. P152-H6d strain comprises a genome comprising about 37, 38, 39, 40, 41, 42, 43, 44, or 45 tRNAs. In certain embodiments, the Christensenella sp. P152-H6d strain comprises a genome comprising about 42 tRNAs.

[0033] Additional bacterial strains of Christensenella sp. strain P152-H6d provided herein include Christensenella strains that exhibit a pattern identical or nearly identical to Christensenella sp. P152-H6d, strain P152-H6d, when analyzed by, for example, DNA fingerprinting techniques. Any DNA fingerprinting technique known in the art may be used to identify strains of Christensenella sp. P152-H6d. DNA fingerprinting techniques include, but are not limited to, pulsed-field gel electrophoresis (PFGE), ribotyping, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), amplified ribosomal DNA restriction analysis (ARDRA), rep-PCR including repetitive extragenic palindrome-PCR (REP-PCR) (repetitive element primed PCR that targets naturally occurring highly conserved repetitive DNA sequences present in multiple copies within the genome), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), BOX-PCR (derived from boxA elements), (GTG)5-PCR, triplicate arbitrarily primed PCR (TAP-PCR), multilocus sequence analysis (MLSA), multilocus sequence typing (MLST), repetitive sequence polymorphism analysis (MLVA), and DNA microarray-based genotyping techniques.

[0034] Additional bacterial strains of Christensenella sp. P152-H6d provided herein include Christensenella strains that exhibit phenotypic similarity to Christensenella sp. P152-H6d, strain P152-H6d. Phenotypic similarity can be based on, for example, cell shape and size, colony morphology (e.g., plate colony size, color, and odor), Gram staining, biochemical tests, pH and temperature optima, sugar fermentation, metabolic capacity (e.g., catalase and / or oxidase status), chemotaxonomic analysis (e.g., polar lipid and lipoquinone composition; see Tindall et al., Int J Syst Evol Microbiol 58, 1737-1745 (2008)), and / or fatty acid methyl ester (FAME) analysis. In some embodiments, the bacterial strain of Christensenella sp. P152-H6d is catalase-positive. In some embodiments, the bacterial strain of Christensenella sp. P152-H6d is oxidase negative. In some embodiments, the bacterial strain of Christensenella sp. P152-H6d is catalase positive and oxidase negative.

[0035] In some embodiments, the bacterial strain of Christensenella sp. P152-H6d is capable of fermenting at least one carbon source selected from the group consisting of glucose (e.g., α-D-glucose), arabinose (e.g., L-arabinose), ribose (e.g., D-ribose), and cyclodextrin (e.g., α-cyclodextrin). In some embodiments, the bacterial strain of Christensenella sp. P152-H6d is capable of fermenting each of glucose (e.g., α-D-glucose), arabinose (e.g., L-arabinose), ribose (e.g., D-ribose), and cyclodextrin (e.g., α-cyclodextrin). In some embodiments, the bacterial strain of Christensenella sp. P152-H6d is capable of producing fructose (e.g., D-fructose), glucosamine (e.g., N-acetyl-D-glucosamine, D-glucosamine), galactose (e.g., D-galactose), mannose (e.g., D-mannose), pectin, rhamnose (e.g., D-rhamnose), trehalose (e.g., D-trehalose), sorbitol (e.g., D-sorbitol), psicose (e.g., D-psicose), zinc The yeast is unable to ferment or substantially ferment at least one carbon source selected from the group consisting of luciitol, malitol, palatinose, sorbose (e.g., L-sorbose), tagatose (e.g., D-tagatose), turanose, glucosaminitol (e.g., N-acetyl-D-glucosaminitol), butyric acid (e.g., β-hydroxybutyric acid), maltose, lactose (e.g., α-D-lactose), sucrose, and cellobiose (e.g., D-cellobiose).In some embodiments, the bacterial strain of Christensenella sp. P152-H6d is capable of producing fructose (e.g., D-fructose), glucosamine (e.g., N-acetyl-D-glucosamine, D-glucosamine), galactose (e.g., D-galactose), mannose (e.g., D-mannose), pectin, rhamnose (e.g., D-rhamnose), trehalose (e.g., D-trehalose), sorbitol (e.g., D-sorbitol), psicose (e.g., D-psicose), dulcitol, malitol, palatinose, sorbose (e.g., L-sorbose), tagatose (e.g., D-tagatose), turanose, glucosaminitol (e.g., N-acetyl-D-glucosaminitol), butyric acid (e.g., β-hydroxybutyric acid), maltose, lactose (e.g., α-D-lactose), sucrose, and cellobiose (e.g., D-cellobiose) cannot be fermented or substantially fermented.

[0036] In some embodiments, the bacterial strain of Christensenella sp. P152-H6d increases or is capable of increasing production of one or more short-chain fatty acids (SCFAs). In some embodiments, the bacterial strain of Christensenella sp. P152-H6d produces or is capable of producing one or more short-chain fatty acids (SCFAs). In some embodiments, the SCFA is butyric acid. In some embodiments, the SCFA is acetic acid. In particular embodiments, the bacterial strain of Christensenella sp. P152-H6d produces both butyric acid and acetic acid.

[0037] In some embodiments, the bacterial strain of Christensenella sp. P152-H6d increases or is capable of increasing the production of at least one anti-inflammatory gene, such as an anti-inflammatory cytokine or chemokine, in a cell, tissue, or subject. Exemplary anti-inflammatory gene products include CCL-18, IL-1Ra, IL-4, IL-6, IL-10, IL-11, IL-13, MCP-1, and TGF-β. For example, in some embodiments, the bacterial strain of Christensenella sp. P152-H6d increases or is capable of increasing the production of IL-10 and / or CCL-18 in a cell, tissue, or subject. In some embodiments, the increased production of anti-inflammatory gene products, such as IL-10 and / or CCL-18, occurs in human cells, such as THP-1 macrophages or monocytes, or PBMCs. For example, contacting a human cell, such as a THP-1 macrophage or PBMC, with Christensenella sp. P152-H6d, e.g., by co-culturing the human cell with Christensenella sp. P152-H6d, can increase the production of IL-10 and / or CCL-18 in the cell by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 750%, at least about 800%, at least about 900%, at least about 1000%, at least about 1200%, at least about 1400%, at least about 1500%, at least about 1600%, at least about 1800%, at least about 2000%, at least about 2500%, at least about 3000%, at least about 3500%, at least about 4000%, at least about 4500%, at least about 5000%, at least about 6500%, at least about 7500%, at least about 10000%, at least about 25000%, at least about 35000%, at least about 45000%, at least about 50000%, at least about 65000%, at least about 75000%, at least about 10000%, at least about 25000%, at least about 35000%, at least about 45000%, at least about 50000%, at least about 50000%, at least about 65000%, at least about 75000%, at least about 100000%, at least about 15000 400%, at least about 500%, at least about 750%, at least about 1000%, about 10% to about 20%, about 10% to about 50%, about 10% to about 100%, about 10% to about 200%, about 10% to about 500%, about 10% to about 1000%, about 20% to about 50%, about 20% to about 100%, about 20% to about 200%, about 20% to about 5 00%, about 20% to about 1000%, about 50% to about 100%, about 50% to about 200%, about 50% to about 500%, about 50% to about 1000%, about 100% to about 200%, about 100% to about 500%, about 100% to about 1000%, about 200% to about 500%, about 200% to about 1000%, or about 500% to about 1000%. In some embodiments, contacting of the human cell with Christensenella sp. P152-H6d occurs in vitro.In another embodiment, contacting the human cell with Christensenella sp. P152-H6d occurs in vivo.

[0038] In some embodiments, the Christensenella sp. P152-H6d bacterial strain is capable of reducing or attenuating or reducing or attenuating the production of at least one pro-inflammatory gene, such as a pro-inflammatory cytokine or chemokine, in a cell, tissue, or subject. In some embodiments, the bacterial strain is capable of reducing or attenuating or reducing or attenuating the production of at least one pro-inflammatory gene, such as a pro-inflammatory cytokine or chemokine, in a cell, tissue, or subject, e.g., in the presence of a pro-inflammatory stimulus. Exemplary proinflammatory gene products include IL-1-β, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-17, IL-21, IL-22, IL-23, IL-27, IFN, CCL-2, CCL-3, CCL-5, CCL-20, CXCL-5, CXCL-10, CXCL-12, CXCL-13, IFN-γ, and TNF-α. For example, in some embodiments, the bacterial strain of Christensenella sp. P152-H6d is capable of reducing or attenuating, or reducing or attenuating, the production of IL-12, such as the IL-12 subunit p40, in a cell, tissue, or subject. In some embodiments, the bacterial strain of Christensenella sp. P152-H6d is capable of reducing or attenuating, or reducing or attenuating, the production of TNF-α in a cell, tissue, or subject. In some embodiments, the decreased or attenuated production of anti-inflammatory gene products, eg, IL-12 and / or TNF-α, occurs in human cells, eg, THP-1 macrophages or monocytes, moDCs, or PBMCs.For example, contacting human cells, such as THP-1 macrophages or PBMCs, with Christensenella sp. P152-H6d, e.g., by co-culturing human cells with Christensenella sp. P152-H6d, reduces or attenuates IL-12 production in the cells by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, about 10% to about 20%, about 10% to about 50%, about 10% to about 100%, about 20% to about 50%, about 20% to about 100%, or about 50% to about 100%, compared to cells (e.g., cells of the same cell type) that have not been contacted, e.g., not co-cultured, with Christensenella sp. P152-H6d. In some embodiments, contacting human cells, e.g., THP-1 macrophages or PBMCs, with Christensenella sp. P152-H6d, e.g., by co-culturing human cells with Christensenella sp. P152-H6d, reduces or attenuates TNF-α production in the cells by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, about 10% to about 20%, about 10% to about 50%, about 10% to about 100%, about 20% to about 50%, about 20% to about 100%, or about 50% to about 100%, compared to cells (e.g., cells of the same cell type) that have not been contacted, e.g., not co-cultured, with Christensenella sp. P152-H6d. In some embodiments, contacting human cells with Christensenella sp. P152-H6d occurs in vitro. In another embodiment, contacting the human cell with Christensenella sp. P152-H6d occurs in vivo.

[0039] The contemplated bacterial strain or mixture of bacterial strains may be capable of reducing or attenuating the production of one or more biomarkers (e.g., serum or stool biomarkers) of an inflammatory condition, such as inflammatory bowel disease (IBD), in a cell, tissue, or subject. For example, lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL) or siderocalin, is a potent bacteriostatic protein stored in neutrophil granules and released at sites of inflammation. High expression of LCN2 by intestinal epithelial cells has been demonstrated in colon biopsies from inflamed areas of IBD patients (Nielsen et al., Gut, 38:414-420 (1996)), and LCN2 has been reported to be one of the 10 most up-regulated genes in both active ulcerative colitis and Crohn's disease (Ostvik et al., Clin, Exp Immunol. 173:502-511 (2013)). Ostvik reported that LCN2 protein is found in both epithelial cells and infiltrating neutrophils, but LCN2 mRNA synthesis occurs only in epithelial cells, indicating that excessive de novo synthesis of LCN2 in IBD is localized to the colonic epithelium. Serum levels of LCN2 are a reliable biomarker of disease activity in UC, demonstrating a higher sensitivity in distinguishing active disease from disease in remission than CRP or white blood cell count (Stallhofer et al., Inflamm. Bowel Dis 21 (10):2327-2340 (2015)). LCN2 / NGAL levels can be assessed in cells, tissues, or subjects contacted with a target bacterial strain or mixture of bacterial strains by measuring the expression and / or concentration of the LCN2 / NGAL gene product in samples, such as plasma, serum, stool, and / or tissues (e.g., colonic tissue), using any method known in the art, including qPCR, ELISA, immunohistochemistry, etc. Other IBD biomarkers that may be reduced or attenuated by the bacterial strains described herein include serum amyloid A protein (SAA). SAA expression is associated with an inflamed colon in IBD patients, and systemic SAA in serum promotes the differentiation of pathogenic Th17 cells (Lee et al., Cell 180, 79-91 (2020)).Other IBD biomarkers that may be reduced or attenuated by the bacterial strains described herein include granulocyte colony-stimulating factor (G-CSF). Margarita et al. evaluated 27 protein biomarkers, including serum cytokines, chemokines, and growth factors, in IBD patients with different endoscopic activity and found that patients with endoscopically active disease exhibited higher serum G-CSF levels (P=0.04) (Medicine. (2019) 98:e17208).

[0040] The Christensenella sp. P152-H6d bacterial strain provided herein can be characterized by its effect on gene product production, such as IL-12 or CCL-18 production, in human cells, such as THP-1 monocytes or macrophages or PBMCs. It is understood that the expressed gene product may have both pro-inflammatory and / or anti-inflammatory activity. Gene product production, such as IL-12 or CCL-18 production, in THP-1 macrophages may be assayed, for example, as follows: THP-1 human macrophages are generated by culturing the THP-1 human monocytic cell line with phorbol 12-myristate 13-acetate (PMA) for 24 hours, followed by culturing with IL-4 and IL-13 (Genin et al., BMC Cancer 15:577 (2015)). The bacterial strain is cultured with THP-1 macrophages in the presence of lipopolysaccharide (LPS) for 24 hours. The production of the gene product is assessed by measuring the concentration of the gene product, e.g., IL-12 or CCL-18, in the cell culture supernatant by ELISA. Gene product production may be assessed as described in Sudhakaran et al., Genes Nutr., 8(6):637-48. Gene product production, e.g., IL-10, IL-12, or CCL-18 production, in PBMCs may be assayed, for example, as follows: Primary PBMCs are isolated from a donor's blood sample using a Percoll gradient (Sim et al., J. Vis. Exp. (112), e54128 (2016)). The bacterial strain is cultured with PBMCs for 24 hours. The production of the gene product is assessed by measuring the concentration of the gene product, e.g., IL-10, IL-12, or CCL-18, in the cell culture supernatant by ELISA.

[0041] Also provided herein are methods for isolating and / or purifying the Christensenella species bacterial strain described herein (Christensenella sp. P152-H6d, i.e., Christensenella californii). In some embodiments, the Christensenella species strain described herein is isolated and / or purified from a biological sample from a mammalian donor. In some embodiments, the mammalian donor is a human, e.g., a healthy human donor. In other embodiments, the mammalian donor is a non-human animal. In some embodiments, the biological sample is any biological sample known in the art to harbor viable microorganisms, such as stool, saliva, blood, skin, intestine, nose, etc. In certain embodiments, the Christensenella species strain described herein is isolated and / or purified from the stool or intestine of a healthy human donor. In some embodiments, the methods for isolating and / or purifying bacterial strains of Christensenella sp. described herein include ex vivo isolating and / or purifying one or more microorganisms from donor biological material (e.g., intestine or stool) and confirming the identity of the one or more microorganisms as strains of Christensenella sp. P152-H6d using methods known in the art for such identification and / or methods described herein. In some embodiments, the identity of the one or more microorganisms as strains of Christensenella sp. P152-H6d is confirmed by genetic or genomic means. For example, as demonstrated in Example 7 below, PCR is used to amplify a region of the genome of the bacterial strain that has high homology to a gene or gene fragment of Christensenella sp. P152-H6d (e.g., a nucleotide sequence selected from any of SEQ ID NOS: 2-28). In some embodiments, the gene is the 16S rRNA gene of Christensenella sp. P152-H6d (SEQ ID NO: 1). In some embodiments, the gene is the relA gene of Christensenella sp. P152-H6d (SEQ ID NO: 33). In other embodiments, a genome sequence (e.g., a partial or full genome sequence) can be used to confirm the identity of one or more microorganisms as a strain of Christensenella sp. P152-H6d, for example, by average nucleotide identity (ANI) at a threshold of >95% ANI.In some embodiments, the method comprises purifying the strain from donor material. In some embodiments, the method further comprises culturing the strain in monoculture.

[0042] Also provided herein are strains of the Anaerostipes caccae species, such as the strain designated herein as Anaerostipes caccae strain P127-A10a, and compositions, e.g., pharmaceutical compositions, comprising such strains.

[0043] The bacterial strains of the species Anaerostipes cacae provided herein include strains containing a 16s rRNA gene sequence having a certain percent identity to SEQ ID NO:45. In some embodiments, the bacterial strain comprises a 16S rRNA gene sequence having at least about 98.00%, about 98.05%, about 98.1%, about 98.15%, about 98.2%, about 98.25%, about 98.3%, about 98.35%, about 98.4%, about 98.45%, about 98.5%, about 98.55%, about 98.6%, about 98.65%, about 98.7%, about 98.75%, about 98.80%, about 98.85%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9% identity to the polynucleotide sequence of SEQ ID NO:45. In certain embodiments, the bacterial strain comprises a 16s rRNA gene sequence identical to SEQ ID NO: 45. In some embodiments, the above-referenced sequence identity is over at least about 70% of SEQ ID NO: 45. In other embodiments, the above-referenced sequence identity is over at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO: 45.

[0044] In certain embodiments, the Anaerostipes cacae bacterial strain provided herein is Anaerostipes cacae strain P127-A10a. A deposit of Anaerostipes cacae P127-A10a was made on March 6, 2020, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Brunswick, Germany). This deposit was assigned accession number DSM33531. The 16S rDNA sequence of Anaerostipes cacae P127-A10a is provided as SEQ ID NO: 45.

[0045] Additional bacterial strains of the Anaerostipes cacae species provided herein include Anaerostipes cacae strains having a DNA-DNA hybridization (DDH) value of about 70% or greater with Anaerostipes cacae strain P127-A10a. In certain embodiments, the Anaerostipes cacae strain has a DNA-DNA hybridization value of greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% with Anaerostipes cacae strain P127-A10a, or any range between any of these values. In certain embodiments, the Anaerostipes cacae strain has a DDH or dDDH value of about 70% or greater with Anaerostipes cacae strain P127-A10a. In some embodiments, the DDH or dDDH value is greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of Anaerostipes cacae strain P127-A10a, or any range between any of these values.

[0046] Additional bacterial strains of the Anaerostipes cacae species provided herein include Anaerostipes cacae strains having an average nucleotide identity (ANI) of 95% or greater with Anaerostipes cacae strain P127-A10a. In some embodiments, the ANI is about 95% or greater, about 95.5% or greater, about 96% or greater, about 96.5% or greater, about 97% or greater, about 97.5% or greater, about 98% or greater, about 98.5% or greater, about 99% or greater, about 99.5% or greater, or 100% with Anaerostipes cacae strain P127-A10a, or any range therebetween.

[0047] Additional bacterial strains of the Anaerostipes cacae species provided herein include Anaerostipes cacae strains having an alignment fraction (AF) of 60% or greater with Anaerostipes cacae strain P127-A10a. In some embodiments, the AF is about 65% or greater, about 70% or greater, about 75% or greater, about 80% or greater, about 85% or greater, about 90% or greater, about 95% or greater, or 100% with Anaerostipes cacae strain P127-A10a, or any range between any of these values. In some embodiments, the AF is calculated by dividing the sum of the lengths of all BBH genes by the sum of the lengths of all genes in genome A. This calculation is performed separately from genome A to genome B and from genome B to genome A.

[0048] In certain embodiments, the Anaerostipes cacae strain comprises a genome having an ANI of about 95% or greater and an AF of about 60% or greater with the genome of Anaerostipes cacae strain P127-A10a. In another specific embodiment, the Anaerostipes cacae strain comprises a genome having an ANI of about 96.5% or greater and an AF of about 60% or greater with the genome of Anaerostipes cacae strain P127-A10a.

[0049] The present disclosure encompasses derivatives of the disclosed bacterial strains. The term "derivative" includes daughter strains (progeny) or strains cultured (subcloned) from the original strain that have been modified in any way (including at the genetic level) without negatively altering the biological activity of the strain.

[0050] II. Composition containing Christensenella sp. P152-H6d In another aspect, provided herein are compositions, e.g., pharmaceutical compositions, comprising a bacterial strain of Christensenella sp. P152-H6d. In some embodiments, the compositions include one or more bacterial strains, including one or more bacterial strains of Christensenella sp. P152-H6d. In some embodiments, the compositions provided herein include a bacterial strain of Christensenella sp. P152-H6d, but do not include any other bacterial strains or species. In other embodiments, the compositions include a bacterial strain of Christensenella sp. P152-H6d and at least one or more additional bacterial strains or species. In some embodiments, at least one additional bacterial strain or species in the composition is a bacterial strain of the Christensenella genus. For example, the composition may include an additional bacterial strain of Christensenella sp. P152-H6d and / or one or more strains of Christensenella species that are not Christensenella sp. P152-H6d. Exemplary additional Christensenella species include C. minuta (Morotomi et al., International Journal of Systematic and Evolutionary Microbiology 62:144-14 (2012)), C. massiliensis (Ndongo et al., New Microbe and New Infect. 12:69-70 (2016)), and C. timonensis (Ndongo et al., New Microbe and New Infect. 13:32-33 (2016)). In other embodiments, the composition may include Christensenella sp. P152-H6d and one or more non-Christensenella bacterial species.

[0051] In some embodiments, the one or more non-Christensenella bacterial species comprises a member of the Anaerostipes genus, e.g., Anaerostipes cacae. An exemplary Anaerostipes cacae strain useful in combination with Christensenella sp. P152-H6d in the compositions provided herein is Anaerostipes cacae strain P127-A10a, deposited under accession number DSM 33531. The 16S rDNA sequence of Anaerostipes cacae P127-A10a is provided as SEQ ID NO: 45. Other Anaerostipes cacae strains useful in combination with Christensenella sp. P152-H6d in the compositions provided herein include strains comprising a 16S rDNA sequence having at least 98% identity to SEQ ID NO: 45. Additional useful strains include Anaerostipes cacae strain DSM14662, Anaerostipes cacae strain 3_2_56FAA, and Anaerostipes cacae isolate MGYG-HGUT-00080.

[0052] In some embodiments, the compositions provided herein comprise at least two, e.g., two or three, bacterial strains, hi some embodiments, the compositions comprise at least two, three, four, five, six, seven, eight, nine, ten, or more than ten bacterial strains. For example, in some embodiments, the composition comprises 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, or 2 to 5 bacterial strains, such as vegetative bacterial strains, or for example, 3 to 10, 3 to 9, 3 to 8, 3 to 7, or 3 to 6 bacterial strains, such as vegetative bacterial strains, or for example, 4 to 10, 4 to 9, 4 to 8, 4 to 7, or 4 to 6 bacterial strains, such as vegetative bacterial strains, or for example, 5 to 10, 5 to 9, 5 to 8, 6 to 9, 6 to 8, 7 to 10, 7 to 9, or 7 to 8 bacterial strains, such as vegetative bacterial strains, or for example, 8 to 10 bacterial strains, such as vegetative bacterial strains. In some embodiments, the composition comprises two or three bacterial strains, such as vegetative bacterial strains.

[0053] Compositions, e.g., pharmaceutical units provided herein, can contain each bacterial strain in any suitable ratio, measured either by total bacterial mass or colony forming units. For example, a disclosed pharmaceutical composition or pharmaceutical unit can contain two strains in a ratio of 0.1:1, 0.2:1, 0.25:1, 0.5:1, 0.75:1, 1:1, 2:1, 3:1, 4:1, 5:1, or 10:1, measured either by total bacterial mass or colony forming units. For example, the disclosed pharmaceutical compositions or pharmaceutical units can contain the three strains in the following ratios by either total bacterial mass or colony forming units: 1:1:1, 1:1:2, 1:1:4, 1:2:1, 1:2:2, 1:2:4, 1:4:1, 1:4:2, 1:4:4, 2:1:1, 2:1:2, 2:1:4, 2:2:1, 2:4:1, 4:1:1, 4:1:2, 4:1:4, 4:2:1, 4:4:1.

[0054] In some embodiments, a composition comprises a bacterial strain of Christensenella sp. P152-H6d, and optionally one or more additional bacterial strains or species, wherein the composition (i) increases production of one or more anti-inflammatory gene products, such as CCL-18, IL-1Ra, IL-4, IL-6, IL-10, IL-11, IL-13, MCP-1, and TGF-β, in human cells, such as THP-1 macrophages or monocytes, moDCs, or PBMCs; and / or (ii) increases production of one or more anti-inflammatory gene products, such as CCL-18, IL-1Ra, IL-4, IL-6, IL-10, IL-11, IL-13, MCP-1, and TGF-β, in human cells, such as THP-1 macrophages or monocytes, moDCs, or PBMCs. For example, reducing or attenuating the production of one or more pro-inflammatory gene products, such as IL-1-β, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-17, IL-21, IL-22, IL-23, IL-27, IFN (e.g., IFN-γ), CCL-2, CCL-3, CCL-5, CCL-20, CXCL-5, CXCL-10, CXCL-12, CXCL-13, and TNF-α in THP-1 macrophages or monocytes or PBMCs. In some embodiments, one or more additional bacterial strains in the composition are each (i.e., individually) capable of (i) increasing the production of one or more anti-inflammatory gene products, e.g., CCL-18, IL-1Ra, IL-4, IL-6, IL-10, IL-11, IL-13, MCP-1, and TGF-β, in human cells, e.g., THP-1 macrophages or monocytes or PBMCs, and / or (ii) increasing the production of one or more anti-inflammatory gene products, e.g., CCL-18, IL-1Ra, IL-4, IL-6, IL-10, IL-11, IL-13, MCP-1, and TGF-β, in human cells, e.g., THP-1 macrophages or monocytes or PBMCs. or reducing or attenuating production of one or more pro-inflammatory gene products in monocytes or PBMCs, such as IL-1-β, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-17, IL-21, IL-22, IL-23, IL-27, IFN, CCL-2, CCL-3, CCL-5, CCL-20, CXCL-5, CXCL-10, CXCL-12, CXCL-13, and TNF-α.

[0055] excipients The bacterial strain Christensenella sp. P152-H6d disclosed herein may be combined with a pharmaceutically acceptable excipient to form a pharmaceutical composition, which can be administered to a patient by any means known in the art. As used herein, the term "pharmaceutically acceptable excipient" is understood to mean one or more buffers, carriers, or excipients suitable for administration to a subject, e.g., a human subject, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. An excipient should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

[0056] Pharmaceutically acceptable excipients include buffers, solvents, dispersion media, coating agents, isotonicity agents, and absorption delaying agents that are compatible with pharmaceutical administration. Pharmaceutically acceptable excipients also include fillers, binders, disintegrants, glidants, lubricants, and any combination thereof. For example, contemplated compositions may contain a pharmaceutical excipient selected from the group consisting of cellulose, polyvinylpyrrolidone, silicon dioxide, stearyl fumarate or a pharmaceutically acceptable salt thereof, lactose, starch, glucose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, magnesium stearate, mannitol, sorbitol, and any combination thereof. Further examples of excipients, carriers, stabilizers, and adjuvants can be found, for example, in Handbook of Pharmaceutical Excipients, 8 th Ed., Edited by PJ Sheskey, WG Cook, and CG Cable, Pharmaceutical Press, London, UK

[2017] . The use of such media and agents for pharmaceutically active substances is well known in the art.

[0057] Stabilized bacterial composition In some embodiments, the Christensenella sp. P152-H6d bacterial strain described herein may be used in any composition in a stabilized form, including, for example, lyophilized (optionally with one or more appropriate cryoprotectants), frozen (e.g., in a standard or supercooled freezer), spray-dried, and / or freeze-dried. Stabilized bacteria (e.g., bacteria stabilized via lyophilization, freezing, spray-drying, or freeze-drying), particularly stabilized anaerobic bacteria, may, in some embodiments, have more advantageous properties than bacteria in culture with respect to administration, e.g., administration of pharmaceutical compositions provided herein. For example, lyophilized bacteria involve a freeze-drying process that removes water from bacterial cells. The resulting lyophilized bacteria, in some embodiments, may have enhanced stability compared to bacterial cultures and thus may be stored for longer periods (i.e., may have an extended shelf life). Additionally, in some embodiments, dehydrated bacterial cells do not grow or reproduce in the stabilized form but maintain viability and may grow and reproduce upon rehydration. In some embodiments, the viability of stabilized anaerobic Christensenella sp. P152-H6d is maintained even when exposed to oxygen, thus facilitating their formulation (e.g., formulation into oral dosage forms) and use as live biopharmaceuticals that retain biological activity. Thus, in certain embodiments, the Christensenella sp. P152-H6d bacterial strains described herein are stabilized (e.g., stabilized via lyophilization, freezing, freeze-drying, or spray-drying) to remain live and viable and retain some, most, or all of their chemical stability and / or biological activity upon storage. Stability can be measured at selected temperature and humidity conditions over a selected period of time. Trend analysis can be used to estimate the expected shelf life before the material is actually stored for that period. For example, for live bacteria, stability can be defined as the time required to lose 1 log of cfu / g of dry preparation under given conditions of temperature, humidity, and duration.

[0058] In some embodiments, a pharmaceutical composition or pharmaceutical unit comprising Christensenella sp. P152-H6d has at most 0.05% of the total mass of soluble ... For example, a pharmaceutical composition or pharmaceutical unit may lose up to 3 log cfu of each bacterial strain present in the pharmaceutical composition or pharmaceutical unit after storage at 4° C. for 6 months, 1 year, or 2 years.

[0059] The Christensenella sp. P152-H6d bacteria disclosed herein may be combined with one or more cryoprotectants. Exemplary cryoprotectants include fructooligosaccharides (e.g., Raftilose® (a fructooligosaccharide derived from inulin)), trehalose, maltodextrin, sodium alginate, proline, glutamic acid, glycine (e.g., glycine betaine), mono-, di-, or polysaccharides (e.g., glucose, sucrose, maltose, lactose), polyols (e.g., mannitol, sorbitol, or glycerol), dextran, DMSO, methylcellulose, propylene glycol, polyvinylpyrrolidone, non-ionic surfactants such as Tween 80, and any combination thereof.

[0060] In some embodiments, cryoprotectants include Raftilose®, maltodextrin, alginate, trehalose, and sucrose, or any combination thereof. In some embodiments, the pharmaceutical composition comprising the bacterial strain of Christensenella sp. P152-H6d further comprises sucrose as a cryoprotectant. In some embodiments, the pharmaceutical composition comprising the bacterial strain of Christensenella sp. P152-H6d further comprises Raftilose®, maltodextrin, alginate, trehalose, and sucrose as cryoprotectants. In some embodiments, the pharmaceutical composition comprising the bacterial strain of Christensenella sp. P152-H6d further comprises Raftilose®, maltodextrin, alginate, trehalose, and sucrose as cryoprotectants.

[0061] In some embodiments, lyophilized powder forms of bacterial strains contemplated herein contain about 10% to about 80% by weight of one or more bacterial strains (e.g., one bacterial strain) and about 20% to about 90% by weight of a cryoprotectant and / or excipient, such as a cryoprotectant and / or excipient selected from the group consisting of Raftilose®, maltodextrin, sodium alginate, trehalose, sucrose, water, and any combination thereof. For example, 5 mg of a contemplated lyophilized powder form of a bacterial strain may contain about 0.5 mg to about 1.5 mg of the bacterial strain, about 1.5 mg to about 2.5 mg of the bacterial strain, about 2.5 mg to about 3.5 mg of the bacterial strain, or about 3.5 mg to about 4.5 mg of the bacterial strain. It can be understood that each lyophilized powder form of a bacterial strain that can form a component of the disclosed compositions may have different excipients and / or amounts of excipients, as well as distinct bacterial strains.

[0062] A pharmaceutical composition should be formulated to be compatible with its intended route of administration. The bacterial compositions disclosed herein can be prepared by any suitable method and can be formulated into various forms and administered by several different means. The compositions can be administered orally, rectally, or enterally, as desired, in formulations containing conventionally accepted carriers, adjuvants, and vehicles. As used herein, "rectal administration" is understood to include administration by enema, suppository, or colonoscopy. The disclosed pharmaceutical compositions can be suitable, for example, for bolus administration or bolus release. In an exemplary embodiment, the disclosed bacterial compositions are administered orally.

[0063] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules. Capsules typically include a core material containing the bacterial composition and a shell wall encapsulating the core material. In some embodiments, the core material includes at least one of a solid, a liquid, and an emulsion. In some embodiments, the shell wall material includes at least one of soft gelatin, hard gelatin, and a polymer. Suitable polymers include, but are not limited to, cellulose-based polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate, and sodium carboxymethylcellulose; acrylic acid polymers and copolymers such as those formed from acrylic acid, methacrylic acid, methyl acrylate, ammoniomethyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate (e.g., copolymers thereof sold under the trade name "Eudragit®"); vinyl polymers and copolymers such as polyvinylpyrrolidone, polyvinyl acetate, polyvinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; and shellac (purified lac). In some embodiments, at least one polymer functions as a taste-masking agent.

[0064] Tablets, pills, etc. may be compressed, multi-compressed, multi-layered, and / or coated. Contemplated coatings may be single or multiple. In one embodiment, contemplated coating materials include at least one of sugars, polysaccharides, and glycoproteins extracted from at least one of plants, fungi, and microorganisms. Non-limiting examples include corn starch, wheat starch, potato starch, tapioca starch, cellulose, hemicellulose, dextran, maltodextrin, cyclodextrin, inulin, pectin, mannan, gum arabic, locust bean gum, mesquite gum, guar gum, karaya gum, ghatti gum, tragacanth gum, funori, carrageenan, agar, alginate, chitosan, or gellan gum. In some embodiments, contemplated coating materials include proteins. In some embodiments, contemplated coating materials include at least one of fats and oils. In some embodiments, at least one of the fat and oil is high-melting. In some embodiments, at least one of the fat and oil is hydrogenated or partially hydrogenated. In some embodiments, at least one of the fat and oil is derived from a plant. In some embodiments, at least one of the fat and oil comprises at least one of a glyceride, a free fatty acid, and a fatty acid ester. In some embodiments, contemplated coating materials comprise at least one edible wax. Contemplated edible waxes may be derived from animals, insects, or plants. Non-limiting examples include beeswax, lanolin, bayberry wax, carnauba wax, and rice bran wax. Tablets and pills may further be prepared with enteric coatings or reverse enteric coatings.

[0065] Alternatively, powders or granules embodying the bacterial compositions disclosed herein can be incorporated into food products. In some embodiments, the contemplated food product is a beverage for oral administration. Non-limiting examples of suitable beverages include water, fruit juices, fruit drinks, artificially flavored drinks, artificially sweetened drinks, carbonated drinks, sports drinks, liquid dairy drinks, shakes, alcoholic drinks, caffeinated drinks, infant formula, and the like. Other suitable means for oral administration include aqueous and non-aqueous solutions, emulsions, suspensions, and solutions and / or suspensions reconstituted from non-effervescent granules containing at least one of a suitable solvent, preservative, emulsifier, suspending agent, diluent, sweetener, colorant, and flavoring agent.

[0066] In some embodiments, the pharmaceutical compositions provided herein comprise (a) Christensenella sp. strain P152-H6d, and (b) a filler (e.g., microcrystalline cellulose, lactose, sucrose, mannitol, or dicalcium phosphate dihydrate), a disintegrant (e.g., polyvinylpyrrolidone, sodium starch glycolate, starch, or carboxymethylcellulose), a flow aid / glidant (e.g., talc or a silica derivative (e.g., colloidal silica such as Cab-O-Sil or Aerosil), and a lubricant (e.g., sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, stearates, talc, liquid paraffin, propylene glycol (PG), PEG 6000, or magnesium sulfate / sodium lauryl sulfate).

[0067] In some embodiments, a contemplated pharmaceutical composition includes (a) Christensenella sp. strain P152-H6d, and (b) a filler (microcrystalline cellulose), a disintegrant (polyvinylpyrrolidone), a flow aid / glidant (silicon dioxide), and a lubricant (sodium stearyl fumarate).

[0068] In some embodiments, contemplated pharmaceutical compositions are formulated as capsules. In some embodiments, the capsules are hydroxypropyl methylcellulose (HPMC) capsules. In some embodiments, the capsules include a banding polymer (e.g., hydroxypropyl methylcellulose (HPMC)) and a banding solvent (e.g., water or ethanol). In some embodiments, the capsules include two banding solvents, water and ethanol. In some embodiments, the capsules are coated with an inverse enteric coating polymer (e.g., amino methacrylate copolymer) and include a surfactant (e.g., sodium lauryl sulfate), a flow aid / glidant (e.g., silicon dioxide), a lubricant (e.g., stearic acid), an anti-adherent (e.g., talc), and a coating solvent (e.g., water). In some embodiments, the capsules are coated with an enteric coating polymer (e.g., poly(methacrylic acid-co-methyl methacrylate)) and further include a plasticizer (e.g., triethyl citrate), an anti-adherent (e.g., talc), a pH adjuster (e.g., ammonia solution), and a coating solvent (e.g., purified water and isopropyl alcohol).

[0069] In some embodiments, the contemplated capsule is a capsule-in-capsule dosage form comprising an inner capsule and an outer capsule. In some embodiments, the inner capsule comprises one or more freeze-dried bacterial strains, a filler (e.g., microcrystalline cellulose, lactose, sucrose, mannitol, dicalcium phosphate dihydrate, or starch), a disintegrant (e.g., polyvinylpyrrolidone, sodium starch glycolate, or carboxymethylcellulose), a flow aid / glidant (e.g., silicon dioxide, talc, or colloidal silica), and a lubricant (e.g., sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, stearates, talc, liquid paraffin, propylene glycol (PG), PEG 6000, or magnesium / sodium lauryl sulfate). In some embodiments, the outer capsule comprises one or more lyophilized bacterial strains, a filler (e.g., microcrystalline cellulose, lactose, sucrose, mannitol, dicalcium phosphate dihydrate, or starch), a disintegrant (e.g., polyvinylpyrrolidone, sodium starch glycolate, or carboxymethylcellulose), a flow aid / glidant (e.g., silicon dioxide, talc, or colloidal silica), and a lubricant (e.g., sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, stearates, talc, liquid paraffin, propylene glycol (PG), PEG 6000, or magnesium / sodium lauryl sulfate).

[0070] In some embodiments, the contemplated capsule is a capsule-in-capsule dosage form comprising an inner capsule and an outer capsule. In some embodiments, the inner capsule comprises one or more freeze-dried bacterial strains, a filler (microcrystalline cellulose), a disintegrant (polyvinylpyrrolidone), a flow aid / glidant (silicon dioxide), and a lubricant (sodium stearyl fumarate). In some embodiments, the outer capsule comprises one or more freeze-dried bacterial strains, a filler (microcrystalline cellulose), a disintegrant (polyvinylpyrrolidone), a flow aid / glidant (silicon dioxide), and a lubricant (sodium stearyl fumarate).

[0071] In some embodiments, the disclosed pharmaceutical unit comprises a dual-component capsule. For example, the dual-component capsule may comprise an inner capsule having a reverse enteric polymer coating and an outer capsule encapsulating the inner capsule, the outer capsule having an enteric polymer coating. The contemplated inner and / or outer capsules may comprise a bacterial strain or a mixture of bacterial strains. For example, the dual-component capsule may comprise an inner capsule having a reverse enteric polymer coating, the inner capsule comprising an inner composition comprising a bacterial strain or a mixture of bacterial strains and one or more pharmaceutical excipients, and an outer capsule having an enteric polymer coating, the inner capsule and the outer composition comprising a bacterial strain or a mixture of bacterial strains and one or more pharmaceutical excipients. The contemplated inner and / or outer compositions may comprise, for example, the Cristensenella sp. P152-H6d strain, and optionally one or more additional strains. The inner and outer compositions may be the same or different.

[0072] Contemplated dual-component capsules may contain a total of about 5 mg to about 60 mg of inner and outer compositions, e.g., about 5 mg to about 50 mg of inner and outer compositions, about 5 mg to about 15 mg of inner and outer compositions, about 5 mg to about 25 mg of inner and outer compositions, or about 25 mg to about 50 mg of inner and outer compositions. Contemplated dual-component capsules may contain a total of about 50 mg to about 120 mg of inner and outer compositions, e.g., about 50 mg to about 75 mg of inner and outer compositions, about 60 mg to about 85 mg of inner and outer compositions, about 50 mg to about 95 mg of inner and outer compositions, or about 25 mg to about 110 mg of inner and outer compositions.

[0073] In some embodiments, the disclosed dual-component capsules include an inner capsule with a reverse enteric polymer coating and an outer capsule with an enteric polymer coating. Each coating allows, for example, biphasic release of the capsule contents (including bacterial strains) at different locations along the gastrointestinal tract. For example, the gastrointestinal tract has been determined to have several distinct regions, each distinctly delimited by a local pH ranging from 1 to 8.2. The normal pH profile of the gastrointestinal tract rises and falls between the stomach and the colon, with pH ranges of 1 to 4 in the stomach, 5.5 to 6.4 in the duodenum, 6.8 to 8.2 in the ileum, and 5.5 to 6.5 in the colon. For example, the distal ileum contains a region with a normal pH of 6.8 to 8.2, but after passing through the ileal valve and entering the cecum and ascending colon, the pH drops sharply from 8.2 to 5.5. As the colon progresses from the proximal to the distal colon, the pH gradually rises again to 8.0. Thus, in some embodiments, the enteric polymer coating of the outer capsule solubilizes at a pH of about 7-8 to allow release in the ileum, and the reverse enteric polymer coating of the inner capsule solubilizes at a pH of about 6.2-6.5 to allow subsequent release in the colon. In some embodiments, the outer capsule maintains its integrity (e.g., no splitting, cracking, or rupture of the capsule shell) for about 2 hours at pH 1.2 and 37°C. In some embodiments, the outer capsule maintains its integrity (e.g., no splitting, cracking, or rupture of the capsule shell) for about 2 hours at pH 5.5 and 37°C. In some embodiments, the outer capsule disintegrates within about 1 hour at pH 7.4 and 37°C. In some embodiments, the inner capsule maintains its integrity (e.g., no splitting, cracking, or rupture of the capsule shell) for up to 1 hour at pH 7.4 and 37°C. In some embodiments, the inner capsule disintegrates within 2 hours at pH 6.5 and 37°C.

[0074] In some embodiments, the inner and / or outer capsule coating is comprised of poly(dl-lactide-co-glycolide), chitosan (Chi) stabilized with PVA (polyvinyl alcohol), lipids, alginate, carboxymethylethylcellulose (CMEC), cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), hydroxypropylmethylcellulose, ethylcellulose, food glaze, a mixture of hydroxypropylmethylcellulose and ethylcellulose, polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP), shellac, a copolymer of methacrylic acid and ethyl acrylate, or a copolymer of methacrylic acid and ethyl acrylate to which methyl acrylate monomer has been added during polymerization. Methyl methacrylate or a copolymer of methacrylic acid and methyl methacrylate is Eudragit® polymer (Evonik Industries, Darmstadt, Germany). These polymers are available as Eudragit® L100 and Eudragit® S100 (anionic copolymers based on methacrylic acid and methyl methacrylate), either alone or in combination. Eudragit® L100 dissolves at about pH 6 or above and contains 46.0% to 50.6% methacrylic acid units per gram of dry matter, while Eudragit® S100 dissolves at about pH 7 or above and contains 27.6% to 30.7% methacrylic acid units per gram of dry matter. Another exemplary group of encapsulating polymers are the polyacrylic acids Eudragit® L and Eudragit® S, optionally combined with Eudragit® RL or RS (copolymers of ethyl acrylate, methyl methacrylate, and methacrylic acid esters with a low content of quaternary ammonium groups). These modified acrylic acids are useful because they can be made soluble at pH 6-7.5, depending on the particular Eudragit chosen and the ratio of Eudragit® S to Eudragit® L, RS, and RL used in the formulation.In some embodiments, a contemplated coating for the inner capsule is comprised of Eudragit EPO® ReadyMix. In some embodiments, a contemplated outer capsule coating is comprised of Eudragit® L100 (methylacrylic acid-methylmethacrylate copolymer (1:1)) and Eudragit® S100 (methylacrylic acid-methylmethacrylate copolymer (1:2)). In some embodiments, contemplated capsules are suitable for sustained or timed release. In some embodiments, contemplated inner and / or outer capsule coatings further comprise a band / seal, e.g., hypromellose, an opacifier, e.g., titanium dioxide, a plasticizer, e.g., triethyl citrate (TEC), or an anti-adherent, e.g., talc.

[0075] Further exemplary capsule-in-capsule formulations are described in US Pat. No. 9,907,755.

[0076] Unit dosage form Pharmaceutical compositions comprising Christensenella sp. P152-H6d disclosed herein can be provided in unit dosage form, i.e., pharmaceutical units. Compositions, such as pharmaceutical units provided herein, can contain any suitable amount of one or more bacterial strains, measured by total mass or by bacterial colony-forming units.

[0077] For example, the disclosed pharmaceutical compositions or dosage units may contain about 10 of each bacterial strain. 3 cfu ~ approx. 10 12 cfu, approximately 10 6 cfu ~ approx. 10 12 cfu, approximately 10 7 cfu ~ approx. 10 12 cfu, approximately 10 8 cfu ~ approx. 10 12 cfu, approximately 10 9 cfu ~ approx. 10 12 cfu, approximately 10 10 cfu ~ approx. 10 12 cfu, approximately 10 11 cfu ~ approx. 10 12 cfu, approximately 10 3cfu ~ approx. 10 11 cfu, approximately 10 6 cfu ~ approx. 10 11 cfu, approximately 10 7 cfu ~ approx. 10 11 cfu, approximately 10 8 cfu ~ approx. 10 11 cfu, approximately 10 9 cfu ~ approx. 10 11 cfu, approximately 10 10 cfu ~ approx. 10 11 cfu, approximately 10 3 cfu ~ approx. 10 10 cfu, approximately 10 6 cfu ~ approx. 10 10 cfu, approximately 10 7 cfu ~ approx. 10 10 cfu, approximately 10 8 cfu ~ approx. 10 10 cfu, approximately 10 9 cfu ~ approx. 10 10 cfu, approximately 10 3 cfu ~ approx. 10 9 cfu, approximately 10 6 cfu ~ approx. 10 9 cfu, approximately 10 7 cfu ~ approx. 10 9 cfu, approximately 10 8 cfu ~ approx. 10 9 cfu, approximately 10 3 cfu ~ approx. 10 8 cfu, approximately 10 6 cfu ~ approx. 10 8 cfu, approximately 10 7 cfu ~ approx. 10 8 cfu, approximately 10 3 cfu ~ approx. 10 7 cfu, approximately 10 6 cfu ~ approx. 10 7 cfu, or about 10 3 cfu ~ approx. 10 6 cfu of the or each bacterial strain in the composition, or 3 cfu, approximately 10 6 cfu, approximately 10 7 cfu, approximately 10 8 cfu, approximately 10 9 cfu, approximately 10 10 cfu, approximately 10 11cfu, or approximately 10 12 It may also contain cfu.

[0078] For example, the disclosed pharmaceutical compositions or dosage units may contain about 10 of each bacterial strain. 3 cfu ~ approx. 10 12 cfu, approximately 10 6 cfu ~ approx. 10 12 cfu, approximately 10 7 cfu ~ approx. 10 12 cfu, approximately 10 8 cfu ~ approx. 10 12 cfu, approximately 10 9 cfu ~ approx. 10 12 cfu, approximately 10 10 cfu ~ approx. 10 12 cfu, approximately 10 11 cfu ~ approx. 10 12 cfu, approximately 10 3 cfu ~ approx. 10 11 cfu, approximately 10 6 cfu ~ approx. 10 11 cfu, approximately 10 7 cfu ~ approx. 10 11 cfu, approximately 10 8 cfu ~ approx. 10 11 cfu, approximately 10 9 cfu ~ approx. 10 11 cfu, approximately 10 10 cfu ~ approx. 10 11 cfu, approximately 10 3 cfu ~ approx. 10 10 cfu, approximately 10 6 cfu ~ approx. 10 10 cfu, approximately 10 7 cfu ~ approx. 10 10 cfu, approximately 10 8 cfu ~ approx. 10 10 cfu, approximately 10 9 cfu ~ approx. 10 10 cfu, approximately 10 3 cfu ~ approx. 10 9 cfu, approximately 10 6 cfu ~ approx. 10 9 cfu, approximately 10 7 cfu ~ approx. 10 9 cfu, approximately 10 8 cfu ~ approx. 10 9 cfu, approximately 10 3cfu ~ approx. 10 8 cfu, approximately 10 6 cfu ~ approx. 10 8 cfu, approximately 10 7 cfu ~ approx. 10 8 cfu, approximately 10 3 cfu ~ approx. 10 7 cfu, approximately 10 6 cfu ~ approx. 10 7 cfu, or approximately 10 3 cfu ~ approx. 10 6 cfu of the bacterial strain in the composition. 3 cfu, approximately 10 6 cfu, approximately 10 7 cfu, approximately 10 8 cfu, approximately 10 9 cfu, approximately 10 10 cfu, approximately 10 11 cfu, or approximately 10 12 It may also contain cfu.

[0079] In some embodiments, the pharmaceutical unit provided contains at least 1 x 10 of each bacterial strain (e.g., a vegetative bacterial strain). 3 colony-forming units, or at least 1 x 10 of each bacterial strain (e.g., vegetative bacterial strain) 4 colony-forming units, or at least 1 x 10 of each bacterial strain (e.g., vegetative bacterial strain) 5 colony-forming units, or at least 1 x 10 of each bacterial strain (e.g., vegetative bacterial strain) 6 colony-forming units, or at least 1 x 10 of each bacterial strain (e.g., vegetative bacterial strain) 7 colony-forming units, or at least 1 x 10 of each bacterial strain (e.g., vegetative bacterial strain) 8 colony-forming units, or at least 1 x 10 of each bacterial strain (e.g., vegetative bacterial strain) 9 Contains colony forming units.

[0080] For example, a disclosed composition (e.g., a pharmaceutical unit, such as a capsule) can contain about 1 mg to about 5 mg (e.g., 2 mg to about 4 mg) of bacterial strain, each of which can be present in a unit, e.g., about 5 mg to about 50 mg of lyophilized powder form of the bacterial strain. For example, a pharmaceutical unit can contain a total of about 30 mg to about 70 mg, about 30 mg to about 60 mg, about 30 mg to about 50 mg, about 30 mg to about 40 mg, about 40 mg to about 70 mg, about 40 mg to about 60 mg, about 40 mg to about 50 mg, about 50 mg to about 70 mg, about 50 mg to about 60 mg, about 80 mg to about 100 mg, about 90 mg to about 110 mg, about 100 mg to about 120 mg, or about 110 mg to about 150 mg of the lyophilized powder form of the bacterial strain. In some embodiments, the pharmaceutical unit contains a total of about 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 100 mg, 120 mg, 130 mg, 140 mg, or 150 mg of the lyophilized powder form of the bacterial strain.

[0081] In some embodiments, the disclosed compositions, e.g., the disclosed pharmaceutical units, may contain about 5 to about 50 mg of each lyophilized powder form of the bacterial strain, e.g., about 5 to about 45 mg, about 5 to about 40 mg, about 5 to about 35 mg, about 5 to about 30 mg, about 5 to about 25 mg, about 5 to about 15 mg, about 5 to about 10 mg, about 10 to about 50 mg, about 10 to about 35 mg of each lyophilized powder form of the bacterial strain (e.g., a vegetative bacterial strain), about 10 to about 20 mg, about 10 to about 15 mg, or about 15 to about 45 mg of each lyophilized powder form of the bacterial strain (e.g., a vegetative bacterial strain). In some embodiments, the disclosed pharmaceutical units contain about 5, about 10, about 15, about 20, about 25, or about 30 mg of each lyophilized powder form of the bacterial strain (e.g., a vegetative bacterial strain). In some embodiments, the disclosed pharmaceutical unit contains about 25 to about 50 mg of a lyophilized powder form of one bacterial strain (e.g., a vegetative bacterial strain) and about 5 mg to about 10 mg of the remaining lyophilized powder forms of the bacterial strains (e.g., vegetative bacterial strains), or about 5 mg to about 15 mg of a lyophilized powder form of one bacterial strain (e.g., a vegetative bacterial strain) and about 5 mg to 10 mg of the remaining lyophilized powder forms of the bacterial strains (e.g., vegetative bacterial strains), for example, about 15 mg of a lyophilized powder form of one bacterial strain (e.g., a vegetative bacterial strain) and about 5 mg of the remaining lyophilized powder forms of the bacterial strains (e.g., vegetative bacterial strains), or about 15 mg to about 25 mg of each of two lyophilized powder forms of the bacterial strains (e.g., vegetative bacterial strains) and about 5 mg to 10 mg of the remaining lyophilized powder forms of the bacterial strains (e.g., vegetative bacterial strains).

[0082] In some embodiments, the pharmaceutical composition or pharmaceutical unit may comprise or be administered in combination with a prebiotic, i.e., a compound or composition that alters the growth, maintenance, activity and / or balance of the intestinal flora (e.g., can enable specific changes in the composition and / or activity of the microbiome). Exemplary prebiotics include complex carbohydrates, complex sugars, resistant dextrin, resistant starch, amino acids, peptides, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, lignin, psyllium, chitin, chitosan, chitosan oligosaccharides, lactitol, gums (e.g., guar gum), high amylose corn starch (HAS), cellulose, β-glucan, hemicellulose, lactulose, mannooligosaccharides, mannanoligosaccharides (MOS), oligofructose-enriched inulin, fructooligosaccharides, oligoglucose, tagatose, transgalactooligosaccharides, pectin, resistant starch, isomaltooligosaccharides, and xylooligosaccharides (XOS). Prebiotics can be found in foods (e.g., gum arabic, guar gum, brown rice, rice bran, barley husk, chicory root, Jerusalem artichoke, dandelion greens, garlic, leeks, onions, asparagus, wheat bran, oat bran, baked beans, whole wheat flour, and bananas) and breast milk. Prebiotics can also be administered in other forms (e.g., capsules or dietary supplements).

[0083] III. Therapeutic uses The compositions and methods disclosed herein can be used to treat various forms of gastrointestinal disorders, inflammatory disorders, skin disorders, and / or dysbiosis in a subject. The present disclosure provides methods of treating gastrointestinal disorders, inflammatory disorders, skin disorders, and / or dysbiosis in a subject. Contemplated methods include administering to the subject a pharmaceutical composition and / or pharmaceutical unit comprising an effective amount of the Christensenella sp. P152-H6d bacterial strain (and optionally one or more additional bacterial strains) disclosed herein, alone or in combination with another therapeutic agent, to treat the gastrointestinal disorder, inflammatory disorder, skin disorder, and / or dysbiosis in the subject.

[0084] As used herein, "treat," "treating," and "treatment" refer to the treatment of a disease in a subject, e.g., a human. Treating a disease includes (a) inhibiting the disease, i.e., preventing its onset, and (b) alleviating the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals, such as humans, companion animals (e.g., dogs, cats, or rabbits), or livestock animals (e.g., cows, sheep, pigs, goats, horses, donkeys, and mules, buffalo, bulls, or camels).

[0085] The exact dosage of a pharmaceutical unit, pharmaceutical composition, or bacterial strain is selected by an individual physician in view of the patient being treated, and it is understood that dosage and administration are generally adjusted to provide the patient being treated with an effective amount of the bacterial agent. As used herein, "effective amount" refers to the amount necessary to elicit a beneficial or desired biological response. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As will be understood by those skilled in the art, the effective amount of a pharmaceutical unit, pharmaceutical composition, or bacterial strain can vary depending on factors such as the desired biological endpoint, the drug being delivered, the target tissue, and the route of administration. Additional factors that may be considered include the severity of the disease state, the age, weight, and sex of the patient being treated, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerability / response to the treatment.

[0086] It is understood that the disclosed bacterial strains or mixtures of bacterial strains may not need to colonize the gastrointestinal tract, e.g., the intestine, of a subject and / or persist in the subject to elicit a beneficial or desired biological response. For example, in some embodiments, the bacterial strains or mixtures of bacterial strains colonize the gastrointestinal tract, e.g., the intestine, of a subject and / or persist in the subject after administration. In some embodiments, the bacterial strains or mixtures of bacterial strains do not colonize the gastrointestinal tract of a subject and / or do not persist in the subject after administration.

[0087] Gastrointestinal disorders include, for example, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), ulcerative proctitis, microscopic colitis, irritable bowel syndrome (IBS, e.g., IBS-c, IBS-m, or IBS-d), functional diarrhea, functional constipation, celiac disease, radiation enteritis, Clostridium difficile (C. difficile) infection (CDI), recurrent C. difficile infection (rCDI), C. difficile-associated diarrhea (CDAD), colitis (e.g., infectious, ischemic, indeterminate, or radiation colitis), ulcers (including gastric, peptic, and duodenal ulcers), gastroesophageal reflux disease (GERD), stomatitis, gastroenteritis, pancreatitis, mucositis (e.g., oral These conditions include: ulcerative colitis, esophagitis, rectal mucositis, gastrointestinal mucositis, nasal mucositis, and proctitis), necrotizing enterocolitis, esophagitis, non-ulcer dyspepsia, chronic intestinal pseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction, duodenogastric reflux, ileusitis, postoperative ileus, heartburn (high acidity in the gastrointestinal tract), constipation (e.g., constipation associated with the use of medications such as opioids, osteoarthritis medications, osteoporosis medications, postoperative constipation, or constipation associated with neuropathy), hemorrhoids, diverticular disease, chronic pancreatitis, blind duct syndrome, gastroparesis (including diabetic and / or idiopathic), diarrhea, dysphagia, fecal incontinence, short bowel syndrome (SBS), intestinal ischemia, infantile reflux, infantile reflex syndrome, cyclic vomiting syndrome, bulbar symptoms, intestinal volvulus, cancer of the gastrointestinal tract, and gastrointestinal allergies. It is contemplated that the compositions and methods disclosed herein can be used to treat any functional gastrointestinal disorder, including, for example, disorders mediated by or otherwise related to brain-gut interactions.

[0088] Inflammatory bowel disease or IBD is used interchangeably herein to refer to bowel diseases that cause inflammation and / or ulcers, including, but not limited to, Crohn's disease and ulcerative colitis. Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases of unknown cause.

[0089] Ulcerative colitis (UC) affects the large intestine. The disease course can be continuous or recurrent, mild or severe. The earliest lesion is an inflammatory infiltrate with abscess formation at the base of the Lieberkuhn crypt. This distension and aggregation of ruptured crypts separates the overlying mucosa from its blood supply, predisposing to ulcers. Symptoms of the disease include cramping, lower abdominal pain, rectal bleeding, and frequent, loose bowel movements with few fecal particles and consisting primarily of blood, pus, and mucus. Acute, severe, or chronic, unremitting ulcerative colitis may require total colectomy.

[0090] Unlike ulcerative colitis, Crohn's disease can affect any part of the intestine. The most prominent feature of Crohn's disease is a reddish-purple, granular, edematous thickening of the intestinal wall. With the onset of inflammation, these granulomas often lose their circumscribed borders and become integrated with the surrounding tissue. Diarrhea and intestinal obstruction are the main clinical features. The course of Crohn's disease, like ulcerative colitis, can be continuous or recurrent, mild or severe, but unlike ulcerative colitis, Crohn's disease is not cured by resection of the involved portion of the intestine. Most patients with Crohn's disease require surgery at some point, but later relapses are common and usually require ongoing medical treatment.

[0091] Inflammatory disorders can be characterized, for example, based on the primary tissue affected, the underlying mechanism of action of the disorder, or the portion of the immune system that is dysregulated or overactive. Examples of inflammatory disorders include inflammation of the lungs, joints, connective tissue, eyes, nose, intestines, kidneys, liver, skin, central nervous system, vasculature, heart, or adipose tissue. In some embodiments, inflammatory disorders that can be treated include inflammation due to infiltration of leukocytes or other immune effector cells or their mediators into affected tissues. In some embodiments, inflammatory disorders that can be treated include inflammation mediated by IgA and / or IgE antibodies. Other relevant examples of inflammatory disorders that can be treated by the present disclosure include inflammation caused by infectious agents, including, but not limited to, viruses, bacteria, fungi, and parasites. In some embodiments, the inflammatory disorder treated is an allergic reaction. In some embodiments, the inflammatory disorder is an autoimmune disease.

[0092] Inflammatory lung disorders include asthma, adult respiratory distress syndrome, bronchitis, pneumonia, pulmonary fibrosis, and cystic fibrosis (which may additionally or alternatively involve the gastrointestinal tract or other tissues). Immune-mediated inflammatory diseases include systemic lupus erythematosus, systemic vasculitis, Sjögren's syndrome, alopecia areata, and systemic sclerosis. Inflammatory joint disorders include rheumatoid arthritis, seronegative spondyloarthropathy including ankylosing spondylitis, juvenile rheumatoid arthritis, osteoarthritis, gouty arthropathy, and other joint disorders. Inflammatory eye disorders include uveitis (including iritis), conjunctivitis, meningitis, scleritis, and keratoconjunctivitis sicca. Inflammatory bowel disorders include Crohn's disease, ulcerative colitis, inflammatory bowel disease, and distal proctitis. Inflammatory skin disorders include disorders associated with cell proliferation, such as psoriasis, eczema, dermatitis (e.g., eczematous dermatitis, atopic and seborrheic dermatitis, allergic or irritant contact dermatitis, fissure eczema, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis, radiation dermatitis, and stasis dermatitis), and acne. Inflammatory diseases of the endocrine system include, but are not limited to, autoimmune endocrine disorders, autoimmune thyroiditis (Hashimoto's disease), inflammation of the liver and adipose tissue associated with type I diabetes, type II diabetes, and acute and chronic inflammation of the adrenal cortex. Inflammatory disorders of the cardiovascular system include, but are not limited to, coronary artery infarction injury, peripheral vascular disease, myocarditis, vasculitis, revascularization of stenosis, atherosclerosis, and vascular disease associated with type II diabetes. Inflammatory diseases of the kidney include, but are not limited to, glomerulonephritis, interstitial nephritis, lupus nephritis, nephritis secondary to Wegener's disease, acute renal failure secondary to acute nephritis, Good Pasteur syndrome, post-obstructive syndrome, and tubular ischemia.Inflammatory disorders of the liver include, but are not limited to, hepatitis (which occurs as a secondary result of viral infection, autoimmune response, drug treatment, toxins, environmental factors, or primary disorders), biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis.Metabolic disorders with inflammatory etiology include, but are not limited to, insulin resistance, metabolic syndrome, obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH).In some embodiments, the inflammatory disorder is an autoimmune disease, such as rheumatoid arthritis, lupus, alopecia, autoimmune pancreatitis, celiac disease, Behcet's disease, Cushing's syndrome, and Graves' disease. In some embodiments, the inflammatory disorder is a rheumatic disorder, such as rheumatoid arthritis, juvenile arthritis, bursitis, spondylitis, gout, scleroderma, Still's disease, and vasculitis. Additional exemplary inflammatory disorders include eosinophilic esophagitis and eosinophilic gastroenteritis.

[0093] Exemplary skin disorders include psoriasis, eczema, dermatitis (e.g., eczematous dermatitis, atopic and seborrheic dermatitis, allergic or irritant contact dermatitis, fissure eczema, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis, radiation dermatitis, and stasis dermatitis), and acne.

[0094] In general, dysbiosis refers to a state of the microbiota or microbiome of the gastrointestinal tract or other body regions, including, for example, mucosal or skin surfaces (or any other microbiome niche), in which the normal diversity and / or function of the ecological network is disrupted. Any disruption of the microbiota from its typical (e.g., ideal) state can be considered dysbiosis, even if such dysbiosis does not result in a detectable decline in health. This dysbiotic state can be unhealthy (e.g., resulting in a disease state), unhealthy only under certain conditions, or preventing a subject from becoming healthy. Dysbiosis can result from a decrease in the diversity of the microbiota population composition, an overgrowth of one or more populations of pathogens (e.g., pathogenic bacterial populations) or pathogenic commensals, the presence and / or overgrowth of commensals that can cause disease only when certain genetic and / or environmental conditions are present in the patient, or a shift to an ecological network that no longer provides a beneficial function to the host and therefore no longer promotes health. Distal dysbiosis includes, but is not limited to, dysbiosis outside the lumen of the gastrointestinal tract.

[0095] Dysbiosis is caused by bacteria of the genera Yersinia, Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella, Klebsiella, Helicobacter, and Haemophilus. It is contemplated that the infectious disease may include infections caused by pathogenic bacteria of a genus selected from the group consisting of: Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, and Bacillus. Further examples of pathogenic bacteria include Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum,botulinum, Clostridium difficile, Clostridium perfringens, enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia coli (LT or ST), Escherichia coli 0157:H7, Helicobacter pylori, Lysteria monocytogenes, Plesiomonas shigelloides, Salmonella typhi, Staphylococcus aureus These include Vibrio aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum beta-lactam-resistant Enterococci (ESBL), vancomycin-resistant Enterococci (VRE), and multidrug-resistant bacteria.

[0096] It is further contemplated that the compositions and methods disclosed herein can be used to treat disorders of the liver, pancreas, or gallbladder.

[0097] In certain embodiments, the compositions and methods disclosed herein can be used to prevent or inhibit weight gain, promote weight loss, and / or reduce excess fat in a subject in need thereof. In some embodiments, a subject in need thereof is over 24 (i.e., 24 kg / m 2 ) or greater. In some embodiments, the subject in need thereof has a BMI of 24 or greater, 25 or greater, 26 or greater, 27 or greater, 28 or greater, 29 or greater, 30 or greater, 31 or greater, 32 or greater, 33 or greater, 34 or greater, 35 or greater, 36 or greater, 37 or greater, 38 or greater, 39 or greater, 40 or greater, or greater than 40. In some embodiments, the subject in need thereof is obese, e.g., as determined by BMI > 25, waist circumference, waist-to-hip ratio, skinfold thickness, bioelectrical impedance, underwater weighting (density measurement), air displacement pulsation, dilution (water measurement), dual energy x-ray absorptiometry (DEXA), computed tomography (CT), magnetic resonance imaging (MRI), or any combination thereof.

[0098] In other embodiments, the compositions and methods disclosed herein may also be useful for preventing one or more of the above-mentioned diseases or conditions when administered as a vaccine composition. In some embodiments, the bacterial strains provided herein are viable. In some embodiments, the bacterial strains are capable of at least partially or totally colonizing the gastrointestinal tract, e.g., the intestine. In some embodiments, the bacterial strains of the present invention are viable and capable of at least partially or totally colonizing the gastrointestinal tract, e.g., the intestine. In other embodiments, the bacterial strains of the present invention may be killed, inactivated, or attenuated. In some embodiments, the compositions may include a vaccine adjuvant. In some embodiments, the compositions are for administration via injection, such as subcutaneous injection.

[0099] IV. Combination Therapy The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. As used herein, the term "administered in combination" is understood to mean that two (or more) different treatments are delivered to a subject during the course of the subject's illness so that the effects of the treatments on the patient overlap at some point. In some embodiments, the delivery of one treatment is still occurring when the second treatment begins, thus resulting in an overlap in administration. This is sometimes referred to herein as "simultaneous" or "co-delivery." In other embodiments, the delivery of one treatment ends before the other treatment begins. In some embodiments, in either case, the combined administration results in more effective treatments. For example, the second treatment is more effective, e.g., a comparable effect is achieved with less of the second treatment, or the second treatment reduces symptoms more than when the second treatment is administered without the first treatment or when an equivalent situation occurs with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, wholly additive, or greater than additive. Delivery may be such that the effects of the first treatment delivered are still detectable when the second treatment is delivered. In some embodiments, the side effects of the first and / or second treatments are reduced due to combined administration.

[0100] In some embodiments, the methods or compositions described herein are administered in combination with one or more additional therapies. In some embodiments, contemplated additional therapies may include aminosalicylates, corticosteroids, tumor necrosis factor (TNF) antagonists, linaclotide, antibiotics, or immunosuppressants (e.g., azathioprine, 6-mercaptopurine, cyclosporine, methotrexate, or tacrolimus (Prograf)). In some embodiments, contemplated additional therapies may include biologic agents (e.g., infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), or etanercept (Enbrel)). It is contemplated that a subject treated with the disclosed methods or compositions may have had an inadequate response to a previous administration of a therapy, e.g., a previous administration of an aminosalicylates, corticosteroid, or biologic agent.

[0101] Additional therapeutic agents suitable for combination therapy with the pharmaceutical compositions or pharmaceutical units described herein include proton pump inhibitors (e.g., pantoprazole (Protonix), lansoprazole (Prevacid), esomeprazole (Nexium), omeprazole (Prilosec), and rabeprazole), H2 blockers (e.g., cimetidine (Tagamet), ranitidine (Zantac), famotidine (Pepcid), and nizatidine (Axid)), prostaglandins (e.g., misoprost L (Cytotec)), sucralfate, and antacids.

[0102] In some embodiments, the pharmaceutical composition or pharmaceutical unit may contain or be administered in combination with a corticosteroid. Corticosteroids are a class of chemicals that include steroid hormones naturally produced in the adrenal cortex of vertebrates and laboratory-synthesized analogs of these hormones. Corticosteroids are involved in a wide range of physiological processes, including stress response, immune response, regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. Exemplary corticosteroids include betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or deflazacort. It is contemplated that subjects treated with the disclosed methods or compositions may have had an inadequate response to previous administration of corticosteroids.

[0103] In some embodiments, the pharmaceutical composition or pharmaceutical unit may contain or be administered in combination with an aminosalicylates. Exemplary aminosalicylates include 4-aminosalicylic acid, balsalazide, olsalazine, sulfasalazine, and mesalazine (5-aminosalicylic acid). It is contemplated that a subject treated with the disclosed methods or compositions may have had an inadequate response to previous administration of mesalamine, for example, oral administration of > 2.4 g / day of mesalamine for at least 8 weeks.

[0104] In some embodiments, the pharmaceutical composition or pharmaceutical unit may comprise or be administered in combination with a tumor necrosis factor (TNF) antagonist. Exemplary TNF antagonists include infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), etanercept (Enbrel), thalidomide (Immunoprin), lenalidomide (Revlimid), pomalidomide (Pomalyst, Imnovid), xanthine derivatives (e.g., pentoxifylline), and bupropion. It is contemplated that subjects treated with the disclosed methods or compositions may have had an inadequate response to previous administration of a TNF antagonist.

[0105] In some embodiments, the pharmaceutical composition or unit may comprise or be administered in combination with an integrin α4β7 antagonist, such as vedolizumab. It is contemplated that a subject treated with the disclosed methods or compositions may have had an inadequate response to previous administration of an integrin α4β7 antagonist.

[0106] In some embodiments, the pharmaceutical composition or pharmaceutical unit may include or be administered in combination with an antibacterial agent, e.g., an antibiotic. The disclosed methods may also include antibiotic pretreatment, e.g., administering an antibiotic to a subject prior to administration of the disclosed pharmaceutical composition or pharmaceutical unit. Exemplary antibiotics for use in combination therapy include vancomycin, metronidazole, gentamicin, colistin, fidaxomicin, telavancin, oritavancin, dalbavancin, daptomycin, cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, ceftobiprole, cipro, levaquin, floxin, tequin, avelox, norflox, tetracycline, minocycline, and oxytetracycline. cycline, doxycycline, amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, methicillin, ertapenem, doripenem, imipenem / cilastatin, meropenem, amikacin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefoxothin, and / or streptomycin.

[0107] In some embodiments, the pharmaceutical composition or unit may include or be administered in combination with an antifungal or antiviral agent. Exemplary antiviral agents include abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, foscarnet, fomivirsen, ganciclovir, indinavir, idoxuridine, lamivudine, lopinavir, maraviroc, MK-2048, nelfinavir, nevirapine, penciclovir, raltegravir, rilpivirine, ritonavir, saquinavir, stavudine, tenofovir, trifluridine, valacyclovir, valganciclovir, vidarabine, ibacitabine, amantadine, oseltamivir, rimantidine, tipranavir, zalcitabine, zanamivir, and zidovudine. Exemplary antifungal agents include natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, and hamycin, miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, and albaconazole, abafungin, terbinafine, naftifine, butenafine, anidulafungin, caspofungin, micafungin, polygodial, benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine, or 5-fluorocytosine, griseofulvin, and haloprogin.

[0108] In some embodiments, the pharmaceutical composition or pharmaceutical unit may contain or be administered in combination with an additional bacterial strain. Exemplary bacterial strains include Christensenella sp. P152-H6d and strains of Christensenella species that are not Christensenella sp. P152-H6d (e.g., C. minuta, C. massiliensis, and C. timonensis). Additional exemplary bacterial strains include strains of the genus Anaerostipes, such as Anaerostipes cacae. An exemplary strain of Anaerostipes cacae is Anaerostipes cacae strain P127-A10a, deposited under accession number DSM 33531. Additional useful strains include Anaerostipes cacae strain DSM 14662, Anaerostipes cacae strain 3_2_56FAA, and Anaerostipes cacae isolate mgYG-HGUT-00080.

[0109] Throughout this specification, when compositions are described as having, including, or comprising specific components, or when processes and methods are described as having, including, or comprising specific steps, it is additionally contemplated that there are compositions of the present disclosure that consist essentially of or consist of the recited components, and that there are processes and methods according to the present disclosure that consist essentially of or consist of the recited process steps.

[0110] In this application, when an element or component is said to be included in and / or selected from a list of recited elements or components, it is to be understood that the element or component can be any one of the recited elements or components or can be selected from a group consisting of two or more of the recited elements or components.

[0111] Furthermore, it should be understood that elements and / or features of the compositions or methods described herein, whether expressly or implicitly stated herein, can be combined in various ways without departing from the spirit and scope of the present disclosure. For example, if a particular compound is referenced, that compound can be used in various embodiments of the disclosed compositions and / or methods, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a manner that facilitates writing and illustrating a clear and concise application, but it is intended and understood that the embodiments can be combined or separated in various ways without departing from the present teachings and disclosure. For example, it is understood that all features described and depicted herein are applicable to all aspects of the present disclosure as described and depicted herein.

[0112] The phrase "at least one of" should be understood to include each of the listed items following the phrase individually, as well as various combinations of two or more of the listed items, unless otherwise understood from context and usage. The phrase "and / or" in connection with more than two listed items should be understood to have the same meaning, unless otherwise understood from context.

[0113] It should be understood that the use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, is generally open-ended and non-limiting and does not, for example, exclude additional, unrecited elements or steps, unless otherwise stated or understood from the context.

[0114] When the term "about" is used before a quantitative value, the present disclosure also includes the particular quantitative value itself unless otherwise specified. As used herein, the term "about" refers to a ±10% variation from the stated value unless otherwise specified or inferred.

[0115] It should be understood that the order of steps or order for performing certain actions is inconsequential so long as the present disclosure remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0116] The use of any and all examples or exemplary language herein, such as "such as" or "including," is intended merely to better illustrate the disclosure and does not imply any limitation on the scope of the disclosure beyond the claims. No language herein should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0117] [Example] The following examples are for illustrative purposes only and are not intended to limit the scope or content of the present disclosure in any way.

[0118] [Example 1] Isolation and purification of Christensenella sp. P152-H6d 1.1 Source Isolate P152-H6d was isolated from a fecal sample of a healthy human donor. The donor underwent extensive laboratory testing to confirm their health, including screening for infectious agents, to minimize the risk of transmissible infection. Serologic screening included HIV-1 / HIV-2 (IgG and EIA), HTLV-I and HTLV-II (Ab), hepatitis A virus (IgM), hepatitis B virus (HBSAg, anti-HBc IgG and IgM), hepatitis C virus (anti-HCV IgG), treponate pallidum (EIA, or RPR if EIA was positive), Strongyloides pallidum Ab, CMV viral load, and EBV viral load. Stool screening included Clostridium difficile toxins A / B (PCR), H. pylori EIA, routine bacterial culture (with enrichment) for enteric pathogens including Salmonella, Shigella, Yersinia, Campylobacter, and Vibrio, Escherichia coli O157 (if stx1 / 2 EIA +ve, perform E. coli O157 culture), Shiga toxin-like toxin stx1 / 2 (Shigella) EIA, vancomycin-resistant Enterococcus (VRE), extended-spectrum beta-lactamase (ESBL)-producing strains, carbapenem-resistant Enterobacteriaceae (CRE), and methicillin-resistant Staphylococcus aureus (Staphylococcus aureus). These included culture-based assays for MRSA, Giardia antigen (EIA), Cryptosporidium antigen (EIA), Cyclospora, Isospora, and Microsporidia (microscopic observation with acid-fast stain), ova and parasites (microscopic observation), rotavirus (EIA), norovirus GI / GII (RT-PCR), and adenovirus 40, 41 EIA.

[0119] 1.2 Isolation and Purification. Dilutions of donor samples were plated onto isolation media. Colonies were picked from isolation media agar plates (YCFAC, BHI supplemented with vitamin K and hemin, TSA supplemented with 5% sheep blood, BUA OxyPras) into 200 μl of BHI + hemin + vitamin K in a 96-well microtiter plate. After growth was visually observed in the 96-well microtiter plate, 20 μl of culture from each well of the 96-well microtiter plate was transferred to a 96-well deep-well plate containing 1 ml of BHI + hemin + vitamin K and then incubated at 37°C. After visual detection of growth, 1 ml of 50% glycerol was added to each well, and 600 μl of the mixture was transferred into a Thermo Fisher Matrix tube plate. Individual cultures were subsequently plated onto isolation media to ensure uniformity of colony morphology. Colonies were observed after two weeks of incubation at 37°C and were clear in appearance and approximately 0.1 mm in diameter. Individual colonies were picked for identification by 16S sequencing and replated onto BUA OxyPras plates. After colonies were visible and a single morphology was observed, a single colony was inoculated into 6 ml of YCFAC medium. After the liquid culture became turbid, matrix plates were prepared by adding 6 ml of 50% glycerol to the liquid culture and aliquoting 120 μl per matrix tube. Purity was confirmed by plating one of the prepared matrix vials onto a BUA OxyPras plate and testing a single colony by 16S sequencing.

[0120] [Example 2] Taxonomic characterization of isolate P152-H6d 2.1 16S sequencing and phylogenetic analysis Taxonomic characterization of the purified isolate P152-H6d was performed using full-length 16S rRNA gene sequencing data. Homology searches were performed against existing publicly available strains in the National Center for Biotechnology Information (NCBI) taxonomic tree database and the SILVA ribosomal RNA database (Max Planck Institute for Marine Microbiology and Jacobs University, Bremen, Germany).

[0121] 2.1.1 16S rRNA Gene Sequencing. Fifty microliters of liquid culture of isolate P152-H6d was denatured at 95°C for 10 minutes. The denatured sample served as a template for PCR amplification of the 16S gene using 16S rRNA primers 27F (SEQ ID NO: 29) and 1492R (SEQ ID NO: 30). Sanger sequencing (Elim Biopharm, Hayward, CA) was performed using four primers (27F, 1492R, 515F (SEQ ID NO: 31), and 907R (SEQ ID NO: 32)) to identify a nearly full-length 16S rRNA gene fragment (SEQ ID NO: 1). The four amplicons were assembled into a single contiguous sequence using DNAbaser (Heracle BioSoft SRL, Arges, Romania) and then searched against the NCBI database using BLASTn.

[0122] 2.1.2 Phylogenetic Analysis. Database matches spanning the entire P152-H6d contig were selected, and a distant relative of the isolate was selected to serve as an outgroup on the phylogenetic tree. The P152-H6d contig and its close relatives from the NCBI 16S database, including the outgroup, were then searched against the ARB SILVA database using the Alignment (SINA v1.2.11), Classification, and Tree services. For searching and classification, sequences with a minimum identity of 92% (15 complete sequences) were used to classify P152-H6d. RaxML (Randomized Axelerated Maximum Likelihood) was used to perform a maximum likelihood search for phylogenetic tree construction (general time-reversible (GTR+Gamma) model with gamma as the rate model for likelihood).

[0123] A BLASTn search against the 16S rRNA gene database on NCBI yielded the closest match (SEQ ID NO: 1) with 97.85% identity across 100% of the 1442 bp sequence length. This match was from Christensenella timonensis, and the next closest match across the entire length, with 96.6% identity, was from Christensenella massiliensis (Figure 1 and Table 1). Brassicibacter thermophilus (96% identity) was used as an outgroup to root the phylogenetic tree.

[0124] [Table 1]

[0125] The three selected sequences from the NCBI database, along with the P152-H6d 16S contig, were searched against the ARB SILVA database. The closest match was found to be 97.98% identical to Christensenella timonensis. The reference 16S rRNA gene length was 1515 bp (ARB ID: FLKP01000001). The identity was reduced to 94.72% across the SEED alignment.

[0126] A maximum likelihood (ML) phylogenetic tree constructed using the closest neighbors of isolate P152-H6d is shown in Figure 1. It indicates that Christensenella is a monophyletic genus with clear taxonomic delineation among its member species, with each individual Christensenella species clearly clustering into a single phylogenetic group. Based on this 16S rRNA gene fragment analysis, isolate P152-H6d is a member of the genus Christensenella. Because 97.89% identity across the full / partial length of the 16S rRNA gene is insufficient to establish species identity (>98.5%), P152-H6d is a member of a new Christensenella species, and its closest known relative is C. timonensis.

[0127] 2.2 Whole genome sequencing and phylogenetic analysis 2.2.1 Sequencing DNA extraction, sequencing, quality filtering, assembly, and annotation were performed by Corebiome, Inc. (Minneapolis, MN). DNA was extracted from isolate P152-H6d using bead beating with 0.1 mm glass bead plates on a QiaCube (Qiagen) using an automated MO Bio PowerFecal (Qiagen) for large-scale processing. Samples were quantified using the Qiant-iT Picogreen dsDNA Assay (Invitrogen). Libraries were prepared using a proprietary procedure adapted from the Nextera Library Prep kit (Illumina) and sequenced on an Illumina NextSeq using a NextSeq 500 / 550 High Output v2 kit (Illumina) with single-end 1 × 150 reads. DNA sequences were filtered for low quality (Q score < 20) and length (< 50), and adapter sequences were trimmed using cutadapt v.1.15 (Martin, EMBnet Journal, [Sl], v. 17, n. 1, pp. 10-12, (2011)).

[0128] 2.2.2 Assembly and Annotation Sequences were assembled using SPAdes v3.11.0 (Bankevich et al., J Comput Biol. 19(5):455-477 (2012)). Protein annotation was performed using Prokka v1.12 (Seemann, Bioinformatics 30(14):2068-2069 (2014)) across 1,000 base pairs of contigs.

[0129] 2.2.3 Quality Assessment Sequencing quality was determined by examining the quality scores generated by FASTQC, with low-quality bases indicated by a score below 20. Assembly quality metrics were generated by QUAST v.4.5 (Gurevich et al., Bioinformatics 29(8):1072-1075 (2013)).

[0130] 2.2.4 Taxonomy Taxonomic identity was generated using appropriate score cutoffs for average nucleotide identity and alignment fraction scores. These scores were calculated using the Joint Genome Institute Microbial Species Identifier (Varghes et al., Nucleic Acids Research 43(14):6761-6771 (2015)) and an internal reference genome database.

[0131] 2.2.5 Genome Characteristics The intrinsic characteristics of isolate P152-H6d genome assembly were compared to those of the closest Christensenella reference, Christensenella timonensis (accession number NZ_FLKP00000000), and are summarized in Table 2 below.

[0132] [Table 2]

[0133] 2.2.6 Whole genome similarity across P152-H6d and other members of Christensenella. The PI genome was compared to each member species of the genus Christensenella to determine the degree of genome similarity, specifically, average nucleotide identity (ANI) and alignment fraction (AF). The results are summarized in Table 3 below.

[0134] [Table 3]

[0135] 2.2.7 Phylogenetic tree analysis Pairwise ANI values ​​between P152-H6d and all Christensenella genomes on Refseq / NCBI, all NCBI genomes with an ANI >74.18 (the ANI value between P152-H6d and the most divergent Christensenella genome, Christensenella sp. Marseille-P3954), and all genomes in the branch of the web of life containing Christensenella (available on the World Wide Web at biocore.github.io / wol / ) were calculated using FastANI (Jain et al., Nat Commun. 9(1):5114 (2018)) and used to create a distance matrix in phylip format. Phylogenetic relationships among these genomes were inferred using the neighbor-joining method with the R package BionNJ and are shown in Figure 2. Branch lengths are proportional to ANI distances. These results support the 16S rRNA-based phylogenetic analysis and indicate that the P152-H6d isolate represents a new Christensenella species.

[0136] [Example 3] Phenotypic characterization of isolate P152-H6d A summary of the physiological and metabolic characteristics of P152-H6d is provided in Table 4 below.

[0137] [Table 4]

[0138] P152-H6d cells are nonmotile, obligate anaerobes, oxidase-negative, and catalase-positive. Catalase activity is noteworthy in the context of inflammatory bowel disease because reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), contribute to epithelial damage and ion transport dysfunction in IBD (key events in inflammatory diarrhea). However, catalase partially prevents and rescues the loss of ion transport properties in DSS colitis, even in the setting of unresolved tissue inflammation. See Barrett and McCole, Clin Exp Pharmacol Physiol. 43(11):1097-1106 (Nov. 2016).

[0139] P152-H6d was evaluated for its ability to utilize 190 different carbon sources and 95 nitrogen sources, as well as its ability to grow over a wide range of pH, using a phenotypic microarray (Biolog, Hayward, CA). As shown in Table 4, P152-H6d can utilize four carbon sources and three nitrogen sources. Carbon sources include glucose, arabinose, ribose, and α-cyclodextrin. P152-H6d can utilize cysteine ​​as a nitrogen source and showed weak growth in the presence of adenine and parabanic acid as nitrogen sources. P152-H6d was capable of growth over a pH range of 5 to 8, with optimal growth observed at pH 7.

[0140] P152-H6d cells were prepared for electron microscopy imaging. Cells were washed twice with PBS and fixed in 4% paraformaldehyde for 30 minutes at room temperature. Fixed cells were washed twice with PBS and resuspended in sterile water. 25 microliters of the sample was applied to an ITO-coated coverslip, 22 x 22 mm thick, #1, 30-60 ohm resistivity (SPI Supplies, catalog number 06471-AB1), and allowed to air dry. Cells were visualized using a Sigma 500 VP FESEM electron microscope. A representative micrograph of P152-H6d is provided in Figure 3. Cells appear as straight, short rods.

[0141] P152-H6d was also evaluated for its ability to form spores. Using two different sporulation induction methods (i.e., heat shock and chemical shock), P152-H6d was found to be non-sporulating (Table 5). Clostridium butyricum (ATCC 19398) was used as a positive control.

[0142] [Table 5]

[0143] P152-H6d was also evaluated for its production of short-chain fatty acids (SCFAs). SCFAs produced by human gut microbes include butyrate, acetate, and propionate, all three of which have been found to contribute to the maintenance of intestinal homeostasis through multiple mechanisms (Lee and Hase, Nat Chem Biol 10(6):416-424 (2014); Hoeppli et al., Front Immunol 6:61 (2015); Koh et al., Cell 165(6):1332-1345 (2016)). The SCFA production profile of P152-H6d was evaluated after 72 hours of growth in batch culture in YCFAC medium. Uninoculated YCFAC medium was used as a negative control. As seen in Figure 4, P152-H6d produces both butyrate and acetate.

[0144] [Example 4] In vitro functional activity of P152-H6d This example describes studies of the activity of P152-H6d in in vitro human macrophage, monocyte, and dendritic cell models.

[0145] 4.1 Preparation of freshly cultured P152-H6d for cell culture assays. Freshly cultured bacteria from overnight cultures of P152-H6d were prepared under anaerobic conditions. Bacteria were centrifuged at 4300 × g for 4 min. Bacteria were washed once with pre-reduced anaerobic PBS (Gibco). Working stocks were prepared by reducing the washed bacteria to a total surface area of ​​approximately 1 × 10^10 μm² using anaerobic PBS.2 Total surface area = number of particles multiplied by the average surface area (μm) measured by a particle counter (Beckman Coulter Counter). 2 Serial 10-fold dilutions were made using anaerobic PBS for the specific assay.

[0146] 4.2 Human Macrophage and Monocyte In Vitro Cytokine and Chemokine Assays. The THP-1 human monocytic cell line (ATCC catalog no. TIB-202) was cultured at 37°C and 5% CO2 in RPMI 1640 medium containing 2.05 mM L-glutamine (Corning) supplemented with 10% heat-inactivated FBS (Corning), 100 IU / mL penicillin, 100 μg / mL streptomycin, and 0.292 mg / mL L-glutamine (Corning). The number of passages was limited to eight. The THP-1 human monocytic cell line was grown to 70–80% confluence. Cells were counted and resuspended in culture medium. 100,000 cells were plated per well in a 96-well plate. THP-1 human macrophages were generated by culturing THP-1 human monocytic cells with 10 ng / mL phorbol 12-myristate 13-acetate (PMA) (InvivoGen) for 24 hours at 37°C and 5% CO, followed by 20 ng / mL IL-4 (R&D Systems) and 20 ng / mL IL-13 (R&D Systems) for 48 hours (Genin et al., BMC Cancer 15:577 (2015)). One day before the experiment, cells were washed and resuspended in antibiotic-free RPMI medium containing 20 ng / mL IL-4 and 20 ng / mL IL-13.

[0147] Working stocks were prepared for freshly cultured P152-H6d bacteria, anaerobic PBS, 500 ng / ml LPS, and a positive control bacterial strain (known to induce proinflammatory cytokines). Each was added to THP-1 macrophages at 10% v / v and centrifuged at 515 × g for 4 minutes to load onto THP-1 cells. Test or control compounds and THP-1 macrophages were co-incubated for 3 hours at 37°C and 5% CO2. The co-culture medium was replaced with fresh RPMI medium supplemented with antibiotics to limit excessive bacterial growth. THP-1 cells were incubated for 15 hours at 37°C and 5% CO2 after medium replacement. THP-1 cell supernatants were collected and analyzed by ELISA. Levels of CCL-18, IL12-p40, and TNFα in the culture supernatants were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits detecting TMB from Biolegend or R&D Systems according to the manufacturer's specifications.

[0148] E. coli LPS, P152-H6d, and control strains were each evaluated for their ability to induce CCL-18, an M2-macrophage-associated chemokine, in THP-1 macrophages. The induction and polarization of M2 macrophages has previously been reported to be a crucial defense mechanism against inflammatory bowel disease and colonic inflammation (Seo et al., Sci. Rep 7(1):851 (2017); Steinbach et al., Inflamm Bowel Dis. 20(1):166-175 (2014)). CCL-18 is a validated marker of M2 macrophages (Genin et al., BMC Cancer 15:577 (2015)). Figure 5A shows a significant increase in CCL-18 production when P152-H6d was cocultured with THP-1 macrophages compared to PBS, E. coli LPS, and immune-stimulating strain controls. Figure 5B shows the dose-dependent response of CCL-18 production to increasing doses of P152-H6d. In contrast, coculture of THP-1 macrophages with P152-H6d did not significantly induce the proinflammatory cytokines IL12-p40 (Figure 6A) and TNFα (Figure 6B). These data indicate that P152-H6d can increase the production of the anti-inflammatory cytokine CCL-18 but not the production of proinflammatory cytokines from human macrophages, indicating the induction and polarization of anti-inflammatory M2 macrophages.

[0149] 4.3 Human monocyte-derived dendritic cells (moDC) in vitro cytokine assay Cryopreserved PBMCs were thawed in a 37°C water bath, diluted with warm RPMI 1640 supplemented with 10% heat-inactivated FBS and L-glutamine, and centrifuged (515 x g, 4 min). Cells were resuspended in PBS buffer containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA, and CD14+ monocytes were isolated by selection using Miltenyi CD14 microbeads according to the manufacturer's instructions. Isolated CD14+ monocytes were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, L-glutamine, penicillin / streptomycin antibiotics, 50 ng / mL recombinant human IL-4 (R&D Systems), and 100 ng / mL recombinant human GM-CSF (Biolegend). Medium was replenished on days 3 and 6. On day 7 after isolation, cells were cultured at 5 × 10 in RPMI 1640 containing 10% heat-inactivated FBS (Tissue Culture Biologicals) and L-glutamine (Corning) supplemented with 0.292 mg / mL L-glutamine (Corning). 5 Diluted to 5 x 10 cells / mL 5 A 100 μL aliquot of cells / mL cell suspension was added to each well in a flat-well 96-well plate and incubated at 37° C. and 5% CO 2 for 24 hours before addition of the test article.

[0150] The bacterial test material (P152-H6d) was 1×10^8μm 2 and 1×10^7μm 2The plates were prepared to a total surface area of ​​100 μg. Test articles, vehicle (PBS) control, and moDCs were co-incubated for 3 hours at 37°C and 5% CO2. The plates were then centrifuged (515 × g, 4 minutes), the medium was removed, and replaced with RPMI 1640 supplemented with 10% heat-inactivated FBS, L-glutamine, and penicillin / streptomycin antibiotics. The culture plates were then incubated for an additional 15 hours at 37°C and 5% CO2. The plates were centrifuged (515 × g, 4 minutes), and the supernatants were collected and analyzed using a custom U-plex multiplex kit from Meso Scale Discovery according to the manufacturer's instructions. Results were averaged from four human donors, with two replicates from each donor.

[0151] As shown in Figure 7, Christensenella sp. P152-H6d induced a significant dose-dependent increase in the production of (A) IL-10 and (B) IL-1RA by human monocyte-derived dendritic cells (moDCs) compared with the production of these anti-inflammatory cytokines induced by vehicle (PBS).

[0152] 4.4 Human PBMC in vitro cytokine assay Trima retentate blood products containing enriched blood mononuclear cells were obtained from anonymous donors through the Pacific Blood Center (San Francisco, CA) and processed within 24 hours of collection. Blood samples tested negative for HIV, HBV, HCV, HTLV, syphilis, West Nile virus, and Zecavirus. PBMCs were isolated using a Ficoll gradient as previously described (Sim et al., J. Vis. Exp. (112), e54128 2016). Briefly, 50 mL of Trima retentate was diluted with 50 mL of sterile PBS (Gibco) and 25 mL was layered over 15 mL of Ficoll-Paque Plus (GE Healthcare) in a 50 mL conical tube. Samples were centrifuged at 450 × g for 30 minutes at room temperature with the brake off. PBMC interphase was collected, washed with PBS, and resuspended in RPMI 1640 containing 2.05 mM L-glutamine (Corning) supplemented with 10% heat-inactivated FBS (Tissue Culture Biologicals) and 0.292 mg / mL L-glutamine (Corning). Cells were maintained by incubation at 37°C and 5% CO2 and used for assay evaluation within 24 hours or frozen for later use. Cells were collected at 5 x 10 in RPMI 1640 supplemented with 50% FBS and 10% DMSO (Sigma Aldrich). 7 Cells were cryopreserved at a concentration of 1000 cells / mL and stored in liquid nitrogen until ready for use.

[0153] Human PBMCs, used immediately after isolation or thawed from cryopreservation, were cultured at 5 × 10 in RPMI 1640 containing 10% heat-inactivated FBS (Tissue Culture Biologicals) and L-glutamine (Corning) supplemented with 0.292 mg / mL L-glutamine (Corning). 6 Diluted to 5 x 10 cells / mL 6 A 100 μL aliquot of cells / mL cell suspension was added to each well in a round-bottom 96-well plate and incubated at 37° C. and 5% CO 2 for 24 hours before addition of the test article.

[0154] Test articles were prepared and added to PBMCs as described above for the moDC assay. After 3 hours of incubation at 37°C with 5% CO2, the plates containing the co-cultures were centrifuged (515 x g, 4 minutes), the medium was removed, and replaced with RPMI 1640 supplemented with 10% heat-inactivated FBS, L-glutamine, and penicillin / streptomycin antibiotics. The culture plates were then incubated for an additional 15 hours at 37°C and 5% CO2. The plates were centrifuged (515 x g, 4 minutes), and the supernatants were collected and analyzed using a custom U-plex multiplex kit from Meso Scale Discovery according to the manufacturer's instructions. Results were averaged from four human donors, with two replicates from each donor.

[0155] As shown in Figure 7, Christensenella sp. P152-H6d induced significantly increased production of (C) IL-10 and (D) MCP1 by human PBMCs compared with the production of anti-inflammatory cytokines induced by vehicle (PBS).

[0156] 4.5 Crohn's disease (CD) fecal microbiota in human THP-1 macrophage in vitro cytokine assays Christensenella sp. P152-H6d was tested for its ability to regulate the production of the proinflammatory cytokine IL-12p40 in THP-1 macrophages in the presence of fecal microbiota from human subjects with Crohn's disease. Aliquots of a glycerol stock solution containing 50% glycerol:50% CD fecal sample were prepared. A working stock was prepared on the day of the experiment by thawing the glycerol stock under anaerobic conditions, followed by washing with pre-reduced anaerobic PBS and resuspension to an optical density (OD600) of 0.3. This working stock of CD microbiota was added to THP-1 macrophages (2x CD feces or 1x CD feces, v / v) with or without freshly cultured P152-H6d working stock (1x P152-H6d or 0.1x P152-H6d, v / v) or anaerobic PBS control. After 4 hours of co-incubation at 37°C and 5% CO2, THP-1 macrophages were washed and resuspended in RPMI medium supplemented with Pen / Strep to remove excess bacteria. THP-1 macrophages were incubated for 24 hours at 37°C and 5% CO2. THP-1 cell supernatants were collected and analyzed for IL-12p40 using ELISA.

[0157] Figure 8 shows that CD fecal flora alone can significantly induce IL-12p40 in THP-1 macrophages, but the addition of Christensenella sp. P152-H6d attenuated IL-12p40 production in a dose-dependent manner, indicating that Christensenella sp. P152-H6d can reduce or attenuate the induction of IL-12p40 in human macrophages in the presence of CD flora.

[0158] [Example 5] In vivo functional activity of P152-H6d Christensenella sp. P152-H6d was tested for efficacy in five different well-documented mouse models of inflammatory disease: (1) imiquimod (IMQ)-induced psoriasis, (2) oxazolone-induced atopic dermatitis, (3) DSS-induced colitis, (4) Citrobacter rodentium-induced colitis, and (5) TNBS-induced colitis.

[0159] 5.1 Imiquimod (IMQ)-induced psoriasis Psoriasis is an immune-mediated chronic inflammatory skin disorder characterized by scaly, reddened skin lesions and thickening of the affected skin, as well as epidermal and / or dermal changes and histological changes. Topical application of imiquimod (IMQ), a Toll-like receptor 7 / 8 activator, is known to induce psoriasis-like skin inflammation in both humans and mice. See, for example, van der Fits L. et al., "Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL23 / IL17 axis; J Immunology, 2009, 182:5836-5845."

[0160] In this study, BALB / c mice received daily topical application of 5% IMQ cream (47 mg / day) to the dorsal skin (approximate area: 4 cm x 2 cm) for six consecutive days. Test articles were administered once daily from 7 days before through termination and included live purified individual bacterial strains or vehicle (bacterial frozen culture) administered by oral gavage once daily approximately 1-2 hours after IMQ application. Animals receiving the positive control received 0.05% clobetasol cream (62.5 mg / day) applied topically approximately 1 hour after IMQ application. Skin evaluations, including evaluation of dorsal skin thickness using an engineering micrometer, were performed daily starting on day 2.

[0161] As shown in Figure 9, administration of Christensenella sp. P152-H6d and clobetasol, but not bacterial strain X, reduced oxazolone-induced skin thickening compared to the vehicle control.

[0162] 5.2 Oxazolone-induced atopic dermatitis Atopic dermatitis, also known as atopic eczema, is a type of inflammation that results in itchy, red, swollen, and cracked skin that thickens over time. Induction of atopic dermatitis in mice through topical application of oxazolone has previously been reported. See, for example, Hatano et al., 2009, "Maintenance of an acidic stratum corneum prevents the emergence of murine atopic dermatitis." J Invest Dermat 129: 1824-1835; and Ishii et al., 2013, "Antipruritic effect of the topical phosphodiesterase 4 inhibitor E6005 ameliorates skin lesions in a mouse atopic dermatitis model." J Pharmacol Exp Ther 346:105-112.

[0163] In this study, on day 0, BALB / c mice were sensitized with 60 μL of 0.3% oxazolone (Ox) applied topically to the dorsal skin. Starting on day 5, animals received a topical Ox challenge (60 μL, 0.3%) on the back once every two days until termination. Test articles were administered once daily from 7 days before until termination and included live, purified individual bacterial strains or vehicle (bacterial frozen culture) administered by oral gavage once daily. Animals receiving the positive control received topical application of 0.05% clobetasol cream (62.5 mg / day) to the back from days 1 through 21. On the day of challenge, the test article and clobetasol were administered 1–2 hours after oxazolone application. Skin evaluations were performed every other day beginning on day 5 and included evaluation of affected skin for erythema or redness and dorsal scaling according to the following scale:

[0164] Skin erythema or redness None = 0 Slightly red =1 Moderately red = 2 Noticeably red = 3 Extremely red = 4

[0165] skin desquamation None = 0 Slightly scaly = 1 Moderately scaling = 2 Markedly desquamating = 3 Very marked desquamation = 4

[0166] As shown in Figure 10, administration of Christensenella sp. P152-H6d and clobetasol, but not bacterial strain X, reduced the oxazolone-induced skin redness clinical score (A - time course, B - AUC) compared to the vehicle control. Similarly, as shown in Figure 11, administration of Christensenella sp. P152-H6d and clobetasol reduced the oxazolone-induced dorsal skin desquamation clinical score (A - time course, B - AUC) compared to the vehicle control.

[0167] 5.3 DSS-induced colitis Colitis is an inflammation of the lining of the colon that can be caused by infection, inflammatory bowel disease (Crohn's disease and ulcerative colitis), ischemic colitis, allergic reactions, and microscopic colitis. Induction of colitis in mice through administration of dextran sulfate sodium (DSS) has been previously reported. See, e.g., Chassaing et al., Dextran sulfate sodium (DSS)-induced colitis in mice. Curr Protoc Immunol. 2014 Feb 4;104:Unit 15.25.

[0168] In this model, colitis was induced in C57B1 / 6 mice via the addition of 3% DSS to drinking water from days 0 to 5, with the exception of untreated control animals. Test articles were administered once daily from day 1 before to termination and included live purified individual bacterial strains or vehicle (bacterial frozen culture) via oral gavage. Animals receiving the positive control received an antibody against IL-12p40 parenterally once every three days starting on day 6. Animals were dosed at the same time on each dosing day and weighed daily.

[0169] As shown in Figure 12, administration of Christensenella sp. P152-H6d and anti-IL-12p40 antibody, but not bacterial strain 1, reduced DSS-induced weight loss compared to vehicle control. As shown in Figure 13 (left panel), administration of Christensenella sp. P152-H6d (P152-H6d) and anti-IL-12p40 antibody reduced colonic proinflammatory cytokines (A) IL-1β, (B) IL-17A, and (C) TNF-α compared to vehicle administration. For each of these inflammatory cytokines, decreased cytokine levels in response to P152-H6d correlated with reduced % body weight loss (Figure 13, right panel).

[0170] In additional DSS studies, administration of Christensenella sp. P152-H6d and anti-IL-12p40 antibody repeatedly reduced DSS-induced weight loss compared to vehicle controls (Figure 14). On day 13, blood was collected from the animals, plasma was extracted, and plasma levels of known IBD disease activity biomarkers were measured by ELISA. As shown in Figure 15, administration of Christensenella sp. P152-H6d and anti-IL-12p40 antibody reduced plasma levels of (A) G-CSF, (B) lipocalin-2 / NGAL, and (C) serum amyloid A (SAA). Furthermore, after sacrificing the animals on day 13, the colons were collected and fixed in paraformaldehyde. The fixed distal colons were embedded in paraffin, sectioned, and placed on slides for H&E staining. Each sample was given a score ranging from 1 to 5 in the categories of subacute inflammation, colonic gland damage / loss, erosion, thickening, and submucosal edema. A graphical histology score is presented in Figure 15D and represents the sum of distal colon scores for all five categories per mouse. Administration of Christensenella sp. P152-H6d and anti-IL-12p40 antibody significantly reduced the histology score compared to vehicle controls.

[0171] 5.4 Citrobacter rodentium-induced colitis Induction of colitis in mice via infection with the natural mouse pathogen Citrobacter rodentium has been previously reported. See, for example, Koroleva et al., Citrobacter rodentium-induced colitis: A robust model to study mucosal immune responses in the gut. J Immunol Methods. 2015 Jun;421:61-72.

[0172] In this model, C57BL / 6 mice were fasted for 3 hours prior to bacterial infection on day 0. On day 0, mice were administered 10 9 Animals were administered CFU of Citrobacter rodentium (DBS100 ATCC 51459). Body weight was monitored regularly throughout the study, and clinical signs were followed (three times weekly). From days 1 to 14, animals received a single daily PO dose of live, purified individual bacterial strains or vehicle. On day 14, 3 to 4 hours (3.5 hours + / - 30 minutes) after the last dose, all animals were terminated, and colons and plasma were harvested for analysis. Three efficacy endpoints were assessed: body weight loss and colon weight and colon length over the study period. Additionally, colon tissue was evaluated for the production of proinflammatory cytokines, including TNF-α, IFN-γ, IL-1β, and IL-21.

[0173] As shown in Figure 16, administration of Christensenella sp. P152-H6d reduced Citrobacter-induced weight loss compared to the vehicle control and bacterial strain Y. A similar effect of Christensenella sp. P152-H6d was observed on colon weight and length, with the colon weight and length of mice treated with bacterial strain Y being similar to those of vehicle-treated mice (Figure 17). As shown in Figure 18, administration of Christensenella sp. P152-H6d (P152-H6d) reduced colonic proinflammatory cytokines (A) IFN-γ, (B) IL-1β, (C) IL-21, and (D) TNF-α compared to vehicle and bacterial strain Y administration.

[0174] Lipocalin-2 / NGAL, a disease activity biomarker for inflammatory bowel disease (Stallhofer et al., Inflamm Bowel Dis 21(10):2327-2340 (2015)), was assessed in plasma samples. Administration of Christensenella sp. P152-H6d reduced plasma levels of lipocalin-2 compared to vehicle control and bacterial strain Y (Figure 19).

[0175] 5.5 TNBS-induced colitis Induction of colitis in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) has been previously reported. See, e.g., Antoniou et al., The TNBS-induced colitis animal model: An overview. Ann Med Surg (Lond) 11: 9-15 (Nov. 2016).

[0176] In this model, colitis was induced in C57B1 / 6 mice on day 0 via rectal administration of 5 mg of TNBS plus 50% ethanol, except for untreated control animals. Vehicle or bacterial test substances (Christensenella sp. P152-H6d, Anaerostipes cacae, and a combination of Christensenella sp. P152-H6d and Anaerostipes cacae, respectively) were administered ad libitum from 3 days before through day 3. Body weight change was assessed for each mouse as the % change from day 0 weight. After sacrificing the animals on day 3, the colons were collected and fixed in paraformaldehyde. The fixed distal colons were embedded in paraffin, sectioned, and placed on slides for H&E staining. Each specimen was given a score of 1 to 5 in the categories of subacute inflammation, colonic gland damage / loss, erosion, thickening, and submucosal edema. Body weight changes are presented in Figure 20A and histology scores are presented in Figure 20B (sum of distal colon scores for all five categories per mouse).

[0177] As shown in Figure 20A, administration of Christensenella sp. P152-H6d alone significantly reduced body weight loss compared to the vehicle control. In contrast, administration of Anaerostipes cacae alone did not reduce body weight loss compared to the vehicle control (a slight enhancement of body weight loss was observed). However, administration of the combination of Christensenella sp. P152-H6d and Anaerostipes cacae resulted in the greatest reduction in body weight loss compared to the vehicle control, surpassing the reduction in body weight loss achieved by Christensenella sp. P152-H6d alone. In light of the effect of Anaerostipes cacae alone in enhancing weight loss, the combined effect of Anaerostipes cacae and Christensenella sp. P152-H6d to significantly reduce weight loss compared to vehicle and Christensenella sp. P152-H6d alone was unexpected, suggesting that Anaerostipes cacae and Christensenella sp. P152-H6d may act synergistically to modulate colitis-related disease activity.

[0178] As shown in Figure 20B, administration of Christensenella sp. P152-H6d alone, and to a lesser extent, administration of Anaerostipes cacae alone, reduced histology scores compared to the vehicle control. However, administration of the combination of Christensenella sp. P152-H6d and Anaerostipes cacae resulted in the greatest reduction in histology scores compared to the vehicle control, significantly exceeding the reduction in histology scores achieved by Christensenella sp. P152-H6d alone or Anaerostipes cacae alone. These results also suggest that Anaerostipes cacae and Christensenella sp. P152-H6d can act synergistically to modulate colitis-related disease activity.

[0179] [Example 6] In vitro functional activity of Christensenella sp. P152-H6d in combination with Anaerostipes cacae To further evaluate the synergistic activity of the combination of Christensenella sp. P152-H6d and Anaerostipes cacae in modulating the above-mentioned TNBS-induced colitis, the combination was tested against each strain individually for its ability to modulate the production of inflammatory cytokines in THP-1 macrophages.

[0180] Working stock solutions were prepared for Christensenella sp. P152-H6d, Anaerostipes cacae, a combination of Christensenella sp. P152-H6d and Anaerostipes cacae, or an anaerobic PBS control. Individual bacterial test substances were added to THP-1 macrophages at 2x and 1x doses, respectively, for Christensenella sp. P152-H6d and Anaerostipes cacae, and combinations were added at 1x + 1x or 0.5x + 0.5x doses, respectively. After 4 hours of co-incubation at 37°C and 5% CO2, THP-1 macrophages were washed and resuspended in RPMI medium supplemented with Pen / Strep to remove excess bacteria. THP-1 macrophages were incubated for 24 hours at 37°C and 5% CO2. THP-1 cell supernatants were collected and analyzed for IL-1β, IL-12p40, and TNF-α using ELISA.

[0181] Figure 21 shows that Anaerostipes cacae alone significantly induced the proinflammatory cytokines IL-1β (A), IL-12p40 (B), and TNF-α (C) from THP-1 macrophages, whereas the addition of Christensenella sp. P152-H6d significantly attenuated the production of each of these cytokines compared to Anaerostipes cacae alone, and the effect was dose-dependent.

[0182] [Example 7] Targeted PCR screening for additional strains of Christensenella sp. P152-H6d 7.1 Fecal Source A fecal sample from a healthy human donor was used for the isolation of the new strain of Christensenella sp. P152-H6d. The donor underwent extensive clinical and laboratory testing as described in Example 1 to confirm their health status.

[0183] 7.2 Fecal Sample Selection. A Christensenella sp. P152-H6d species-specific primer pair was designed against the relA gene of P152-H6d (SEQ ID NO: 33) and selected due to its relatively low homology to the relA genes of three other members of the Christensenella genus: C. massiliensis (SEQ ID NO: 34), C. minuta (SEQ ID NO: 35), and C. timonensis (SEQ ID NO: 36). Sequence alignment of the four relA sequences was performed using Clustal Omega, and primers were designed from regions of the Christensenella sp. P152-H6d relA gene with multiple mismatches to the other three Christensenella species. As shown in Table 6, the relA gene from Christensenella sp. P152-H6d was 79–85% identical to the genes from C. massiliensis, C. minuta, and C. timonensis.

[0184] [Table 6]

[0185] Two primer pairs, Ch_relA_1 and Ch_relA_2, were designed for real-time PCR screening of fecal samples from healthy donors to identify samples for the isolation of additional Christensenella species strains. Briefly, genomic DNA was isolated from feces using a commercially available kit (Qiagen PowerSoil Pro) and used as a template in real-time PCR amplification (20 μl reaction volume, SYBR Green detection). Genomic DNA isolated from Christensenella sp. P152-H6d was used as a positive control (1 e5 copies / reaction). The positive fecal sample with the lowest threshold cycle for both primer pairs was selected and used in the isolation of additional Christensenella sp. P152-H6d strains. Dilutions of the positive fecal sample were plated on isolation medium, and colonies were picked from the isolation medium agar plate into 96-well microtiter plates containing 200 ml of liquid medium. After growth was visually observed in the 96-well microtiter plates, 10 μl of culture medium was removed from each well and processed for PCR-based screening.

[0186] 7.3 PCR Screening Assay. A third relA primer pair, Ch_relA_AA_2, was designed for PCR screening of isolates of Christensenella sp. P152-H6d to identify them. The Ch_relA_AA_2 amplicon is larger (589 bp) than the two relA real-time PCR amplicons (105 and 121 bp) and was designed for easier detection on a 2% agarose gel. Primer specificity was tested using 3 μl of culture medium or gDNA (approximately 1 e5 copies) from Christensenella sp. P152-H6d, C. massiliensis DSM 102344, C. minuta DSM 22607, and C. timonensis DSM 102800, respectively. To enhance the robustness of the screen, a genus-specific primer pair (Ch_Fred) was designed from a conserved region within the NAD(P)H flavin reductase gene (Fred, SEQ ID NOs: 37–40). Confirmation of the specificity of the Christensenella sp. P152-H6d species-specific primer and the Christensenella genus-specific primer is shown in Figure 22A (Ch = C. sp. P152-H6d, Cmi = C. minuta, Cma = C. massiliensis, and Ct = C. timonensis). PCR screening was performed in a 96-well PCR plate using 3 μl of culture medium as template in a 25 μl PCR reaction (Phusion master mix, NEB, 40 cycles). To identify positive isolates, 15 μl of the PCR reaction was run on a 2% agarose gel along with controls. All isolates were further purified as follows.

[0187] 7.4 Purification and Confirmation by 16S Sequencing. 20 ml of culture from PCR-positive wells of a 96-well microtiter plate was transferred to a 96-well deep-well plate containing 1 ml of liquid medium and subsequently incubated at 37°C. After visual detection of growth, 1 ml of 50% glycerol was added to each well, and 600 μl of the mixture was transferred to a Thermo Fisher Matrix tube plate. Individual cultures were subsequently plated onto isolation media for colony morphology uniformity. Colonies were observed after 2 weeks of incubation at 37°C and were clear in appearance and approximately 0.1 mm in diameter. Individual colonies were picked for identification by 16S sequencing and replated onto agar plates. After colonies were visible and a single morphology was observed, a single colony was plated into 6 ml of YCFAC medium. After the liquid culture became turbid, 6 ml of 50% glycerol was added to the liquid culture and matrix plates were prepared by aliquoting 120 μl per matrix tube. Purity was confirmed by plating agar plates from one of the prepared matrix vials and testing single colonies by species- and genus-specific PCR (Figure 22B) and 16S rDNA Sanger sequencing. Four new isolates, P235-A1a, P235-A3a, P237-A7a, and P237-B12a, were identified, all with 100% 16S rDNA sequence identity to Christensenella sp. P152-H6d (SEQ ID NOS: 41-44, Table 7).

[0188] [Table 7]

[0189] Incorporation by Reference The entire disclosure of each patent and scientific document referred to herein is incorporated by reference for all purposes.

[0190] equivalent The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the foregoing embodiments are to be considered in all respects illustrative rather than limiting of the disclosure set forth herein. The scope of the present disclosure is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalents of the claims are intended to be embraced therein.

Claims

1. 1. A composition comprising: A bacterial strain of the same species as Christensenella sp. P152-H6d deposited under accession number DSM33237, which comprises the 16s rRNA gene sequence of SEQ ID NO: 1; and Excipients, diluents, and / or carriers wherein the bacterial strain is freeze-dried or spray-dried.

2. The composition of claim 1, wherein the bacterial strain shares at least 70% DNA-DNA hybridization with Christensenella sp. P152-H6d deposited under accession number DSM33237.

3. The composition of claim 1 or 2, wherein the bacterial strain comprises a genome having at least 95% average nucleotide identity (ANI) with the genome of Christensenella sp. P152-H6d deposited under accession number DSM33237.

4. 4. The composition of any one of claims 1 to 3, wherein the bacterial strain comprises a genome having an average nucleotide identity (ANI) of at least 96.5% and an alignment fraction (AF) of at least 60% with the genome of Christensenella sp. P152-H6d deposited under accession number DSM33237.

5. 5. The composition of any one of claims 1 to 4, wherein the bacterial strain is Christensenella sp. P152-H6d deposited under accession number DSM33237.

6. 6. The composition of any one of claims 1 to 5, wherein the bacterial strain is capable of increasing the production of an anti-inflammatory gene product by human cells.

7. The composition of claim 6, wherein the anti-inflammatory gene product is selected from the group consisting of CCL-18, IL-10, IL-1RA, and MCP-1.

8. 8. The composition of any one of claims 1 to 7, wherein the bacterial strain is capable of reducing or attenuating the production of inflammatory gene products by human cells.

9. 9. The composition of claim 8, wherein the inflammatory gene product is selected from the group consisting of IL12-p40, IL-1β, IL-17A, IL-21, IFN-γ, and TNF-α.

10. 10. The composition of any one of claims 6 to 9, wherein the human cells are selected from the group consisting of THP-1 macrophages, moDCs, and PBMCs.

11. 11. The composition of any one of claims 1 to 10, wherein the bacterial strain is capable of reducing or attenuating the production of one or more serum biomarkers selected from the group consisting of lipocalin-2 / NGAL, serum amyloid A (SAA), and granulocyte colony-stimulating factor (G-CSF) in a cell, tissue, or subject.

12. 12. The composition of any one of claims 1 to 11, further comprising one or more additional bacterial strains.

13. 13. The composition of claim 12, wherein the one or more additional bacterial strains comprise a strain of Anaerostipes caccae.

14. 14. The composition of claim 13, wherein the Anaerostipes cacae strain is Anaerostipes cacae strain P127-A10a deposited under accession number DSM 33531.

15. 1. A pharmaceutical composition comprising: A bacterial strain mixture comprising a bacterial strain of the same species as Christensenella sp. P152-H6d deposited under accession number DSM33237, the bacterial strain comprising a 16s rRNA gene sequence of SEQ ID NO: 1, wherein each bacterial strain in the bacterial strain mixture is in lyophilized form, and the pharmaceutical composition contains at least 1 x 10 8 a bacterial strain mixture having viable bacterial organisms; and pharmaceutically acceptable excipients A pharmaceutical composition comprising:

16. 16. The pharmaceutical composition of claim 15, wherein the bacterial strain mixture further comprises a bacterial strain of the species Anaerostipes cacae.

17. 17. The composition of any one of claims 1 to 16, formulated as an enteric formulation.

18. 17. The composition of any one of claims 1 to 16, formulated as a capsule, tablet, caplet, pill, troche, lozenge, powder, or granule.

19. 17. The composition of any one of claims 1 to 16, formulated as a suppository, suspension, emulsion, or gel.

20. At least 1 x 10 3 20. The composition of any one of claims 1 to 19, comprising a CFU of a bacterial strain.

21. 21. The composition of any one of claims 1 to 20, comprising a therapeutically effective amount of the bacterial strain sufficient to prevent or treat the disorder when administered to a subject.

22. 22. The composition of claim 21, wherein the disorder is selected from the group consisting of inflammatory disorders, gastrointestinal disorders, inflammatory bowel disease, cancer, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, insulin deficiency, insulin resistance-related disorders, impaired glucose tolerance, pre-diabetes, diabetes, high body mass index (BMI), excess fat, obesity, overweight, cardiovascular disease, atherosclerosis, hyperlipidemia, hyperglycemia, dyslipidemia, and hypertension.

23. 23. The composition of claim 22, wherein the gastrointestinal disorder is selected from the group consisting of ulcerative colitis, Crohn's disease, and irritable bowel syndrome.

24. 24. The composition of any one of claims 1 to 23, wherein the excipient is selected from the group consisting of a filler, a binder, a disintegrant, and any combination thereof.

25. 24. The composition of any one of claims 1 to 23, wherein the excipient is selected from the group consisting of cellulose, polyvinylpyrrolidone, silicon dioxide, stearyl fumarate or a pharmaceutically acceptable salt thereof, and any combination thereof.

26. 26. The composition of any one of claims 1 to 25, further comprising a cryoprotectant.

27. 27. The composition of claim 26, wherein the cryoprotectant is selected from the group consisting of fructooligosaccharides, trehalose, and combinations thereof.

28. 28. The composition of any one of claims 1 to 27 for bolus administration or bolus release.

29. 29. The composition of any one of claims 1 to 28, wherein the bacterial strain is capable of at least partially colonizing the intestine of a human subject.

30. 30. The composition of any one of claims 1 to 18 and 20 to 29 for oral delivery to a subject.

31. 31. The composition of any one of claims 1 to 30, wherein the bacterial strain is viable.

32. 32. The composition of any one of claims 1 to 31, comprising at least one or more additional bacterial strains.

33. 33. A food product comprising a composition according to any one of claims 1 to 18 and 20 to 32.

34. 33. The composition of any one of claims 1 to 32 for treating dysbiosis in a subject, wherein a therapeutically effective amount of the composition is administered to the subject.

35. 33. The composition of any one of claims 1 to 32 for modifying the gut microbiome in a subject, wherein a therapeutically effective amount of the composition is administered to the subject.

36. 33. The composition of any one of claims 1 to 32 for treating a gastrointestinal disorder in a subject, wherein a therapeutically effective amount of the composition is administered to the subject.

37. 37. The composition of claim 36, wherein the gastrointestinal disorder is ulcerative colitis (UC), Crohn's disease, or irritable bowel syndrome.

38. 33. The composition of any one of claims 1 to 32 for treating an inflammatory disorder in a subject, wherein a therapeutically effective amount of the composition is administered to the subject.

39. 25. The composition of any one of claims 1 to 24 for treating a skin disorder in a subject, wherein a therapeutically effective amount of the composition is administered to the subject.

40. 40. The composition of claim 39, wherein the skin disorder is selected from the group consisting of psoriasis, eczema, dermatitis, and acne.

41. 33. The composition of any one of claims 1 to 32 for treating non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, insulin deficiency, insulin resistance-related disorders, impaired glucose tolerance, pre-diabetes, diabetes, high body mass index (BMI), excess fat, obesity, overweight, cardiovascular disease, atherosclerosis, hyperlipidemia, hyperglycemia, dyslipidemia, or hypertension in a subject, wherein a therapeutically effective amount of the composition is administered to the subject.

42. 42. The composition of any one of claims 34 to 41, wherein a prebiotic is further administered to the subject.

43. 43. The composition of any one of claims 34 to 42, wherein the subject is selected from the group consisting of humans, companion animals, and livestock animals.

44. 41. The composition of claim 40, wherein the eczema and dermatitis comprises one or more of eczematous dermatitis, atopic and seborrheic dermatitis, allergic or irritant contact dermatitis, fissure eczema, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis, radiation dermatitis, and stasis dermatitis.

Images