Methods and compositions for treating gastrointestinal and inflammatory disorders

Abstract

The present disclosure generally relates to bacterial strains and bacterial strain mixtures, such as Eubacterium, Bacteroides and Roseburia bacterial strains and mixtures thereof. The present disclosure further relates to methods of using bacterial strains and bacterial strain mixtures to treat gastrointestinal disorders (e.g., ulcerative colitis, inflammatory disorders and / or dysbiosis).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 62 / 730,792 filed on September 13, 2018, U.S. Provisional Patent Application Serial No. 62 / 734,495 filed on September 21, 2018, U.S. Provisional Patent Application Serial No. 62 / 747,391 filed on October 18, 2018, and U.S. Provisional Patent Application Serial No. 62 / 782,666 filed on December 20, 2018.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The name of this ASCII copy created on December 19, 2018 is ASP-056_SL.txt and the size is 17,078,089 bytes. Background Art

[0005] The gastrointestinal (GI) tract and other organ systems are complex biological systems that include communities of many different organisms, including different bacterial strains. Hundreds of different species may form symbiotic communities in the GI tract and other organs of a healthy person. Furthermore, the microorganisms present in the gut not only play a vital role in digestive health, but also influence the immune system.

[0006] Perturbations or imbalances in biological systems, such as the gastrointestinal tract, may include changes in the types and numbers of bacteria in the gut, which may lead to the development of a disease state or may be an indicator of a disease state. Patients with certain diseases, such as gastrointestinal disorders, may have a different microbiota profile than healthy people. For example, ulcerative colitis (UC) is associated with alterations and imbalances in the structure of the colonic microbial community (dysbiosis), with reduced bacterial species diversity and stability over time. In such dysbiotic environments, microbiota function may be altered, leading to increased susceptibility to pathogens, changes in metabolic profiles, or the induction of pro-inflammatory signals that can lead to local or systemic inflammation or autoimmunity.

[0007] Ulcerative colitis (UC) is an inflammatory bowel disease (IBD), an idiopathic chronic inflammatory disorder affecting the colon. It is characterized by recurrence and remission of varying degrees of mucosal inflammation starting from the rectum and reaching the proximal colon. Drugs used to treat UC include mesalazine, corticosteroids, oral immunosuppressive drugs, and monoclonal antibodies against TNF-α and α4β7 lymphocyte integrins. Aminosalicylates (mesalazine, 5-ASA) are standard treatments for mild to moderately active UC. However, a large number of mild to moderately active UC patients do not respond adequately to first-line treatment with mesalazine alone, so it is necessary to introduce more toxic immunosuppressive drugs. For example, systemic immunosuppressants (including biological drugs) are commonly used to treat moderate to severe UC patients who do not respond adequately to treatment with mesalazine (5-ASA) alone. Up to 15% of UC patients require colectomy (Ungaro et al., Lancet 389 (10080): 1756-1770 (2017)).

[0008] A significant number of patients suffering from gastrointestinal disorders fail to respond adequately to first-line treatments, highlighting the need in the art for pharmaceutical compositions and methods that can effectively treat this subset of patients. Summary of the invention

[0009] The present disclosure generally relates to bacterial strains and bacterial strain mixtures, such as Eubacterium, Bacteroides and Roseburia bacterial strains and mixtures thereof. The disclosed bacterial strains or bacterial strain mixtures can be used to treat gastrointestinal disorders and / or inflammatory disorders, including, for example, dysbiosis and / or immune-mediated inflammatory disorders, such as ulcerative colitis, Crohn's disease and other forms of inflammatory bowel disease (IBD).

[0010] For example, in one aspect, a pharmaceutical composition is provided herein, comprising: a bacterial mixture comprising at least two bacterial species, each of which is selected from: Bacteroides species, Eubacterium species and Roseburia species; and a pharmaceutically acceptable excipient. In certain embodiments, the bacterial mixture considered comprises Bacteroides species, Eubacterium species and Roseburia species. In certain embodiments, the bacterial mixture considered is essentially composed of Bacteroides species, Eubacterium species and Roseburia species. In certain embodiments, the Bacteroides species in the bacterial mixture is Bacteroides thetaiotaomicron. In certain embodiments, the Eubacterium species in the bacterial mixture is Eubacterium hallii. In certain embodiments, the Roseburia species in the bacterial mixture is Roseburia hominis. In certain embodiments, the bacterial mixture considered is in the form of freeze-dried powder. In certain embodiments, the disclosed bacterial mixture can increase the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or PBMCs). In certain embodiments, the disclosed bacterial mixture can reduce or attenuate the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17, and / or TNF-α) in human cells (e.g., THP macrophages or PBMC cells). In certain embodiments, the disclosed bacterial mixture can reduce or prevent or increase the barrier integrity of a human cell (e.g., epithelial cell) monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α). In certain embodiments, the disclosed bacterial mixture can induce or increase autophagy in human cells (e.g., THP-1 macrophages). In certain embodiments, the disclosed bacterial mixture can reduce or attenuate the production of lipocalin-2 / NGAL in cells, tissues, or subjects.

[0011] In another aspect, provided herein is a method of treating a gastrointestinal disorder in a subject in need thereof. The contemplated method comprises administering to the subject an effective amount of a disclosed pharmaceutical composition, pharmaceutical unit, or bacterial strain.

[0012] In some embodiments, the method comprises administering to the subject at least two bacterial strains, each bacterial strain being selected from a Bacteroides species strain, a Eubacterium species strain, and a Roseburia species strain. In certain embodiments, each bacterial strain is in the form of a freeze-dried powder.

[0013] Contemplated gastrointestinal disorders include, for example, dysbiosis and / or immune-mediated inflammatory disorders, such as ulcerative colitis, Crohn's disease, and other forms of inflammatory bowel disease (IBD).

[0014] In another aspect, provided herein is a method for treating ulcerative colitis (UC) in a subject in need thereof. The contemplated method comprises administering to the subject an effective amount of a disclosed pharmaceutical composition, a pharmaceutical unit, or a bacterial strain. The contemplated subject may have had an inadequate response to administration of a previous UC treatment (e.g., mesalamine). For example, the contemplated subject may have previously taken ≥2.4 g / day of mesalamine orally for at least 8 weeks.

[0015] In another aspect, provided herein is a method of treating dysbiosis in a subject in need thereof. The contemplated method comprises administering to the subject an effective amount of a disclosed pharmaceutical composition, pharmaceutical unit, or bacterial strain. In certain embodiments, the subject also suffers from a gastrointestinal disorder.

[0016] In another aspect, the present invention provides a method for altering the intestinal microbiome of a subject in need thereof. The contemplated method comprises administering to the subject an effective amount of a disclosed pharmaceutical composition, pharmaceutical unit, or bacterial strain. In certain embodiments, the subject also suffers from a gastrointestinal disorder.

[0017] In another aspect, provided herein is a method of treating an inflammatory disorder in a subject in need thereof. The contemplated method comprises administering to the subject an effective amount of a disclosed pharmaceutical composition, pharmaceutical unit, or one or more bacterial strains.

[0018] Contemplated methods may further include, for example, administering a corticosteroid to the subject, pre-treating the subject with an antibiotic, administering an antibiotic to the subject, or administering a prebiotic to the subject.

[0019] These and other aspects and features of the disclosure are described in the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present disclosure may be more fully understood with reference to the following drawings.

[0021] Figure 1 Short-chain fatty acid (SCFA) production profiles of individual and mixed consortium batch cultures of Consortium 1 (C1) strains are depicted. The levels of lactate, acetate, propionate, and butyrate in the supernatants of batch cultures of individual C1 strains and mixed consortiums of all three strains were analyzed by HPLC (ABPDU Berkeley CA). The average amounts of SCFA detected from two independent experiments for individual strains BTH01 (A), EHA01 (B), and RHO01 (C) and mixed consortiums of all three strains (D) are shown.

[0022] Figure 2The abundance of each C1 strain in individual and mixed-biota fed-batch cultures is depicted. C1 strains were grown in replicates either alone or in mixed-biota. Samples were taken at the indicated time points and a portion of the culture was transferred to fresh medium for fed-batch culture. The abundance of each strain, as represented by the number of copies / ml sample, was determined by species-specific qPCR for strains grown alone (A) and in mixed-biota (B).

[0023] Figure 3 Cross-feeding between EHA01 and BTH01 is depicted. EHA01 and BTH01 strains were grown alone or co-cultured with starch added as the sole carbon source in the growth medium. After 48 hours of batch culture, the copy number of each strain was determined by species-specific qPCR (A), and the production of butyrate in the spent medium of each culture was analyzed using HPLC (B). In another experiment, the EHA01 strain was grown in Peptone-yeast extract (PY) growth medium without any carbon source, PY medium with starch added (PY starch), and PY medium with starch and 50% filter-sterilized spent medium (from a BTH01 culture previously grown for 48 hours). The OD of each of the three cultures is shown as (C).

[0024] Figure 4 Effects of freshly cultured C1 strain and mixed flora on HT29-MTX-E12 barrier integrity are depicted. Percent change in TEER (ohms x cm) between 0 and 24 hours after addition of TNF-α to the basal compartment is plotted for 10% v / v freshly cultured mixed flora of (A) BTH01, EHA01, and RHO01, (B) EHA01, (C) RHO01, (D) BTH01, and (E) a non-protective unrelated bacterial strain negative control. 2 ). PBS control was set as baseline. Each test subject was evaluated in 6 replicates, and the results are representative of at least two independent experiments. *p value < 0.05, student t-test.

[0025] Figure 5The effects of newly cultured C1 strains and mixed flora on human THP-1 macrophage and PBMC IL-10 are depicted. THP-1 macrophage supernatants were collected after the assay was completed, and (A) the IL-10 concentration in the culture supernatant was plotted for 10%, 3.3% and 1.0% v / v working solutions of each individual strain (BTH01, EHA01 and RHO01), mixed flora of the three strains, or PBS (LPS only) control. Each test subject was evaluated in 4 replicates, and the results represent at least two independent experiments. Primary human PBMC supernatants were collected after the assay was completed, and (B) the IL-10 concentration in the culture supernatant from PBMC donor 1 and PBMC donor 2 was plotted for 10% or 1% v / v working solutions of individual strains or mixed flora. Each test subject was evaluated in 4 replicates, and the results represent at least two independent experiments. *p value <0.05, student t-test.

[0026] Figure 6 The effects of freshly cultured C1 strains and mixed flora on CCL-18 in human THP-1 macrophages are depicted. THP-1 macrophage supernatants were collected after the assay was completed, and CCL-18 concentrations in the culture supernatants were plotted for 10%, 3.3%, and 1.0% v / v working solutions of each individual strain (BTH01, EHA01, and RHO01), a mixed flora of the three strains, or a PBS control. Each test subject was evaluated in 4 replicates, and the results are representative of at least two independent experiments. *p value <0.05 student t-test.

[0027] Figure 7 Effects of freeze-dried C1 strain and mixed flora on HT29-MTX-E12 barrier integrity are depicted. Percent change in TEER (ohms x cm) between 0 and 24 hours after addition of TNF-α to the basal compartment is plotted for 10% v / v working solutions of freeze-dried (A) mixed flora of BTH01, EHA01, and RHO01, (B) EHA01, (C) RHO01, (D) BTH01, and (E) a non-protective negative control of an unrelated bacterial strain. 2 ). PBS control was set as baseline. Each test subject was evaluated in 6 replicates, and the results are representative of at least two independent experiments. *p value < 0.05 student t-test.

[0028] Figure 8Depict the in vitro effects of freeze-dried individual C1 bacterial strains or mixed flora on the production of IL-10 cytokines and CCL-18 chemokines in the THP-1 human macrophage model and the production of IL-10 in the human PBMC model. THP-1 macrophage supernatants were collected at the completion of the assay, and (A) the concentration of IL-10 in the culture supernatant was plotted for each individual freeze-dried strain (BTH01, EHA01, and RHO01), a mixed flora of the three strains, or a PBS (LPS only) control at 10%, 3.3%, and 1.0% v / v working solution. Primary human PBMC supernatants were collected at the completion of the assay, and (B) the IL-10 concentration in the culture supernatant from PBMC donor 1 and PBMC donor 2 was plotted for 10% or 1% v / v working solutions of individual freeze-dried strains or mixed flora. THP-1 macrophage supernatants were collected at the end of the assay and (C) CCL-18 concentrations in culture supernatants were plotted against 10% and 1.0% v / v working solutions of each individual freeze-dried strain (BTH01, EHA01, and RHO01), a mixed population of the three strains, or a PBS (LPS only) control. Each test subject was evaluated in 4 replicates and the results are representative of at least two independent experiments.

[0029] Fig. 9 Depicted are the effects of freshly cultured C1 mixed flora in the presence of UC fecal microbiota in an in vitro HT29-MTX-E12 human epithelial barrier assay and an in vitro cytokine assay in human THP-1 macrophages. (A) Percent change in TEER (ohms x cm) between time 0 and 24 hours is plotted for the indicated test subjects. 2 ). PBS control was set to relative maximum. Each test subject was evaluated in 6 replicates, and the results are representative of at least two independent experiments. (B) THP-1 macrophage supernatants were collected at the end of the assay, and IL-10 concentrations in culture supernatants were plotted against PBS control and UC fecal microbiota with specified v / v amounts of C1 mixed flora added. Each test subject was evaluated in 4 replicates, and the results are representative of at least two independent experiments. *p value <0.05 student t-test.

[0030] Fig.10Depicted are the in vitro effects of individual C1 bacterial strains or mixed flora (2- and 3-strains) on induction of autophagy and IL-10 cytokine production in a THP-1 human macrophage model. THP-1 human macrophages and supernatants were collected, processed, and analyzed after incubation with 10% v / v working solutions of each test subject. (A) Mean fluorescence intensity corresponding to the degree of autophagy induction in THP-1M2 macrophages is shown for each individual freeze-dried strain (EHA01, RHO01, and BTH01), paired combinations of the three strains (EHA01+RHO01, RHO01+BTH01, and EHA01+BTH01), mixed flora of the three strains together, or PBS (LPS only) control. The PBS control was set as the baseline. Each test subject was evaluated in 4 replicates, and the results are representative of at least two independent experiments. (B) IL-10 concentrations in culture supernatants from THP-1M1 macrophages are shown for each individual freeze-dried strain (EHA01, RHO01, and BTH01), pairwise combinations of the three strains (EHA01+RHO01, RHO01+BTH01, and EHA01+BTH01), a mixed population of the three strains together, or a PBS (LPS only) control. Each test subject was evaluated in 4 replicates, and the results are representative of at least two independent experiments.

[0031] Fig.11 The effects of individual strains of C1 on the percent body weight change in a DSS-induced colitis mouse model are depicted. Groups of mice were used in a standard DSS-induced colitis model, including controls (N=5), test subjects (N=12), and vehicle controls (N=20). Mice were treated with 3% DSS from days 0-5. From day 1 to day 21, mice were treated with 1×10 of each of the three C1 bacterial strains daily. 9 Each group of mice was treated with an oral dose of VCC or a negative control bacterial strain that did not show in vitro activity. Each group of mice was administered PBS as a vehicle control, or an antibody against IL-12p40 (Q3D IP) was administered as a positive control for efficacy. The daily weight change over time (d0-21) of each test group is shown together with control mice (black dashed line), vehicle control (grey, circle) and anti-IL-12p40 (black dashed line, square). (A) EHA01, (B) RHO01, (C) BTH01, and (D) negative control microorganisms. Error bars are shown as SEM.

[0032] Fig.12Depicted are cytokine levels in colonic tissue at day 21 of DSS-induced colitis. Cytokine levels were assessed by ELISA after homogenization of colonic tissue in lysis buffer. Levels were normalized to mg of tissue analyzed. Each test subject was assessed in N=12 animals. *p value < 0.05; **p value < 0.001; ***p value < 0.005; Mann-Whitney nonparametric test.

[0033] Fig.13 The effects of C1 bacterial mixed flora and individual strains on the percent body weight change in a DSS-induced colitis mouse model are depicted. Groups of mice were used in a standard DSS-induced colitis model, including controls (N=5), individual test subjects (N=12), mixed flora (N=24), mixed flora plus anti-IL-12p40 antibody (N=12), and vehicle control (N=20). Mice were treated with 3% DSS in drinking water from days 0-5. From days 1-19, each group of mice received the following treatments daily: oral doses of 3×10 9 Individual C1 bacterial strains of VCC, 1 × 10 of each strain 9 VCC C1 mixed flora or contains 1×10 9 Individual strains of VCC and C1 mixed flora of anti-IL-12p40 antibody (10 mg / kg in PBS, administered IP Q3D from day 6). Groups of mice were administered PBS as a vehicle negative control or anti-IL-12p40 antibody alone (Q3D IP) as a positive control to observe efficacy. Daily body weight changes over time for each test group (d0-l9) are shown together with control mice (black dashed line), vehicle control (grey, circles) and anti-IL-12p40 (black dashed line, squares). (A) EHA01, (B) RHO01, (C) BTH01, (D) C1 and (E) C1 + anti-IL-12p40. (F) AUC analysis of C1 + anti-IL-12p40. Error bars are shown as SEM.

[0034] Fig.14 Depicted are cytokine levels in colonic tissues at day 19 of DSS-induced colitis. Cytokine levels were assessed by ELISA after homogenization of colonic tissues in lysis buffer. Levels were normalized to mg of tissue analyzed. *p value < 0.05; **p value < 0.001; Mann-Whitney nonparametric test.

[0035] Fig.15Figure 20 shows the levels of lipocalin-2 / NGAL in plasma at day 19 of DSS-induced colitis. Lipocalin-2 / NGAL was assessed by ELISA and levels were normalized to ml of plasma analyzed. Differences between experimental groups were determined statistically using one-way ANOVA and Tukey post hoc test. (A) Lipocalin-2 / NGAL plasma levels; (B) Decrease (%) in lipocalin-2 / NGAL plasma levels compared to body weight (BW). (r = -0.5129; 95% confidence interval = -0.6502 to -0.3432; R 2 =0.2631; P value: P (two-tailed) = <0.0001; significant (α = 0.05)).

[0036] Fig.16 A process flow diagram for producing freeze-dried powdered form of bacterial strains is depicted.

[0037] Fig.17 Depicted Schematic diagram of a capsule within a capsule.

[0038] Fig.18 A flowchart of the manufacturing process of a capsule within a capsule is depicted. DETAILED DESCRIPTION

[0039] The present disclosure generally relates to bacterial strains and bacterial strain mixtures, such as Eubacterium, Bacteroides and Roseburia bacterial strains and mixtures thereof. For example, in one aspect, a pharmaceutical composition is provided herein, comprising: a bacterial mixture comprising at least two bacterial species, each of which is selected from Bacteroides species, Eubacterium species and Roseburia species; and a pharmaceutically acceptable excipient. In certain embodiments, the bacterial mixture considered comprises Bacteroides species, Eubacterium species and Roseburia species. In certain embodiments, the bacterial mixture considered is essentially composed of Bacteroides species, Eubacterium species and Roseburia species. In certain embodiments, the Bacteroides species in the bacterial mixture is Bacteroides thetaiotaomicron. In certain embodiments, the Eubacterium species in the bacterial mixture is Eubacterium hallii. In certain embodiments, the Roseburia species in the bacterial mixture is Roseburia hominis.

[0040] In another aspect, provided herein is a pharmaceutical composition comprising: a bacterial mixture comprising at least two bacterial species, each of which is selected from a Bacteroides species, a Eubacterium species, and a Roseburia species, wherein the bacterial mixture is capable of: (i) increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 monocytes or macrophages or peripheral blood mononuclear cells (PBMCs)); (ii) reducing or attenuating at least one pro-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP macrophages or PBMCs); (e.g., IL-6, IL-12, IL-17 and / or TNF-α); (iii) reducing or preventing or increasing the barrier integrity of a human cell (e.g., epithelial cell) monolayer (e.g., HT29MTX-E12 cell monolayer treated with TNF-α) (e.g., barrier integrity measured by transepithelial electrical resistance (TEER)); (iv) inducing or increasing autophagy in human cells (e.g., THP-1 monocytes or macrophages); and / or (v) reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue or subject; and a pharmaceutically acceptable excipient. In another aspect, a drug unit is provided herein, comprising: a bacterial mixture comprising at least two bacterial species, each of which is selected from a Bacteroides species, a Eubacterium species and a Roseburia species, and wherein the drug unit has, for example, at least 1×10 6 , at least 1×10 7 , at least 1×10 8 , at least 1×10 9 , at least 1×10 10 , at least 1×10 11 or at least 1×10 12 living organisms (e.g., cfu); and a pharmaceutically acceptable excipient. In certain embodiments, the bacterial strain mixture contemplated comprises at least about 1×10 of a Bacteroides species strain. 9 viable organisms (e.g., cfu), at least about 1×10 of a strain of Eubacterium spp. 9 viable organisms (e.g., cfu) and / or at least about 1×10 of a Roseburia species strain 9 living organisms (e.g., cfu) or any combination thereof.

[0041] The present disclosure further provides a pharmaceutical composition comprising a strain of Eubacterium species and a pharmaceutically acceptable excipient. In certain embodiments, the contemplated composition further comprises a strain of Bacteroides species and / or a strain of Roseburia species. In some embodiments, the strain of Eubacterium species is a strain of Eubacterium hallii.

[0042] The present disclosure further provides a pharmaceutical composition comprising a Bacteroides species strain and a pharmaceutically acceptable excipient. In certain embodiments, the contemplated composition further comprises a Eubacterium species strain and / or a Roseburia species strain. In some embodiments, the Bacteroides species strain is a strain of Bacteroides thetaiotaomicron.

[0043] The present disclosure further provides a pharmaceutical composition comprising a Roseburia species strain and a pharmaceutically acceptable excipient. In certain embodiments, the contemplated composition further comprises a Bacteroides species strain and / or a Eubacterium species strain. In some embodiments, the Roseburia species strain is Roseburia hominis.

[0044] In another aspect, the present invention provides a human Roseburia bacterial strain that is capable of: (i) increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP macrophages or monocytes or PBMCs); (iii) reducing or preventing or increasing the barrier integrity of a human cell (e.g., epithelial cell) monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue or subject. In another aspect, provided herein is a strain of Bacteroides thetaiotaomicron that is capable of: (i) increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP macrophages or monocytes or PBMCs); (iii) reducing or preventing or increasing the barrier integrity of a human cell (e.g., epithelial cell) monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue or subject. In another aspect, the present invention provides a strain of Eubacterium hallii bacteria that is capable of: (i) increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP macrophages or monocytes or PBMCs); (iii) reducing or preventing or increasing the barrier integrity of a human cell (e.g., epithelial cell) monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue or subject.

[0045] The present disclosure further relates to pharmaceutical compositions or units and methods for using the disclosed bacterial strains and / or bacterial strain mixtures to treat gastrointestinal disorders and inflammatory disorders, including, for example, dysbiosis and / or immune-mediated inflammatory disorders, such as ulcerative colitis, Crohn's disease and other forms of inflammatory bowel disease (IBD).

[0046] I. Strains and strain mixtures

[0047] The term "species" as used herein refers to a taxonomic entity generally defined by genomic sequence and phenotypic characteristics. A "strain" is a specific example of a species isolated and purified according to conventional microbiological techniques. Contemplated bacterial species and / or strains include live and / or viable, as well as killed, inactivated or attenuated. In addition, contemplated bacterial strains include both vegetative and non-spore-forming forms of the bacteria.

[0048] The present disclosure encompasses derivatives of the disclosed bacterial strains.The term "derivative" includes progeny strains (progeny) or strains cultured from the original strain (subclones) but modified in some way (including at the genetic level) without negatively altering the biological activity of the strain.

[0049] rRNA, 16S rDNA, 16S rRNA, 16S, 18S, 18S rRNA, and 18S rDNA refer to nucleic acids that make up or encode components of ribosomes. There are two subunits in the ribosome called the small subunit (SSU) and the large subunit (LSU). Ribosomal RNA genes (rDNA) and their complementary RNA sequences are widely used to determine evolutionary relationships between organisms because they are variable but conserved enough to allow for molecular comparisons across organisms.

[0050] In embodiments, the 16S rDNA sequence of the 30S SSU can be used for molecular-based prokaryotic taxonomic assignments. For example, 16S sequences can be used for phylogenetic reconstruction because they are generally highly conserved but contain specific hypervariable regions that have sufficient nucleotide diversity to distinguish between genera and species of most bacteria. Although 16S rDNA sequence data has been used to provide taxonomic assignments, closely related bacterial strains assigned to the same genus species may exhibit different biological phenotypes.

[0051] The identity of the bacterial strain under consideration can be characterized by 16S rRNA, 16S rDNA or whole genome sequence analysis. For example, in certain embodiments, the bacterial strain under consideration may include 16S rRNA, 16S rDNA and / or a genomic sequence with a specific % identity to a reference sequence. The sequence of the bacterial strain described herein, such as the 16s rRNA gene sequence or genomic sequence (such as a whole genome sequence or a fragment or overlapping group thereof) of the bacterial strain, can be obtained by any sequencing method known in the art, including, for example, Sanger sequencing. The Illumina platform is an example of a sequencing technology for identifying bacterial strains. The Illumina platform is based on amplifying DNA on a solid surface (such as a flow cell) using folded PCR and anchor primers (such as capture oligonucleotides). In order to use the Illumina platform for sequencing, bacterial DNA is fragmented, and a joint is added to the end of the fragment. By capturing an oligonucleotide that can hybridize with the joint end of the fragment, the DNA fragment is attached to the surface of the flow cell channel. The DNA fragment is then extended and bridge amplified. After multiple rounds of solid phase amplification and denaturation, millions of spatially fixed nucleic acid clusters or single-stranded nucleic acid colonies are produced. Each cluster may contain about hundreds to one thousand copies of single-stranded DNA molecules of the same template. The Illumina platform uses synthetic sequencing, in which sequencing nucleotides containing detectable markers (e.g., fluorophores) are continuously added to 3' hydroxyls. After nucleotide incorporation, a laser specific to the wavelength of the labeled nucleotide can be used to excite the marker. Capture images and record the identity of the nucleotide bases. These steps can be repeated to sequence the remaining bases. Sequencing according to the technology is described in, for example, U.S. Patent Publication Application Nos. 2011 / 0009278, 2007 / 0014362, 2006 / 0024681, 2006 / 0292611 and U.S. Patent Nos. 7,960,120, 7,835,871, 7,232,656 and 7,115,200. Another example of sequencing technology that can be used to identify bacterial strains is the SOLiD technology of Applied Biosystems from Life Technologies Corporation (Carlsbad, Calif.). In SOLiD sequencing, bacterial DNA may be sheared into fragments and adapters may be ligated to the ends of the fragments to generate a library. A population of clonal beads may be prepared in a microreactor containing template, PCR components, beads, and primers. After PCR, the template may be denatured and bead enrichment may be performed to isolate the beads with extended primers. The template on the selected beads is 3' modified and can be covalently bound to the slide. The sequence can be determined by sequential hybridization and ligation with several primers. A set of four fluorescently labeled two-base probes compete for ligation with the sequencing primer. Multiple ligation, detection, and cleavage cycles are performed, and the number of cycles determines the final read length.Another example of a sequencing technology used to identify bacterial strains is Ion Torrent sequencing. In this technology, bacterial DNA is sheared into fragments and then oligonucleotide adapters are ligated to the ends of the fragments. The fragments are then attached to a surface and the H released during base incorporation is measured. + ions, each base in the fragment can be detected. For example, this technology is described in U.S. Patent Publication Nos. 2009 / 0026082, 2009 / 0127589, 2010 / 0035252, 2010 / 0137143 and 2010 / 0188073.

[0052] After obtaining the polynucleotide sequence of the bacterial strain (e.g., 16s rRNA gene sequence or genomic sequence), sequence identity can be determined in a variety of ways within the technical scope 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 used by blastp, blastn, blastx, tblastn and tblastn (Karlin et al., PROC. NATL. ACAD. SCI. USA 87: 2264-2268 (1990); Altschul, J. MOL. EVOL. 36, 290-300 (1993); Altschul et al., NUCLEIC ACIDSRES. 25: 3389-3402 (1997)) programs is tailor-made for sequence similarity searches. For a discussion of basic issues in searching sequence databases, see Altschul et al., NATURE GENETICS 6:119-129 (1994). One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences. The search parameters for histograms, descriptions, alignments, expectations (i.e., statistically significant thresholds for reporting matches to database sequences), cutoffs, matrices, and filtering are all set by default. The default scoring matrix used by blastp, blastx, tblastn, and tblastn is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919). The four blastn parameters can be adjusted as follows: Q = 10 (gap creation penalty); R = 10 (gap extension penalty); wink = 1 (a penalty for each wink in the query). thposition to generate word hits); and gapw=16 (setting the window width within the gapped alignment). The equivalent Blastp parameter settings may be Q=9; R=2; wink=1; gapw=32. Searches can also be performed using NCBI (National Center for Biotechnology Information) BLAST advanced option parameters (e.g.: -G, gap opening cost [integer]: default = 5 (nucleotides) / 11 (proteins); -E, gap extension cost [integer]: default = 2 for nucleotides / default = 1 for proteins; -q, nucleotide mismatch penalty [integer]: default = -3; -r, reward for nucleotide match [integer]: default = 1; -e, expectation [real number]: default = 10; -W, word size [integer]: default = 11 nucleotides / megablast 28 / protein 3; -y, Dropoff (X) for blast extension (in bits): default = 20 for blastn, 7 for others; -X, X dropoff value for gapped alignments (in bits): default = 15 for all programs, not for blastn; and -Z, final X dropoff value for gapped alignments (in bits): 50 for blastn, 25 for others). The Bestfit comparison between sequences provided in the GCG software package version 10.0 uses the DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty), the equivalent settings in protein comparisons are GAP=8 and LEN=2.

[0053] In certain embodiments, the bacterial strain under consideration may include a DNA-DNA hybridization (DDH) value relative to a reference bacterial strain. Any method known in the art for determining a DNA-DNA hybridization value may be used to assess the degree of DNA-DNA hybridization, including but not limited to the spectrophotometric method for determining renaturation rate described in De Ley et al. (J Biochem 12 133-142 (1970)), which is slightly modified in hybridization temperature (Gavini et al., Ecology in Health and Disease 12 40-45 (2001)); and Grimont et al., Curr Microbiol 4, 325-330 (1980) and Rossello-Mora, Molecular Identification, Systematics and Population Structure of Prokaryotes pp. 23-50 (2006) descriptions. In some embodiments, the extent of DNA-DNA hybridization is determined by digital DNA-DNA hybridization (dDDH) analysis, for example, using the Genome-Genome Distance Calculator online tool (see Meier-Kolthoff et al., BMC Bioinformatics 14:60 (2013)).

[0054] In certain embodiments, the bacterial strain under consideration may include an average nucleotide identity (ANI) relative to a reference bacterial strain. It is well known that the average nucleotide identity (ANI) of shared genes between two strains is a powerful means of comparing genetic relatedness between strains, and an ANI value of about 95% corresponds to a 70% DNA-DNA hybridization standard for determining species. See, e.g., Konstantinidis and Tiedje, Proc Natl Acad Sci USA, 102 (7): 2567-72 (2005); and Goris et al., Int J Syst Evol Microbiol. 57 (Pt l): 81-91 (2007). The ANI between two bacterial genomes is calculated by pairwise comparison of all sequences shared between any two strains and can be determined using any of a number of publicly available ANI tools, including but not limited to OrthoANI with 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); and Varghese et al., Nucleic Acids Research, 43 (14): 6761-6771 (2015). In a particular embodiment, the ANI between two bacterial genomes can be determined by averaging the nucleotide identities of orthologous genes identified as bidirectional best hits (BBHs). The protein-coding genes of the first genome (genome A) and the second genome (genome B) were compared at the nucleotide level using a similarity search tool such as NSimScan (Novichkov et al., Bioinformatics 32(15):2380-23811 (2016)). The results were then filtered to retain only BBHs that showed at least 70% sequence identity over at least 70% of the shorter sequence length in each BBH pair. The ANI of genome A to genome B was defined as the percent identity of all BBHs multiplied by the sum of the alignment lengths divided by the sum of the lengths of the BBH genes.

[0055] In certain embodiments, the bacterial strain under consideration may comprise an alignment score (AF) relative to a reference bacterial strain. In some embodiments, the genetic factor 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 in two directions: from genome A to genome B and from genome B to genome A.

[0056] In some embodiments, the bacterial strain or bacterial strain mixture considered (i) reduces or weakens or can reduce or weaken the production of at least one pro-inflammatory gene product (e.g., pro-inflammatory cytokines or chemokines) in cells, tissues, or subjects; and / or (ii) increases or can increase the production of at least one anti-inflammatory gene product (e.g., anti-inflammatory cytokines or chemokines) in cells, tissues, or subjects. Exemplary pro-inflammatory 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, and TNF-α. For example, in some embodiments, bacterial strains or bacterial strain mixtures reduce or weaken the production of IL12 (e.g., IL12 subunit p40) in cells, tissues, or subjects. Exemplary anti-inflammatory gene products include CCL-18, IL-1Ra, IL-4, IL-6, IL-10, IL-11, IL-13 and TGF-β. For example, the bacterial strain or bacterial strain mixture contemplated increases the production of IL-10 and / or CCL-18 in cells, tissues or subjects. It should be understood that the gene product can have both pro-inflammatory and / or anti-inflammatory activity.

[0057] The bacterial strain or bacterial strain mixture contemplated may be characterized by having an effect on gene product production (e.g., IL-10, IL-12, or CCL-18 production) in immune cells, such as macrophages (e.g., THP-1 macrophages) or PBMCs (including lymphocytes (T cells, B cells, NK cells) and monocytes). In vivo, the major sources of IL-10 include helper T cells, monocytes, macrophages, and dendritic cells, however, a variety of immune effector cell types are capable of producing IL-10, IL-12, or CCL-18 in certain circumstances. 10, including B cells, cytotoxic T cells, NK cells, mast cells, and granulocytes such as neutrophils and eosinophils. Gene product production in macrophages (e.g., IL-10, IL-12, or CCL-18) can be analyzed, for example, as follows. THP-1 human macrophages are prepared by culturing THP-1 human monocyte cell line with phorbol 12-myristate 13-acetate (PMA) for 24 hours, optionally followed by IL-4 and IL-13, as previously described (Genin et al., BMC Cancer 15:577 (2015)). In the presence of lipopolysaccharide (LPS), bacterial strains or bacterial strain mixtures are cultured with THP-1 macrophages for 24 hours. Gene product production is assessed by measuring the concentration of gene products (e.g., IL-10, IL-12, or CCL-18) in cell culture supernatants with ELISA. Gene product production can also be performed according to Sudhakaran et al., Genes Nutr., 8(6):637-48(2013). Gene product production in PBMC (e.g., IL-10, IL-12, or CCL-18 production) can be analyzed, for example, as follows. Primary PBMCs are isolated from donor blood samples using a percoll gradient (Sim et al., J. Vis. Exp. (112), e54128 (2016)). Bacterial strains or bacterial strain mixtures are cultured with PBMCs for 24 hours. Gene product production is assessed by measuring the concentration of gene products (e.g., IL-10, IL-12, or CCL-18) in cell culture supernatants using ELISA.

[0058] The bacterial strain or bacterial strain mixture contemplated may (i) reduce or prevent damage to the integrity of an epithelial barrier (e.g., an epithelial cell monolayer) or increase the integrity of an epithelial barrier (e.g., an epithelial cell monolayer), or be capable of reducing or preventing damage to the integrity of an epithelial barrier (e.g., an epithelial cell monolayer) or increasing the integrity of an epithelial barrier (e.g., an epithelial cell monolayer); and / or (ii) increase or be capable of increasing the production of at least one pro-barrier integrity gene (e.g., ZO-1) in a cell, tissue, or subject. In some embodiments, the epithelial barrier is an intestinal barrier, such as an intestinal mucosal barrier. The intestinal epithelium is organized into a 20 μm monolayer, comprising 5 different cell types: enterocytes, endocrine cells, M cells, goblet (mucus) cells, and Paneth cells. Enterocytes are the most representative cell type, acting as a physical barrier that inhibits the transport of intestinal contents in the internal tissues. They are connected by intercellular junctions, characterized by transmembrane proteins that interact with neighboring cells and intracellular proteins associated with the cytoskeleton. The bacterial strain or bacterial strain mixture under consideration may be characterized by the effect on the integrity of the epithelial barrier (e.g., HT29MTX-E12 cell monolayer or Caco-2 monolayer, or any intestinal epithelial cell or cell line monolayer). In certain embodiments, the bacterial strain or bacterial strain mixture under consideration may be characterized by the effect on the barrier integrity of a human epithelial cell monolayer (e.g., HT29MTX-E12 cell monolayer or Caco-2 cell monolayer) treated with TNF-α. The barrier integrity of the HT29MTX-E12 cell monolayer treated with TNF-α may be determined, for example, as follows. HT29MTX-E12 cells are inoculated into a Transwell flat plate system for 18-21 days to form a polarized monolayer as described previously (Hall et al., Journal of Pediatric Surgery 48: 353-358 (2013)). The bacterial strain or bacterial strain mixture is added to the top layer of the Transwell, and TNF-α is subsequently added to the basal layer of the Transwell, thereby simulating inflamed intestines. Monolayer integrity was assessed by measuring the transepithelial electrical resistance (TEER) across the cell barrier at 0 and 24 hours after addition of TNF-α. Barrier integrity can also be analyzed as described by Pontier et al., J. Pharm. Sci., 90(10):1608-19 (2001).

[0059] In a specific embodiment, for the identification of a bacterial strain or mixture of bacterial strains that reduces or prevents damage to the integrity of an epithelial barrier (e.g., an epithelial cell monolayer) or improves its integrity, the bacterial strain or mixture of bacterial strains is identified as being able to reduce or prevent damage to or improve the integrity of an intestinal barrier (e.g., an intestinal mucosal barrier). Therefore, on the other hand, methods for selecting a bacterial strain or mixture of bacterial strains having intestinal barrier protective properties are provided herein. Methods for selecting a bacterial strain or mixture of bacterial strains for treating gastrointestinal disorders, dysbiosis and / or inflammatory disorders described herein are also provided herein. In some embodiments, the method comprises the following steps: (a) contacting an epithelial cell monolayer with a bacterial strain or a mixture of bacterial strains in vitro; (b) contacting the epithelial cell monolayer with a proinflammatory compound in the presence of the bacterial strain or the mixture of bacterial strains; and (c) measuring the transepithelial electrical resistance (TEER) across the cell monolayer; wherein the increase in TEER across the cell monolayer identifies the bacterial strain or the mixture of bacterial strains as having intestinal barrier protective properties, and / or can be used to treat gastrointestinal disorders, dysbiosis and / or inflammatory disorders compared to a cell monolayer (e.g., a cell monolayer of the same cell type) that is in contact with a proinflammatory compound but not with a bacterial strain or a mixture of bacterial strains. In some embodiments, the epithelial cell monolayer is a human epithelial cell monolayer. In some embodiments, the epithelial cell monolayer is a mucosal epithelial cell monolayer. In some embodiments, the epithelial cell monolayer is a HT29MTX-E12 cell monolayer or a Caco-2 cell monolayer. In some embodiments, the bacterial strain or the mixture of bacterial strains contacts the top side of the epithelial cell monolayer. In some embodiments, contacting the epithelial cell monolayer with the bacterial strain or mixture of bacterial strains occurs at least 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 minutes prior to contacting the epithelial cell layer with the pro-inflammatory compound. In some embodiments, the pro-inflammatory compound is selected from 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. In some embodiments, measurement of TEER across a cell monolayer occurs 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and / or more than 24 hours after contacting the epithelial cell monolayer with a proinflammatory compound.In some embodiments, the increase in TEER across a cell monolayer is at least 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or greater than 100% compared to a cell monolayer contacted with a pro-inflammatory compound but not contacted with a bacterial strain or a mixture of bacterial strains.

[0060] The bacterial strain or bacterial strain mixture contemplated may induce or increase or be capable of inducing or increasing autophagy in cells (e.g., antigen presenting cells, epithelial cells, myeloid cells, intestinal epithelial cells, macrophages (e.g., THP-1 macrophages), PBMCs (including lymphocytes (T cells, B cells, NK cells) and monocytes), dendritic cells, mast cells, and granulocytes such as neutrophils and / or eosinophils). Autophagy is an intracellular degradation system in which dysfunctional proteins and organelles are degraded. In this process, aggregated dysfunctional proteins are wrapped by a double membrane to form autophagosomes. The induction of autophagy in cells (e.g., THP-1 macrophages) can be analyzed as follows. THP-1 human macrophages are prepared by culturing a THP-1 human monocyte cell line with phorbol 12-myristate 13-acetate (PMA) for 24 hours, optionally followed by IL-4 and IL-13 as previously described (Genin et al., BMC Cancer 15:577 (2015)). The bacterial strain or bacterial strain mixture is cultured with THP-1 macrophages in the presence of lipopolysaccharide (LPS) for at least 1, 2, 3 or more hours (e.g., up to 24 hours). Autophagy can be analyzed by detecting autophagosome formation, for example, by staining the cells with a fluorescent dye that accumulates in the membrane specific for the autophagosome, e.g. (Enzo Life Sciences) or DAPGreen (Dojindo Molecular Technologies, Inc.). Autophagy can also be detected by any of the methods described by Puleston et al., Cold Spring Harb Protoc; doi: 10.1101 / pdb.top070391.

[0061] Therefore, in another aspect, the present invention provides a method for selecting a bacterial strain or a mixture of bacterial strains for treating gastrointestinal disorders, dysbiosis and / or inflammatory disorders, the method comprising the following steps: (a) contacting a cell with a bacterial strain or a mixture of bacterial strains in vitro; and (b) measuring autophagy in the cell; wherein the increase in autophagy in the cell compared to a cell (e.g., a cell of the same cell type) that has not been contacted with the bacterial strain or mixture of bacterial strains identifies the bacterial strain or mixture of bacterial strains as being useful for treating gastrointestinal disorders, dysbiosis and / or inflammatory disorders. In some embodiments, the cell is a human cell. In some embodiments, the cell is an antigen presenting cell, an epithelial cell, a myeloid cell, an intestinal epithelial cell, a macrophage (e.g., a THP-1 macrophage), a PBMC (including lymphocytes (T cells, B cells, NK cells) and monocytes), a dendritic cell, a mast cell or a granulocyte (e.g., a neutrophil or an eosinophil). In some embodiments, the monocyte or macrophage is a THP-1 monocyte or macrophage. In some embodiments, the gastrointestinal disorder is inflammatory bowel disease, including ulcerative colitis and Crohn's disease. In some embodiments, contacting occurs in the presence of lipopolysaccharide (LPS). In some embodiments, contacting occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more than 24 hours before measuring autophagy.

[0062] The bacterial strain or bacterial strain mixture considered can reduce or weaken or can reduce or weaken the production of lipocalin-2 / NGAL in cells, tissues or subjects.Lipocalin-2 (LCN2), also known as neutrophil gelatinase-related lipocalin (NGAL) or siderocalin (siderocalin), is an effective antibacterial protein stored in neutrophil granules and released at inflammatory sites.The high LCN2 expression of intestinal epithelial cells has been confirmed in colon biopsies from the inflammatory areas of IBD patients (Nielsen et al., Gut, 38:414-420 (1996)), and LCN2 has been reported as one of the 10 most upregulated genes in both active ulcerative colitis and Crohn's disease (Ostvik et al., Clin Exp Immunol.173:502-511 (2013)). Ostvik reports that, although LCN2 protein is found in both epithelial cells and infiltrating neutrophils, LCN2 mRNA synthesis occurs only in epithelial cells, indicating that the excessive de novo synthesis of LCN2 in IBD is located in the colonic epithelium. The serum level of LCN2 has been shown to be a reliable biomarker of disease activity in UC, which can distinguish active disease from remission disease, with a sensitivity higher than CRP or white blood cell count (Stallhofer et al., Inflamm Bowel Dis 21 (10): 2327-2340 (2015)). The level of LCN2 / NGAL can be measured by any method known in the art (including qPCR, ELISA, immunohistochemistry, etc.) The expression and / or concentration of LCN2 / NGAL gene products in samples (e.g., plasma, serum, feces and / or tissue (e.g., colon tissue)) are evaluated in cells, tissues or subjects contacted with the bacterial strain or bacterial strain mixture under consideration.

[0063] As described below, the cell bank preparation / propagation bacterial strains contemplated herein can be prepared, for example, in culture medium for isolation and preparation of master cell banks without animal-derived components.

[0064] For example, contemplated bacterial strains for use in the disclosed bacterial strain mixtures, pharmaceutical compositions or units, or methods include strains of Bacteroides species. Exemplary Bacteroides species include B. acidifaciens, B. barnesiaes, B. caccae, B. caecicola, B. caecigallinarum, B. cellulosilyticus, B. cellulosolvens, B. clarus, B. coagulans, B. coprocola, B. coprophilus, B. coprosuis, B. di stasonis), B.dorei, B.eggerthii, B.gracilis, B.faecichinchillae, B.faecis, B.finegoldii, B.fluxus, B.fragilis, B.galacturonicus, B.gallinaceum, B.gallinarum, B.goldsteinii, B.graminisolvens, B.helcogene, B.intestinalis, B.luti, B.masseillais Bacillus massiliensis, Bacillus melaninogenicus, Bacillus nordii, Bacillus oleiciplenus, Bacillus oris, Bacillus ovatus, Bacillus paurosaccharolyticus, Bacillus pectinophilus, Bacillus plebeius, Bacillus polypragmatus, Bacillus propionicifaciens, Bacillus putredinis, Bacillus pyogenes s), B.reticulotermitis, B.rodentium, B.salanitronis, B.salyersiae, B.sartorii, B.sediment, B.stercoris, B.suis, B.tectus, B.thetaiotaomicron, B.uniformis, B.vulgatus, B.xylanisolvens and B.xylanolyticusxylanolyticus.Those skilled in the art will recognize that the genus Bacteroides can undergo taxonomic reorganization. Thus, contemplated Bacteroides species are intended to include Bacteroides species that have been renamed and / or reclassified, as well as those that may subsequently be renamed and / or reclassified.

[0065] In certain embodiments, the contemplated Bacteroides species strain is a Bacteroides thetaiotaomicron strain. For example, the contemplated Bacteroides thetaiotaomicron strain genome may comprise a 16s rRNA gene sequence having a nucleotide sequence of SEQ ID NO: 2, or a 16s rRNA gene sequence having a nucleotide sequence of SEQ ID NO: 3. NO: 2 nucleotide sequence at least about 70.0%, about 80.0%, about 90.0%, about 91.0%, about 92.0%, about 93.0%, about 94.0%, about 95.0%, about 96.0%, about 97.0%, about 98.0%, about 98.1%, about 98.2%, about 98.3%, about 98.4%, about 98.5%, about 98.6%, about 98.7%, about 98.8%, about 98.9%, about 99.0%, 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% identical 16s rRNA gene sequence. In some embodiments, the above sequence identity spans at least about 70% of SEQ ID NO: 2. In other embodiments, the sequence identity spans 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:2.

[0066] In some embodiments, the contemplated Bacteroides thetaiotaomicron strain comprises a genome sequence (e.g., a whole genome sequence or a fragment or contig thereof) having a certain % identity to one or more of SEQ ID NOs: 33-390. The contemplated Bacteroides thetaiotaomicron strain genome may comprise a nucleotide sequence of any one of SEQ ID NOs: 33-390, or a nucleotide 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 a nucleotide sequence of any one of SEQ ID NOs: 33-390. The contemplated Bacteroides thetaiotaomicron strain genome may comprise the nucleotide sequence of any one of SEQ ID NOs: 33-390, or any of the nucleotide sequences 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 the nucleotide sequence of any one of SEQ ID NOs: 33-390.

[0067] In some embodiments, the contemplated Bacteroides thetaiotaomicron strain comprises a whole genome sequence that has at least about 70% identity to the sum of all genomic contigs represented by SEQ ID NOs:33-390 across at least 70% of its genome. In some embodiments, the whole genome sequence has at least about 75%, 80%, 85%, 90%, 95%, or greater than 95% identity to the sum of all genomic contigs represented by SEQ ID NOs:33-390. In some embodiments, the above sequence identity spans at least 75%, 80%, 85%, 90%, 95%, or greater than 95% of the whole genome sequence of the bacterial strain. In some embodiments, the contemplated Bacteroides thetaiotaomicron strain comprises a whole genome sequence that includes a coding region that has at least about 70% identity to a coding region within the sum of all genomic contigs represented by SEQ ID NOs:33-390 across at least 70% of its genome. In some embodiments, the coding region within the whole genome sequence has at least about 75%, 80%, 85%, 90%, 95% or greater than 95% identity with the coding region within the sum of all genomic contigs represented by SEQ ID NO: 33-390. In some embodiments, the above sequence identity spans at least 75%, 80%, 85%, 90%, 95% or greater than 95% of the coding region within the whole genome sequence of the bacterial strain.

[0068] In certain embodiments, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) increases or is capable of increasing the production of at least one anti-inflammatory gene product in a cell, tissue, or subject. For example, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) increases the production of IL-10 and / or CCL-18 in human cells (e.g., THP-1 macrophages or monocytes or PBMCs). For example, contacting human cells (e.g., THP-1 macrophages or PBMCs) with a Bacteroides species strain (e.g., a Bacteroides thetaiotaomicron strain) can increase the production of IL-10 and / or CCL-18 in the cells relative to cells that have not been contacted with the strain (e.g., cells of the same cell type) 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 750%, at least about 1000%, about 10% to about 20%, about 10% to about 50%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 51 ... From about 10% to about 100%, from about 10% to about 200%, from about 10% to about 500%, from about 10% to about 1000%, from about 20% to about 50%, from about 20% to about 100%, from about 20% to about 200%, from about 20% to about 500%, from about 20% to about 1000%, from about 50% to about 100%, from about 50% to about 200%, from about 50% to about 500%, from about 50% to about 1000%, from about 100% to about 200%, from about 100% to about 500%, from about 100% to about 1000%, from about 200% to about 500%, from about 200% to about 1000%, or from about 500% to about 1000%. In some embodiments, contacting of the human cell with the Bacteroides species strain occurs in vitro, for example, the human cell is cultured with the Bacteroides species strain. In some embodiments, contacting the human cell with the Bacteroides sp. strain occurs in vivo.

[0069] In certain embodiments, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product in a cell, tissue, or subject. For example, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) reduces or attenuates the production of IL-6, IL-12, IL-17, and / or TNF-α in human cells (e.g., THP-1 macrophages or monocytes or PBMCs). For example, contacting a human cell (e.g., a THP-1 macrophage or PBMC) with a Bacteroides species strain (e.g., a Bacteroides thetaiotaomicron strain) can reduce or attenuate the production of IL-6, IL-12, IL-17, and / or TNF-α in the cell relative to a cell (e.g., a cell of the same cell type) that has not been contacted with the strain 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%, about 50% to about 100%. In some embodiments, contacting a human cell with a Bacteroides species strain occurs in vitro, for example, a human cell is cultured with a Bacteroides species strain. In some embodiments, contacting a human cell with a Bacteroides species strain occurs in vivo.

[0070] In certain embodiments, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) reduces or prevents disruption of the barrier integrity of the mucosal epithelium or increases the barrier integrity of the mucosal epithelium, or is capable of reducing or preventing disruption of the barrier integrity of the mucosal epithelium or increasing the barrier integrity of the mucosal epithelium. For example, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) can reduce or prevent disruption of the barrier integrity of a HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α) or increase the barrier integrity of a HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α). For example, culturing a contemplated Bacteroides species strain (e.g., a Bacteroides thetaiotaomicron strain) with a HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α) can reduce or prevent disruption of barrier integrity or increase barrier integrity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 58 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, about 20% to about 100%, about 40% to about 100%, about 60% to about 100%, about 80% to about 100%, about 20% to about 80%, about 40% to about 80%, about 60% to about 80%, about 20% to about 60%, about 40% to about 60%, about 20% to about 40%, for example as measured by transepithelial electrical resistance (TEER).

[0071] In certain embodiments, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) induces or increases or is capable of inducing or increasing autophagy in a cell, tissue, or subject. For example, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) induces or increases autophagy in human cells (e.g., THEM macrophages). For example, contacting a human cell (e.g., a THP-1 macrophage) with a Bacteroides species strain (e.g., a Bacteroides thetaiotaomicron strain) can induce or increase autophagy in the cell relative to a cell not contacted with the strain (e.g., a cell of the same cell type) by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, 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%, about 20% to about 200%, about 50% to about 100%, or about 50% to about 200%. In some embodiments, contacting a human cell with a Bacteroides species strain occurs in vitro, for example, a human cell is cultured with a Bacteroides species strain. In some embodiments, contacting a human cell with a Bacteroides species strain occurs in vivo.

[0072] In certain embodiments, the contemplated Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) reduces or attenuates or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject. For example, contacting a human cell with a Bacteroides species strain (e.g., Bacteroides thetaiotaomicron strain) can reduce or attenuate the production of lipocalin-2 / NGAL 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%, 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% relative to cells not contacted with the strain (e.g., cells of the same cell type). In some embodiments, contacting a human cell with a Bacteroides species strain occurs in vitro, for example, a human cell is cultured with a Bacteroides species strain. In some embodiments, contacting a human cell with a Bacteroides species strain occurs in vivo.

[0073] In certain embodiments, the Bacteroides thetaiotaomicron strain contemplated is a strain identified herein as BTH01. According to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, the Bacteroides thetaiotaomicron strain BTH01 (P118-A12a) was deposited at DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstraβe 7B, 38124 Brunswick, Germany) on September 14, 2018. The deposit was given accession number DSM-32919. The 16s rRNA gene sequence of the Bacteroides thetaiotaomicron strain BTH01 is provided in the present application as SEQ ID NO: 2, and the genome sequence of the Bacteroides thetaiotaomicron strain BTH01 is provided in the present application as SEQ ID NO: 33-390.

[0074] In certain embodiments, the contemplated Bacteroides thetaiotaomicron strain has a DNA-DNA hybridization (DDH) value equal to or greater than about 70% with the Bacteroides thetaiotaomicron strain BTH01 (P118-A12a). In certain embodiments, the Bacteroides thetaiotaomicron strain is a strain having a DNA-DNA hybridization greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% with the Bacteroides thetaiotaomicron strain BTH01 (P118-A12a). In certain embodiments, the contemplated Bacteroides thetaiotaomicron strain has an average nucleotide identity (ANI) equal to or greater than 95% with the Bacteroides thetaiotaomicron strain BTH01 (P118-A12a). In certain embodiments, the ANI is equal to or greater than about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, or 100%. In certain embodiments, the contemplated Bacteroides thetaiotaomicron strain has an alignment score (AF) equal to or greater than 60% with Bacteroides thetaiotaomicron strain BTH01 (P118-A12a). In certain embodiments, the AF is equal to or greater than about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100%.

[0075] For example, contemplated bacterial strains for use in the disclosed bacterial strain mixtures, pharmaceutical compositions or units, or methods include Eubacterium sp. strains. Exemplary Eubacterium species include E. aggregans, E. angustum, E. barkeri, E. brachy, E. budayi, E. callanderi, E. cellulosolvens, E. combesii, E. coprostanoligenes, E. dolichum, E. eligens, E. hallii, E. infirmum, E. limosum, E. minutum, E. multiforme, E. nitritogenes, E. nodatum, E. oxidoreducens, E. plexicaudatum, E. pyruvativorans, E. ramulus, E. rectale, E. ruminantium, E. saphenum, E. siraeum, E. sulci, E. tarantellae, E. tenue, E. tortuosum, E. uniforme, E. ventriosum, E. xylanophilum, and E. yurii. Those skilled in the art will recognize that Eubacterium can undergo taxonomic recombination. Thus, contemplated Eubacterium species are intended to include Eubacterium species that have been renamed and / or reclassified, as well as those that may subsequently be renamed and / or reclassified. For example, contemplated Eubacterium hallii include those reclassified as Anaerobutyricum hallii (Shetty et al., Int J Syst Evol Microbiol. 68:3741-3746 (2018)).

[0076] In certain embodiments, the contemplated Eubacterium species strain is a Eubacterium hallii strain. For example, the contemplated Eubacterium hallii strain genome may comprise a 16s rRNA gene sequence having a nucleotide sequence of SEQ ID NO: 3, or a 16s rRNA gene sequence having a nucleotide sequence of SEQ ID NO: 4. NO:3 nucleotide sequence at least about 70.0%, about 80.0%, about 90.0%, about 91.0%, about 92.0%, about 93.0%, about 94.0%, about 95.0%, about 96.0%, about 97.0%, about 98.0%, about 98.1%, about 98.2%, about 98.3%, about 98.4%, about 98.5%, about 98.6%, about 98.7%, about 98.8%, about 98.9%, about 99.0%, 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% identical 16s rRNA gene sequence. In some embodiments, the above sequence identity spans at least about 70% of SEQ ID NO:3. In other embodiments, the sequence identity spans 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:3.

[0077] In some embodiments, a contemplated Eubacterium hallii strain comprises a genomic sequence (e.g., a whole genome sequence or a fragment or contig thereof) having a certain % identity to one or more of SEQ ID NOs: 391-523. A contemplated Eubacterium hallii strain genome may comprise a nucleotide sequence of any one of SEQ ID NOs: 391-523, or a nucleotide 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 a nucleotide sequence of any one of SEQ ID NOs: 391-523. The genome of the contemplated Eubacterium hallii strain may comprise the nucleotide sequence of any one of SEQ ID NOs: 391-523, or any of the nucleotide sequences 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 the nucleotide sequence of any one of SEQ ID NOs: 391-523.

[0078] In some embodiments, the contemplated Eubacterium hallii strain comprises a whole genome sequence that has at least about 70% identity to the sum of all genomic contigs represented by SEQ ID NOs: 391-523 across at least 70% of its genome. In some embodiments, the whole genome sequence has at least about 75%, 80%, 85%, 90%, 95% or greater than 95% identity to the sum of all genomic contigs represented by SEQ ID NOs: 391-523. In some embodiments, the above sequence identity spans at least 75%, 80%, 85%, 90%, 95% or greater than 95% of the whole genome sequence of the bacterial strain. In some embodiments, the contemplated Eubacterium hallii strain comprises a whole genome sequence that comprises a coding region that has at least about 70% identity to the coding region within the sum of all genomic contigs represented by SEQ ID NOs: 391-523 across at least 70% of its genome. In some embodiments, the coding region within the whole genome sequence has at least about 75%, 80%, 85%, 90%, 95% or greater than 95% identity with the coding region within the sum of all genomic contigs represented by SEQ ID NO: 391-523. In some embodiments, the above sequence identity spans at least 75%, 80%, 85%, 90%, 95% or greater than 95% of the coding region within the whole genome sequence of the bacterial strain.

[0079] In certain embodiments, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) increases or is capable of increasing the production of at least one anti-inflammatory gene product in a cell, tissue, or subject. For example, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) increases the production of IL-10 and / or CCL-18 in human cells (e.g., THP-1 macrophages or monocytes or PBMCs). For example, contacting human cells (e.g., THP-1 macrophages or PBMCs) with a Eubacterium species strain (e.g., a Eubacterium hallii strain) can increase the production of IL-10 and / or CCL-18 in the cells relative to cells that have not been contacted with the strain (e.g., cells of the same cell type) 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 750%, at least about 1000%, about 10% to about 20%, about 10% to about 50%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 500%, about 5 From about 10% to about 100%, from about 10% to about 200%, from about 10% to about 500%, from about 10% to about 1000%, from about 20% to about 50%, from about 20% to about 100%, from about 20% to about 200%, from about 20% to about 500%, from about 20% to about 1000%, from about 50% to about 100%, from about 50% to about 200%, from about 50% to about 500%, from about 50% to about 1000%, from about 100% to about 200%, from about 100% to about 500%, from about 100% to about 1000%, from about 200% to about 500%, from about 200% to about 1000%, or from about 500% to about 1000%. In some embodiments, contacting of the human cell with the Eubacterium species strain occurs in vitro, for example, the human cell is cultured with the Eubacterium species strain. In some embodiments, contacting the human cell with the Eubacterium sp. strain occurs in vivo.

[0080] In certain embodiments, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product in a cell, tissue, or subject. For example, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) reduces or attenuates the production of IL-6, IL-12, IL-17, and / or TNF-α in human cells (e.g., THP-1 macrophages or monocytes or PBMCs). For example, contacting a human cell (e.g., THP-1 macrophage or PBMC) with a strain of Eubacterium species (e.g., a strain of Eubacterium hallii) can reduce or attenuate the production of IL-6, IL-12, IL-17, and / or TNF-α in the cell relative to cells not contacted with the strain (e.g., cells of the same cell type) 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%, about 50% to about 100%. In some embodiments, contacting of a human cell with a strain of Eubacterium species occurs in vitro, e.g., a human cell is cultured with a strain of Eubacterium species. In some embodiments, contacting of a human cell with a strain of Eubacterium species occurs in vivo.

[0081] In certain embodiments, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) reduces or prevents damage to the barrier integrity of the mucosal epithelium or increases the barrier integrity of the mucosal epithelium, or is capable of reducing or preventing damage to the barrier integrity of the mucosal epithelium or increasing the barrier integrity of the mucosal epithelium. For example, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) reduces or prevents damage to the barrier integrity of a HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α) or increases the barrier integrity of a HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α). For example, a contemplated Eubacterium species strain (e.g., a Eubacterium hallii strain) cultured with a HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α) can reduce or prevent disruption of barrier integrity or increase barrier integrity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 15 ... 0%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, about 20% to about 100%, about 40% to about 100%, about 60% to about 100%, about 80% to about 100%, about 20% to about 80%, about 40% to about 80%, about 60% to about 80%, about 20% to about 60%, about 40% to about 60%, about 20% to about 40%, for example as measured by transepithelial electrical resistance (TEER).

[0082] In certain embodiments, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) induces or increases or is capable of inducing or increasing autophagy in a cell, tissue, or subject. For example, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) induces or increases autophagy in human cells (e.g., THP-1 macrophages). For example, contacting a human cell (e.g., a THP-1 macrophage) with a strain of a Eubacterium species (e.g., a strain of Eubacterium hallii) can induce or increase autophagy in the cell relative to a cell (e.g., a cell of the same cell type) that is not contacted with the strain by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, 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%, about 20% to about 200%, about 50% to about 100%, or about 50% to about 200%. In some embodiments, contacting a human cell with a strain of a Eubacterium species occurs in vitro, e.g., a human cell is cultured with a strain of a Eubacterium species. In some embodiments, contacting a human cell with a strain of a Eubacterium species occurs in vivo.

[0083] In certain embodiments, the contemplated Eubacterium species strain (e.g., Eubacterium hallii strain) reduces or attenuates or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject. For example, contacting a human cell with a Eubacterium species strain (e.g., Eubacterium hallii strain) can reduce or attenuate the production of lipocalin-2 / NGAL in the cell relative to cells not contacted with the strain (e.g., cells of the same cell type) 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%. In some embodiments, the contacting of the human cell with the Eubacterium species strain occurs in vitro, such as by incubating the human cell with the Eubacterium species strain. In some embodiments, the contacting of the human cell with the Bacteroides species strain occurs in vivo.

[0084] In certain embodiments, the Eubacterium hallii strain under consideration is the strain identified herein as EHA01. Eubacterium hallii strain EHA01 (P168-F1a) was deposited with DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstraβe 7B, 38124 Brunswick, Germany) on September 14, 2018 under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The deposit was assigned accession number DSM-32920. The 16s rRNA gene sequence of Eubacterium hallii strain EHA01 is provided herein as SEQ ID NO: 3, and the genomic sequence of Eubacterium hallii strain EHA01 is provided herein as SEQ ID NO: 391-523.

[0085] In certain embodiments, the DNA-DNA hybridization (DDH) value of the contemplated Eubacterium hallii strain and Eubacterium hallii strain EHA01 (P168-F1a) is equal to or greater than about 70%. In certain embodiments, the DNA-DNA hybridization of the Eubacterium hallii strain and Eubacterium hallii strain EHA01 (P168-F1a) is greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98% or about 99%. In certain embodiments, the average nucleotide identity (ANI) of the contemplated Eubacterium hallii strain and Eubacterium hallii strain EHA01 (P168-F1a) is equal to or greater than 95%. In certain embodiments, the ANI is equal to or greater than about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or 100%. In certain embodiments, the comparison score (AF) of the contemplated Eubacterium hallii strain to Eubacterium hallii strain EHA01 (P168-Fla) is equal to or greater than 60%. In certain embodiments, the AF is equal to or greater than about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or 100%.

[0086] For example, bacterial strains considered for use in disclosed bacterial strain mixtures, pharmaceutical compositions, or units or methods include strains of the genus Roseburia. Exemplary Roseburia species include cecal Roseburia (R.cecicola), fecal Roseburia (R.faecis), human Roseburia (R.hominis), intestinal Roseburia (R.intestinalis) and glucosaccharolytic Roseburia (R.inulinivorans). Those skilled in the art will recognize that the genus Roseburia may undergo taxonomic restructuring. Therefore, it is intended that Roseburia species that have been renamed and / or reclassified and those that may subsequently be renamed and / or reclassified are included in the Roseburia species considered.

[0087] In certain embodiments, the Roseburia species strain under consideration is a Roseburia hominis strain. For example, the Roseburia hominis strain genome under consideration may comprise a 16s rRNA gene sequence having a nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having a nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence of NO:1 has a 16s rRNA gene sequence that is at least about 70.0%, about 80.0%, about 90.0%, about 91.0%, about 92.0%, about 93.0%, about 94.0%, about 95.0%, about 96.0%, about 97.0%, about 98.0%, about 98.1%, about 98.2%, about 98.3%, about 98.4%, about 98.5%, about 98.6%, about 98.7%, about 98.8%, about 98.9%, about 99.0%, 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% identical. In some embodiments, the sequence identity spans at least about 70% of SEQ ID NO: 1. In other embodiments, the sequence identity spans 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.

[0088] In some embodiments, the Roseburia hominis strain contemplated comprises a genomic sequence (e.g., a whole genome sequence or a fragment or contig thereof) having a certain % identity to one or more of SEQ ID NOs: 4-32. The Roseburia hominis strain genome contemplated may comprise a nucleotide sequence of any one of SEQ ID NOs: 4-32, or a nucleotide 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 the nucleotide sequence of any one of said SEQ ID NOs: 4-32. The contemplated Roseburia hominis strain genome may comprise the nucleotide sequence of each of SEQ ID NOs: 4-32, or each of the nucleotide sequences 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 the nucleotide sequence of each of said SEQ ID NOs: 4-32.

[0089] In some embodiments, the Roseburia hominis strain under consideration comprises a whole genome sequence that has at least about 70% identity with the sum of the whole genome contigs represented by SEQ ID NO:4-32 in at least 70% of its genome. In some embodiments, the whole genome sequence has at least about 75%, 80%, 85%, 90%, 95% or greater than 95% identity with the sum of the whole genome contigs represented by SEQ ID NO:4-32. In some embodiments, the above sequence identity spans at least 75%, 80%, 85%, 90%, 95% or greater than 95% of the whole genome sequence of the bacterial strain. In some embodiments, the Roseburia hominis strain under consideration comprises a whole genome sequence that comprises a coding region that has at least about 70% identity with the coding region within the sum of the whole genome contigs represented by SEQ ID NO:4-32 in at least 70% of all coding regions across its genome. In some embodiments, the coding region within the whole genome sequence has at least about 75%, 80%, 85%, 90%, 95% or greater than 95% identity with the coding region within the sum of all genomic contigs represented by SEQ ID NO: 4-32. In some embodiments, the above sequence identity spans at least 75%, 80%, 85%, 90%, 95% or greater than 95% of the coding region within the whole genome sequence of the bacterial strain.

[0090] In certain embodiments, the contemplated Roseburia species strain (e.g., human Roseburia strain) increases or is capable of increasing the production of at least one anti-inflammatory gene product in a cell, tissue, or subject. For example, the contemplated Roseburia species strain (e.g., human Roseburia strain) increases the production of IL-10 and / or CCL-18 in human cells (e.g., THP-1 macrophages or monocytes or PBMCs). For example, contacting human cells (e.g., THP-1 macrophages or PBMCs) with the Roseburia species strain (e.g., a Roseburia hominis strain) can increase the production of IL-10 and / or CCL-18 in the cells relative to cells (e.g., cells of the same cell type) that are not contacted with the strain 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 750%, at least about 1000%, about 10% to about 20%, about 10% to about 5 ...10 In some embodiments, the contacting of the human cell with the Roseburia species strain occurs in vitro, for example, by incubating the human cell with the Roseburia species strain. In some embodiments, said contacting of said human cell with said Roseburia sp. strain occurs in vivo.

[0091] In certain embodiments, the contemplated Roseburia species strain (e.g., human Roseburia strain) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product in a cell, tissue, or subject. For example, the contemplated Roseburia species strain (e.g., human Roseburia strain) reduces or attenuates the production of IL-6, IL-12, IL-17, and / or TNF-α in human cells (e.g., THP-1 macrophages or monocytes or PBMCs). For example, contacting a human cell (e.g., THP-1 macrophage or PBMC) with a Roseburia species strain (e.g., a human Roseburia strain) can reduce or attenuate the production of IL-6, IL-12, IL-17, and / or TNF-α in the cell relative to a cell (e.g., a cell of the same cell type) that is not contacted with the strain 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%. In some embodiments, the contacting of the human cell with the Roseburia species strain occurs in vitro, such as by incubating the human cell with the Roseburia species strain. In some embodiments, the contacting of the human cell with the Roseburia species strain occurs in vivo.

[0092] In certain embodiments, the contemplated Roseburia species strain (e.g., human Roseburia strain) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of the mucosal epithelium. For example, the contemplated Roseburia species strain (e.g., human Roseburia strain) reduces or prevents disruption, or increases the barrier integrity of a HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α). For example, incubating a HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α) with a Roseburia species strain (e.g., human Roseburia strain) can reduce or prevent disruption, or increase barrier integrity (e.g., barrier integrity measured by transepithelial electrical resistance (TEER)) by at least about 10%, at least about 20%, to at least about 10%, to at least about 20%, to at least about 10%, to at least about 20%, to at least about 10%, to at least about 20%, to at least about 10%, to at least about 20%, to at least about 10%, to at least about 20%, to at least about 20%, to at least about 20%, to at least about 3 ... At least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, about 20% to about 100%, about 40% to about 100%, about 60% to about 100%, about 80% to about 100%, about 20% to about 80%, about 40% to about 80%, about 60% to about 80%, about 20% to about 60%, about 40% to about 60%, or about 20% to about 40%.

[0093] In certain embodiments, the contemplated Roseburia species strain (e.g., human Roseburia strain) induces or increases or is capable of inducing or increasing autophagy in a cell, tissue, or subject. For example, the contemplated Roseburia species strain (e.g., human Roseburia strain) induces or increases autophagy in human cells (e.g., THP-1 macrophages). For example, contacting a human cell (e.g., THP-1 macrophage) with the Roseburia species strain (e.g., human Roseburia strain) can induce or increase autophagy 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 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, 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%, about 20% to about 200%, about 50% to about 100%, or about 50% to about 200%, relative to a cell (e.g., a cell of the same cell type) that is not contacted with the strain. In some embodiments, the contacting of the human cell with the Roseburia species strain occurs in vitro, such as by incubating the human cell with the Roseburia species strain. In some embodiments, said contacting of said human cell with said Roseburia sp. strain occurs in vivo.

[0094] In certain embodiments, the contemplated Roseburia species strain (e.g., human Roseburia strain) reduces or attenuates or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject. For example, contacting a human cell with a Roseburia species strain (e.g., human Roseburia strain) can reduce or attenuate the production of lipocalin-2 / NGAL 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%, 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% relative to cells not contacted with the strain (e.g., cells of the same cell type). In some embodiments, the contacting of the human cell with the Roseburia species strain occurs in vitro, such as by incubating the human cell with the Roseburia species strain. In some embodiments, said contacting of said human cell with said Bacteroides sp. strain occurs in vivo.

[0095] In certain embodiments, the Roseburia hominis strain under consideration is a strain identified herein as RHO01. Roseburia hominis strain RHO01 (P127-H9a) was deposited with DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstraβe 7B, 38124 Brunswick, Germany) on September 14, 2018 under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. The deposit is assigned accession number DSM-32921. The 16s rRNA gene sequence of Roseburia hominis strain RHO01 is provided herein as SEQ ID NO: 1, and the genomic sequence of Roseburia hominis strain RHO01 is provided herein as SEQ ID NO: 4-32.

[0096] In certain embodiments, the DNA-DNA hybridization (DDH) value of the human Roseburia strain considered to be human Roseburia strain RHO01 (P127-H9a) is equal to or greater than about 70%. In certain embodiments, the DNA-DNA hybridization of the human Roseburia strain to the human Roseburia strain RHO01 (P127-H9a) is greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98% or about 99%. In certain embodiments, the average nucleotide identity (ANI) of the human Roseburia strain considered to be human Roseburia strain RHO01 (P127-H9a) is equal to or greater than 95%. In certain embodiments, the ANI is equal to or greater than about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or 100%. In certain embodiments, the comparison score (AF) of the Roseburia hominis strain under consideration to the Roseburia hominis strain RHO01 (P127-H9a) is equal to or greater than 60%. In certain embodiments, the AF is equal to or greater than about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or 100%.

[0097] In some embodiments, a contemplated bacterial strain mixture may, for example, include at least 2 (eg, 2 or 3) bacterial strains disclosed herein.

[0098] For example, a contemplated bacterial strain mixture may include: (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain. For example, in certain embodiments, a contemplated bacterial strain mixture includes: (i) a Bacteroides species strain and a Eubacterium species strain; (ii) a Bacteroides species strain and a Eubacterium hallii strain; (iii) a Bacteroides species strain and an EHA01 strain; (iv) a Bacteroides thetaiotaomicron strain and a Eubacterium species strain; (v) a Bacteroides thetaiotaomicron strain and an Eubacterium hallii strain; (vi) a Bacteroides thetaiotaomicron strain and an EHA01 strain; (vii) a BTH01 strain and a Eubacterium species strain; (viii) a BTH01 strain and an Eubacterium hallii strain; or (ix) a BTH01 strain and an EHA01 strain. In certain embodiments, the bacterial strain mixture contemplated: (i) increases or is capable of increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induces or increases, or is capable of inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0099] For example, a contemplated bacterial strain mixture may include: (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Roseburia species strain, such as a Roseburia hominis strain, such as a RHO01 strain. For example, in certain embodiments, a contemplated bacterial strain mixture includes: (i) a Bacteroides species strain and a Roseburia species strain; (ii) a Bacteroides species strain and a Roseburia hominis strain; (iii) a Bacteroides species strain and a RHO01 strain; (iv) a Bacteroides thetaiotaomicron strain and a Roseburia species strain; (v) a Bacteroides thetaiotaomicron strain and a Roseburia hominis strain; (vi) a Bacteroides thetaiotaomicron strain and a RHO01 strain; (vii) a BTH01 strain and a Roseburia species strain; (viii) a BTH01 strain and a Roseburia hominis strain; or (ix) a BTH01 strain and a RHO01 strain. In certain embodiments, the bacterial strain mixture contemplated: (i) increases or is capable of increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induces or increases, or is capable of inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0100] For example, a contemplated bacterial strain mixture may include: (i) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and (ii) a Roseburia species strain, such as an Homo Roseburia strain, such as an RHO01 strain. For example, in certain embodiments, a contemplated bacterial strain mixture includes: (i) a Eubacterium species strain and a Roseburia species strain; (ii) a Eubacterium species strain and a Homo Roseburia strain; (iii) a Eubacterium species strain and an RHO01 strain; (iv) a Eubacterium hallii strain and a Roseburia species strain; (v) a Eubacterium hallii strain and a Homo Roseburia strain; (vi) a Eubacterium hallii strain and an RHO01 strain; (vii) an EHA01 strain and a Roseburia species strain; (viii) an EHA01 strain and an Homo Roseburia strain; or (ix) an EHA01 strain and an RHO01 strain. In certain embodiments, the bacterial strain mixture contemplated: (i) increases or is capable of increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induces or increases, or is capable of inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0101] For example, the bacterial strain mixture contemplated may include: (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as an RHO01 strain. For example, in certain embodiments, the bacterial strain mixture contemplated includes: (i) a Bacteroides species strain, a Eubacterium species strain, and a Roseburia species strain; (ii) a Bacteroides species strain, a Eubacterium species strain, and a Roseburia hominis strain; (iii) a Bacteroides species strain, a Eubacterium species strain, and a RHO01 strain; (iv) a Bacteroides species strain, a Eubacterium hallii strain, and a Roseburia species strain; (v) a Bacteroides species strain, a Eubacterium hallii strain, and a Roseburia hominis strain; (vi) a Bacteroides species strain, a Eubacterium hallii strain, and a RHO01 strain; (vii) strains of Bacillus species, EHA01 strains, and Roseburia species; (viii) Bacteroides species, EHA01 strains, and Roseburia hominis strains; (ix) Bacteroides species, EHA01 strains, and RHO01 strains; (x) Bacteroides thetaiotaomicron strains, Eubacterium species, and Roseburia species; (xi) Bacteroides thetaiotaomicron strains, Eubacterium species, and Roseburia hominis strains; (xii) Bacteroides thetaiotaomicron strains, Eubacterium species, and RHO01 strains; (xiii) Bacteroides thetaiotaomicron strains, Eubacterium hallii strains, and Roseburia species; (xiv ) Bacteroides thetaiotaomicron, Eubacterium hallii and Roseburia hominis strains; (xv) Bacteroides thetaiotaomicron, Eubacterium hallii and RHO01 strain; (xvi) Bacteroides thetaiotaomicron, EHA01 and Roseburia spp. strains; (xvii) Bacteroides thetaiotaomicron, EHA01 and Roseburia hominis strains; (xviii) Bacteroides thetaiotaomicron, EHA01 and RHO01 strains; (xix) BTH01 strain, Eubacterium spp. strains and Roseburia spp. strains; (xx) BTH01 strain, Eubacterium spp. strains and Roseburia hominis strains; (xxi) strain, and RHO01; (xxii) BTH01, Eubacterium hallii, and Roseburia species; (xxiii) BTH01, Eubacterium hallii, and Roseburia hominis; (xxiv) BTH01, Eubacterium hallii, and RHO01; (xxv) BTH01, EHA01, and Roseburia species; (xxvi) BTH01, EHA01, and Roseburia hominis; or (xxvii) BTH01, EHA01, and RHO01.In certain embodiments, the bacterial strain mixture contemplated: (i) increases or is capable of increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induces or increases, or is capable of inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0102] For example, a contemplated bacterial strain mixture may consist essentially of: (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; and (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain. For example, in certain embodiments, a contemplated bacterial strain mixture consists essentially of: (i) a Bacteroides species strain and a Eubacterium species strain; (ii) a Bacteroides species strain and a Eubacterium hallii strain; (iii) a Bacteroides species strain and an EHA01 strain; (iv) a Bacteroides thetaiotaomicron strain and a Eubacterium species strain; (v) a Bacteroides thetaiotaomicron strain and a Eubacterium hallii strain; (vi) a Bacteroides thetaiotaomicron strain and an EHA01 strain; (vii) a BTH01 strain and a Eubacterium species strain; (viii) a BTH01 strain and a Eubacterium hallii strain; or (ix) a BTH01 strain and an EHA01 strain. In certain embodiments, the bacterial strain mixture contemplated: (i) increases or is capable of increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induces or increases, or is capable of inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0103] For example, a contemplated bacterial strain mixture may consist essentially of: (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; and (ii) a Roseburia species strain, such as a Roseburia hominis strain, such as a RHO01 strain. For example, in certain embodiments, a contemplated bacterial strain mixture consists essentially of: (i) a Bacteroides species strain and a Roseburia species strain; (ii) a Bacteroides species strain and a Roseburia hominis strain; (iii) a Bacteroides species strain and a RHO01 strain; (iv) a Bacteroides thetaiotaomicron strain and a Roseburia species strain; (v) a Bacteroides thetaiotaomicron strain and a Roseburia hominis strain; (vi) a Bacteroides thetaiotaomicron strain and a RHO01 strain; (vii) a BTH01 strain and a Roseburia species strain; (viii) a BTH01 strain and a Roseburia hominis strain; or (ix) a BTH01 strain and a RHO01 strain. In certain embodiments, the bacterial strain mixture contemplated: (i) increases or is capable of increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induces or increases, or is capable of inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0104] For example, a contemplated bacterial strain mixture may consist essentially of: (i) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and (ii) a Roseburia species strain, such as an Homo Roseburia strain, such as an RHO01 strain. For example, in certain embodiments, a contemplated bacterial strain mixture consists essentially of: (i) a Eubacterium species strain and a Roseburia species strain; (ii) a Eubacterium species strain and a Homo Roseburia strain; (iii) a Eubacterium species strain and a RHO01 strain; (iv) a Eubacterium hallii strain and a Roseburia species strain; (v) a Eubacterium hallii strain and a Homo Roseburia strain; (vi) a Eubacterium hallii strain and a RHO01 strain; (vii) an EHA01 strain and a Roseburia species strain; (viii) an EHA01 strain and a Homo Roseburia strain; or (ix) an EHA01 strain and a RHO01 strain. In certain embodiments, the bacterial strain mixture contemplated: (i) increases or is capable of increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induces or increases, or is capable of inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0105] For example, the bacterial strain mixture contemplated may consist essentially of: (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as an RHO01 strain. For example, in certain embodiments, the bacterial strain mixture contemplated consists essentially of: (i) a Bacteroides species strain, a Eubacterium species strain, and a Roseburia species strain; (ii) a Bacteroides species strain, a Eubacterium species strain, and a Roseburia hominis strain; (iii) a Bacteroides species strain, a Eubacterium species strain, and a RHO01 strain; (iv) a Bacteroides species strain, a Eubacterium hallii strain, and a Roseburia species strain; (v) a Bacteroides species strain, a Eubacterium hallii strain, and a Roseburia hominis strain; (vi) a Bacteroides species strain, a Eubacterium hallii strain, and a RHO01 strain; ( vii) Bacteroides species, EHA01 strain, and Roseburia species strain; (viii) Bacteroides species, EHA01 strain, and Roseburia hominis strain; (ix) Bacteroides species, EHA01 strain, and RHO01 strain; (x) Bacteroides thetaiotaomicron strain, Eubacterium species, and Roseburia species strain; (xi) Bacteroides thetaiotaomicron strain, Eubacterium species, and Roseburia hominis strain; (xii) Bacteroides thetaiotaomicron strain, Eubacterium species, and RHO01 strain; (xiii) Bacteroides thetaiotaomicron strain, Eubacterium hallii strain, and Roseburia species strain; (xiv) Bacteroides thetaiotaomicron, Eubacterium hallii and Roseburia hominis strains; (xv) Bacteroides thetaiotaomicron, Eubacterium hallii and RHO01 strain; (xvi) Bacteroides thetaiotaomicron, EHA01 and Roseburia spp. strains; (xvii) Bacteroides thetaiotaomicron, EHA01 and Roseburia hominis strains; (xviii) Bacteroides thetaiotaomicron, EHA01 and RHO01 strains; (xix) BTH01 strain, Eubacterium spp. strains and Roseburia spp. strains; (xx) BTH01 strain, Eubacterium spp. strains and Roseburia hominis strains; (xx (xxiv) BTH01 strain, Eubacterium hallii strain and RHO01 strain; (xxv) BTH01 strain, EHA01 strain and Roseburia species strain; (xxvi) BTH01 strain, EHA01 strain and Roseburia human strain; or (xxvii) BTH01 strain, EHA01 strain and RHO01 strain.In certain embodiments, the bacterial strain mixture contemplated: (i) increases or is capable of increasing the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduces or attenuates or is capable of reducing or attenuating the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduces or prevents disruption, or increases, or is capable of reducing or preventing disruption, or is capable of increasing the barrier integrity of an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induces or increases, or is capable of inducing or increasing autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0106] The bacterial strain mixture contemplated may, for example, comprise or consist essentially of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 bacterial strains. In some embodiments, one or more strains in the bacterial strain mixture are vegetative. In some embodiments, all strains of the bacterial strain mixture are vegetative. For example, in certain embodiments, the disclosed bacterial strain mixture comprises or consists essentially of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3 bacterial strains; or, for example, may comprise or consist essentially of 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5 or 3 to 4 bacterial strains; or, for example, may comprise or consist essentially of 4 to In some embodiments, the disclosed bacterial strain mixture comprises or consists essentially of 2 or 3 bacterial strains. In some of the above embodiments, the bacterial strain mixture comprises at least two bacterial strains, each of which is selected from: a Bacteroides species strain (e.g., a Bacteroides thetaiotaomicron strain), a Eubacterium species (e.g., a Eubacterium hallii strain), and a Roseburia species (e.g., a Roseburia hominis strain); and further comprises or consists essentially of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 additional bacterial strains. In some embodiments, the additional bacterial strains include a Bacteroides species strain, a Eubacterium species strain, and / or a Roseburia species strain, and / or a strain other than a Bacteroides species strain, a Eubacterium species strain, and / or a Roseburia species strain.In some embodiments, the additional bacterial strains are selected based on their ability alone to: (i) increase the production of at least one anti-inflammatory gene product (e.g., IL-10 and / or CCL-18) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (ii) reduce or attenuate the production of at least one pro-inflammatory gene product (e.g., IL-6, IL-12, IL-17 and / or TNF-α) in human cells (e.g., THP-1 macrophages or monocytes or PBMCs); (iii) reduce or prevent disruption of, or increase the barrier integrity of, an epithelial cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α); (iv) induce or increase autophagy in human cells (e.g., THP-1 macrophages); and / or (v) reduce or attenuate, or be capable of reducing or attenuating, the production of lipocalin-2 / NGAL in a cell, tissue, or subject.

[0107] In some embodiments, the contemplated bacterial strain mixture increases or is capable of increasing the production of at least one anti-inflammatory gene product in a cell, tissue, or subject. For example, the contemplated bacterial strain mixture, for example, increases the production of IL-10 and / or CCL-18 in human cells (e.g., THP-1 macrophages or monocytes or PBMCs). For example, contacting human cells (e.g., THP-1 macrophages or PBMCs) with the bacterial strain mixture can increase the production of IL-10 and / or CCL-18 in the cells relative to cells (e.g., cells of the same cell type) that are not contacted with the bacterial strain mixture 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 750%, at least about 1000%, about 10% to about 20%, about 10% to about 50%, about 1 0% 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 500%, 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, the contacting of the human cell with the mixture of bacterial strains occurs in vitro, for example, by incubating the human cell with the mixture of bacterial strains. In some embodiments, the contact of the human cell with the bacterial strain mixture occurs in vivo. In some embodiments, the bacterial strains of the bacterial strain mixture increase or are capable of increasing the production of at least one anti-inflammatory gene product in a synergistic manner. For example, the production of at least one anti-inflammatory gene product induced by the bacterial strain mixture is greater than the production induced by each bacterial strain in the mixture alone and / or the production induced by each individual bacterial strain added together. In some embodiments, the production of at least one anti-inflammatory gene product induced by the bacterial strain mixture is greater than the production induced by each bacterial strain in the mixture alone and / or the production induced by each individual bacterial strain added together by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100%.

[0108] In some embodiments, the bacterial strain mixture considered reduces or weakens or can reduce or weaken the production of at least one pro-inflammatory gene product in a cell, tissue or subject. For example, the bacterial strain mixture considered reduces or weakens the production of IL-6, IL-12, IL-17 and / or TNF-α in human cells such as THP-1 macrophages or monocytes or PBMCs. For example, contacting human cells such as THP-1 macrophages or PBMCs with a bacterial strain mixture can reduce or weaken the production of IL-6, IL-12, IL-17 and / or TNF-α in the cell relative to cells (e.g., cells of the same cell type) that are not contacted with a bacterial strain mixture 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%. In some embodiments, contacting of human cells with a mixture of bacterial strains occurs in vitro, for example, by incubating human cells with a mixture of bacterial strains. In some embodiments, contacting of human cells with a mixture of bacterial strains occurs in vivo. In some embodiments, the bacterial strains of the mixture of bacterial strains reduce or attenuate the production of at least one pro-inflammatory gene product in a synergistic manner. For example, the reduction or attenuation of the production of at least one pro-inflammatory gene product induced by the mixture of bacterial strains is greater than the reduction or attenuation induced by each bacterial strain in the mixture alone, and / or the reduction or attenuation induced by each individual bacterial strain added together. In some embodiments, the reduction or attenuation in the production of at least one pro-inflammatory gene product induced by the bacterial strain mixture is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater than 100% greater than the reduction or attenuation induced by each bacterial strain in the mixture alone and / or the reduction or attenuation induced by each bacterial strain together.

[0109] In some embodiments, the contemplated bacterial strain mixture reduces or prevents disruption of the barrier integrity of the mucosal epithelium or improves the barrier integrity of the mucosal epithelium, or is capable of reducing or preventing disruption of the barrier integrity of the mucosal epithelium or improving the barrier integrity of the mucosal epithelium. For example, the contemplated bacterial strain mixture reduces or prevents disruption of the barrier integrity of the HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α) or improves the barrier integrity of the HT29MTX-E12 cell monolayer. For example, incubating the contemplated bacterial strain mixture with the HT29MTX-E12 cell monolayer (e.g., a HT29MTX-E12 cell monolayer treated with TNF-α) can reduce or prevent disruption of the barrier integrity or improve the barrier integrity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 40 %, at least about 70%, at least about 80%, at least about 90%, at least about 100%, about 20% to about 100%, about 40% to about 100%, about 60% to about 100%, about 80% to about 100%, about 20% to about 80%, about 40% to about 80%, about 60% to about 80%, about 20% to about 60%, about 40% to about 60%, or about 20% to about 40% (e.g., as measured by transepithelial electrical resistance (TEER)).

[0110] In some embodiments, the bacterial strain mixture considered induces or increases or can induce or increase autophagy in cells, tissues or subjects.For example, the bacterial strain mixture considered induces or increases autophagy in human cells such as THP-1 macrophages.For example, human cells such as THP-1 macrophages are contacted with a bacterial strain mixture to make the autophagy in the cell relative to the cell (for example, the same cell type) not contacted with the bacterial strain mixture induce or increase at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, 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%, about 20% to about 200%, about 50% to about 100% or about 50% to about 200%. In some embodiments, the contact of human cells with a mixture of bacterial strains occurs in vitro, for example, human cells are incubated with a mixture of bacterial strains. In some embodiments, the contact of human cells with a mixture of bacterial strains occurs in vivo. In some embodiments, the bacterial strains of a mixture of bacterial strains increase autophagy in a synergistic manner. For example, the autophagy induced by a mixture of bacterial strains is higher than the autophagy induced by each bacterial strain alone in the mixture, and / or the autophagy induced by each single bacterial strain together. In some embodiments, the autophagy induced by a mixture of bacterial strains is higher than the autophagy induced by each bacterial strain alone in the mixture and / or the autophagy induced by each single bacterial strain together. At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100%.

[0111] In some embodiments, the bacterial strain mixture contemplated reduces or attenuates, or is capable of reducing or attenuating the production of lipocalin-2 / NGAL in a cell, tissue, or subject. For example, contacting a human cell with a bacterial strain mixture can reduce or attenuate the production of lipocalin-2 / NGAL in the cell relative to a cell (e.g., of the same cell type) that is not contacted with a bacterial strain mixture 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%. In some embodiments, contact of a human cell with a bacterial strain mixture occurs in vitro, for example, a human cell is incubated with a bacterial strain mixture strain. In some embodiments, contact of a human cell with a bacterial strain mixture occurs in vivo.

[0112] II. Pharmaceutical Composition / Unit

[0113] Bacteria disclosed herein can 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 refer to one or more buffers, carriers or excipients suitable for administration to a subject, such as a human subject, without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit / risk ratio. An excipient should be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and is harmless to the recipient.

[0114] Pharmaceutically acceptable excipients include buffers, solvents, dispersion media, coatings, isotonic agents and absorption delay agents compatible with drug administration. Pharmaceutically acceptable excipients also include fillers, binders, disintegrants, glidants, lubricants and any combination thereof. For example, the composition considered may include a pharmaceutical excipient selected from: cellulose, polyvinyl pyrrolidone, silicon dioxide, stearyl fumaric acid or its pharmaceutically acceptable salt, lactose, starch, glucose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, magnesium stearate, mannitol, sorbitol and any combination thereof. For further examples of excipients, carriers, stabilizers and adjuvants, see Handbook of Pharmaceutical Excipients, 8th Ed., PJ Sheskey, WG Cook and CG Can edit, Pharmaceutical Press, London, UK. Such media and reagents are known in the art for the use of pharmaceutically active substances.

[0115] In certain embodiments, bacterial strains described herein can be used in any composition in a stable form, including, for example, a lyophilized state (with optional one or more suitable cryoprotectants), frozen (e.g., in a standard or ultra-cold refrigerator), spray-dried and / or freeze-dried. In certain embodiments, stable bacteria (e.g., by freeze-drying, freezing, spray-drying or freeze-drying), and especially stable anaerobic bacteria can have advantageous properties over cultured bacteria in terms of application (e.g., using a pharmaceutical composition provided herein). For example, freeze-dried bacteria relate to a freeze-drying process that removes water from bacterial cells. In certain embodiments, compared with bacterial cultures, the resulting freeze-dried bacteria can have enhanced stability, so that a longer time (i.e., extended shelf life) can be stored. In addition, in certain embodiments, in a stable form, dehydrated bacterial cells do not grow or reproduce, but remain viable, and can grow and reproduce when rehydrated. In certain embodiments, even when exposed to oxygen, the viability of stable anaerobic bacteria is maintained, thereby facilitating its preparation (e.g., being formulated into an oral dosage form) and being used as a living biological therapeutic product that maintains biological activity. Therefore, in a specific embodiment, the bacterial strains described herein are stable (e.g., by lyophilization, freezing, freeze drying or spray drying), alive and active, and retain some, most or all of their chemical stability and / or biological activity when stored. Stability can be measured under selected temperature and humidity conditions and in a selected time period. Trend analysis can be used to estimate the expected shelf life of the material before the actual storage of the time period. For live bacteria, for example, stability can be measured by the time spent by losing 0.5 or 1 log cfu / g of the dried preparation under predetermined temperature and / or humidity conditions. Alternatively, stability can be defined as the time spent measuring the decline of a specific biological function per unit of dried preparation according to biological function.

[0116] In certain embodiments, the pharmaceutical composition or pharmaceutical unit under consideration loses at most 0.5 log cfu, 1 log cfu, 1.5 log cfu, 2 log cfu, 2.5 log cfu, 3 log cfu, 3.5 log cfu, 4 log cfu, 4.5 log cfu, 5 log cfu, 5.5 log cfu, 6 log cfu, 6.5 log cfu, 7 log cfu, 7.5 log cfu, 8 log cfu, 8.5 log cfu, 9 log cfu, 9.5 log cfu, or 10 log cfu of each bacterial strain present in the pharmaceutical composition or pharmaceutical unit when the pharmaceutical composition or pharmaceutical unit is stored at 4°C or -20°C for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years. For example, a contemplated pharmaceutical composition or pharmaceutical unit may lose up to 3 log cfu of each bacterial strain present in the pharmaceutical composition or pharmaceutical unit when stored at 4°C for 6 months, 1 year or 2 years.

[0117] The bacteria disclosed herein can be combined with one or more cryoprotectants. Exemplary cryoprotectants include fructooligosaccharides (e.g., ), trehalose, maltodextrin, sodium alginate, proline, glutamic acid, glycine (e.g., glycine betaine), monosaccharides, disaccharides or polysaccharides (e.g., glucose, sucrose, maltose, lactose), polyols (e.g., mannitol, sorbitol or glycerol), dextran, DMSO, methylcellulose, propylene glycol, polyvinyl pyrrolidone, non-ionic surfactants such as Tween 80 and / or any combination thereof.

[0118] In certain embodiments, contemplated cryoprotectants include Maltodextrin, alginate, trehalose and sucrose, or any combination thereof. Exemplary contemplated pharmaceutical compositions include sucrose as a cryoprotectant and include one or more strains selected from: (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; and (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a Roseburia species strain, such as a Roseburia hominis strain. Exemplary contemplated pharmaceutical compositions include as a cryoprotectant maltodextrin, alginate, trehalose and sucrose, and comprising one or more strains selected from the group consisting of: (i) a strain of Bacteroides species, such as a strain of Bacteroides thetaiotaomicron, such as a strain of BTH01; (ii) a strain of Eubacterium species, such as a strain of Eubacterium hallii, such as a strain of EHA01; and / or (iii) a strain of Roseburia species, such as a strain of Roseburia hominis, such as a strain of RHO01. Exemplary contemplated pharmaceutical compositions include as a cryoprotectant maltodextrin, alginate and trehalose, and include one or more strains selected from the group consisting of: (i) a Bacteroides species strain, e.g., a Bacteroides thetaiotaomicron strain, e.g., a BTH01 strain; (ii) a Eubacterium species strain, e.g., a Eubacterium hallii strain, e.g., an EHA01 strain; and / or (iii) a Roseburia species strain, e.g., a Roseburia hominis strain, e.g., an RHO01 strain.

[0119] For example, as contemplated herein, the lyophilized powder form of the bacterial strain may include about 10% to about 80% (by weight) of one or more bacterial strains (e.g., a 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 oligofructose, maltodextrin, sodium alginate, trehalose, sucrose, water, and / or a combination thereof. For example, 5 mg of the lyophilized powder form of the bacterial strain contemplated may include 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 to about 3.5 mg of the bacterial strain, or about 3.5 mg to about 4.5 mg of the bacterial strain. It is understood that each lyophilized powder form of the bacterial strain that can form the components of the disclosed composition can each have different excipients and / or excipient amounts, as well as discrete bacterial strains.

[0120] The pharmaceutical composition should be prepared to be compatible with its expected route of administration. The bacterial composition of consideration disclosed herein can be prepared by any suitable method, and can be formulated into various forms and applied by a variety of different modes. The composition of consideration can be administered orally, rectally or enterally as required with a preparation containing conventional acceptable carriers, adjuvants and solvents. As used herein, "rectal administration" should be understood to include administration by enema, suppository or colonoscopy. The disclosed pharmaceutical composition can, for example, be suitable for bolus administration or bolus release. In an exemplary embodiment, the disclosed bacterial composition is administered orally.

[0121] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules. Capsules typically include a core material comprising a bacterial composition and a shell wall encapsulating the core material. In some embodiments, the core material comprises at least one of a solid, a liquid, and an emulsion. In some embodiments, the shell wall material comprises at least one of soft gelatin, hard gelatin, and a polymer. Suitable polymers include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC), methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose succinate, and sodium carboxymethyl cellulose; acrylic acid polymers and copolymers, such as those formed by acrylic acid, methyl methacrylate, ammonium methacrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate (e.g., those sold under the trade name and ethylene-vinyl acetate copolymers; and shellac (purified lac). In some embodiments, at least one polymer is used as a taste masking agent.

[0122] Tablets, pills, etc. can be compressed, multi-compressed, multi-layered and / or coated. The coating considered can be single or multiple. In one embodiment, the coating material considered comprises at least one of sugar, polysaccharide and glycoprotein 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, sweet bean gum, guar gum, guar gum, karaya gum, gum ghatti, tragacanth, Funori, carrageenan, agar, alginate, chitosan or gellan gum. In some embodiments, the coating material considered comprises protein. In some embodiments, the coating material considered comprises at least one of fat and oil. In some embodiments, at least one of fat and oil is high temperature molten. In some embodiments, at least one of fat and oil is hydrogenated or partially hydrogenated. In some embodiments, at least one of fat and oil is derived from plants. In some embodiments, at least one of fat and oil comprises at least one of glyceride, free fatty acid and fatty acid ester. In some embodiments, the coating material considered comprises at least one edible wax. The edible wax considered can be derived from animals, insects or plants. Non-limiting examples include beeswax, lanolin, bayberry wax, carnauba wax and rice bran wax. Tablets and pills can be prepared with enteric coating or reverse enteric coating in addition.

[0123] Alternatively, powders or particles embodying the bacterial composition disclosed herein can be incorporated into food. In some embodiments, the food under consideration is a beverage for oral administration. Non-limiting examples of suitable beverages include water, fruit juice, fruit drinks, artificially flavored beverages, artificially sweetened beverages, carbonated beverages, sports drinks, liquid dairy products, milk shakes, alcoholic beverages, caffeinated beverages, infant formula milk powder, etc. Other suitable modes for oral administration include aqueous and non-aqueous solutions, emulsions, suspensions and / or solutions and / or suspensions reconstituted by non-effervescent particles, which include at least one suitable solvent, preservative, emulsifier, suspending agent, diluent, sweetener, coloring agent and flavoring agent.

[0124] In certain embodiments, pharmaceutical compositions contemplated include: (a) a mixture of freeze-dried bacterial strains (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as an RHO01 strain; 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 silicon dioxide derivative (e.g., colloidal silicon dioxide such as Cab-O-Sil or Aerosil)), and a lubricant (e.g., sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, a stearate, talc, liquid paraffin, propylene glycol (PG), PEG6000, or magnesium / sodium lauryl sulfate).

[0125] In certain embodiments, contemplated pharmaceutical compositions include: (a) a mixture of freeze-dried bacterial strains (i) a Bacteroides species strain, e.g., a Bacteroides thetaiotaomicron strain, e.g., a BTH01 strain; (ii) a Eubacterium species strain, e.g., a Eubacterium hallii strain, e.g., an EHA01 strain; and / or (iii) a Roseburia species strain, e.g., a Roseburia hominis strain, e.g., an RHO01 strain; and (b) a filler (microcrystalline cellulose), a disintegrant (polyvinyl pyrrolidone), a flow aid / glidant (silicon dioxide), and a lubricant (sodium stearyl fumarate).

[0126] In certain embodiments, the pharmaceutical composition considered is formulated as a capsule. In certain embodiments, the capsule is a hydroxypropyl methylcellulose (HPMC) capsule. In certain embodiments, the capsule includes a sealing (banding) polymer (e.g., hydroxypropyl methylcellulose (HPMC)) and a sealing solvent (e.g., water or ethanol). In certain embodiments, the capsule includes two sealing solvents, water and ethanol. In certain embodiments, the capsule is coated with a reverse enteric coating polymer (e.g., aminomethyl acrylate copolymer), and includes 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., talcum powder) and a coating solvent (e.g., water). In certain embodiments, the capsule is coated with an enteric coating polymer (e.g., poly (methacrylic acid-to-methyl methacrylate)), and also includes a plasticizer (e.g., triethyl citrate), an anti-adherent (e.g., talcum powder), a pH regulator (e.g., ammonia solution) and a coating solvent (e.g., purified water and isopropanol).

[0127] In certain embodiments, the capsule contemplated is a capsule-in-capsule dosage form, which includes an inner capsule and an outer capsule. In certain embodiments, the inner capsule includes a mixture of freeze-dried bacterial strains (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as an RHO01 strain, a filler (e.g., microcrystalline cellulose, lactose, sucrose, mannitol, dicalcium phosphate dihydrate or starch), a disintegrant (e.g., polyvinyl pyrrolidone, sodium starch glycolate or carboxymethyl cellulose), a flow aid / glidant (e.g., silicon dioxide, talc or colloidal silicon dioxide), and a lubricant (e.g., sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, a stearate salt, talc, liquid paraffin, propylene glycol (PG), PEG 6000 or magnesium / sodium lauryl sulfate). In certain embodiments, the outer capsule includes a mixture of freeze-dried bacterial strains (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as an RHO01 strain, a filler (e.g., microcrystalline cellulose, lactose, sucrose, mannitol, dicalcium phosphate dihydrate, or starch), a disintegrant (e.g., polyvinyl pyrrolidone, sodium starch glycolate, or carboxymethyl cellulose), a flow aid / glidant (e.g., silicon dioxide, talc, or colloidal silicon dioxide), and a lubricant (e.g., sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, a stearate salt, talc, liquid paraffin, propylene glycol (PG), PEG 6000, or magnesium / sodium lauryl sulfate).

[0128] In certain embodiments, the capsule contemplated is a capsule-in-capsule dosage form, which includes an inner capsule and an outer capsule. In certain embodiments, the inner capsule includes a freeze-dried bacterial strain (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as a mixture of the RHO01 strain, a filler (microcrystalline cellulose), a disintegrant (polyvinyl pyrrolidone), a flow aid / glidant (silicon dioxide), and a lubricant (sodium stearyl fumarate). In certain embodiments, the outer capsule includes a mixture of freeze-dried bacterial strains (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as an RHO01 strain, a filler (microcrystalline cellulose), a disintegrant (polyvinyl pyrrolidone), a flow aid / glidant (silicon dioxide) and a lubricant (sodium stearyl fumarate).

[0129] In certain embodiments, the disclosed drug unit includes a two-component capsule. For example, the two-component capsule may include an inner capsule and an outer capsule encapsulating the inner capsule, wherein the inner capsule has a reverse enteric polymer coating, and wherein the outer capsule has an enteric polymer coating. The inner and / or outer capsules contemplated may contain a bacterial strain or a mixture of bacterial strains. For example, the two-component capsule may include an inner capsule having an inner composition comprising a bacterial strain or a mixture of bacterial strains and one or more pharmaceutical excipients, wherein the inner capsule has a reverse enteric polymer coating, and an outer capsule encapsulating the inner capsule and an outer composition comprising a bacterial strain or a mixture of bacterial strains and one or more pharmaceutical excipients, wherein the outer capsule has an enteric polymer coating. The inner and / or outer compositions contemplated may, for example, include: (i) a strain of a Bacteroides species, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a strain of a Eubacterium species, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a strain of a Roseburia species, such as a Roseburia hominis strain, such as an RHO01 strain. The inner composition and the outer composition may be the same or different.

[0130] The contemplated two-component capsules may include a total of about 5 mg to about 60 mg of the inner and outer composition, for example, a total of about 5 mg to about 50 mg of the inner and outer composition, a total of about 5 mg to about 15 mg of the inner and outer composition, a total of about 5 mg to about 25 mg of the inner and outer composition, or a total of about 25 mg to about 50 mg of the inner and outer composition. The contemplated two-component capsules may include a total of about 50 mg to about 120 mg of the inner and outer composition, for example, a total of about 50 mg to about 75 mg of the inner and outer composition, a total of about 60 mg to about 85 mg of the inner and outer composition, a total of about 50 mg to about 95 mg of the inner and outer composition, or a total of about 25 mg to about 110 mg of the inner and outer composition.

[0131] In certain embodiments, the disclosed two-component capsule comprises an inner capsule having a reverse enteric polymer coating and an outer capsule having an enteric polymer coating. For example, each respective coating allows for biphasic release of the contents of the capsule (including bacterial strains) at different locations along the gastrointestinal tract. For example, it has been determined that the gastrointestinal tract has several regions clearly demarcated by local pH ranging from 1 to 8.2. The normal pH distribution of the gastrointestinal tract rises and falls between the stomach and the colon, with a pH range of 1-4 in the stomach, 5.5-6.4 in the duodenum, 6.8-8.2 in the ileum, and 5.5-6.5 in the colon. For example, although the distal ileum contains an area where the pH is usually between 6.8 and 8.2, the pH drops sharply from 8.2 to 5.5 after passing through the ileocecal valve into the cecum and ascending colon. In the process from the proximal colon to the distal colon, the pH value gradually increases again to 8.0. Therefore, in certain embodiments, the enteric polymer coating of the outer capsule dissolves at a pH of about 7 to 8, thereby allowing release in the ileum, and the reverse enteric polymer coating of the inner capsule dissolves at a pH of about 6.2 to 6.5, thereby allowing subsequent release in the colon. In certain embodiments, the outer capsule maintains integrity (for example, there is no crack, crack or rupture of the capsule shell) for about 2 hours at pH 1.2 and 37°C. In certain embodiments, the outer capsule maintains integrity (for example, there is no crack, crack or rupture of the capsule shell) for about 2 hours at pH 5.5 and 37°C. In certain embodiments, the outer capsule disintegrates in about 1 hour at pH 7.4 and 37°C. In certain embodiments, the inner capsule maintains integrity (for example, there is no crack, crack or rupture of the capsule shell) for up to 1 hour at pH 7.4 and 37°C. In certain embodiments, the inner capsule disintegrates in 2 hours at pH 6.5 and 37°C.

[0132] In certain embodiments, the inner and / or outer capsule coating consists 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, copolymers of methacrylic acid and ethyl acrylate, or copolymers of methacrylic acid and ethyl acrylate with methacrylic acid monomers added during polymerization. Methyl methacrylate or copolymers of methacrylic acid and ethyl acrylate may be used as the monomers. The polymers are obtained from Evonik Industries, Darmstadt, Germany. For example, L100 and S100 (anionic copolymer based on methacrylic acid and methyl methacrylate). L100 dissolves at a pH of about pH 6 or above and contains 46.0% to 50.6% methacrylic acid units per gram of dry matter; S100 dissolves at a pH of about pH 7 or above and contains 27.6% to 30.7% methacrylic acid units per gram of dry matter. Another exemplary class of encapsulating polymers is polyacrylic acid. L and S, which may optionally be RL or RS (copolymers of ethyl acrylate, methyl methacrylate and low levels of methacrylate with quaternary ammonium groups). These modified acrylic acids are useful because they can be made soluble at pH values ​​of 6 to 7.5, depending on the particular Eudragit selected, and on the amount of methacrylate used in the formulation. S and In certain embodiments, the coating of the inner capsule is contemplated to be made of Eudragit ReadyMix. In certain embodiments, the outer capsule coating contemplated is composed of L100 (methacrylic acid-methyl methacrylate copolymer (1:1)) and S100 (methacrylic acid-methyl methacrylate copolymer (1:2)). In certain embodiments, the contemplated capsules are suitable for extended or timed release. In certain embodiments, the contemplated inner and / or outer capsule coatings also include 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).

[0133] Additional exemplary capsule-in-capsule formulations are described in US Pat. No. 9,907,755.

[0134] Pharmaceutical compositions comprising the bacterial strains disclosed herein can be present in unit dosage form, ie, pharmaceutical units. Compositions, such as pharmaceutical units provided herein, can include any suitable amount of each bacterial strain, measured by the total mass of bacteria or colony forming units.

[0135] For example, a disclosed pharmaceutical composition or unit may include about 10 3 cfu to about 10 12 cfu, about 10 6 cfu to about 10 12 cfu, about 10 7 cfu to about 10 12 cfu, about 10 8 cfu to about 10 12 cfu, about 10 9 cfu to about 10 12 cfu, about 10 10 cfu to about 10 12 cfu, about 10 11 cfu to about 10 12 cfu, about 10 3 cfu to about 10 11 cfu, about 10 6 cfu to about 10 11 cfu, about 10 7 cfu to about 10 11 cfu, about 10 8 cfu to about 10 11 cfu, about 10 9 cfu to about 10 11 cfu, about 10 10 cfu to about 10 11 cfu, about 10 3 cfu to about 10 10 cfu, about 10 6 cfu to about 10 10 cfu, about 10 7 cfu to about 10 10cfu, about 10 8 cfu to about 10 10 cfu, about 10 9 cfu to about 10 10 cfu, about 10 3 cfu to about 10 9 cfu, about 10 6 cfu to about 10 9 cfu, about 10 7 cfu to about 10 9 cfu, about 10 8 cfu to about 10 9 cfu, about 10 3 cfu to about 10 8 cfu, about 10 6 cfu to about 10 8 cfu, about 10 7 cfu to about 10 8 cfu, about 10 3 cfu to about 10 7 cfu, about 10 6 cfu to about 10 7 cfu or about 10 3 cfu to about 10 6 cfu, or may include about 10 3 cfu, about 10 6 cfu, about 10 7 cfu, about 10 8 cfu, about 10 9 cfu, about 10 10 cfu, about 10 11 cfu or about 10 12 cfu of bacterial strain or each bacterial strain.

[0136] For example, a disclosed pharmaceutical composition or unit may include about 10 3 cfu to about 10 12 cfu, about 10 6 cfu to about 10 12 cfu, about 10 7 cfu to about 10 12 cfu, about 10 8 cfu to about 10 12 cfu, about 10 9 cfu to about 10 12 cfu, about 10 10 cfu to about 10 12 cfu, about 10 11 cfu to about 10 12 cfu, about 103 cfu to about 10 11 cfu, about 10 6 cfu to about 10 11 cfu, about 10 7 cfu to about 10 11 cfu, about 10 8 cfu to about 10 11 cfu, about 10 9 cfu to about 10 11 cfu, about 10 10 cfu to about 10 11 cfu, about 10 3 cfu to about 10 10 cfu, about 10 6 cfu to about 10 10 cfu, about 10 7 cfu to about 10 10 cfu, about 10 8 cfu to about 10 10 cfu, about 10 9 cfu to about 10 10 cfu, about 10 3 cfu to about 10 9 cfu, about 10 6 cfu to about 10 9 cfu, about 10 7 cfu to about 10 9 cfu, about 10 8 cfu to about 10 9 cfu, about 10 3 cfu to about 10 8 cfu, about 10 6 cfu to about 10 8 cfu, about 10 7 cfu to about 10 8 cfu, about 10 3 cfu to about 10 7 cfu, about 10 6 cfu to about 10 7 cfu or about 10 3 cfu to or about 10 6 cfu, or may include about 10 3 cfu, about 10 6 cfu, about 10 7 cfu, about 10 8 cfu, about 10 9 cfu, about 10 10 cfu, about 10 11 cfu or about 10 12cfu of: (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as an RHO01 strain.

[0137] In certain embodiments, provided drug units contain at least 1 x 10 3 CFU of each bacterial strain (e.g., vegetative bacterial strain), or at least 1 x 10 4 Colony forming units of a bacterial strain (e.g., a vegetative bacterial strain), or at least 1 x 10 5 Colony forming units of a bacterial strain (e.g., a vegetative bacterial strain), or at least 1 x 10 6 CFU of each bacterial strain (e.g., vegetative bacterial strain), or at least 1 x 10 7 CFU of each bacterial strain (e.g. vegetative bacterial strain), or at least 1 x 10 8 CFU of each bacterial strain (e.g. vegetative bacterial strain), or at least 1 x 10 9 Colony forming units of each bacterial strain (eg, vegetative bacterial strain).

[0138] Compositions, such as pharmaceutical units provided herein, can include each bacterial strain in any suitable ratio, which is measured by the total mass of bacteria or colony forming units. For example, the disclosed pharmaceutical compositions or units can include 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 according to the total mass of bacteria or colony forming units. For example, the disclosed pharmaceutical composition or unit may include three strains, for example, (i) a Bacteroides species strain, such as a Bacteroides thetaiotaomicron strain, such as a BTH01 strain; (ii) a Eubacterium species strain, such as a Eubacterium hallii strain, such as an EHA01 strain; and / or (iii) a Roseburia species strain, such as a Roseburia hominis strain, such as an RHO01 strain, in a ratio of 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. For example, the disclosed pharmaceutical composition or unit may include (i) a strain of Bacteroides species, such as a strain of Bacteroides thetaiotaomicron, such as a strain of BTH01; (ii) a strain of Eubacterium species, such as a strain of Eubacterium hallii, such as a strain of EHA01; and (iii) a strain of Roseburia species, such as a strain of Roseburia hominis, such as a strain of RHO01, in a ratio of 1:1:1 based on the total mass of bacteria or colony forming units. For example, the disclosed pharmaceutical composition or unit may include about 1 x 10 9 The invention relates to a method for treating a genus of the present invention and comprising the following living organisms (e.g., cfu) of each of the following: (i) a Bacteroides species strain, e.g., a Bacteroides thetaiotaomicron ...TH01 strain; (ii) a Eubacterium species strain, e.g., a Eubacterium hallii strain, e.g., a EHA01 strain; and (iii) a Roseburia species strain, e.g., a Roseburia hominis strain, e.g., a RHO01 strain.

[0139] For example, disclosed compositions (e.g., pharmaceutical units such as capsules) can include about 1 mg to about 5 mg (e.g., 2 mg to about 4 mg) of each individual bacterial strain, which can be present in the unit individually, for example, in about 5 mg to about 50 mg of a lyophilized powder of the bacterial strain. For example, a pharmaceutical unit can include 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 a lyophilized powder of the bacterial strain. In certain embodiments, the pharmaceutical unit comprises 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 bacterial strain in lyophilized powder form.

[0140] In certain embodiments, the disclosed compositions, e.g., the disclosed drug units, can include about 5 to about 50 mg of each bacterial strain in the form of a lyophilized powder, 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 bacterial strain in the form of a lyophilized powder, about 10 to about 20 mg, about 10 to about 15 mg, or about 15 to about 45 mg of each bacterial strain in the form of a lyophilized powder. In certain embodiments, the disclosed drug units contain about 5, about 10, about 15, about 20, about 25, or about 30 mg of each bacterial strain in the form of a lyophilized powder. In certain embodiments, the disclosed drug unit includes about 25 to about 50 mg of one bacterial strain in the form of a lyophilized powder and about 5 mg to about 10 mg of the remaining bacterial strains in the form of lyophilized powder, or about 5 to about 15 mg of one bacterial strain in the form of a lyophilized powder and about 5 to 10 mg of the remaining bacterial strains in the form of lyophilized powder, for example, about 15 mg of one bacterial strain in the form of a lyophilized powder and about 5 mg of the remaining bacterial strains in the form of lyophilized powder, or about 15 mg to about 25 mg of each of two bacterial strains in the form of lyophilized powder and about 5 mg to 10 mg of the remaining bacterial strains in the form of lyophilized powder. In some embodiments, one or more (e.g., all) bacterial strains in the form of lyophilized powder are vegetative.

[0141] III. Therapeutic Uses

[0142] The compositions and methods disclosed herein can be used to treat various forms of gastrointestinal diseases, inflammatory diseases and / or dysbiosis in subjects. The present disclosure provides a method for treating gastrointestinal diseases, inflammatory diseases and / or dysbiosis in subjects. The intended method includes administering an effective amount of pharmaceutical compositions, drug units and / or bacterial strains disclosed herein to subjects alone or in combination with another therapeutic agent to treat gastrointestinal diseases, inflammatory diseases and / or dysbiosis in subjects. The intended method includes administering at least 2 nutritional bacterial strains to subjects, each of which is selected from: Bacteroides species strains, Eubacterium species strains and Roseburia species strains. In certain embodiments, Bacteroides species is Bacteroides thetaiotaomicron. In certain embodiments, Eubacterium species is Eubacterium hallii. In certain embodiments, Roseburia species is Roseburia hominis.

[0143] As used herein, "treat" (treating) refers to treating a disease in a subject, such as a human. It includes: (a) inhibiting the disease, i.e., preventing its development; 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 to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals, such as humans, pets (e.g., dogs, cats, or rabbits), or livestock (e.g., cows, sheep, pigs, goats, horses, donkeys and mules, buffaloes, bulls, or camels).

[0144] It should be understood that the exact dosage of the drug unit, pharmaceutical composition or bacterial strain is selected by the individual physician according to the patient to be treated, and generally, the dosage and administration are adjusted to provide an effective amount of the bacterial agent to the patient to be treated. As used herein, "effective amount" refers to the amount necessary to cause a beneficial or desired biological response. The effective amount can be administered in one or more administrations, applications or dosages, and is not intended to be limited to a specific formulation or route of administration. As will be appreciated by those of ordinary skill in the art, the effective amount of the drug unit, pharmaceutical composition or bacterial strain can vary according to factors such as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. Other factors that may be considered include the severity of the disease state; the age, weight and sex of the patient to be treated; diet, time and frequency of administration; drug combination; reaction sensitivity; and tolerance / reaction to treatment.

[0145] It should be understood that the disclosed bacterial strains or bacterial strain mixtures may not require colonization of the intestinal tract of the subject and / or persist in the subject to cause a beneficial or desired biological response. For example, in certain embodiments, the bacterial strain or bacterial strain mixture colonizes or partially colonizes in the intestinal tract of the subject and / or persists in the subject after administration. In certain embodiments, the bacterial strain or bacterial strain mixture does not colonize in the intestinal tract of the subject and / or persists in the subject after administration.

[0146] Gastrointestinal diseases 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 infection (CDI), recurrent Clostridium difficile infection (rCDI), Clostridium difficile associated diarrheal disease (CDAD), colitis (e.g., infectious, ischemic, indeterminate, or radiation colitis), ulcers (including gastric ulcers, peptic ulcers, and duodenal ulcers), gastroesophageal reflux disease (GERD), pouchitis, gastroenteritis, pancreatitis, mucositis (e.g., oral mucositis, gastrointestinal mucositis, nasal mucositis and proctitis), necrotizing enterocolitis, esophagitis, non-ulcer dyspepsia, chronic intestinal pseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction, duodenogastric reflux, ileus inflammation, 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 neurological diseases), hemorrhoids, diverticular disease, chronic pancreatitis, blind loop syndrome, gastroparesis (including diabetic and / or idiopathic), diarrhea, dysphagia, fecal incontinence, short bowel syndrome (SBS), intestinal ischemia, infantile reflux, infantile rumination syndrome, cyclic vomiting syndrome, globus hystericus, volvulus, gastrointestinal cancer and gastrointestinal allergy. It is expected that the compositions and methods disclosed herein can be used to treat any functional gastrointestinal disorder, including, for example, diseases mediated by or associated with brain-gut interactions.

[0147] Inflammatory bowel disease or IBD, used interchangeably herein, refers to intestinal diseases that cause inflammation and / or ulcers and include, but are not limited to, Crohn's disease and ulcerative colitis.Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases of unknown etiology.

[0148] Ulcerative colitis (UC) affects the large intestine. The course of the disease may be continuous or relapsing, mild or severe. The earliest lesions are inflammatory infiltrates that form abscesses at the bottom of the Leyden crypts. The coalescence of these swollen and ruptured crypts tends to separate the overlying mucosa from its blood supply, resulting in ulcers. Symptoms of the disease include cramping, lower abdominal pain, rectal bleeding, and frequent, loose bowel movements that consist primarily of blood, pus, and mucus with few fecal particles. For acute, severe, or chronic persistent ulcerative colitis, total colectomy may be necessary.

[0149] Unlike ulcerative colitis, Crohn's disease can affect any part of the intestine. The most prominent feature of Crohn's disease is a granular, reddish-purple, edematous thickening of the intestinal wall. As inflammation progresses, these granulomas often lose their limiting borders and fuse with the surrounding tissue. Diarrhea and intestinal obstruction are the main clinical features. Like ulcerative colitis, the course of Crohn's disease can be continuous or relapsing, mild or severe, but unlike ulcerative colitis, Crohn's disease cannot be cured by removing the relevant part of the intestine. Most patients with Crohn's disease require surgery at some point, but subsequent relapses are common and usually require continued medical treatment.

[0150] Inflammatory diseases can be characterized, for example, based on the affected primary tissue, the potential mechanism of action of the disease, or the disordered or overactive immune system part. Examples of inflammatory diseases include inflammation of the lungs, joints, connective tissue, eyes, nose, intestines, kidneys, liver, skin, central nervous system, vascular system, heart, or adipose tissue. In certain embodiments, inflammatory diseases that can be treated include inflammation caused by infiltration of leukocytes or other immune effector cells or their mediators into the affected tissue. In certain embodiments, inflammatory diseases that can be treated include inflammation mediated by IgA and / or IgE antibodies. Other related examples of inflammatory diseases 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 certain embodiments, the inflammatory disease treated is an allergic reaction. In certain embodiments, inflammatory diseases are autoimmune diseases.

[0151] Pulmonary diseases include asthma, adult respiratory distress syndrome, bronchitis, lung inflammation, pulmonary fibrosis and cystic fibrosis (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 diseases include rheumatoid arthritis, seronegative spondyloarthropathies (including ankylosing spondylitis), juvenile rheumatoid arthritis, osteoarthritis, gouty arthritis and other arthritic diseases. Ocular inflammatory diseases include uveitis (including iritis), conjunctivitis, episcleritis, scleritis and keratoconjunctivitis sicca. Inflammatory bowel disease includes Crohn's disease, ulcerative colitis, inflammatory bowel disease and distal proctitis. Inflammatory skin diseases include diseases associated with cell proliferation, such as psoriasis, eczema, dermatitis (e.g., eczematous dermatitis, local and seborrheic dermatitis, allergic or irritant contact dermatitis, chapped eczema, photoallergic dermatitis, phototoxic dermatitis, radiation dermatitis, and stasis dermatitis) and acne. Inflammatory diseases of the endocrine system include, but are not limited to, autoimmune endocrinopathy, autoimmune thyroiditis (Hashimoto's disease), type I diabetes, liver and adipose tissue inflammation associated with type II diabetes, and acute and chronic inflammation of the adrenal cortex. Inflammatory diseases of the cardiovascular system include, but are not limited to, coronary artery infarction lesions, peripheral vascular disease, myocarditis, vasculitis, stenotic angiogenesis, atherosclerosis, and vascular diseases 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, Goodpasture syndrome, post-obstructive syndrome, and renal tubular ischemia. Inflammatory diseases of the liver include, but are not limited to, hepatitis (caused by viral infection, autoimmune reaction, drug therapy, toxins, environmental factors, or as secondary consequences caused by primary diseases), biliary atresia, primary biliary cirrhosis and primary hepatitis sclerosing cholangitis. Metabolic diseases with inflammatory causes include insulin resistance, metabolic syndrome, obesity, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic fatty liver disease (NASH). In certain embodiments, inflammatory diseases are autoimmune diseases, such as rheumatoid arthritis, lupus, alopecia, autoimmune pancreatitis, celiac disease, Behcet's disease, Cushing's syndrome and Grave's disease. In certain embodiments, inflammatory diseases are rheumatoid diseases, such as rheumatoid arthritis, juvenile arthritis, bursitis, spondylitis, gout, scleroderma, Still's disease and vasculitis. Other exemplary inflammatory diseases include eosinophilic esophagitis and eosinophilic gastroenteritis.

[0152] Generally, dysbiosis refers to the state of the microbiota or microbiome of the intestinal tract or other body regions, including, for example, mucosal or skin surfaces (or any other microbiota niche) in which the normal diversity and / or function of the ecological network are disrupted. Any disruption from the typical (e.g., ideal) state of the microbiota can be considered dysbiosis, even if this dysbiosis does not result in a detectable reduction in health. This dysbiosis state may be unhealthy (e.g., leading to a disease state), or may be unhealthy only under certain conditions, or may prevent the subject from becoming healthier. Dysbiosis may be caused by a reduction in the diversity of the microbiota composition, the overgrowth of one or more pathogens (e.g., pathogenic bacteria groups) or pathogenic bacteria groups, the presence and / or overgrowth of symbiotic organisms that can cause disease (only when the patient has certain genetic and / or environmental disorders, or when turning to no longer providing beneficial functions to the host and therefore no longer promoting a healthy ecological network). Distal dysbiosis includes, but is not limited to, dysbiosis outside the gastrointestinal lumen.

[0153] It is expected that dysbiosis may include infection with pathogenic bacteria of the genus selected from the group consisting of Yersinia, Vibrio, Treponema pallidum, Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella, Klebsiella, Helicobacter pylori, Haemophilus, Francisella, Escherichia, Ehrlichia, Enterococcus, Rickettsia, Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, and Bacillus. Other examples of pathogenic bacteria include Aeromonas hydrophila, Campylobacter fetus, Pseudomonas shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium 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, Listeria monocytogenes, Pseudomonas shigelloides, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum β-lactam-resistant Enterococci (ESBL), vancomycin-resistant Enterococci (VRE), and multidrug-resistant bacteria.

[0154] It is also contemplated that the compositions and methods disclosed herein may be used to treat diseases of the liver, pancreas, or gall bladder.

[0155] IV. Combination Therapy

[0156] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or methods. As used herein, the term "combination" administration is understood to refer to delivering two (or more) different treatments to a subject during the subject's disease, so that the effects of treatment on the patient overlap at a certain time point. In certain embodiments, when the delivery of the second treatment is started, the delivery of the first treatment is still being carried out, so that there is overlap in the application. It is sometimes referred to as "simultaneous" or "synchronous delivery" in this article. In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In certain embodiments of any one case, due to combined administration, the treatment is more effective. For example, the second treatment is more effective, for example, compared with the situation of applying the second treatment in the absence of the first treatment, the same effect is observed with less second treatment, or the second treatment alleviates symptoms to a greater extent, or observes a situation similar to the first treatment method. In certain embodiments, delivery makes the reduction of symptoms or other parameters related to the disease greater than the reduction observed by delivering the first treatment in the absence of another treatment. The effects of the two treatments can be partially cumulative, completely cumulative, or greater than cumulative. Delivery can make it possible to still detect the effect of the first treatment delivered when the second treatment is delivered. In certain embodiments, side effects of the first and / or second treatment are reduced as a result of the combined administration.

[0157] In certain embodiments, the methods or compositions described herein are administered in combination with one or more additional therapies, such as one or more anti-inflammatory agents. In certain embodiments, the additional therapies contemplated may include aminosalicylate, corticosteroids, tumor necrosis factor (TNF) antagonists, linaclotide, antibiotics or immunosuppressants (e.g., azathioprine, 6-mercaptopurine, cyclosporine, methotrexate or tacrolimus (Prograf)). In certain embodiments, the additional therapies contemplated may include biologics (e.g., infliximab (Remicade), adalimumab (Humira), certolizumab (Cimzia), golimumab (Simponi) or etanercept (Enbrel)). It is contemplated that the subject treated with the disclosed method or composition may be insufficiently responsive to the previously administered therapy (e.g., previously administered aminosalicylate, corticosteroids or biologics).

[0158] Other therapeutic agents suitable for combination therapy with the pharmaceutical compositions or 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., misoprostol (Cytotec)), sucralfate, and antacids.

[0159] In certain embodiments, pharmaceutical composition or unit can include corticosteroid or be used in combination therewith.Corticosteroid is a class of chemical substances, including steroid hormones produced naturally in the adrenal cortex of vertebrates and analogs of these hormones synthesized in the laboratory.Corticosteroid participates in a wide range of physiological processes, including stress response, immune response and inflammation regulation, carbohydrate metabolism, protein catabolism, blood electrolyte levels and behavior.Exemplary corticosteroids include betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone or deflazacort.Expected experimenter treated with disclosed method or composition may be under-reacted to the corticosteroid used before.

[0160] In certain embodiments, pharmaceutical composition or unit may include aminosalicylate or be used in combination therewith.Exemplary aminosalicylate includes 4-aminosalicylic acid, balsalazide, olsalazine, sulfasalazine and mesalazine (5-aminosalicylic acid).Expected subject treated with disclosed method or composition may be under-reacted to mesalazine (e.g., previously orally administered>2.4g / day mesalazine at least 8 weeks) used previously.

[0161] In certain embodiments, the pharmaceutical composition or unit may include a tumor necrosis factor (TNF) antagonist or be administered in combination therewith. Exemplary TNF antagonists include infliximab (Remicade), adalimumab (Humira), certolizumab (Cimzia), golimumab (Simponi), etanercept (Enbrel), thalidomide (Immunoprin), lenalidomide (Revlimid), pomalidomide (Pomalyst, Imnovid), xanthine derivatives (e.g., pentoxifylline) and bupropion. It is expected that the subject treated with the disclosed method or composition may not respond adequately to the TNF antagonist previously administered.

[0162] In certain embodiments, a pharmaceutical composition or unit may include or be administered in combination with an integrin α4β7 antagonist (eg, vedolizumab).It is contemplated that a subject treated with the disclosed methods or compositions may have had an inadequate response to a prior administration of an integrin α4β7 antagonist.

[0163] In certain embodiments, the pharmaceutical composition or unit can include an antimicrobial agent (e.g., an antibiotic) or be administered in combination therewith. The disclosed method can include pre-treatment with an antibiotic, such as administering an antibiotic to a subject prior to administering the disclosed pharmaceutical composition or unit. Exemplary antibiotics for combined therapy include vancomycin, metronidazole, gentamicin, colistin, fidaxomicin, telavancin, oritavancin, dalbavancin, daptomycin, cephalexin, cefuroxime, cefoperazone, cefazolin, cephalosporin, cefaclor, cefoperazone, cefoxitin, cefuroxime, cefuroxime, cefuroxil, cefuroxil, cefuroxime, ciprofloxacin, levofloxacin, ofloxacin, gatifloxacin, moxifloxacin, norfloxacin, tetracycline, minocycline, soil metronidazole, doxycycline, amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, methicillin, ertapenem, doripenem, imipenem / cilastatin, meropenem, amikacin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, cefixime, cefdinir, cefoperazone, cefotaxime, ceftazidime, ceftriaxone, cefoxitin, and / or streptomycin.

[0164] In certain embodiments, a 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, dananavir, delavirdine, didanosine, docosin, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, foscarnet, fomivirsen, ganciclovir, indinavir, herpes, lamivudine, lopinavir, maraviroc, MK-2048, nefimavir, nevirapine, penciclovir, ramivudine, rilpivirine, ritonavir, saquinavir, stavudine, tenofovir trifluridine, valacyclovir, valganciclovir, vidarabine, ibacitabine, amantadine, oseltamivir, ramitidine, tipranavir, zalcitabine, zanamivir, and zidovudine. Exemplary antifungal agents include natamycin, cytomycin, filipin, nystatin, amphotericin B, candidiasis, and hamycin, miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxconazole, sertaconazole, sukiconazole, tioconazole, fluconazole, itraconazole, itraconazole, ivaconazole, lavconazole, posaconazole, voriconazole, terconazole, and albaconazole, abafungin, terbinafil, naftifine, butenafine, anidulafungin, caspofungin, micafungin, polygonal dialdehyde, benzoic acid, ciclopirox, tosylate, undecylenic acid, flucytosine or 5-flucytosine, griseofulvin, and halopronin.

[0165] In certain embodiments, the pharmaceutical composition or unit may include 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 microflora (e.g., a compound or composition that causes a specific change in the composition and / or activity of the microbiome). Exemplary prebiotics include complex carbohydrates, complex sugars, resistant dextrins, resistant starches, amino acids, peptides, nutritional compounds, biotin, polydextrose, oligofructose (FOS), galacto-oligosaccharides (GOS), inulin, lignin, psyllium, chitin, chitosan, chitooligosaccharides, lactitol, gums (e.g., guar gum), high amylose corn starch (HAS), cellulose, β-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), inulin rich in oligofructose, oligofructose, oligoglucose, tagatose, trans-galactose, pectin, resistant starch, isomaltooligosaccharides, and xylo-oligosaccharides (XOS). Prebiotics can be found in foods such as gum arabic, guar seeds, brown rice, rice bran, barley hulls, chicory root, Jerusalem artichoke, dandelion greens, garlic, leeks, onions, asparagus, wheat bran, oat bran, baked beans, whole wheat flour, and bananas and in breast milk. Prebiotics can also be administered in other forms such as capsules or dietary supplements.

[0166] Throughout the 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 also contemplated that there are compositions of the disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the disclosure that consist essentially of, or consist of, the recited processing steps.

[0167] In the present application, when it is stated that an element or component is included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from two or more of the recited elements or components.

[0168] In addition, it should be understood that, without departing from the spirit and scope of the present disclosure, the elements and / or features of the compositions or methods described herein can be combined in various ways, whether it is clear or implicit in this article. For example, in the case of referring to a specific compound, unless otherwise understood from the context, the compound can be used in various embodiments of the compositions of the present disclosure and / or the methods of the present disclosure. In other words, within the application, embodiments have been described and described in a manner that enables clear and concise writing and describing applications, but it is intended and will be understood that, without departing from this teaching and disclosure, embodiments can be combined or separated in various ways. For example, it will be understood that all features described and described herein can be applied to all aspects of the present disclosure described and described herein.

[0169] It should be understood that, unless otherwise understood from the context and usage, the expression "at least one" includes each of the recited objects and various combinations of two or more recited objects after the expression. Unless otherwise understood from the context, the expression "and / or" related to three or more recited objects should be understood to have the same meaning.

[0170] Unless otherwise specifically stated or understood from the context, the use of the terms "including", "having" or "comprising", including their grammatical equivalents, should generally be understood to be open and non-limiting, for example, not excluding other unrecited elements or steps.

[0171] When the term "about" is used before a quantitative value, unless specifically stated otherwise, the present disclosure also includes the specific quantitative value itself. As used herein, unless otherwise stated or inferred, the term "about" refers to a difference of ±10% from the nominal value.

[0172] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present disclosure remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

[0173] The use of any and all examples or exemplary language (e.g., "such as" or "including") herein is intended only to better illustrate the present disclosure and does not limit the scope of the present disclosure unless otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the present disclosure.

[0174] Example

[0175] The following examples are illustrative only and are not intended to limit the scope or content of the present disclosure in any way.

[0176] Example 1 - Overview of the bacterial strain selection process for mixed bacterial colony 1 (C1)

[0177] Mixed flora 1 (C1), a live biotherapeutic product for development as a treatment for gastrointestinal diseases (e.g., dysbiosis and / or immune-mediated inflammatory diseases such as ulcerative colitis, Crohn's disease and other forms of inflammatory bowel disease (IBD)), comprises a mixed flora of three bacterial strains, namely Bacteroides thetaiotaomicron BTH01, Eubacterium hallii EHA01 and Roseburia hominis RHO01.

[0178] Target candidate strains were isolated, purified, and characterized from healthy donor stool samples using standard anaerobic microbiological procedures. All stool donors underwent comprehensive clinical and laboratory testing to confirm health status, including screening for infectious agents to minimize the risk of transmissible infection. Serological screening included HIV-1 / HIV-2 (IgG and EIA), HTLY-1 and HTLY-II (Ab), hepatitis A virus (IgM), hepatitis B virus (HBSAg, anti-HBc IgG and IgM), hepatitis C virus (anti-HCV IgG), Treponema pallidum (EIA or RPR if EIA is positive), anti-Streptozoa monoclonal antibody, CMV viral load, and EBV viral load. Stool screening includes Clostridium difficile toxin A / B (PCR), routine bacterial culture (and enrichment) for enteric pathogens including Helicobacter pylori EIA, Salmonella, Shigella, Yersinia, Campylobacter, and Vibrio, Escherichia coli 0157 (if stxl / 2 EIA+ve, culture for E. coli 0157), Shiga-like toxin stxl / 2 (Shigella) EIA, culture-based assays for vancomycin-resistant enterococci (VRE), extended-spectrum beta-lactamase (ESBL) producers, carbapenem-resistant Enterobacteriaceae (CRE), and methicillin-resistant Staphylococcus aureus (MRSA), Giardia antigen (EIA), Cryptosporidium antigen (EIA), Cyclospora, isospora, and microsporidia (microscopy with acid-fast rapid stain), ova and parasites (microscopy), rotavirus (EIA), norovirus GI / GII (RT-PCR), and adenovirus40,41 EIA.

[0179] 16S RNA sequencing is used to classify and identify candidate strains during the selection and screening process. The target bacterial strains were extensively characterized by phenotype and genotype. Based on these data, a subset of candidate therapeutic strains was selected to further evaluate their potential for regulating inflammation and intestinal barrier integrity in cell-based in vitro functional assays and in vivo mouse DSS colitis models (Chassaing et al., Curr Proloc Immunol.; 04: Unit 15.25 (2014)). Based on the results of these experiments, three bacterial strains belonging to the following species groups were selected: Bacteroides thetaiotaomicron, Eubacterium hallii, and Roseburia hominis. The 16S RNA sequences of RHO01, BTH01, and EHA01 are provided in SEQ ID NOs: 1, 2, and 3, respectively. The taxonomic identity of each bacterial strain was confirmed via whole genome sequencing (WGS). The entire genome sequence of RHO01 is provided in SEQ ID NOs: 4-32. The entire genome sequence of BTH01 is provided in SEQ ID NOs: 33-390. The entire genome sequence of EHA01 is provided in SEQ ID NOs: 391-523. Strains BTH01, EHA01 and RHO01 were deposited at DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstraβe 7B, 38124 Brunswick, Germany) on September 14, 2018 under the Budapest Treaty under the DSM deposit numbers BTH01 (DSM-32919), EHA01 (DSM-32920) and RHO01 (DSM-32921), respectively.

[0180] Throughout the Examples, and particularly in Examples 2-6, information and data are provided to support the explanations for the Bacteroides thetaiotaomicron strain (BTH01), Eubacterium hallii strain (RHA01), and Roseburia hominis strain (RHO01) selected for development of C1.

[0181] Example 2-Taxonomic and phenotypic characterization of C1 bacterial strains

[0182] A live biotherapeutic product mixed bacterial population 1 (C1) was prepared by mixing three bacterial strains, namely BTH01, EHA01 and RHO01. Table 1 shows some physiological and metabolic characteristics of the C1 bacterial strains BTH01, EHA01 and RHO01. Gram staining of the three C1 bacterial strains showed that EHA01 was Gram-positive, while BTH01 and RHO01 were both Gram-negative. All three strains were obligate anaerobes.

[0183] In order to investigate the characteristics that are often found to be different in strains of the same bacterial species, the C1 bacterial strain was subjected to a carbon source utilization analysis. The ability of the BTH01, EHA01 and RHO01 strains to metabolize 190 carbon sources and 95 nitrogen sources was evaluated using a phenotypic microarray (Biolog, Hayward, CA). As shown in Table 1, BTH01 appears to be the most versatile, utilizing 34 unique carbon sources, while EHA01 is the least versatile, utilizing 8 unique carbon sources. Each of the BTH01, EHA01 and RHO01 strains is able to metabolize some unique carbon sources that are not reported in the literature for their respective species. BTH01 was able to utilize D-mannose, maltose, aD-glucose, D-galactose, lactulose, maltotriose, N-acetyl-bD-mannosamine, N-acetyl-D-glucosamine, D-trehalose, aD-lactose, sucrose, turanose, gentiobiose, D-melibiose, D-fructose, L-fucose, L-arabinose, D-glucuronic acid, D-xylose, L-rhamnose, D-galacturonic acid, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, glycogen, laminin, mannan, pectin, N-acetyl-D-galactosamine, b-methyl-D-galactoside, D-arabinose, maltitol, palatinose, and D-raffinose. RHO01 can utilize D-mannose, maltose, aD-glucose, N-acetyl-D-glucosamine, D-trehalose, aD-lactose, sucrose, turanose, gentiobiose, D-cellobiose, amygdalin, arbutin, D-melezitose, salcinin, D-glucosamine and b-methyl-D-glucoside. EHA01 can utilize D-mannose, maltose, α-D-glucose, D-galactose, lactulose, maltotriose, N-acetyl-bD-mannosamine and L-tartaric acid.

[0184] The microbiota plays an important role in bile acid metabolism. Analysis of the microbiota of IBD patients showed an overall reduction in biodiversity, with a particular reduction in bacteria from the phyla Bacteroidetes and Firmicutes, which are responsible for approximately 44% of bile hydrolase activity in the intestine (Baars et al., Microorganisms 3(4):641-666 (2015)).

[0185] Therefore, restoring bile acid metabolism with microorganisms may be a potential approach for therapeutic intervention. Since conjugated bile salts have antimicrobial activity, bacteria have evolved to produce bile salt hydrolases (BSH) to neutralize this adverse activity. Many bacterial strains, including Bacteroides species, are tolerant to physiological concentrations of bile (Pumbwe et al., Microb Pathog. 43 (2-3): 78-87 (2007)).

[0186] To analyze the bile tolerance of C1 bacterial strains BTH01, EHA01 and RHO01, growth assays were performed in media with bile concentrations ranging from 0.2% to 1.0%. The results are shown in Table 1. BTH01 could reach 85% of the maximum OD at bile concentrations up to 0.6% in the growth medium compared to the growth of the control without added bile. RHO01 also showed bile tolerance up to 35% of the maximum OD in the presence of 0.4% bile. EHA01 was sensitive to bile and showed growth inhibition even in the presence of 0.2% bile.

[0187] Table 1: Taxonomic identity and phenotypic characteristics of C1 bacterial strains

[0188]

[0189]

[0190] N = Gram-negative; P = Gram-positive; WGS = whole genome sequencing

[0191] 1 190 unique carbon sources tested

[0192] 2 The physiological concentration of bile salts in the human intestine is usually 0.1%-1.3% (Pumbwe et al., 2007)

[0193] 3 In the presence of 0.6% bile in the medium, a maximum OD of 85% was observed; there was an 8-hour lag to the exponential phase.

[0194] 4 In the presence of 0.4% bile in the medium, a maximum OD of 35% was observed with a lag of 5 hours to the exponential phase.

[0195] 5 RHO01 growth is not supported by minimal medium used for nitrogen source utilization assays

[0196] The literature indicates that all three C1 representative species are non-spore forming. Using two different methods of inducing sporulation (i.e., heat shock and chemical shock), no C1 bacterial strain was found to form spores. The positive control Clostridium butyricum (ATCC 19398) strain obtained from ATCC produced spores by both test methods shown in Table 2.

[0197] Table 2: Assessment of sporulation

[0198]

[0199] Each bacterial strain from the Research Cell Bank (RCB) was tested for susceptibility / resistance to a panel of clinically relevant antibiotics using the agar dilution minimum inhibitory concentration (MIC) method and showed that each strain (RCB) was susceptible to at least two clinically relevant antibiotics (Table 3A).

[0200] Table 3A: Phenotypic antibiotic susceptibility results of the studied cell banks

[0201]

[0202] N / A = no interpretive criteria available; N / T = not tested; R = resistant; S = susceptible

[0203] 1 No growth on required CLSI agar; tested using Etests

[0204] Example 3-Whole genome sequencing (WGS) analysis of C1 bacterial strain

[0205] Each bacterial strain genome (BTH01, EHA01 and RHO01) was WGS was performed to confirm taxonomic identity and to assess genomic evidence for the presence of antibiotic resistance genes, virulence factors, and toxins, including evidence for associated mobile genetic elements.

[0206] Example 3.1 - Genomic Analysis Method

[0207] Taxonomic identity was assessed using the Joint Genome Institute's Microbial Species Identifier, which compared average nucleotide identity (ANI) with a curated genome database and was assigned to a taxonomy if the ANI exceeded 96.5% and the alignment length between the query and reference genomes was >70% (Varghese el al., Nucleic Acids Research, 43(l4):676l-677l (2015)).

[0208] Candidate bacterial strain genomes were compared to three selected databases using BLAST: VFDB to determine the presence of putative virulence factors (including toxins) (Chen et al., Nucleic Acids Res. 2005 Jan 1; 33 (2005)), PATRIC (Mao et al., Bioinformatics, 31 (2): 252-258 (2015)) and PHIDIAS (Xianget. al., Genome Biol. 8 (7): R150 (2007)). Matches with e-values ​​exceeding 1e-7 and 70% identity within 70% of the database reference sequences were retained (Mao et al., 2015).

[0209] Putative antibiotic resistance genes were identified using the Resistance Gene Identifier / CARD software package.

[0210] IslandViewer 4 (Bertelli et al., Nucleic Acids Res. 45 (Wl): W30-W35 (2017)) and Virsorter (for phages; Roux et al., eLife. 2015; 4: e08490 (2015)) were used to predict mobile genetic elements (MGEs) within the genomes of candidate bacterial strains to assess the theoretical potential for gene transfer. IslandViewer 4 used the default threshold for MGE detection and retained phage predictions classified as “confident” by Virsorter.

[0211] Example 3.2 - Results and Interpretation

[0212] The taxonomic assignments of candidate bacterial species exceeded the 96.5% ANI threshold for BTH01 (ANI = 99.8%), EHA01 (ANI = 97.5%), and RHO01 (ANI = 98.1%).

[0213] Although sequence analysis showed antibiotic resistance in BTH01 and RHO01, no genomic evidence of antibiotic resistance was detected in EHA01. To confirm the clinical relevance of these data, empirical testing for antibiotic resistance / sensitivity was performed in the C1 research cell bank. The results are shown in Table 3A. The choice of antibiotics was based on the relevant antibiotic sensitivity patterns of the bacterial groups under consideration. Importantly, all three candidate bacterial strains showed sensitivity to most of the tested antibiotics. Although nucleic acid-based technologies may become useful tools for predicting antibiotic resistance in the future, there is currently no evidence to support the use of WGS to reveal antibiotic sensitivity; problems remain with method standardization, accuracy, and clinical interpretability (Ellington et al, Clin Microbiol Infect. 23 (l): 2-22 (2017)).

[0214] In our evaluation of virulence factors, no genes directly associated with host mortality (e.g., genes specifically encoding toxins or secretion systems) were detected.

[0215] The identified virulence factors are related to metabolic functions or tolerance to physiological conditions within the host, which, when impaired by in vitro mutagenesis, reduce the virulence of the pathogen by impairing growth or survival. Therefore, it is not unexpected that the genomes of commensal bacteria associated with the host contain homologs of these genes, and therefore these data are not considered to be clinically relevant safety risks.

[0216] In our assessment of MGE, using our criteria, no genomic region of any strain was identified to be derived from prophage. In addition, the presence of phage and prophage of bacterial candidate strains has been tested by EM analysis (± mitomycin C induction). No phage or inducible prophage was detected. As shown in Table 3B, although other features of MGE were detected, many types of MGE cannot be transmitted between bacteria (e.g., intracellular MGE; Siguier et al., FEMS Microbiology Reviews, 38 (5): 865-891 (2013)).

[0217] Table 3B. Presence of predicted antimicrobial resistance genes and flanking putative mobile genetic elements (MGEs)

[0218]

[0219] Example 4 - Short-chain fatty acid (SCFA) production profile of C1 bacterial strains grown alone or as mixed consortia

[0220] Short-chain fatty acids (SCFAs) produced by human intestinal microorganisms include butyrate, acetate, and propionate, all of which have been found to help maintain 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)). Butyrate is mainly produced by Clostridium species IV and XIVa, including EHA01 and RHO01. On the other hand, Bacteroides species are the main producers of acetate and propionate. After 72 hours of batch culture in YCFAC medium, the SCFA production profile of each C1 bacterial strain was evaluated. Figure 1 As shown in A, acetate and propionate were the most abundant SCFAs produced by BTH01. On the other hand, as expected, acetate and butyrate were the most abundant SCFAs produced by EHA01 and RHO01 ( Figure 1 B, C). None of the three C1 bacterial strains produced significant amounts of lactic acid. When grown as a mixed consortium in batch culture, acetate and butyrate were the most abundant SCFAs produced (Figure ID).

[0221] It is known that certain microbial strains produce compounds or toxins that inhibit the growth of other microorganisms. There are examples of such antimicrobial compounds produced by strains belonging to the intestinal flora (Spinler et al., Anaerobe. 14 (3): 166-71 (2008); Arief et al., Benef Microbes. 6 (4): 603-13 (2015); Renye Jr. et ah, Biotechnol Lett. (11): 1947-1954 (2016)). In each of these embodiments, the production of antimicrobial compounds resulted in significant inhibition of the growth of sensitive strains in a short period of time (18-48 hours). Therefore, in order to exclude any antagonistic relationship between C1 bacterial strains, a batch feeding culture experiment was carried out. After standardizing the inoculated cell count, each of the C1 bacterial strains was grown alone or in a mixed flora. After about 48 hours, the culture was transferred to a fresh medium for 5 consecutive days. The 48-hour fed-batch transfer time was chosen based on previously determined growth rates of individual strains in YCFAC medium (data not shown), with the rationale that each strain would have enough time to reach maximum OD. Samples from replicate cultures on day 0 (just after inoculation), day 2, and day 5 were analyzed by qPCR to assess the abundance of each of the C1 strains. Figure 2 As shown in A, all three C1 strains grew to similar extents over time in individual cultures. When grown in mixed consortia, the abundance of all three C1 strains remained at or above the inoculum level during the 5-day fed-batch culture ( Figure 2 B), indicating that there is no toxicity / growth inhibition relationship between the C1 strains. In the day 5 samples, EHA01 levels were higher than BTH01 and RHO01 levels, which is most likely due to the higher growth rate of EHA01 than BTH01 and RHO01 in the YCFAC medium used in this experiment.

[0222] Example 5-C1 Elucidation of symtrophy between bacterial strains

[0223] The stability and function of microbial communities depend on nutritional interactions between community members, such as the mutual trophy of essential small molecules synthesized by population subsets (Seth and Taga, 2014). Starch (a polymer of glucose) is the most abundant polysaccharide in the human diet and is present in many foods, including cereals, legumes, and potatoes (Diet and Health: Implications for Reducing Chronic Disease Risk, National Academies Press (ETS), 1989)). In a typical Western diet, 33%-50% of calorie intake is in the form of carbohydrates. Starch accounts for about 17%-25% of total carbohydrates (britannica.com / science / carbohydrate / Role-in-human-nutrition, available on the World Wide Web).

[0224] To investigate whether C1 bacterial strains exhibit syntrophy, metabolic traits of individual strains were identified that would allow these strains to syntrophy with each other. For example, Bacteroides species encode complex starch utilization systems (Wexler et al., Microb Genom. 3(11)(2017)). This includes extracellular secreted enzymes that produce starch digestion products such as oligomers, trimers (maltotriose), dimers (maltose), and glucose monomers. From the experiments listed above to determine carbon source utilization by C1 strains, it is clear that EHA01 can utilize starch digestion products. However, based on the literature and available sequenced genomes, it is expected that Eubacterium hallii species do not express starch degrading enzymes.

[0225] like Figure 3 As shown in A, qPCR-based copy number calculations indicate that EHA01 exhibits inferior growth compared to BTH01 in the presence of starch as the sole carbon source provided in the growth medium. The copy number of EHA01 increases when grown in co-culture with BTH01 ( Figure 3 A) A concomitant 25% increase in butyrate production was also observed for EHA01 when grown in co-culture with BTH01 in the presence of starch as the sole carbon source ( Figure 3 B).

[0226] To determine whether products secreted by BTH01, including starch-degrading enzymes, could promote the growth of EHA01 in the presence of starch as the sole carbon source in the growth medium, EHA01 medium was supplemented with filtered supernatant from spent medium from a previous growth of BTH01. Figure 3 As shown in C, the growth of EHA01 in the medium containing starch as the sole carbon source was promoted only when BTH01 supernatant was added.

[0227] It has been reported that Eubacterium hallii produces and secretes vitamin B12, which is essential for the growth of other intestinal microorganisms, including Bacteroides thetaiotaomicron species, which sequester vitamin B12 from the extracellular environment (Engels et al, Front Microbiol. 7:713 (2016)). Therefore, vitamin B12-mediated syntrophy may promote the growth of BTH01 when co-cultured with EHA01, as indicated by the increase in BTH01 copy number in co-culture compared to BTH01 grown in separate cultures ( Figure 3 A).

[0228] In summary, these results indicate syntrophic and mutualistic effects of co-culture of BTH01 and EHA01. Roseburia species are also able to utilize starch as a sole carbon source. When grown on starch as the sole carbon source, RHA01 and EHA01 showed similar syntrophy (data not shown).

[0229] Example 6 - C1 bacterial strain and in vitro and in vivo functional activity of C1

[0230] This example describes the study of the activity of the C1 bacterial strain alone and as a mixed flora in several in vitro and in vivo models, which may provide mechanistic insights into potential clinical benefits.

[0231] Example 6.1. Preparation of freshly cultured bacterial strains and mixed bacterial consortia working stocks for cell culture assays

[0232] Prepare fresh bacterial cultures by overnight culture under anaerobic conditions. Use a rotator to mix and grow EHA01 and RHO01, while BTH01 is not mixed. Centrifuge the bacteria at 4300 x g for 4 minutes. Wash the bacteria once with pre-reduced anaerobic PBS (Gibco). Resuspend the washed bacteria in anaerobic PBS to an optical density of OD600 = 0.3 (-10 8 CFU / ml) to prepare a working stock. A 10-fold serial dilution was performed with anaerobic PBS for specific determination. A mixed population of three bacteria was prepared by mixing the three separate working stocks by volume at 1:1:1. Freeze-dried bacteria were prepared under anaerobic conditions using the same preparation method used to make the C1 drug substance. The freeze-dried bacteria were resuspended in the pre-reduced anaerobic PBS as described above to prepare a working stock of freeze-dried bacteria.

[0233] Example 6.2 - HT29-MTX-E12 Human Epithelial Barrier In Vitro Assay

[0234] HT29-MTX-E12 human epithelial cell line (Sigma Aldrich cat#1200401-1VL) was cultured at 37°C and 5% CO2 using high glucose DMEM containing 4.00 mM L-glutamine and 4500 mg / L sodium pyruvate (HyClone), supplemented with 10% FBS (Tissue Culture Biologicals), 100 I.U / mL penicillin, 100 pg / mL streptomycin, and 0.292 mg / mL L-glutamine (Corning). The number of passages was limited to 6 passages. In the top compartment of the HTS 96-well Transwell plate, type 1 collagen (SigmaAldrich) was applied to the rat tail with 0.4 pm of microscopic transparent polyester film (Corning). The HT29-MTX-E12 human cell line was cultured in a T-175 tissue culture flask until 70-80% confluent. Remove cells and count with 0.25% trypsin 2.21mM EDTA. 30,000 cells are plated in the top compartment of a Transwell plate pre-coated with DMEM culture medium as described above, and the same culture medium is filled in the base reservoir of the Transwell plate. The cells are cultured at 37 ° C and 5% CO2 for 18 days until a confluent monolayer as described above is formed (Hall et al, Journal of Pediatric Surgery 48: 353-358 (2013)). The culture medium in the top compartment and the base reservoir is replaced with new culture medium every two days. One day before the experiment, the top compartment insert containing the confluent monolayer of HT29-MTX-E12 is transferred to the HTS Transwell-96 well receiving plate (Coming). The cells are washed and resuspended with DMEM culture medium without antibiotics, and the cells are filled into the base compartment of the Transwell receiving plate.

[0235] The working solutions of fresh culture or lyophilized bacteria, mixed flora, or anaerobic PBS controls were added at 10% v / v to the top compartment of a transwell plate containing a confluent HT29-MTX-E12 monolayer. The test article was separated into a monolayer by centrifugation at 515 x g for 4 minutes. The HT29-MTX-E12 monolayer was incubated with the test article at 37°C and 5% CO2 for 1 hour. 100 ng / ml TNF-α (InvivoGen) was added to the basal compartment of the transwell plate. At 0 and 24 hours after the addition of TNF-α, the transepithelial electrical resistance (TEER) across the cell barrier was measured using an STX100C electrode connected to an EVOM2 V / Ω (TEER) meter (World Precision Instruments).

[0236] Example 6.3 - In vitro cytokine and chemokine assays of human macrophages and monocytes

[0237] THP-1 human monocytic cell line (ATCC cat#TIB-202) was cultured at 37°C and 5% CO2 using RPMI 1640 containing 2.05 mM L-glutamine (Corning), supplemented with 10% heat-inactivated FBS (Tissue Culture Biologicals), 100 I.U / mL penicillin, 100 pg / mL streptomycin, and 0.292 mg / mL L-glutamine (Corning). The number of passages was limited to 8 passages. The THP-1 human monocytic cell line was grown until 70-80% confluence. The cells were counted and resuspended in culture medium. 100,000 cells per well were plated onto a 96-well plate. THP-1 human macrophages were prepared by culturing THP-1 human monocytes and 10 ng / mL phorbol 12-myristate 13-acetate (PMA) (InvivoGen) for 24 hours at 37°C and 5% CO2, followed by culturing with 20 ng / mL IL-4 (R&D Systems) and 20 ng / mL IL-13 (R&D Systems) for 48 hours as previously described (Genin et al, BMC Cancer. 15:577 (2015)). One day before the experiment, the cells were washed and resuspended in RPMI medium containing 20 ng / mL IL-4 and 20 ng / mL IL-13 without antibiotics.

[0238] The working stock of the described fresh culture or lyophilized bacteria, mixed flora or anaerobic PBS control was added to THP-1 macrophages at 10% v / v and centrifuged at 515 x g for 4 minutes to THP-1 cells. 100 ng / ml of LPS (InvivoGen) was added simultaneously with the test article. The test article, LPS and THP-1 macrophages were incubated at 37°C and 5% CO2 for 24 hours. THP-1 cell supernatants were collected and analyzed by ELISA. The IL-10 and CCL-18 levels in the quantitative culture supernatants were detected with TMB according to the manufacturer's instructions using a commercial enzyme-linked immunosorbent assay (ELISA) kit from Biolegend or R&D Systems.

[0239] Example 6.4 - In vitro cytokine assay of human PBMC

[0240] Trima residual blood products containing concentrated blood mononuclear cells are obtained from anonymous donors by Blood Centers of the Pacific (San Francisco, CA) and processed within 24 hours of collection. Blood samples were tested for HIV, HBV, HCV, HTLV, syphilis, West Nile virus and Zika virus negative. As previously described, PBMCs were separated using a ficoll gradient (Sim et al., J. Vis. Exp. (112), e54128 (2016)). In short, 50 mL of Trima residuals were diluted with 50 mL of sterile PBS (Gibco) and 25 mL were covered on 15 mL of Ficoll-Paque Plus (GE Healthcare) in a 50 mL conical tube. The sample was centrifuged at room temperature for 30 minutes at 450 x g and stopped without braking. 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 stored by incubation at 37°C and 5% CO2 and used for assay evaluation within 24 hours or frozen for later use. 7 The cells were cryopreserved at a concentration of 10 cells / mL in RPMI1640 supplemented with 50% FBS and 10% DMSO (Sigma Aldrich) and stored in liquid nitrogen until use.

[0241] Human PBMCs isolated or used immediately after thawing from cryopreservation were diluted to 1×10 in RPMI 1640 containing L-glutamine (Corning) supplemented with 10% heat-inactivated FBS (Tissue Culture Biologicals) and 0.292 mg / mL L-glutamine (Corning). 6 cells / mL. 6 100 pL aliquots of the 10 cells / mL cell suspension were added to each well of a flat-well 96-well plate and incubated at 37°C and 5% CO2 for 24 hours before adding the test article.

[0242] Test articles were prepared and added as described in the other assays.After incubation for 24 h at 37°C and 5% CO2, the plates containing the co-cultures were centrifuged (515 xg; 4 min) and the supernatants were collected and analyzed by ELISA as described above.

[0243] Example 6.5 - Freshly cultured C1 in HT29-MTX-E12 human epithelial barrier assay

[0244] Human ulcerative colitis (UC) patients experience symptoms of increased intestinal permeability, which can be restored by reducing levels of the inflammatory cytokine TNF-α using anti-TNF-α therapy (Michielan et al., Mediators Inflamm. 2015:628157 (2015)).

[0245] Finding new therapies that can reduce intestinal permeability and barrier disruption remains an important goal in the development of colitis treatments. As a model of intestinal barrier disruption and permeability, the functional activity of each C1 bacterial strain (BTH01, EHA01 and RHO01) and a mixed flora of the three strains on human epithelial cells was evaluated in the presence of TNF-α. The confluent HT29-MTX-E12 human epithelial cell line forms a polarized monolayer and produces mucin, which are two major characteristics of human primary intestinal epithelial cells (Dolan et ah, PLoS One.7(10):e47300(2012)). Figure 4 The results showed that when three freshly cultured C1 bacterial strains were incubated with HT29-MTX-E12 monolayers alone and as a mixed flora in the presence of TNF-α, TEER (a measure of cell monolayer integrity) was significantly increased compared to PBS controls. This suggests that C1 bacterial strains alone and as a mixed flora can protect human epithelial cells from cytokine-induced barrier damage and can reduce intestinal barrier permeability.

[0246] Example 6.6 - In vitro cytokine and chemokine assays of freshly cultured C1 bacterial strains and mixed bacterial flora in human macrophages and monocytes

[0247] IL-10 is an anti-inflammatory cytokine released by immune cells essential for intestinal homeostasis (Kole et al, Curr Top Microbiol Immunol. 380: 19-38 (2014)). Mice with IL-10 gene deletion (Wilson et al., PLoS Pathog. (8): el002171 (2011)) and humans with IL-10 loss-of-function mutations (Bisborough et al, Am J Gastroenterol Suppl 3: 27-37 (2016)) have increased susceptibility to UC development. Intestinal macrophages that produce IL-10 are protective against infection with the enteric pathogen C. rodentium, which induces colitis (Krause et al, Nature Communications 6: 7055 (2015)). Therapies targeting mechanisms that increase IL-10 levels have shown promise, but lack sufficient efficacy (Li and He, World J Gastroenterol. 10 (5): 620-5 (2004)). Improvements in IL-10-boosting therapy remain an attractive strategy for therapies targeting colitis. The ability of each individual bacterial strain, as well as all three strains together as a mixed consortium, to induce IL-10 production by human THP-1 macrophages was assessed in the presence of LPS and primary human PBMCs. Figure 5 Shown are the dose-dependent significant increases in the production of the anti-inflammatory cytokine IL-10 by (A) THP-1 macrophages and (B) primary human PBMCs induced by each freshly cultured bacterial strain alone or a mixed consortium of the three strains. The results indicate that the C1 bacterial strain can induce the production of anti-inflammatory IL-10 in both human THP-1 macrophages and primary human monocytes.

[0248] The ability of each freshly cultured C1 bacterial strain or a mixed flora of the three strains to induce CCL-18 (a chemokine associated with M2 macrophages) was evaluated with THP-1 macrophages. It has been previously reported that the induction and polarization of M2 macrophages is a key mechanism to prevent inflammatory bowel disease and colon 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 proven marker of M2 macrophages (Genin et al., BMC Cancer 15: 577 (2015)). Figure 6The results show that when individual strains EHA01 and RHO01 and a mixed consortium of three strains, EHA01, RHO01 and BTH01, were co-cultured with THP-1 macrophages, a significant dose-dependent increase in CCL-18 production was observed compared to a PBS control; BTH01 alone did not induce CCL-18. These data indicate that two of the three C1 bacterial strains and the mixed consortium can increase CCL-18 production by human macrophages, suggesting the induction and polarization of anti-inflammatory M2 macrophages.

[0249] Example 6.7 - Lyophilized C1 in the HT29-MTX-E12 human epithelial barrier assay

[0250] Results using freshly cultured C1 bacterial strains showed that the strains alone and as mixed flora protected human intestinal epithelial cells from TNF-α-induced barrier damage. Since the C1 bacterial strains were prepared as lyophilized drug substances for oral delivery as therapeutic agents, lyophilized forms of the C1 bacterial strains were tested to ensure that they retained their barrier protective effects. Each C1 strain was prepared as a lyophilized powder using the same method used to prepare the C1 drug substance. Figure 7 showed that the TEER of individual lyophilized C1 bacterial strains or a mixed population of all 3 species was significantly increased when incubated with HT29-MTX-E12 monolayers in the presence of TNF-α compared to PBS controls. This suggests that the lyophilized form of the C1 bacterial strain retains the protective effect and reduces intestinal epithelial permeability when tested on human epithelial cells in the presence of TNF-α-induced barrier disruption.

[0251] Example 6.8 - Lyophilized C1 bacterial strain in in vitro cytokine and chemokine assays in human macrophages and monocytes

[0252] Similar to the results of the human intestinal epithelial barrier assay, C1 alone and all three strains as a mixed consortium in lyophilized form retained the ability to induce the anti-inflammatory cytokine IL-10 and the M2 macrophage-associated chemokine CCL-18 in human THP-1 macrophages ( Figure 8 A, IL-10 and Figure 8 C, CCL-18). The lyophilized form of C1 also retained the ability to induce IL-10 in primary human PBMCs ( Figure 8 B).

[0253] Example 6.9 - UC fecal microbiota in the HT29-MTX-E12 human epithelial barrier in vitro assay and human THP-1 macrophage in vitro cytokine assay

[0254] Aliquots of glycerol stocks consisting of 50% glycerol:50% UC fecal samples were prepared by thawing the glycerol stocks under anaerobic conditions on the day of the experiment, then washing and resuspending in pre-reduced anaerobic PBS to an optical density of OD600 = 0.3 (-10 8 CFU / ml) to prepare a working stock. This UC working stock was added to the top compartment of a Transwell plate containing a confluent HT29-MTX-E12 monolayer (10% v / v) and a working stock of a freshly cultured C1 mixed flora (10% v / v) or an anaerobic PBS control (10% v / v). TEER assays were performed as described without the addition of TNFα. Similarly, the UC working stock was added to THP-1 macrophages (10% v / v) with or without a freshly cultured C1 mixed flora working stock (10% v / v) or an anaerobic PBS control. After incubation for 4 hours at 37°C and 5% CO2, the THP-1 macrophages were washed and resuspended in RPMI medium supplemented with Pen / Strep to remove excess bacteria. THP-1 macrophages were incubated at 37°C and 5% CO2 for 24 hours, and THP-1 cell supernatants were collected and analyzed for IL-10 by ELISA.

[0255] In the HT29-MTX-E12 human intestinal epithelial barrier assay, the protective ability of the C1 mixed flora against barrier damage caused by UC fecal flora was tested. Fig. 9 A shows that the addition of UC fecal flora alone reduced the TEER of the HT29-MTX-E12 monolayer, indicating that the microorganisms in the UC fecal flora can destroy the monolayer and increase its permeability. When the HT29-MTX-E12 monolayer was co-incubated with UC fecal flora and freshly cultured C1 mixed flora, the reduction of TEER by UC fecal flora was significantly inhibited ( Fig. 9 A). This suggests that the C1 mixed microbiota can protect human intestinal epithelial cells from damage caused by UC fecal microbiota and reduce barrier permeability.

[0256] The ability of the C1 mixed microbiota to induce the anti-inflammatory cytokine IL-10 in THP-1 macrophages in the presence of UC fecal microbiota was tested. Fig. 9 B shows that UC fecal flora alone can induce IL-10 by THP-1 macrophages, but the addition of C1 mixed flora significantly enhanced the production of IL-10 in a dose-dependent manner. This suggests that C1 mixed flora can increase the induction of IL-10 by human macrophages in the presence of UC flora.

[0257] Example 6.10-C1 bacterial strain in human macrophage autophagy and IL-10 assay Autophagy is an intracellular process whereby unwanted or damaged cytoplasmic components (such as proteins and organelles) are degraded within lysosomes (Doria el al, N Engl J Med 368 (19): 1845 (2013)). This process plays an important role in innate and adaptive immunity and in maintaining homeostasis, and serves as a defense mechanism against invading pathogens. Autophagy dysfunction can lead to a variety of pathological consequences, including tumor progression, pathogen hypervirulence, and neurodegeneration, and has been shown to be associated with the pathogenesis of several autoimmune and inflammatory diseases, such as systemic lupus erythematosus, psoriasis, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis (Yin et al, Front Immunol. 2018; 9: 1512 (2018)). Inhibition of autophagy accelerates the progression of certain inflammatory and autoimmune diseases by promoting the production of inflammatory cytokines (Yin et al, 2018). Autophagy regulation shows promise in the treatment of Crohn's disease (Nys et al., Nat Rev Gastroenterol Hepatol. (7): 395-401 (2013)), and several approved Crohn's disease therapies work by inducing autophagy, including 5-ASA (mesalazine / sulfasalazine; see Wu et al, PLoS ONE 7: e37572 (2012); and Fiorucci et al., Br. J. Pharmacol. 150: 996-1002 (2007)); and anti-TNF-a (infliximab, adalimumab, cetuximab polyethylene glycol; see Nys et al, 2013). C1 bacterial strains EHA01, RHO01, and BTH01 were tested individually, in pairwise combinations, and together in a single mixed consortium for their ability to induce autophagy and IL-10 production in human THP-1 macrophages.

[0258] In order to evaluate autophagy, M2 macrophages were prepared as follows. THP-1 human monocytic cell line (ATCC cat#TIB-202) was cultured at 37°C and 5% CO2 with RPMI 1640 containing 2.05mM L-glutamine (Corning), supplemented with 10% heat-inactivated FBS (Tissue Culture Biologicals), 100I.U / mL penicillin, 100pg / mL streptomycin and 0.292mg / mL L-glutamine (Corning). The number of passages was limited to 8 passages. THP-1 human monocytic cell line was grown until 70-80% confluence. Cells were counted and resuspended in culture medium. 100,000 cells per well were plated on 96-well plates. THP-1 human macrophages were prepared by culturing THP-1 human monocytes with 10 ng / mL phorbol 12-myristate 13-acetate (PMA) (InvivoGen) for 24 hours at 37°C and 5% CO2, followed by culturing with 20 ng / mL IL-4 (R&D Systems) and 20 ng / mL IL-13 (R&D Systems) for 48 hours as previously described (Genin et al, BMC Cancer. 15:577 (2015)).

[0259] To evaluate IL-10, M1 macrophages were prepared as follows. THP-1 human monocytic cell line (ATCC cat#TIB-202) was cultured at 37°C and 5% CO2 using RPMI 1640 containing 2.05mM L-glutamine (Corning), supplemented with 10% heat-inactivated FBS (Tissue Culture Biologicals), 100I.U / mL penicillin, 100pg / mL streptomycin, and 0.292mg / mL L-glutamine (Corning). The number of passages was limited to 8 passages. THP-1 human monocytic cell line was grown until 70-80% confluence. Cells were counted and resuspended in culture medium. 100,000 cells per well were plated on a 96-well plate. THP-1 human macrophages were prepared by culturing THP-1 human monocytes with 10 ng / mL phorbol 12-myristate 13-acetate (PMA) (InvivoGen) for 24 hours, followed by washing and incubation with antibiotic-free RPMI at 37°C and 5% CO2 for 48 hours.

[0260] For the autophagy and IL-10 assays, working stocks of freshly cultured or frozen bacteria alone (100% EHA01, RHO01, or BTH01), mixed consortia (1:1 mix of 2 strains (EHA01:RHO01; RHO01:BTH01; and EHA01:BTH01) and 1:1:1 mix of 3 strains (EHA01:RHO01:BTH01)), and anaerobic PBS controls were prepared and each bacterial working stock was normalized to 10 by surface area using a Coulter counter. 8 pm 2 / ml. Each test substance was added to THP-1 macrophages at 10% v / v and separated onto THP-1 cells by centrifugation at 515x g for 4 minutes. 500ng / ml LPS (InvivoGen) was added simultaneously with the test substance. The test substance, LPS and THP-1 macrophages were incubated at 37°C and 5% CO2 for 3 hours. The co-culture medium was replaced with fresh RPMI and 100I.U / mL penicillin, 100pg / mL streptomycin. The cells were incubated overnight at 37°C and 5% CO2. M1 macrophage supernatants were collected and analyzed by ELISA. IL-10 in the culture supernatant was quantified by TMB detection using a commercial enzyme-linked immunosorbent assay (ELISA) kit from Biolegend or R&D Systems according to the manufacturer's instructions. M2 macrophages were cultured using The Autophagy Detection Kit (Enzo) was used to detect, process and analyze the autophagy level.

[0261] To compare the difference between autophagy or IL-10 production induced by the mixed consortium and the total induction by each individual strain of the mixed consortium, the values ​​measured by the single strain test items were normalized to biomass because these test items contained approximately two-fold (2x) or three-fold (3x) increases in biomass of the specific strain relative to the two-strain or three-strain mixed consortium, respectively.

[0262] Biomass normalization is carried out as follows. For comparison with the mixed flora of 2-strains, the measured values ​​of each of the two corresponding single strain test objects are added and divided by two (i.e., averaged). For comparison with the mixed flora of 3-strains, the measured values ​​of each of the three corresponding single strain test objects are added and divided by 3 (i.e., averaged). The measured values ​​of each mixed flora are then compared with the single strain average values ​​corresponding to it (see "% increase (mixed flora vs single strain average value)" in Table 4 and Table 5).

[0263] like Fig.10As shown in A, and in the table below, autophagy was induced in THP-1 macrophages by adding the following strains compared to the addition of PBS: the EHA01, RHO01 and BTH01 strains, respectively (Table 4A); each pairwise combination of the three strains (EHA01+RHO01 in Table 4B; RHO01+BTH01 in Table 4C; EHA01+BTH01 in Table 4D); and a mixed population of all three strain combinations (EHA01+RHO01+BTH01 in Table 4A). It is noteworthy that the biomass was normalized, and each pairwise combination as well as the three-strain combination as a mixed population induced autophagy to a greater extent than the combination induction of each member strain alone (see "% increase (mixed population vs single strain average)" in Tables 4A-D).

[0264] Table 4A.3 Mixed bacterial flora: Eubacterium hallii + Roseburia hominis + Bacteroides thetaiotaomicron induces autophagy

[0265]

[0266] Table 4B.2 Mixed bacterial flora: Eubacterium hallii + Roseburia hominis induces autophagy

[0267]

[0268] Table 4C.2 Mixed bacterial flora: Roseburia hominis + Bacteroides thetaiotaomicron induce autophagy

[0269]

[0270] Table 4D.2 Mixed bacterial flora: Eubacterium hallii + Bacteroides thetaiotaomicron induce autophagy

[0271]

[0272] like Fig.10 As shown in FIG. 5B and in the table below, IL-10 production was also induced in THP-1 macrophages by adding the following strains compared to those with PBS: the EHA01, RHO01, and BTH01 strains, respectively (Table 5A); each pairwise combination of the three strains (EHA01+RHO01 in Table 5B; RHO01+BTH01 in Table 5C; EHA01+BTH01 in Table 5D); and a mixed population of all three strain combinations (EHA01+RH001+BTH01 in Table 5A). Similar to the results of autophagy induction, biomass was normalized, and each pairwise combination as well as the three-strain combination as a mixed population induced IL-10 production to a greater extent than the combined induction of each member strain alone (see "% increase (mixed population vs. single strain average)" in Tables 5A-D).

[0273] Table 5A.3 Mixed bacterial flora: Eubacterium hallii + Roseburia hominis + Bacteroides thetaiotaomicron induces IL-10

[0274]

[0275] Table 5B.2 Mixed bacterial flora: Eubacterium hallii + Roseburia hominis induces IL-10

[0276] Test object pg / ml IL-10 protein (average) PBS 1.00 100% EHA01 1517.12 100% RHO01 147.85 2 strains mixed flora: 50% EHA01, 50% RHO01 1484.24 Average value of a single strain (normalized to biomass) 832.48 % increase (mixed flora vs. single strain average) 78.29

[0277] Table 5C.2 Mixed bacterial flora: Roseburia hominis + Bacteroides thetaiotaomicron induce IL-10

[0278]

[0279]

[0280] Table 5D.2 Mixed bacterial flora: Eubacterium hallii + Bacteroides thetaiotaomicron induce IL-10

[0281]

[0282] These results demonstrate the ability of each C1 bacterial strain to induce autophagy and IL-10 production in THP-1 macrophages individually. In addition, the unexpected increase in induction observed from each two-strain and three-strain mixed consortia compared to the combined induction of each member strain alone suggests that EHA01, RH001, and BTH01 interact synergistically in these mixed consortia to affect their biological activities.

[0283] Example 6.11 - C1 bacterial strain alone reduces disease severity in the DSS-induced colitis model

[0284] Lyophilized forms of C1 bacterial strains EHA01, RHO01, and BTH01 were tested in a well-validated mouse model of DSS-induced colitis (Chassaing et al., Curr Protoc Immunol:, 104: Unit 15.25 (2014)) to evaluate efficacy in conditions of chemically induced intestinal damage. Mice were treated with 3% DSS in drinking water for 5 days to induce intestinal mucosal damage, and then DSS was removed for the remaining 21 days of the study. During the entire 21-day study, 1x10 9 Daily doses of VCC were administered with lyophilized bacterial strains ( Fig.11 ). Antibodies against IL-12p40 were administered parenterally as a positive control for efficacy.

[0285] Three efficacy endpoints were assessed on days 12 and 21 of the study: weight loss over time, colitis severity scores, and stool consistency scores (measured by endoscopy). Each of the three individual C1 strains and the anti-IL-12p40 antibody reduced DSS-induced weight loss compared with vehicle control, whereas microbes without in vitro gut barrier activity did not. Fig.11 AD, Table 6). Similar effects of the three C1 strains and the anti-IL-12p40 positive control on colitis severity scores and stool consistency scores were observed (Table 8).

[0286] Example 6.11.1 - C1 bacterial strain alone prevents weight loss in the DSS-induced colitis model

[0287] All groups administered the three C1 bacterial strains EHA01, RHO01 and BTH01 showed significant improvements in weight loss compared to vehicle and negative microbial controls ( Fig.11 AC). These differences in the severity of weight loss were most pronounced on days 7-16, which marks the peak of intestinal damage and recovery in the colitis model. On day 21, animals administered the test article BTH01 regained body weights similar to the baseline positive control group administered anti-IL-12p40 ( Fig.11 C). Negative control microbes did not show any positive effect on weight loss compared to vehicle control ( Fig.11 D). These observations were also reflected in the quantified AUC, which summarizes the percent change in weight from study day 0 (Table 6). The protection conferred by the individual strains reached statistical significance on days between days 8-21.

[0288] Table 6: Evaluation of body weight changes (area under the curve) in the DSS colitis model

[0289] Test Group Weight change mean AUC original 74.03±11.4 3%DSS+carrier -88.11±16.64 3%DSS+EHA01 -62.20±16.42 3%DSS+RHO01 -52.07±16.42 3%DSS+BTH01 -22.6±29.8 3%DSS+negative control microorganisms -87.38±16.42 3%DSS+anti-IL-12p40 -1.348±18.66

[0290] UC was calculated using the Prism area under the curve algorithm applying the trapezoidal rule to a nonlinear regression of the percent change in weight. Errors are expressed as SEM.

[0291] Table 7 shows a complete summary of the daily weight loss percentage and statistics for each test group, which shows the daily weight loss percentage mean ± SEM for each test item (N = 12), vehicle negative control (N = 20) and anti-IL-12p40 positive control (N = 12). All test items included in the table were compared to the vehicle control simultaneously using the Kruskal-Wallis test and the two-level step-up method of Bejamini, Keieger and Yekutieli to correct for multiple comparisons by controlling the false discovery rate, so that Q values ​​< 0.05 were considered statistically significant. When analyzed using one-way analysis of variance, the results were similar.

[0292] Table 7: Mean percent body weight loss and statistical analysis for studies on DSS-induced colitis

[0293]

[0294] Example 6.11.2 - C1 bacterial strain improves endoscopic colitis scores and stool consistency in the DSS-induced colitis model

[0295] On days 12 and 21, all groups underwent endoscopy to assess the severity of colitis and stool consistency, a hallmark of intestinal inflammation. The severity of colitis was scored using a 0-4 scale (0 = normal; 1 = loss of vascularity; 2 = loss of vascularity and looseness; 3 = looseness and erosion; 4 = ulceration and bleeding). In addition, stool consistency was scored during endoscopy using the parameters defined in Table 8.

[0296] Table 8: Stool consistency

[0297] Score describe 0 Normal, intact spherical shape 1 Feces are loose, soft and stable in shape 2 Loose, watery stools with unusual shapes 3 Watery or diarrhea 4 Bloody diarrhea

[0298] As shown in Table 9, the mean endoscopic scores were reduced for animals administered EHA01 and RHO01 on day 12 and for all animals administered the test articles on day 21 relative to vehicle control. The mean stool consistency scores were also reduced for animals administered EHA01 and RHO01 on day 12 and for all animals administered the test articles on day 21 compared to animals treated with vehicle control.

[0299] Table 9: Record of the effects of the C1 strain and controls on endoscopic scores and stool consistency measured on study days 12 and 21

[0300]

[0301] Stool consistency and colitis severity were scored as described. Errors are expressed as SEM.

[0302] Example 6.11.3-C1 bacterial strain alone reduces IL-17A and IL-12y in colonic tissue in a DSS-induced colitis model

[0303] DSS-induced colitis is characterized by intestinal inflammation associated with an increase in tissue proinflammatory cytokines including IL-12p40 and IL-17A. In addition to disease scores, levels of inflammatory cytokines in colonic tissue were assessed at the end of the study on day 21 ( Fig.12 Evaluation of these cytokines at the colon tissue level in groups administered three individual C1 bacterial strains, EHA01, RHO01 and BTH01, as well as an anti-IL-12p40 positive control, demonstrated a significant increase in IL-12p40 relative to the vehicle control ( Fig.12 A) and IL-17A ( Fig.12 Each of the three individual C1 strains significantly prevented the DSS-induced increase in IL-17A, and strains EHA01 and BTH01 also significantly reduced colon tissue IL-12p40, with RHO01 showing a decreasing trend.

[0304] Example 6.12 - Mixed bacterial flora reduces disease severity in a DSS-induced colitis model

[0305] Mice were treated with 3% DSS in drinking water for five days to induce intestinal mucosal damage, and then DSS was removed for the remaining 19 days of the study. 9 VCC rehydrated lyophilized forms of each individual C1 strain of the C1 mixed flora.

[0306] Throughout the study, 3x10 9 The doses of VCC were tested against individual bacterial strains EHA01, RHO01, and BTH01 in lyophilized form. The C1 mixed bacterial population (comprising 1x10 9 VCC in a rehydrated lyophilized form) in combination with anti-IL-12p40 antibody (10 mg / kg in PBS, IP dosing Q3D starting on day 6) ( Fig.13 ).An antibody against IL-12p40 was administered parenterally as a positive control for efficacy. The primary efficacy endpoint of this study was weight loss over time.

[0307] A complete summary of the daily percent body weight loss and statistics is presented in Table 10, which shows the mean ± SEM of the daily percent body weight loss for each test article (N=12), mixed flora (N=24), vehicle negative control (N=20), and anti-IL-12p40 positive control (N=12).

[0308] All test items included in the table were compared simultaneously to vehicle controls using the Kruskal-Wallis test and the two-level step-up method of Bejamini, Keieger, and Yekutieli to correct for multiple comparisons by controlling the false discovery rate, so that a Q value < 0.05 was considered statistically significant. When analyzed using one-way ANOVA, the results were similar.

[0309] Table 10: Mean Percent Body Weight Loss and Statistical Analysis from DSS-Induced Colitis Studies

[0310]

[0311] Example 6.12.1-C1 Bacterial Mixed Flora Prevents Weight Loss in DSS-Induced Colitis Model

[0312] Compared with vehicle control, C1 mixed flora showed protection against DSS-induced weight loss similar to that observed with anti-IL-12p40 antibody ( Fig.13 D). The protective effect observed from the C1 mixed flora and anti-IL-12p40 was statistically significant from the peak of intestinal damage on day 10 until the end of the study on day 19. Individual strains EHA01, RHO01 and BTH01 also showed a protective effect against weight loss compared to vehicle control ( Fig.13 AC). Except for BTH01, which achieved statistically significant protection against weight loss at days 14-17, protection by individual strains in all treatment groups reached statistical significance at days 10 and 17. Co-administration of C1 mixed flora with anti-IL-12p40 showed protection against DSS-induced weight loss compared to vehicle control, C1 mixed flora alone, and anti-IL-12p40 alone ( Fig.13 E), as shown in the summary of the percent change in body weight AUC from day 0 of the study ( Fig.13 F).

[0313] Example 6.12.2-C1 mixed bacterial flora reduces colon tissue IL-17A, TNF-α and IL-6 in the DSS-induced colitis model

[0314] Evaluation of cytokines in colonic tissue of mice administered C1 mixed flora demonstrated a reduction in the pro-inflammatory cytokines IL-17A, TNF-α, and IL-6 relative to vehicle controls ( Fig.14 ). The anti-IL-12p40 positive control also showed protection against DSS-induced increases in these proinflammatory cytokines, which was statistically significant for IL-17A.

[0315] Example 6.12.3-C1 Bacterial Mixture Reduces Plasma Lipoprotein-2 / NGAL Levels in DSS-Induced Colitis Model

[0316] Lipocalin-2, a disease activity biomarker for inflammatory bowel disease (Stallhofer et al., Inflamm Bowel Dis 21(l0):2327-2340 (2015)), was assessed in the plasma of mice that were administered the EHA01, RHO01, and BTH01 strains alone, the C1 mixed flora, anti-IL-12p40 alone, and anti-IL-12p40 in combination with the C1 mixed flora ( Fig.15 Administration of each of the individual strains EHA01, RHO01 and BTH01, C1 mixed flora alone and in combination with anti-IL-12p40 resulted in a decrease in plasma levels of lipocalin-2 compared to vehicle control, wherein it was observed that the combination of C1 mixed flora with anti-IL-12p40 showed a greater decrease than C1 mixed flora alone or anti-IL-12p40 alone ( Fig.15 A). Differences between experimental groups were statistically determined using one-way ANOVA and Tukey post hoc test. Fig.15 B shows that the decrease in lipocalin-2 plasma levels was associated with the percent change in body weight, where in the DSS model, a greater decrease in lipocalin-2 corresponded to a reduced weight loss (r = -0.5129; 95% confidence interval = -0.6502 to -0.3432; R 2 =0.2631; P value: P (two-sided test) = <0.0001; significant (α = 0.05)).

[0317] Example 7 - Clinical Studies

[0318] A first-in-human study was conducted to investigate the safety / tolerability, pharmacokinetics and pharmacodynamics of the investigational drug C1. C1 comprises commensal intestinal bacterial strains derived (purified and monocultured) from the feces of healthy human donors. As described in the previous examples herein, the bacterial strains were selected based on their potential to modulate mucosal inflammation and barrier function that are dysregulated in UC patients.

[0319] The study is an adaptively designed, two-cohort, dose-escalation study designed to evaluate the safety and efficacy of 1-5 C1 capsules administered orally daily for 8 weeks. C1 was administered as monotherapy or in combination with oral mesalamine (standard of care) to subjects with mild to moderately active UC who had evidence of an inadequate response to ongoing mesalamine therapy. An inadequate response to mesalamine was defined as the presence of signs and / or symptoms of active UC at the screening visit despite receiving >2.4 g / day of oral mesalamine therapy with or without topical (rectal) mesalamine (mesalamine suppository 1000 mg PR QHS or mesalamine enema 4 g PR QHS) for greater than 4 weeks.

[0320] The study will enroll 44 subjects in two consecutive, non-overlapping dose groups (Group A and Group B). The first group (Group A) will randomly assign 20 subjects to receive one C1 or placebo capsule per day in a 1:1 ratio. The second group (Group B) will randomly assign 24 subjects to receive a maximum of 5 C1 or placebo capsules per day in a 3:1 ratio once a day.

[0321] The inclusion criteria included the following: 1) Male or female aged 18-70 years.

[0322] 2) Establish the diagnosis of UC at least 3 months before screening based on clinical history, exclusion of infectious causes, and characteristic endoscopic and histological findings.

[0323] 3) Active UC involving both colon and rectal disease, with at least 15 cm of the colon involved; patients with isolated ulcerative proctitis (disease no more than 15 cm from the anal verge) were excluded. 4) Modified Mayo score of 3-8 based on sigmoidoscopy or full colonoscopy (if no colonoscopy was performed in the past 24 months) less than 2 weeks from randomization of the subject, with a score of less than 2 points in each individual category of the modified Mayo scoring system and an endoscopic score greater than 1 point in the most affected area. Although subjects who underwent full colonoscopy or sigmoidoscopy had disease progression beyond this level, only the sigmoid colon should be scored up to approximately 30-40 cm. 5) Patients with active disease (defined by clinical and endoscopic criteria) at the screening visit despite continued daily oral mesalamine >2.4 g with or without topical (rectal) mesalamine (mesalamine 1000 mg PR QHS or mesalamine enema 4 g PR QHS) treatment for more than 4 weeks. 6) During the screening and treatment phases of the study, patients were on stable doses of oral mesalamine and concomitant topical (rectal) mesalamine (if applicable). 7) During the screening and treatment phases of the study, patients were on stable doses of oral corticosteroids <15 mg prednisone / day or equivalent.

[0324] The study drug will be packaged as unit doses in plastic tubes with screw caps. The tubes containing the capsules and 1 g silica gel desiccant sachets will be placed in heat-sealed aluminum foil bags.

[0325] Place foil pouches of seven (7) unit doses of study drug into outer cartons to form weekly packages. Provide eight (8) weeks of study drug per patient in two separate sets of kits. The first kit [1 of 2] outer carton contains five (5) weeks of study drug. The second kit [2 of 2] outer carton contains three (3) weeks of study drug.

[0326] Study medications must be kept refrigerated (2-8°C) in a secure location at the study site. Storage conditions (e.g., temperature and other requirements) for study medications will be identified on the study medication label and in investigator site documentation. Study medications must not be frozen.

[0327] Each non-placebo capsule contains 1x 10 of each targeted bacterial strain. 9 Live microorganisms. Subjects in Group A were administered 1 capsule per day for 8 weeks. Subjects in Group B were administered up to 5 capsules per day for 8 weeks. Subjects were instructed to swallow the study drug capsule intact with a full glass of water once a day in the morning after an overnight fast. After ingesting the study drug, they were to wait at least 60 minutes before consuming the first solid or liquid meal of the day (other than water).

[0328] Safety was assessed based on the incidence of severity-graded treatment-emergent adverse events, including serious adverse events.

[0329] The primary efficacy assessment was the percentage of subjects in clinical remission at the end of the 8-week treatment period using the modified Mayo score (MMS).

[0330] The Mayo scoring system for assessing the activity of ulcerative colitis is described in Rutgeerts et al., N Engl J Med. 353(23): 2462-76 (2005). The Mayo scoring system includes stool frequency, rectal bleeding findings on endoscopic examination, and the physician's overall assessment. The subscore for stool frequency is 0-3, where 0 = the patient's normal number of stools, 1 = 1-2 stools more than normal, 2 = 3-4 stools more than normal, and 3 = 5 or more stools more than normal. Each patient serves as his or her own control to determine the degree of abnormality in stool frequency. The subscore for rectal bleeding is 0-3, where 0 = no blood, 1 = blood streaks in the stool less than half the time, 2 = obvious blood in the stool most of the time, and 3 = blood passing alone. The daily bleeding score represents the most severe bleeding of the day. Endoscopic findings were sub-scored on a scale of 0-3, with 0 = normal or inactive disease, 1 = mild disease (erythema, decreased vascualr pattern), 2 = moderate disease (marked erythema, lack of vascular pattern, friability, erosion), and 3 = severe disease (spontaneous bleeding, ulceration). The physician's global assessment was sub-scored on a scale of 0-3, with 0 = normal, 1 = mild disease, 2 = moderate disease, and 3 = severe disease. The physician's global assessment confirmed the other three criteria, the patient's daily memory of abdominal discomfort and overall sense of well-being, as well as other observations, such as physical findings and the patient's presentation status.

[0331] The modified Mayo score (MMS) excludes the PGA component of the Mayo score. The modified Mayo score ranges from 0 to 9, with higher scores indicating more severe disease. Clinical remission will be measured using the modified Mayo score (MMS), which is based on a subscore of "0" for rectal bleeding and a subscore of 0 or 1 for endoscopic mucosal appearance and stool frequency, with at least a 1-point decrease from baseline in stool frequency.

[0332] The primary efficacy assessment is endoscopic improvement at the end of the 8-week treatment period using the Ulcerative Colitis Endoscopic Index of Severity (UCEIS). Improvement in endoscopic disease severity will be assessed using the change from baseline in the UCEIS score. The UCEIS includes 3 descriptors of endoscopic severity, each with a specific definition and scored on a simple visual analog scale with a score of 0 representing normal. The descriptors are vascular pattern (3 grades), bleeding (4 grades), and erosions and ulcers (4 grades). The final score calculated from the simple sum of the individual scores ranges from 0 to 8, with higher scores indicating more severe disease.

[0333] This study also uses UC PRO2 to collect patient-reported outcome data related to underlying disease status. In addition to the above clinical assessments, the study also includes the collection of longitudinal stool samples, and mucosal biopsy samples at baseline and end of the study to understand changes in the composition (taxonomy and phenotype) of the gut microbiota and potentially correlate it with clinical outcomes.

[0334] Concomitant treatments / medications, i.e., those given between the study randomization date and the end-of-study treatment visit, were given only if medically necessary. Examples of such medications include rescue medications for UC disease activity exacerbations, systemic antibiotics for treatment of concurrent infections, or gastric acid reducers such as H2 blockers or proton pump inhibitors. These include: an increase in the dose of oral corticosteroids; introduction of oral corticosteroids; introduction of topical corticosteroids; an increase in the dose of oral mesalamine; introduction of topical mesalamine; and introduction of other oral 5-ASA formulations and prodrugs, including sulfasalazine, olsalazine, or balsalazide. Rescue medications include parenteral corticosteroids, 6-MP, azathioprine, low-dose methotrexate, intravenous immunoglobulin (IVIG), anti-TNF agents, natalizumab, vedolizumab, cyclosporine, tacrolimus, tofacitinib, other commercially available biologics, or experimental drugs.

[0335] Example 8 - Preparation of bacterial strain composition: fermentation, harvesting and lyophilization

[0336] A flow chart of an exemplary GMP process is shown in Fig.15 One or more bottles of each bacterial strain MCB were used to prepare the inoculum. The resulting culture was used to inoculate the fermentor. After fermentation, the cells were harvested by tangential flow filtration (TFF) and / or centrifugation, mixed with the selected cryoprotectant detailed in Table 11, and then distributed into trays, frozen (-70 ± 10 ° C), and subsequently freeze-dried.

[0337] Table 11: Cryoprotectants used for each C1 component

[0338]

[0339] Example 9 - Capsule-in-Capsule

[0340] In this example, C1 is formulated into a capsule-in-capsule dosage form (e.g., ) for oral administration. Fig.16 Shows Schematic diagram of the design. In this example, both the inner and outer capsules are filled with the same C1 formulation, and both the inner and outer capsules are banded before coating. The drug release properties are provided by the functional coating as follows:

[0341] (a) an external enteric-coated capsule that provides gastric resistance in the stomach and proximal small intestine, and then releases its powder-filled formulation and the contents of the coated inner capsule into the distal ileum. The enteric polymer is designed to dissolve as the intestinal pH increases to the critical pH for dissolution of the enteric polymer; and

[0342] (b) Internally reverse enteric coated capsules that are intended to release the contents of their powder fill formulation in the proximal colon. Reverse enteric polymers are designed to dissolve as the intestinal pH decreases to the critical pH for reverse enteric polymer dissolution. Reverse enteric polymers take advantage of the pH drop in the ileocecal portion of the intestine.

[0343] Human scintigraphic studies were previously performed that demonstrated that release was achieved at the intended anatomical target.

[0344] for Fig.17 In the depicted embodiment, the powder fill formulation includes three separately lyophilized bacterial compositions. The formulated concentration of each bacterial strain is targeted to be 1:1 in each C1 The capsule dosage is about 3×10 8 About 3×10 10 A living organism (i.e., a single C1 The powder fill formulation also contains other typical pharmaceutical excipients (e.g., fillers, disintegrants, glidants, and lubricants).

[0345] The unit dose package consists of a single Capsules. As an extra precaution during early research, capsules were inserted into protective plastic sleeves before being placed in foil pouches.

[0346] Example 10 - Composition

[0347] The components of C1 used in the exemplary capsule-in-capsule formulation are shown in Table 12. In this embodiment, the powder-filled formulation includes three separate freeze-dried bacterial strain compositions. The powder-filled formulation also contains other typical pharmaceutical excipients (e.g., fillers, disintegrants, glidants, and lubricants). The inner and outer capsules are composed of hydroxypropyl methylcellulose (HPMC), and the size of the inner capsule is smaller than that of the outer capsule. The sealing polymer is also composed of HPMC, which is used to seal each capsule and provide a suitable surface across the seam before coating. The coating polymer determines the functional performance of the capsule-in-capsule. The outer capsule is coated with a mixture of a specified enteric polymer (poly (methacrylic acid-to-methyl methacrylate) 1:1 and poly (methacrylic acid-to-methyl methacrylate) 1:2), which provides gastric resistance in the stomach and proximal small intestine, and then releases the contents of its powder-filled formulation and coated inner capsule into the distal ileum. The inner capsule is coated with a reverse enteric polymer (amino methacrylic acid copolymer (2:1:1)), which releases the contents of the powder-filled formulation into the proximal colon.

[0348] Table 12: Components of an exemplary C1 capsule-in-capsule formulation

[0349]

[0350]

[0351] 1 C1 bacterial strains BTH01, EHA01, and RHO01 (including cryoprotectants) will be used in C1 Powder fill formulation for capsules.

[0352] 2 These solvents are removed during processing.

[0353] Example 11 - Preparation of an Exemplary C1 Capsule-in-Capsule Formulation

[0354] Example C1 The preparation flow chart of capsules is as follows Fig.16 The exemplary steps include:

[0355] distribute

[0356] The freeze-dried bacterial strain composition was removed from frozen storage and allowed to equilibrate to room temperature before distribution. Active and inactive materials were accurately weighed.

[0357] Powder Screening and Mixing

[0358] The powders may be filtered to remove lumps and then blended in a conventional bin blender (eg, V-blender). The glidant and lubricant excipients may be pre-blended separately with the appropriate amount of filler before adding to the blender.

[0359] Encapsulation (inner capsule)

[0360] Separate the inner HPMC capsule manually or using semi-automatic equipment. Accurately fill the specified amount of powder mixture into each capsule body manually or using semi-automatic equipment, and then close each capsule.

[0361] Capsule sealing (inner capsule)

[0362] The inner capsule is tape sealed using a hydroalcoholic HPMC solution and then dried. Circumferential sealing is performed at the capsule seam using semi-automatic or automatic equipment.

[0363] Capsule coating (inner capsule)

[0364] The inner capsules were coated with a reverse enteric polymer (amino methacrylate copolymer) solution using conventional pan coating equipment. The capsules were coated until the target weight gain was achieved.

[0365] Encapsulation (outer capsule)

[0366] The outer HPMC capsule is separated manually or using semi-automatic equipment. The sealed / coated inner capsule is placed in each capsule body. The prescribed amount of powder mixture is then accurately filled into the same body of each capsule manually or using semi-automatic equipment, and then each capsule is closed.

[0367] Capsule sealing (outer capsule)

[0368] The outer capsule is band-sealed using a hydroalcoholic HPMC solution and then dried. A circumferential seal is made at the capsule seam using semi-automatic or automatic equipment.

[0369] Capsule coating (outer capsule)

[0370] The outer capsules were coated with an enteric polymer (poly(methacrylic acid-co-methyl methacrylate)) solution using conventional pan coating equipment. The capsules were coated until the target weight gain was achieved.

[0371] Package

[0372] Each capsule-in-capsule unit dose is packaged in a heat-sealed foil bag containing a silica gel desiccant bag. Alternatively, the capsule can be inserted into a protective plastic sleeve before being placed in the foil bag. The packaged capsule-in-capsule is stored at 5°C.

[0373] Incorporated by Reference

[0374] The entire disclosure of each patent and scientific document mentioned in this application is incorporated by reference for all purposes.

[0375] Equivalent method

[0376] Without departing from the spirit or essential features of the present disclosure, the present disclosure may be implemented in other specific forms. Therefore, the foregoing embodiments should be considered to be illustrative in all respects, rather than limiting the disclosure described herein. Therefore, the scope of the present disclosure is indicated by the appended claims rather than the foregoing description, and all changes within the meaning and equivalent range of the claims are intended to be included therein.

Claims

1. A pharmaceutical composition comprising: A vegetative bacterial mixture comprising at least two bacterial species, wherein the bacterial species are selected from the group consisting of Bacteroides thetaiotaomicron ( Bacteroides thetaiotaomicron ) BTH01 strain, whose deposit number is DSM-32919, Eubacterium hallii ( Eubacterium hallii ) EHA01 strain, whose deposit number is DSM-32920 and Roseburia hominis ( Roseburia hominis ) RHO01 strain, which has the deposit number DSM-32921, and wherein the bacterial mixture is in a freeze-dried form; and Pharmaceutically acceptable excipients. 2 . The pharmaceutical composition according to claim 1 , wherein the bacterial mixture comprises the Bacteroides thetaiotaomicron strain, the Eubacterium hallii strain and the Roseburia hominis strain.

3. The pharmaceutical composition according to claim 1, wherein the bacterial mixture consists of the Bacteroides thetaiotaomicron strain, the Eubacterium hallii strain and the Roseburia hominis strain.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical excipient is selected from cellulose, polyvinyl pyrrolidone, silicon dioxide, stearyl fumarate or a pharmaceutically acceptable salt thereof, and any combination thereof; or is selected from fillers, binders, disintegrants and any combination thereof.

5. The pharmaceutical composition according to any one of claims 1-3, wherein the pharmaceutical composition further comprises a cryoprotectant.

6. The pharmaceutical composition according to claim 5, wherein the cryoprotectant is selected from fructooligosaccharides, trehalose and mixtures thereof.

7. The pharmaceutical composition according to claim 6, wherein the fructooligosaccharide is 8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is suitable for bolus administration or bolus release.

9. The pharmaceutical composition of any one of claims 1-3, wherein the composition is suitable for rectal delivery to a subject.

10. The pharmaceutical composition of any one of claims 1-3, wherein the composition is suitable for oral delivery to a subject.

11. The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is formulated into capsules, tablets, pills, lozenges, powders or granules.

12. The pharmaceutical composition according to any one of claims 1-3, wherein the composition is formulated as a caplet or a lozenge.

13. The pharmaceutical composition according to claim 11, wherein the composition is formulated into a capsule. The pharmaceutical composition according to claim 13 , wherein the capsule is a two-component capsule.

15. The pharmaceutical composition according to claim 14, wherein the two-component capsule is a capsule within a capsule.

16. The pharmaceutical composition according to claim 15, wherein the capsule-in-capsule comprises: an inner capsule comprising an inner pharmaceutical composition comprising the vegetative bacterial mixture, wherein the inner capsule has a reverse enteric polymer coating; and an outer capsule encapsulating the inner capsule and comprising an outer composition comprising the vegetative bacterial mixture, wherein the outer capsule has an enteric polymer coating.

17. A pharmaceutical composition comprising: A bacterial mixture comprising at least two bacterial species, the bacterial species being selected from the group consisting of Bacteroides thetaiotaomicron BTH01 strain, deposited as DSM-32919, Eubacterium hallii EHA01 strain, deposited as DSM-32920 and Roseburia hominis RHO01 strain, deposited as DSM-32921, and wherein the bacterial mixture is capable of increasing the production of IL-10 and / or CCL-18 and / or improving intestinal barrier integrity and / or increasing autophagy in human cells and / or reducing or attenuating the production of lipocalin-2 / NGAL in cells, tissues or subjects; and Pharmaceutically acceptable excipients.

18. A drug unit comprising: A bacterial strain mixture comprising Bacteroides thetaiotaomicron BTH01 strain, which has a deposit number of DSM-32919, Eubacterium hallii EHA01 strain, which has a deposit number of DSM-32920, and Roseburia hominis RHO01 strain, which has a deposit number of DSM-32921, wherein each bacterial strain is in a freeze-dried form, and wherein the drug unit has at least 1×10 8 living bacterial organisms; and Pharmaceutically acceptable excipients.

19. The pharmaceutical unit according to claim 18, wherein the bacterial strain mixture comprises vegetative bacteria comprising at least 1×10 9 Bacteroides species strains, at least 1 × 10 9 living organisms of Eubacterium species and at least 1 × 10 9 living organisms of a strain of Roseburia species.

20. The pharmaceutical unit of claim 18, wherein the bacterial strain mixture further comprises a cryoprotectant selected from the group consisting of fructooligosaccharides, trehalose, and mixtures thereof.

21. A pharmaceutical unit according to any one of claims 18 to 20, wherein the unit is a capsule.

22. The pharmaceutical unit according to claim 21, wherein the unit is a two-component capsule.

23. The medication unit of claim 22, wherein the two-component capsule is a capsule within a capsule.

24. The medication unit of claim 23, wherein the capsule-in-capsule comprises: an inner capsule comprising an inner pharmaceutical composition comprising the bacterial strain mixture, wherein the inner capsule has a reverse enteric polymer coating; and The inner capsule and an outer capsule comprising an outer composition comprising the bacterial strain mixture are encapsulated, wherein the outer capsule has an enteric polymer coating.

25. A bacterial strain of Roseburia hominis capable of increasing the production of IL-10 and / or CCL-18 in human THP-1 macrophages and / or PBMCs, wherein the strain is the RHO01 strain with the deposit number of DSM-32921.

26. A bacterial strain of Bacteroides thetaiotaomicron capable of increasing the production of IL-10 and / or CCL-18 in human THP-1 macrophages and / or PBMCs, wherein the strain is the BTH01 strain, and its deposit number is DSM-32919.

27. A bacterial strain of Eubacterium hallii capable of increasing the production of IL-10 and / or CCL-18 in human THP-1 macrophages and / or PBMCs, wherein the strain is the EHA01 strain with the deposit number of DSM-32920.

28. Use of the pharmaceutical composition of any one of claims 1-17, the pharmaceutical unit of any one of claims 18-24, or the bacterial strain of any one of claims 25-27 for the preparation of a medicament for treating ulcerative colitis (UC) in a subject in need thereof.

29. Use of at least two bacterial strains in the preparation of a medicament for treating ulcerative colitis (UC) in a subject, wherein the bacterial strains are selected from the group consisting of Bacteroides thetaiotaomicron BTH01 strain, which has a deposit number of DSM-32919, Eubacterium hallii EHA01 strain, which has a deposit number of DSM-32920, and Roseburia hominis RHO01 strain, which has a deposit number of DSM-32921, and wherein each of the bacterial strains is in a freeze-dried form.

30. Use of the pharmaceutical composition of any one of claims 1-17, the pharmaceutical unit of any one of claims 18-24, or the bacterial strain of any one of claims 25-27 for the preparation of a medicament for treating ulcerative colitis in a subject, wherein the subject has an inadequate response to the administration of mesalazine.

31. The use according to any one of claims 28-30, wherein the medicament further comprises a combination with a corticosteroid.

32. The use according to any one of claims 28-30, wherein the medicament further comprises a combination of pre-treatment with an antibiotic.

33. The use according to any one of claims 28-30, wherein the medicament further comprises a combination with a prebiotic.

34. The use according to any one of claims 28-30, wherein the subject is selected from a human, a companion animal or a livestock animal.

35. The use according to claim 34, wherein the subject is a human.

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