# Patent

## Title
Probiotic compositions containing clostridiales for inhibiting inflammation

## Abstract

Pharmaceutical compositions containing microbial entities are described herein. The pharmaceutical compositions may optionally contain or be used in conjunction with one or more prebiotics. Uses of the pharmaceutical compositions to treat or prevent disorders of the local or systemic microbiome in a subject are also provided.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 62/084,536, filed Nov. 25, 2014; U.S. Provisional Patent Application No. 62/084,537, filed Nov. 25, 2014; U.S. Provisional Patent Application No. 62/084,540, filed Nov. 25, 2014; U.S. Provisional Patent Application No. 62/117,632, filed Feb. 18, 2015; U.S. Provisional Patent Application No. 62/117,637, filed Feb. 18, 2015; U.S. Provisional Patent Application No. 62/117,639, filed Feb. 18, 2015; U.S. Provisional Patent Application No. 62/162,562, filed May 15, 2015; and U.S. Provisional Patent Application No. 62/257,714, filed Nov. 19, 2015. The entire contents of each of the foregoing applications are incorporated herein by reference.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 25, 2015, is named 126383_02102_SL.txt and is 4,147,425 bytes in size.
BACKGROUND
Humans and other mammals have numerous microbial niches, and interventions to modulate the microbiota thereof have been focused on antibiotics (which effect largely non-specific eradication of the microbiota in an effort to target a pathogen), probiotics (largely in the form of lactic acid-producing bacteria in food products), prebiotics (stimulatory materials, primarily carbohydrates, that increase bacterial growth and/or activity), and synbiotics (combinations of prebiotics and probiotics) (see, e.g., WO 2011/022542). Autoimmune and inflammatory diseases are characterized by an inappropriate immunological intolerance or an abnormal immune response, and affect up to 50 million Americans. Current treatments for such conditions, such as immunosuppressant drugs, carry a risk of dangerous systemic side effects such as infection, organ damage, and the development of new autoimmunities. There is therefore a need for improved diagnostic and prognostic measures, preventative measures, and treatments for autoimmune and inflammatory diseases.
A healthy microbiota provides the host with multiple benefits, including colonization resistance to a broad spectrum of pathogens, essential nutrient biosynthesis and absorption, and immune stimulation that maintains a healthy gut epithelium and an appropriately controlled systemic immunity. In settings of ‘dysbiosis’ or disrupted symbiosis, microbiota functions can be lost or deranged, resulting in increased susceptibility to pathogens, altered metabolic profiles, or induction of proinflammatory signals that can result in local or systemic inflammation or autoimmunity. Thus, the intestinal microbiota plays a significant role in the pathogenesis of many diseases and disorders, including a variety of pathogenic infections distal to the gastrointestinal tract. Therefore, in response to the need for durable, efficient, and effective compositions and methods for treatment of immune and inflammatory diseases by way of restoring or enhancing microbiota functions, the present invention provides compositions and methods for treatment and prevention of immune and inflammatory conditions associated with dysbiosis, including dysbiosis distal to the gastrointestinal tract.
SUMMARY OF THE INVENTION
Disclosed herein are therapeutic compositions containing probiotic, non-pathogenic bacterial populations and networks thereof, for the prevention, control, and treatment of diseases, disorders and conditions, in particular immune and inflammatory diseases. In some embodiments, the therapeutic compositions contain prebiotics, e.g., carbohydrates, in conjunction with microbial populations and/or networks thereof. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous dysbiotic diseases, disorders and conditions, such as immune and inflammatory disease.
In one aspect, the invention provides a pharmaceutical composition comprising an isolated population of anti-inflammatory bacterial cells of the order Clostridiales capable of decreasing the secretion of a pro-inflammatory cytokine and/or increasing the secretion of an anti-inflammatory cytokine by a population of human peripheral blood mononuclear cells (PBMCs), and a pharmaceutically acceptable excipient. In one embodiment, the secretion of a pro-inflammatory cytokine by a population of PBMCs is induced by Enterococcus faecalis. 
In one embodiment, the anti-inflammatory bacterial cells are of the family Lachnospiraceae. In another embodiments, the anti-inflammatory bacterial cells are of the genus Blautia, Clostridium, Eubacterium, or Ruminococcus. In one the anti-inflammatory bacterial cells are of the genus Blautia. In another embodiment, the anti-inflammatory bacterial cells are of a species selected from the group consisting of Blautia coccoides, Blautia faecis, Blautia glucerasea, Blautia hansenii, Blautia hyrogenotrophica, Blautia luti, Blautia obeum, Blautia producta, Blautia schinkii, Blautia sp. M25, Blautia stercoris, Blautia wexlerae, Blautia uncultured bacterium clone BKLE_a03_2, Blautia uncultured bacterium clone SJTU_B_14_30, Blautia uncultured bacterium clone SJTU_C_14_16, Blautia uncultured bacterium clone S1-5, and Blautia uncultured PAC000178_s. In one embodiment, the anti-inflammatory bacterial cells are of the species Ruminococcus gnavus. In another embodiment, the anti-inflammatory bacterial cells are of the species Eubacterium rectale. 
In one embodiment, the anti-inflammatory bacterial cells comprise a bacterial cell in vegetative form. In another embodiment, the anti-inflammatory bacterial cells comprise a bacterial cell in spore form.
In one embodiment, the isolated population of anti-inflammatory bacterial cells further comprises a bacterial cell belonging to a bacterial strain set forth in Table 1, Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, or Table 1F.
In one embodiment, the pharmaceutical composition comprises a prebiotic. In one embodiment, the prebiotic comprises a monomer or polymer selected from the group consisting of arabinoxylan, xylose, soluble fiber dextran, soluble corn fiber, polydextrose, lactose, N-acetyl-lactosamine, glucose, and combinations thereof. In another embodiment, the prebiotic comprises a monomer or polymer selected from the group consisting of galactose, fructose, rhamnose, mannose, uronic acids, 3′-fucosyllactose, 3′-sialylactose, 6′-sialyllactose, lacto-N-neotetraose, 2′-2′-fucosyllactose, and combinations thereof. In one embodiment, the prebiotic comprises a monosaccharide selected from the group consisting of arabinose, fructose, fucose, galactose, glucose, mannose, D-xylose, xylitol, ribose, and combinations thereof. In another embodiment, the prebiotic comprises a disaccharide selected from the group consisting of xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose, and combinations thereof. In yet another embodiment, the prebiotic comprises a polysaccharide, wherein the polysaccharide is xylooligosaccharide.
In one embodiment, the prebiotic comprises a sugar selected from the group consisting of arabinose, fructose, fucose, lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose, xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose, xylooligosaccharide, and combinations thereof. In one embodiment, the sugar is xylose.
In one embodiment, the pro-inflammatory cytokine is selected from the group consisting of IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof.
In one embodiment, the anti-inflammatory cytokine is selected from the group consisting of IL-10, IL-13, IL-4, IL-5, TGFβ, and combinations thereof.
In one embodiment, the pharmaceutical composition is formulated for oral administration. In another embodiment, the pharmaceutical composition is formulated for rectal administration.
In one embodiment, the anti-inflammatory bacterial cells decrease the secretion of a pro-inflammatory cytokine and/or increase the secretion of an anti-inflammatory cytokine by a population of human peripheral blood mononuclear cells (PBMCs) in vitro.
In another aspect, the invention provides a method for reducing inflammation in a subject, the method comprising administering a pharmaceutical composition of the invention to thereby reduce inflammation in the subject.
In one embodiment, the subject has an autoimmune or inflammatory disorder. In one embodiment, the autoimmune or inflammatory disorder is selected from the group consisting of graft-versus-host disease (GVHD), an inflammatory bowel disease (IBD), ulterative colitis, Crohn's disease, multiple sclerosis (MS), systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, Sjögren's syndrome, and Celiac disease.
In one embodiment, the pharmaceutical composition is administered orally. In another embodiment, the pharmaceutical composition is administered rectally.
In one embodiment, administration of the pharmaceutical composition reduces inflammation in the gastrointestinal tract of the subject. In another embodiment, administration of the pharmaceutical composition reduces inflammation at a site distal to the gastrointestinal tract of the subject. In one embodiment, the distal site is the placenta, the spleen, the skin the liver, the uterus, the blood, an eye/conjunctiva, the mouth an ear, the nose, a lung, the liver, the pancreas, the brain, the embryonic sac, or vagina of the subject. In another embodiment, the distal site is the circulatory system, the reproductive tract, the cardiovascular system, the nervous system, or a combination thereof.
In one embodiment, the subject has a dysbiosis. In one embodiment, the dysbiosis is a gastrointestinal dysbiosis. In another embodiment, the dysbiosis is a distal dysbiosis.
In one embodiment, the anti-inflammatory bacterial cells of the pharmaceutical composition engraft in the gastrointestinal tract of the subject.
In one embodiment, the method further comprises administering a prebiotic to the subject.
BRIEF DESCRIPTION OF THE TABLES
Table 1 provides a list of Operational Taxonomic Units (OTU) with taxonomic assignments made to Genus, Species, and Phylogenetic Clade. Clade membership of bacterial OTUs is based on 16S sequence data. Clades are defined based on the topology of a phylogenetic tree that is constructed from full-length 16S sequences using maximum likelihood methods familiar to individuals with ordinary skill in the art of phylogenetics. Clades are constructed to ensure that all OTUs in a given clade are: (i) within a specified number of bootstrap supported nodes from one another, and (ii) within 5% genetic similarity. OTUs that are within the same clade can be distinguished as genetically and phylogenetically distinct from OTUs in a different clade based on 16S-V4 sequence data, while OTUs falling within the same clade are closely related. OTUs falling within the same clade are evolutionarily closely related and may or may not be distinguishable from one another using 16S-V4 sequence data. Members of the same clade, due to their evolutionary relatedness, play similar functional roles in a microbial ecology such as that found in the human gut. Compositions substituting one species with another from the same clade are likely to have conserved ecological function and therefore are useful in the present invention. All OTUs are denoted as to their putative capacity to form spores and whether they are a Pathogen or Pathobiont (see Definitions for description of “Pathobiont”). NIAID Priority Pathogens are denoted as ‘Category-A’, ‘Category-B’, or ‘Category-C’, and Opportunistic Pathogens are denoted as ‘OP’. OTUs that are not pathogenic or for which their ability to exist as a pathogen is unknown are denoted as ‘N’. The ‘SEQ ID Number’ denotes the identifier of the OTU in the Sequence Listing File and ‘Public DB Accession’ denotes the identifier of the OTU in a public sequence repository. See, e.g., WO 2014/121304.
Table 1A provides a list of exemplary bacteria useful in the present invention.
Table 1B provides a list of exemplary bacteria useful in the present invention.
Table 1C provides a list of exemplary bacteria useful in the present invention.
Table 1D provides a list of exemplary bacteria useful in the present invention.
Table 1E provides a list of exemplary bacteria useful in the present invention. These bacteria are preferably down-modulated in a subject.
Table 1F provides a list of exemplary bacteria that may be used in the invention. These bacteria are preferably up-modulated in a subject.
Table 2A lists species identified as “germinable” and “sporulatable” by colony picking approach.
Table 2B lists species identified as “germinable” using 16S colony picking approach.
Table 2C lists species identified as “sporulatable” using 16s-V4 NGS approach. See, e.g., WO 2014/121304.
Table 3 lists anaerobic bacterial species tested for carbon source usage (Biolog plates).
Table 4 lists exemplary prebiotics/carbon sources.
Table 5 provides bacterial species detected at low frequency in vaginal samples from vancomycin-treated mice (day 6) that were not present in untreated mice (day 0).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph depicting serum endotoxin levels (EU/ml) over time following treatment with xylose. Treatment of mice with xylose alone reduces basal levels of serum endotoxin (day 14 vs day 0). Antibiotic treatment (Ciprofloxacin (cipro) or enrofloxacin (enro)) leads to an increase in serum endotoxin levels (measured 2 days after a 5 day course, at day 0) with a return to baseline by day 14. Xylose counteracts the endotoxin increase caused by cipro but not enro antibiotic treatment.
FIG. 2(a-o) is a panel of graphs showing the time course of Th1 related cytokines that were released by human peripheral mononuclear cells (PBMCs) incubated with Ruminococcus gnavus (Epv 1), Eubacterium rectale (Epv 2), Blautia luti (Epv 3), Blautia wexlerae (Epv 5) and Enterococcus faecalis (Epv 8), or combinations of each bacterium with E. faecalis. Amounts of interferon gamma (IFN-γ), IL-12p70, IL-6, IL-2 and TNF—α that were released in culture supernatants by PBMCs were measured after 24, 48 and 72 hours. a) IFN-γ concentration (pg/ml) after 24 hours. b) IFN-γ concentration (pg/ml) after 48 hours. c) IFN-γ concentration (pg/ml) after 72 hours. d) IL-12p70 concentration (pg/ml) after 24 hours. e) IL-12p70 concentration (pg/ml) after 48 hours. f) IL-12p70 concentration (pg/ml) after 72 hours. g) IL-6 concentration (pg/ml) after 24 hours. h) IL-6 concentration (pg/ml) after 48 hours. i) IL-6 concentration (pg/ml) after 72 hours. j) IL-2 concentration (pg/ml) after 24 hours. k) IL-2 concentration (pg/ml) after 48 hours. 1) IL-2 concentration (pg/ml) after 72 hours. m) TNFα concentration (pg/ml) after 24 hours. n) TNFα concentration (pg/ml) after 48 hours. o) TNFα concentration (pg/ml) after 72 hours.
FIG. 3(a-i) is a panel of graphs showing the time course of Th2 related cytokines that were released by human PBMCs incubated with R. gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv 5) and E. faecalis (Epv 8), or combinations of each bacterium with E. faecalis. Amounts of IL-13, IL-4 and IL-5 that were released in culture supernatants by PBMCs were measured after 24, 48 and 72 hours. a) IL-13 concentration (pg/ml) after 24 hours. b) IL-13 concentration (pg/ml) after 48 hours. c) IL-13 concentration (pg/ml) after 72 hours. d) IL-4 concentration (pg/ml) after 24 hours. e) IL-4 concentration (pg/ml) after 48 hours. f) IL-4 concentration (pg/ml) after 72 hours. g) IL-5 concentration (pg/ml) after 24 hours. h) IL-5 concentration (pg/ml) after 48 hours. i) IL-5 concentration (pg/ml) after 72 hours.
FIG. 4(a-i) is a panel of graphs showing the time course of Th9, Th17 and Treg cytokines that were released by human PBMCs incubated with R. gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv 5) and E. faecalis (Epv 8), or combinations of each bacterium with E. faecalis. Amounts of IL-9, IL-17 and IL-10 that were released in culture supernatants by PBMCs were measured after 24, 48 and 72 hours. a) IL-9 concentration (pg/ml) after 24 hours. b) IL-9 concentration (pg/ml) after 48 hours. c) IL-9 concentration (pg/ml) after 72 hours. d) IL-17 concentration (pg/ml) after 24 hours. e) IL-17 concentration (pg/ml) after 48 hours. f) IL-17 concentration (pg/ml) after 72 hours. g) IL-10 concentration (pg/ml) after 24 hours. h) IL-10 concentration (pg/ml) after 48 hours. i) IL-10 concentration (pg/ml) after 72 hours.
FIG. 5(a-x) is a panel of graphs showing the time course of monocyte, macrophage and neutrophil-derived inflammatory cytokines that were released by human PBMCs incubated with R. gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv 5) and E. faecalis (Epv 8), or combinations of each bacterium with E. faecalis. Amounts of monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP1β), macrophage inflammatory protein 1α (MIP1a), regulated on activation, normal T expressed and secreted protein (RANTES), interleukin-1α (IL-1α), interleukin-1β (IL1β), interferon α2 (IFN-α2) and interleukin-8 (IL-8) that were released in culture supernatants by PBMCs were measured after 24, 48 and 72 hours. a) MCP-1 concentration (pg/ml) after 24 hours. b) MCP-1 concentration (pg/ml) after 48 hours. c) MCP-1 concentration (pg/ml) after 72 hours. d) MIP1β concentration (pg/ml) after 24 hours. e) MIP1β concentration (pg/ml) after 48 hours. f) MIP1β concentration (pg/ml) after 72 hours. g) MIP1α concentration (pg/ml) after 24 hours. h) MIP1α concentration (pg/ml) after 48 hours. i) MIP1α concentration (pg/ml) after 72 hours. j) RANTES concentration (pg/ml) after 24 hours. k) RANTES concentration (pg/ml) after 48 hours. 1) RANTES concentration (pg/ml) after 72 hours. m) IL-1α concentration (pg/ml) after 24 hours. n) IL-1α concentration (pg/ml) after 48 hours. o) IL-1α concentration (pg/ml) after 72 hours. p) IL1β concentration (pg/ml) after 24 hours. q) IL1β concentration (pg/ml) after 48 hours. r) IL1β concentration (pg/ml) after 72 hours. s) IFN-α2 concentration (pg/ml) after 24 hours. t) IFN-α2 concentration (pg/ml) after 48 hours. u) IFN-α2 concentration (pg/ml) after 72 hours. v) IL-8 concentration (pg/ml) after 24 hours. w) IL-8 concentration (pg/ml) after 48 hours. x) IL-8 concentration (pg/ml) after 72 hours.
FIG. 6(a-d) is a panel of graphs showing the secreted levels of cytokines IFNγ (Ifng), IL-12p70, IL-1α(IL-1a), IL-6, IL-8, MCP1, MIP1α(MIP1a), MIP1β (MIP1b), TNFα (TNFa), IL-10, IL-13, IL-9, IL-4, IL-5, IL-17α (IL-17A) and IL-2 produced by PBMCs in the presence of a) R. gnavus, b) B. wexlerae, c) E. rectale and d) B. luti, alone or in combination with E. faecalis (Epv 8), relative to levels secreted following treatment with E. faecalis alone for 24 hours (E. faecalis=100%).
FIG. 7(a-p) is a panel of graphs that show the effect of R. gnavus (Epv1) on cytokine concentration (pg/ml) either alone or in combination with Epv 8 (E. faecalis) on cytokine production by human PBMCs (pg/ml). a) IL-6, b) IFN-γ, c) IL-13, d) IL-10, e) IL-12p70, f) MCP-1, g) IL-8, h) IL17A, i) IL-α, j) IL-9, k) IL-2, l) IL-4, m) IL-5, n) MIP-1α, o) MIP-1β, p) TNF-α.
FIG. 8(a-p) is a panel of graphs that show the effect of E. rectale (Epv2) on cytokine concentration (pg/ml) either alone or in combination with Epv 8 (E. faecalis) on cytokine production by human PBMCs (pg/ml). a) IL-6, b) IFN-γ, c) IL-13, d) IL-10, e) IL-12p70, f) MCP-1, g) IL-8, h) IL17A, i) IL-α, j) IL-9, k) IL-2, l) IL-4, m) IL-5, n) MIP-1α, o) MIP-1β, p) TNF-α.
FIG. 9(a-p) is a panel of graphs that show the effect of B. luti (Epv3) on cytokine concentration (pg/ml) either alone or in combination with Epv 8 (E. faecalis) on cytokine production by human PBMCs (pg/ml). a) IL-6, b) IFN-γ, c) IL-13, d) IL-10, e) IL-12p70, f) MCP-1, g) IL-8, h) IL17a, i) IL-α, j) IL-9, k) IL-2, l) IL-4, m) IL-5, n) MIP-1α, o) MIP-1β, p) TNF-α.
FIG. 10(a-p) is a panel of graphs that show the effect of B. wexlarae) on cytokine concentration (pg/ml) either alone or in combination with Epv 8 (E. faecalis) on cytokine production by human PBMCs (pg/ml). a) IL-6, b) IFN-γ, c) IL-13, d) IL-10, e) IL-12p70, f) MCP-1, g) IL-8, h) IL17α, i) IL-α, j) IL-9, k) IL-2, l) IL-4, m) IL-5, n) MIP-1α, o) MIP-1β, p) TNF-α.
FIG. 11(a-d) is a panel of graphs showing that (a-b) EPV3 is capable of inducing a desirable anti-inflammatory cytokine profile for treating or preventing GVHD and (c-d) EPV5 induces a suboptimal profile for GVHD.
FIG. 12(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv6 (Clostridium leptum).
FIG. 13(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv15 (Blautia faecis).
FIG. 14(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv20 (Blautia/Ruminococcus obeum ATCC 29174).
FIG. 15(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv21 (Blautia producta ATCC 27340).
FIG. 16(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv22 (Blautia coccoides ATCC 29236).
FIG. 17(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv23 (Blautia hydrogenotrophica ATCC BAA-2371).
FIG. 18(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv24 (Blautia Hansenii ATCC27752).
FIG. 19(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv35 (Eubacterium rectale).
FIG. 20(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv47 (previously uncultured Blautia, similar to GQ898099_s S1-5).
FIG. 21(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv51 (previously uncultured Blautia, similar to SJTU_C_14_16).
FIG. 22(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv52 (Blautia wexlerae (SJTU_B_09_77)).
FIG. 23(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv54 (Blautia luti ELU0087-T13-S-NI_000247).
FIG. 24(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv64 (Blautia wexlerae WAL 14507).
FIG. 25(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv78 (Blautia obeum).
FIG. 26(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv102 (Ruminococcus gnavus).
FIG. 27(a-b) depicts the production of (a) pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4, IL-13) cytokines by human PBMCs following treatment with Epv114 (Blautia luti (BlnIX)).
FIG. 28(a-d) presents results from flow cytometry analysis of T cell populations in human PBMCs incubated in the presence of various commensal bacteria, determined using flow cytometry. A) Proportion of Treg cells)(CD25+CD127lo B) Proportion of Th17 cells (CXCR3−CCR6+); C) Proportion of Th1 cells (CXCR3+CCR6−); D) Proportion of Th2 cells (CXCR3−CCR6−). Bacterial strains are as follows: Epv 1: R. gnavus; Epv 3: B. luti; Epv 2: E. rectale; Epv 5: B. wexlerae; Epv. 8: E. faecalis; Epv 20: B. obeum; Epv 21: B. producta; Epv 24: B. hansenii. The results are shown as percent (%) of CD3ε+CD4+cells.
FIG. 29(a-u) presents the preferred carbon sources utilized by various commensal bacteria. (a) R. gnavus; (b) E. rectale; (c) C. leptum; (d) B. luti; (e) B. wexlerae; (f) B. faecis; (g) B. obeum; (h) B. producta; (i) B. coccoides; (j) B. hydrogenotrophica; (k) B. hansenii; (1) B. luti Blnl X; (m) B. luti ELU; (n) R. gnavus; (o) B. faecis; (p) R. torques; (q) B. wexlerae WAL14507; (r) B. wexlerae SJTU; (s) SJTU1416; (t) GQ8980099; (u) E. rectale. 
FIG. 30 graphically depicts levels of serum IFNγ before, during, and after treatment with a prebiotic formulation containing xylose.
FIG. 31 is a graph that shows the change in Chao1 diversity (indicator of community richness) over time in subjects administered xylose three times per day (TID) at 1, 2, 8, 12.5 or 15 grams.
FIG. 32 depicts the impact of oral vancomycin on the microbiome of the gut and the vagina, by principal component analysis (PCA).



DETAILED DESCRIPTION
Disclosed herein are therapeutic compositions (e.g., pharmaceutical compositions) containing bacterial entities (e.g., anti-inflammatory bacterial cells) and optionally containing a prebiotic for the prevention, control, and treatment of immune and inflammatory diseases, disorders and conditions. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in treating or preventing numerous immune and inflammatory diseases and gastrointestinal diseases, disorders and conditions associated with a dysbiosis.
The microbes that inhabit the human gastrointestinal tract, skin, lungs, vagina, and other niches are starting to be understood and appreciated for their roles in human health and disease (see, e.g., Human Microbiome Project Consortium (2012) NATURE 486(7402): 207-14). Aspects of the invention are based, in part, on the realization that, although autoimmune and inflammatory diseases are often attributed to genetic mutations, these conditions are also influenced by microbes. It is also appreciated that, because microbes not only interact with the host but with one another, the immunomodulatory behavior of microbes can depend on relationships between microbes. For example, a microbial network in a given niche may comprise diverse microbes that all accomplish one or more of the same functions, or may instead comprise diverse microbes that all individually contribute to accomplish one or more functions. For example, microbes in a given niche may influence and/or regulate the immunomodulatory behavior of other microbes in the same niche, or in a distal niche. In another example, microbes in a given niche may compete with one another for nutrients or space.
Microbes may influence the risk, progression, or treatment efficacy of an autoimmune or inflammatory disease. In certain aspects, microbes play a role in the prevention of an autoimmune or inflammatory disease or in the suppression of an innate or adaptive immune response. Microbes may also stimulate an inflammatory immune response to contribute to, increase the risk of, or worsen the symptoms of an autoimmune or inflammatory disease. Some microbes may be associated with lower disease severity or mortality.
Also disclosed herein are compositions and methods for the prevention and/or treatment of autoimmune and inflammatory diseases in human subjects.
DEFINITIONS
As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, “a compound” includes mixtures of compounds.
The term “about” or “approximately” means within an acceptable r range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, or within 5-fold, or within 2-fold, of a value.
As used herein, the term “purified bacterial preparation” refers to a preparation that includes “isolated” bacteria or bacteria that have been separated from at least one associated substance found in a source material or any material associated with the bacteria in any process used to produce the preparation.
A “bacterial entity” includes one or more bacteria. Generally, a first bacterial entity is distinguishable from a second bacterial entity.
As used herein, the term “formation” refers to synthesis or production.
As used herein, the term “inducing” means increasing the amount or activity of a given material as dictated by context.
As used herein, the term “depletion” refers to reduction in amount of.
As used herein, a “prebiotic” refers to an ingredient that allows specific changes both in the composition and/or activity in the gastrointestinal microbiota that may (or may not) confer benefits upon the host. In some embodiments, a prebiotic can be a comestible food or beverage or ingredient thereof. Prebiotics may include complex carbohydrates, amino acids, peptides, minerals, or other essential nutritional components for the survival of the bacterial composition. Prebiotics include, but are not limited to, amino acids, biotin, fructooligosaccharide, galactooligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic and carregenaan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber) and xylooligosaccharides.
As used herein, “predetermined ratios” refer to ran determined or selected in advance.
As used herein, “germinable bacterial spores” are spores capable of forming vegetative cells in response to a particular cue (e.g., an environmental condition or a small. molecule).
As used herein, “detectably present” refers to presence in an amount that can be detected using assays provided herein or otherwise known in the art that exist as of the filing date.
As used herein, “augmented” refers to an increase in amount and/or localization within to a point where it becomes detectably present.
As used herein, “fecal material” refers to a solid waste product of digested food and includes feces or bowel washes.
As used herein, the phrase “host cell response” refers to a response produced by a cell of a host organism.
As used herein, a “mammalian subject protein” refers to a protein produced by a mammalian subject and encoded by the mammalian subject genome. The term mammalian subject protein includes proteins that have been post-translationally processed and/or modified.
As used herein, the term “food-derived” refers to a protein or carbohydrate found in a consumed food.
As used herein, the term “biological material” refers to a material produced by a biological organism.
As used herein, the term “detection moiety” refers to an assay component that functions to detect an analyte.
As used herein, the term “incomplete network” refers to a partial network that lacks at least one of the entire set of components needed to carry out one or more network functions.
As used herein, the term “supplemental” refers to something that is additional and non-identical.
As used herein, a composition is “substantially free” of microbes when microbes are absent or undetectable as determined by the use of standard genomic and microbiological techniques. A composition is “substantially free” of a prebiotic or immunostimulatory carbohydrate when non-microbial carbohydrates are absent or undetectable as determined by the use of standard biochemical techniques (e.g., dye-based assays).
Microbial agents (individual or populations of microbes, microbial networks or parts of networks, or microbial metabolites) are considered to be “exogenous” to a subject (e.g., a human or non-human animal), a cell, tissue, organ or other environment of a human or non-human animal, if said subject, or said cell, tissue, organ or other environment of the subject, does not contain detectable levels of the microbial agent.
A microbial agent or population thereof is “heterologous” or “heterologously contained” on or in a host environment when, e.g., the microbial agent or population is administered or disposed on, or in the host, or host environment in a number, concentration, form or other modality that is not found in the host prior to administration of the microbial agent or population, or when the microbial agent or population contains an activity or structural component different from a host that does not naturally have the microbial agent within the target environment to which the microbe is administered or thereafter disposed.
As used herein, the term “antioxidant” is understood to include any one or more of various substances such as beta-carotene (a vitamin. A precursor), vitamin C, vitamin E, and selenium) that inhibit oxidation or reactions promoted by Reactive Oxygen Species (“ROS”) and other radical and non-radical species. Additionally, antioxidants are molecules capable of slowing or preventing the oxidation of other molecules. Non-limiting examples of antioxidants include astaxanthin, carotenoids, coenzyme Q10 (“CoQ10”), flavonoids, glutathione, Goji (wolfberry), hesperidin, lactowolfberry, lignan, lutein, lycopene, polyphenols, selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, or combinations thereof.
“Backbone network ecology” or simply “backbone network” or “backbone” are compositions of microbes that form a foundational composition that can be built upon or subtracted from to optimize a network ecology or functional network ecology to have specific biological characteristics or to comprise desired functional properties, respectively. Microbiome therapeutics can be comprised of these “backbone networks ecologies” in their entirety, or the “backbone networks” can be modified by the addition or subtraction of “R-groups” to give the network ecology desired characteristics and properties. “R-groups” can be defined in multiple terms including, but not limited to: individual OTUs, individual or multiple OTUs derived from a specific phylogenetic clade or a desired phenotype such as the ability to form spores, or functional bacterial compositions that comprise. “Backbone networks” can comprise a computationally derived network ecology in its entirety, or can comprise subsets of the computationally-derived network ecology that represent key nodes in the network that contribute to efficacy such as but not limited to a composition of Keystone OTUs. The number of organisms in a human gastrointestinal tract, is indicative of the functional redundancy of a healthy gut microbiome ecology (see, e.g., The Human Microbiome Consortia (2012). This redundancy makes it highly likely that non-obvious subsets of OTUs or functional pathways (i.e., “backbone networks”) are critical to maintaining states of health and/or catalyzing a shift from a dysbiotic state to one of health. One way of exploiting this redundancy is through the substitution of OTUs that share a given clade (see below) or by adding members of a clade not found in the backbone network.
“Bacterial composition” refers to a consortium of microbes comprising two or more OTUs. Backbone network ecologies, functional network ecologies, network classes, and core ecologies are all types of bacterial compositions. As used herein, bacterial composition includes a therapeutic microbial composition, a prophylactic microbial composition, a spore population, a purified spore population, or an ethanol treated spore population.
“Bacterial translocation” refers to the passage of one or more bacteria across the epithelial layer of any organ of a human or non-human animal.
“Network ecology” refers to a consortium of clades or OTUs that co-occur in some number of subjects. As used herein, a “network” is defined mathematically by a graph delineating how specific nodes (i.e., clades or OTUs) and edges (connections between specific clades or OTUs) relate to one another to define the structural ecology of a consortium of clades or OTUs. Any given network ecology will possess inherent phylogenetic diversity and functional properties.
A network ecology can also be defined in terms of its functional capabilities where for example the nodes would be comprised of elements such as, but not limited to, enzymes, clusters of orthologous groups (COGS; http.//www.ncbi.nlm.nih.gov books/NBK21090/), or KEGG Orthology Pathways (www.genome.jp/kegg/); these networks are referred to as a “functional network ecology”. Functional network ecologies can be reduced to practice by defining the group of OTUs that together comprise the functions defined by the functional network ecology.
The terms “network class” “core network” and “network class ecology” refer to a group of network ecologies that in general are computationally determined to comprise ecologies with similar phylogenetic and/or functional characteristics. A network class therefore contains important biological features, defined either phylogenetically or functionally, of a group (i.e., a cluster) of related network ecologies. One representation of a core network ecology is a designed consortium of microbes, typically non-pathogenic bacteria, that represents core features of a set of phylogenetically or functionally related network ecologies seen in many different subjects. In many occurrences, a core network, while designed as described herein, exists as a network ecology observed in one or more subjects. Core network ecologies are useful for reversing or reducing a dysbiosis in subjects where the underlying, related network ecology has been disrupted.
“Bacterial translocation” refers to the passage of one or more bacteria across the epithelial layer of any organ of a human or non-human animal.
“Clade” refers to the OTUs or members of a phylogenetic tree that are downstream of a statistically valid node in a phylogenetic tree. The clade comprises a set of terminal leaves in the phylogenetic tree (i.e., tips of the tree) that are a distinct monophyletic evolutionary unit and that share some extent of sequence similarity. Clades are hierarchical, in one embodiment, the node in a phylogenetic tree that is selected to define a clade is dependent on the level of resolution suitable for the underlying data used to compute the tree topology.
The “colonization” of a host organism includes the non-transitory residence of a bacterium or other microscopic organism. As used herein, “reducing colonization” of a host subject's gastrointestinal tract or vagina (or any other microbiota niche) by a pathogenic or non-pathogenic bacterium includes a reduction in the residence time of the bacterium in the gastrointestinal tract or vagina, as well as a reduction in the number o concentration) of the bacterium in the gastrointestinal tract or vagina, or adhered to the luminal surface of the gastrointestinal tract. The reduction in colonization can be permanent or occur during a transient period of time. Measuring reductions of adherent pathogens can be demonstrated directly, e.g., by determining pathogenic burden in a biopsy sample, or reductions may be measured indirectly, e.g., by measuring the pathogenic burden in the stool of a mammalian host.
A “combination” of two or more bacteria includes the physical co-existence of the two bacteria, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the two bacteria.
“Cytotoxic” activity of bacterium includes the ability of a bacterium kill a cell (e.g., a host cell or a bacterial cell). A “cytostatic” activity of a bacterium includes the ability to inhibit (e.g., partially or fully) the growth, metabolism, and/or proliferation of a cell (e.g., a bacterial cell or a host cell).
“Dysbiosis” refers to a state of the microbiota or microbiome of the gut or other body area, including mucosal or skin surfaces (or any other microbiota niche) in which the normal diversity and/or function of the ecological network is disrupted. Any disruption from the preferred (e.g., ideal.) state of the microbiota can be considered a dysbiosis, even if such dysbiosis does not result in a detectable decrease in health. This state of dysbiosis may be unhealthy (e.g., result in a diseased state), it may be unhealthy under only certain conditions, or it may prevent a subject from becoming healthier. Dysbiosis may be due to a decrease in diversity of the microbiota population composition, the overgrowth of one or more population of pathogens (e.g., a population of pathogenic bacteria) or pathobionts, the presence of and/or overgrowth of symbiotic organisms able to cause disease only when certain genetic and/or environmental conditions are present in a patient, or a shift to an ecological. network that no longer provides a beneficial function to the host and therefore no longer promotes health. A state of dysbiosis may lead to a disease or disorder (e.g. a gastrointestinal disease, disorder or condition), or the state of dysbiosis may lead to a disease or disorder (e.g., a gastrointestinal disease, disorder or condition) only under certain conditions, or the state of dysbiosis may prevent a subject from responding to treatment or recovering from a disease or disorder (e.g., a gastrointestinal disease, disorder or condition).
The term “distal” generally is used in relation to the gastrointestinal tract, specifically the intestinal lumen, of a human or other mammal. Thus, a “distal dysbiosis” includes a dysbiosis outside of the lumen of the gastrointestinal tract, and a “distal microbiota” includes a microbiota outside of the lumen of the gastrointestinal tract. In specified instances, the term “distal” may be used in relation to the site of administration, engraftment, or colonization of a composition, e.g., a probiotic composition, of the invention. For example, if a probiotic composition is administered vaginally, a “distal” effect of the composition would occur outside the vagina.
“Gastrointestinal dysbiosis” refers to a state of the microbiota or microbiome of the gut in which the normal diversity and/or function of the ecological network or niche is disrupted. The term “gut” as used herein is meant to refer to the entire gastrointestinal or digestive tract (also referred to as the alimentary canal) and it refers to the system of organs within multi-cellular animals which takes in food, digests it to extract energy and nutrients, and expels the remaining waste. As used herein the term “gastrointestinal tract” refers to the entire digestive canal, from the oral cavity to the rectum. The term “gastrointestinal tract” includes, but is not limited to, mouth and proceeds to the esophagus, stomach, small intestine, large intestine, rectum and, finally, the anus.
“Germinant” is a material or composition, or a physical-chemical process, capable of inducing the germination of vegetative bacterial cells from dormant spores, or the proliferation of vegetative bacterial cells, either directly or indirectly in a host organism and/or in vitro.
“Inhibition” of a pathogen or non-pathogen encompasses the inhibition of any desired function or activity of the pathogen or non-pathogen by the probiotic, e.g., bacterial, compositions of the present invention. Demonstrations of inhibition, such as a decrease in the growth of a pathogenic bacterial cell population or a reduction in the level of colonization of a pathogenic bacterial species are provided herein and otherwise recognized by one of ordinary skill in the art. Inhibition of a pathogenic or non-pathogenic bacterial population's “growth” may include inhibiting an increase in the size of a pathogenic or non-pathogenic bacterial cell population and/or inhibiting the proliferation (or multiplication) of a pathogenic or non-pathogenic bacterial cell population. Inhibition of colonization of a pathogenic or non-pathogenic bacterial species may be demonstrated by measuring and comparing the amount or burden of the bacterial species before and after a treatment. An “inhibition” or the act of “inhibiting” includes the total cessation and partial reduction of one or more activities of a pathogen, such as growth, proliferation, colonization, and function. As used herein, inhibition includes cytostatic and/or cytotoxic activities. Inhibition of function includes, for example, the inhibition of expression of a pathogenic gene product (e.g., the genes encoding a toxin and/or toxin biosynthetic pathway, or the genes encoding a structure required for intracellular invasion (e.g., an invasive pilus)) induced by the bacterial composition.
“Isolated” encompasses a bacterium or other entity or substance (e.g., a bacterial population or a prebiotic) that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated bacteria includes, for example, those bacteria that are cultured, even if such cultures are not monocultures. Isolated bacteria may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated bacteria are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified” refer to a bacterium or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A bacterium or a bacterial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the bacterium or bacterial population, or by passage through culture. A purified bacterium or bacterial population may contain other materials up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90%, and still be considered “isolated.” In some embodiments, purified bacteria and bacterial populations are more than about 80%, about 85%, about 90%, about 91%, about 9?%, about 93%, about 94%, about 95%, about 96%, about 97;), about 98;), about 99%, or more than about 99% pure. In the instance of bacterial compositions provided herein, the one or more bacterial types present in the composition can be independently purified from one or more other bacteria produced and/or present in the material or environment containing the bacterial type. In some embodiments, bacterial compositions and the bacterial components thereof are purified from residual habitat products. In some embodiments, pharmaceutical compositions (e.g., bacterial compositions) contain a defined mixture of isolated bacteria. For example, in some embodiments, the pharmaceutical composition (e.g., probiotic composition) contains no more than 100 bacterial species. For example, in some embodiments, the pharmaceutical composition contains no more than 75 bacterial species. In other embodiments, the pharmaceutical composition contains no more than 50 bacterial species, e.g., no more than 40 bacterial species, no more than 30 bacterial species, no more than 25 bacterial species, no more than 20 bacterial species, no more than 15 bacterial species, no more than 10 bacterial species, etc. In other embodiments, the pharmaceutical composition contains no more than 10 bacterial species, e.g., 10 bacterial species, 9 bacterial species, 8 bacterial species, 7 bacterial species, 6 bacterial species, 5 bacterial species, 4 bacterial species, 3 bacterial species, 2 bacterial species, 1 bacterial species. In some embodiments, the pharmaceutical composition contains defined quantities of each bacterial species. In an exemplary embodiment, the pharmaceutical composition contains isolated bacterial populations that are not isolated from fecal matter.
“Keystone OTU” or “keystone function” refers to one or more OTUs or functional pathways (e.g., KEGG or COG pathways) that are common to many network ecologies or functional network ecologies and are members of networks that occur in many subjects (i.e.,′′are pervasive). Due to the ubiquitous nature of keystone OTUs and their associated functional pathways, they are central to the function of network ecologies in healthy subjects and are often missing, or at reduced levels, in subjects with disease. Keystone OTUs and their associated functions may exist in low, moderate, or high abundance in subjects. A “non-keystone OTU” or non-keystone function refers to an OTU or function that is observed in a network ecology or a functional network ecology, that is not observed in a keystone OTU or function.
“Microbiota” refers to the community of microorganisms that occur (sustainably or transiently) in and/or on a subject, (e.g, a mammal such as a human), including, but not limited to, eukaryotes (e.g., protozoa), archaea, bacteria, and viruses (including bacterial viruses, i.e., a phage).
“Microbiome” refers to the genetic content of the communities of microbes that live in and on the human body, both sustainably and transiently, including eukaryotes, archaea, bacteria, and viruses (including, e.g., bacterial viruses (i.e., phage)), wherein “genetic content” includes genomic DNA, RNA such as ribosomal RNA, the epigenome, plasmids, and all other types of genetic information.
“Microbial carriage” or simply “carriage” refers to the population of microbes inhabiting a niche within or on a subject (e.g., a human subject). Carriage is often defined in terms of relative abundance. For example, OTU1 comprises 60% of the total microbial carriage, meaning that OTU1 has a relative abundance of 60% compared to the other OTUs in the sample from which the measurement is made. Carriage is most often based on genomic sequencing data where the relative abundance or carriage of a single OTU or group of OTUs is defined by the number of sequencing reads that are assigned to that OTU/s relative to the total number of sequencing reads for the sample.
“Microbial augmentation” refers to the establishment or significant increase of a population of microbes that are (i) absent or undetectable (as determined by the use of standard genomic, biochemical and/or microbiological techniques) from the administered therapeutic microbial composition, (ii) absent, undetectable, or present at low frequencies in the host niche (as an example: gastrointestinal tract, skin, anterior-nares, or vagina) before the delivery of the microbial composition, and (iii) are found, i.e., detectable, after the administration of the microbial composition or significantly increase, for instance 2-fold, 5-fold, 1×102, 1×103, 1×104, 1×105, 1×106, 1×107, or greater than 1×108, in cases where they are present at low frequencies. The microbes that comprise an augmented ecology can be derived from exogenous sources, such as food and the environment, or grow out from micro-niches within the host where they reside at low frequency.
The administration of the therapeutic composition (e.g., pharmaceutical composition) can induce an environmental shift in the target niche that promotes favorable conditions for the growth of commensal microbes. In the absence of treatment with a therapeutic microbial composition (e.g., a pharmaceutical composition comprising a bacterial cell population), with or without one or more prebiotics, the host can be constantly exposed to these microbes; however, sustained growth and the positive health effects associated with the stable population of increased levels of the microbes comprising the augmented ecology are not observed.
“Microbial engraftment” or simply “engraftment” refers to the establishment of OTUs comprising a therapeutic microbial composition in a target niche. In one embodiment, the OTUs are absent in the treated host prior to treatment. The microbes that comprise the engrafted ecology are found in the therapeutic microbial composition and establish as constituents of the host microbial ecology upon treatment. Engrafted OTUs can establish for a transient period of time, or demonstrate long-term stability in the microbial ecology that populates the host post-treatment with a therapeutic microbial composition. The engrafted ecology can induce an environmental shift in the target niche that promotes favorable conditions for the growth of commensal microbes capable of catalyzing a shift from a dysbiotic ecology to one representative of a healthy state.
“Ecological niche” or simply “niche” refers to the ecological space that an organism or group of organisms (e.g., a bacterial population) occupies. Niche describes how an organism or population or organisms responds to the distribution of resources, physical parameters (e.g., host tissue space) and competitors (e.g., by growing when resources are abundant, and/or when predators, parasites and pathogens are scarce) and how it in turn alters those same factors (e.g., by limiting access to resources by other organisms, acting as a food source for predators and/or as a consumer of prey).
“Pathobionts” or “opportunistic pathogens” refers to symbiotic organisms able to cause disease only when certain genetic and/or environmental conditions are present in a subject.
“Phylogenetic tree” refers to a graphical representation of the evolutionary relationships of one genetic sequence to another that is generated using a defined set of phylogenetic reconstruction algorithms (e.g., parsimony, maximum likelihood, or Bayesian). Nodes in the tree represent distinct ancestral sequences and the confidence of any node is provided by a bootstrap or Bayesian posterior probability, which measures branch uncertainty.
As used herein, the term minerals is understood to include boron, calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, or combinations thereof.
To be free of “non-comestible products” means that a bacterial composition or other material provided herein does not have a substantial amount of a non-comestible product, e.g., a product or material that is inedible, harmful or otherwise undesired in a product suitable for administration, e.g., oral administration, to a human subject. Non-comestible products are often found in preparations of bacteria from the prior art.
“Operational taxonomic units,” “OTU” (or plural “OTUs”) refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species. In some embodiments the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. In 16S embodiments, OTUs that share ≧97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU (see, e.g., Claesson et al. (2010) NUCLEIC ACIDS RES. . 38: e200; Konstantinidis et al. (2006) PHILOS. TRANS. R. SOC. LOND. B. BIOL. SCI. 361: 1929-1940). In embodiments involving the complete genome, MLSTs, specific genes, or sets of genes OTUs that share ≧95% average nucleotide identity are considered the same OTU (see, e.g., Achtman and Wagner (2008) NAT. REV. MICROBIOL. 6: 431-440; Konstantinidis et al. (2006)). OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof. Such characterization employs, e.g., WGS data or a whole genome sequence.
The term “phylogenetic diversity” refers to the biodiversity present in a given network ecology, core network ecology or network class ecology based on the OTUs that comprise the network. Phylogenetic diversity is a relative term, meaning that a network ecology, core network or network class that is comparatively more phylogenetically diverse than another network contains a greater number of unique species, genera, and taxonomic families. Uniqueness of a species, genera, or taxonomic family is generally defined using a phylogenetic tree that represents the genetic diversity all species, genera, or taxonomic families relative to one another. In another embodiment phylogenetic diversity may be measured using the total branch length or average branch length of a phylogenetic tree.
Phylogenetic diversity may be optimized in a bacterial composition by including a wide range of biodiversity.
“Phylogenetic tree”, “rDNA”, “rRNA”, “16S-rDNA”, “16S-rRNA”, “16S”, “16S sequencing”, “16S-NGS”, “18S”, “18S-rRNA”, “18S-rDNA”, “18S sequencing”, and “18S-NGS” refer to the nucleic acids that encode for the RNA subunits of the ribosome, rDNA refers to the gene that encodes the rRNA that comprises the RNA subunits. There are two RNA subunits in the ribosome termed the small subunit (SSU) and large subunit (LSU); the RNA genetic sequences (rRNA) of these subunits are related to the gene that encodes them (rDNA) by the genetic code. rDNA genes and their complementary RNA sequences are widely used for determination of the evolutionary relationships amount organisms as they are variable, yet sufficiently conserved to allow cross organism molecular comparisons.
Typically 16S rDNA sequence (approximately 1542 nucleotides in length) of the 30S SSU is used for molecular-based taxonomic assignments of prokaryotes and the 18S rDNA sequence (approximately 1869 nucleotides in length) of 40S SSU is used for eukaryotes. The bacterial 16S rDNA is used in reconstructing the evolutionary relationships and sequence similarity of one bacterial isolate to another using phylogenetic approaches. 16S sequences are used for phylogenetic reconstruction as they are in general highly conserved, but contain specific hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most bacteria.
The “V1-V9 regions” of the 16S rRNA refers to the first through ninth hypervariable regions of the 16S rRNA gene that are used for genetic typing of bacterial samples. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively using numbering based on the E. coli system of nomenclature (see, e.g., Brosius et al. (1978) PROC. NAT'L. ACAD. SCI. USA 75(10): 4801-4805). In some embodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU. In one embodiment, the V1, V2, and V3 regions are used to characterize an OTU. In another embodiment, the V3, V4, and V5 regions are used to characterize an OTU. In another embodiment, the V4 region is used to characterize an OTU. A person of ordinary skill in the art can identify the specific hypervariable regions of a candidate 16S rRNA by comparing the candidate sequence in question to a reference sequence and identifying the hypervariable regions based on similarity to the reference hypervariable regions, or alternatively, one can employ Whole Genome Shotgun (WGS) sequence characterization of microbes or a microbial community.
“Residual habitat products” refers to material derived from the habitat for microbiota within or on a human or animal. For example, microbiota live in feces in the gastrointestinal tract, on the skin itself, in saliva, mucus of the respiratory tract, or secretions of the genitourinary tract (i.e., biological matter associated with the microbial community). Substantially free of residual habitat products means that the bacterial composition no longer contains the biological matter associated with the microbial environment on or in the human or animal subject and is 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, 90% free, 85% free, 80% free, 75% free, 70% free, 65% free, or 60% free of any contaminating biological matter associated with the microbial community. Residual habitat products can include abiotic materials (including undigested food) or it can include unwanted microorganisms. Substantially free of residual habitat products may also mean that the bacterial composition contains no detectable cells from a human or animal and that only microbial cells are detectable. In one embodiment, substantially free of residual habitat products may also mean that the bacterial composition contains no detectable viral (e.g., bacterial viruses (i.e., phage)), fungal, or mycoplasmal contaminants. In another embodiment, it means that fewer than 1×10−2%, 1×10−3%, 1×10−4%, 1×10−5%, 1×10−6%, 1×10−7%, 1×10−8% of the viable cells in the bacterial composition are human or animal, as compared to microbial cells. There are multiple ways to accomplish this degree of purity, none of which are limiting. For example, contamination may be reduced by isolating desired constituents through multiple steps of streaking to single colonies on solid media until replicate (such as, but not limited to, two) streaks from serial single colonies have shown only a single colony morphology. Alternatively, reduction of contamination can be accomplished by multiple rounds of serial dilutions to single desired cells (e.g., a dilution of 10−8 or 10−9), such as through multiple 10-fold serial dilutions. This can further be confirmed by showing that multiple isolated colonies have similar cell shapes and Gram staining behavior. Other methods for confirming adequate purity include genetic analysis (e.g., PCR and DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.
The term “subject” refers to any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, kangaroos, and transgenic non-human animals. The subject may be suffering from a dysbiosis, including, but not limited to, an infection due to a gastrointestinal pathogen or may be at risk of developing or transmitting to others an infection due to a gastrointestinal pathogen. Synonyms used herein include “patient” and “animal.” In some embodiments, the subject or host may be suffering from a dysbiosis, that contributes to or causes a condition classified as graft-versus-host disease, Crohn's disease, Celiac disease, inflammatory bowel disease, ulcerative colitis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, or type I diabetes. In some embodiments, the host may be suffering from metabolic endotoxemia, altered metabolism of primary bile acids, immune system activation, or an imbalance or reduced production of short chain fatty acids including, for example, butyrate, propionate, acetate, and a branched chain fatty acid.
The term “phenotype” refers to a set of observable characteristics of an individual entity. As example an individual subject may have a phenotype of “health” or “disease”. Phenotypes describe the state of an entity and all entities within a phenotype share the same set of characteristics that describe the phenotype. The phenotype of an individual results in part, or in whole, from the interaction of the entities genome and/or microbiome with the environment.
“Spore” or “endospore” refers to an entity, particularly a bacterial entity, which is in a dormant, non-vegetative and non-reproductive stage. Spores are generally resistant to environmental stress such as radiation, desiccation, enzymatic treatment, temperature variation, nutrient deprivation, and chemical disinfectants.
A “spore population” refers to a plurality of spores present in a composition. Synonymous terms used herein include spore composition, spore preparation, ethanol-treated spore fraction and spore ecology. A spore population may be purified from a fecal donation, e.g., via ethanol or heat treatment, or a density gradient separation, or any combination of methods described herein to increase the purity, potency and/or concentration of spores in a sample. Alternatively, a spore population may be derived through culture methods starting from isolated spore former species or spore former OTUs or from a mixture of such species, either in vegetative or spore form.
A “sporulation induction agent” is a material or physical-chemical process that is capable of inducing sporulation in a bacterium, either directly or indirectly, in a host organism and/or in vitro.
To “increase production of bacterial entities” includes an activity or a sporulation induction agent. “Production” includes conversion of vegetative bacterial cells into spores and augmentation of the rate of such conversion, as well as decreasing the germination of bacteria in spore form, decreasing the rate of spore decay in vivo, or ex vivo, or to increasing the total output of spores (e.g., via an increase in volumetric output of fecal material).
“Synergy” or “synergistic interactions” refers to the interaction or cooperation of two or more microbes to produce a combined effect greater than the sum of their separate effects. In one embodiment, “synergy” between two or more microbes can result in the inhibition of a pathogens ability to grow.
As used herein the term “vitamin” is understood to include any of various fat-soluble or water-soluble organic substances (non-limiting examples include vitamin A, Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin or niacinamide), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), Vitamin B7 (biotin), Vitamin B9 (folic acid), and Vitamin B12 (various cobalamins; commonly cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, K1 and K2 (i.e., MK-4, MK-7), folic acid and biotin) essential in minute amounts for normal growth and activity of the body and obtained naturally from plant and animal foods, or synthetically made, pro-vitamins, derivatives, and/or analogs.
As used herein, the term “antioxidant” is understood to include any one or more of various substances such as beta-carotene (a vitamin A precursor), vitamin C, vitamin E, and selenium) that inhibit oxidation or reactions promoted by reactive oxygen species (“ROS”) and other radical and non-radical species. Additionally, antioxidants are molecules capable of slowing or preventing the oxidation of other molecules. Non-limiting examples of antioxidants include astaxanthin, carotenoids, coenzyme Q10 (“CoQ10”), flavonoids, glutathione, Goji (wolfberry), hesperidin, lactowolfberry, lignan, lutein, lycopene, polyphenols, selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, or combinations thereof.
“Graft versus host disease” as used herein is an immunological disorder in which the immune cells of a transplant attack the tissues of a transplant recipient and may lead to organ dysfunction.
“Acute GVHD” as used herein is GVHD that prevents within the first 100 days of transplant.
“Chronic GVHD” as used herein is GVHD that prevents after the first 100 days of transplant.
“Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
“Treatment”, “treat”, or “treating”, mean a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from native levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. Therefore, in the disclosed methods, treatment′′ can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the disease progression. For example, a disclosed method for reducing the effects of GVHD is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject with GVHD when compared to native levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between, as compared to native or control levels. It is understood and herein contemplated that “treatment” does not necessarily refer to a cure of the disease or condition, but an improvement in the outlook of a disease or condition (e.g., GVHD).
As used herein “preventing” or “prevention” refers to any methodology where the disease state does not occur due to the actions of the methodology (such as, for example, administration of a pharmaceutical composition as described herein). In one aspect, it is understood that prevention can also mean that the disease is not established to the extent that occurs in untreated controls. For example, there can be a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the establishment of disease.
As used herein, the term “recipient” refers to the subject that receives a bone marrow or a solid organ transplantation.
Pharmaceutical Compositions of the Invention
Disclosed herein are pharmaceutical compositions, e.g., probiotic compositions, comprising a population of bacterial cells, e.g., an immunomodulatory bacterial cell population, such as an anti-inflammatory bacterial population, with or without one or more prebiotics, for the prevention, control, and treatment of inflammation, autoimmune and inflammatory disorders, dysbiosis, e.g., gastrointestinal or distal dysbiosis, disorders associated with dysbiosis, and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious for the treatment, prevention, reduction of onset and amelioration of inflammation, autoimmune and inflammatory disorders, dysbiosis, e.g., gastrointestinal or distal dysbiosis, disorders associated with dysbiosis, and for general nutritional health. These pharmaceutical compositions are formulated as provided herein, and administered to mammalian subjects using the methods as provided herein. In some embodiments, the compositions described herein are formulated for oral administration. In other embodiments, the compositions described herein are formulated for rectal administration.
In one embodiment, therapeutic compositions (e.g., pharmaceutical compositions) are provided for the treatment, prevention, reduction of onset, and amelioration of, inflammation or one or more symptom of an autoimmune or inflammatory disorder, dysbiosis, e.g., gastrointestinal or distal dysbiosis, or a disorder associated with dysbiosis. As used herein, “therapeutic” compositions include compositions that function in a prophylactic (e.g., preventative) manner. Generally, the population is provided in an amount effective to treat (including to prevent) a disease, disorder or condition associated with or characterized by inflammation, dysbiosis, e.g., gastrointestinal or distal dysbiosis, inflammation, or an autoimmune or inflammatory disorder. Such treatment may be effective to reduce the severity of at least one symptom of the dysbiosis, e.g., gastrointestinal or distal dysbiosis, or an autoimmune or inflammatory disorder. Such treatment may be effective to modulate the microbiota diversity present in the mammalian recipient.
In some embodiments, the population of anti-inflammatory bacterial cells is a purified population of bacterial cells. In some embodiments, said purified population of bacterial cells is isolated from a mammalian source. In some embodiments, said purified population of bacterial cells is isolated from a human source. In some embodiments, said purified population of bacterial cells is isolated from the skin of a human source. In some embodiments, said purified population of bacterial cells is isolated from the gastrointestinal tract of a human source. In some embodiments, said purified population of bacterial cells is isolated from the fecal matter of a subject. In some embodiments, said purified population of bacterial cells is isolated from human fecal matter. In other embodiments, said purified population of bacterial cells is not isolated from human fecal matter. In some embodiments, said purified population of bacterial cells is not derived from human fecal matter.
In embodiments, the pharmaceutical compositions (e.g., probiotic compositions) contain immunomodulatory bacterial cells (e.g., anti-inflammatory bacterial cells), which are capable of altering the immune activity of a mammalian subject. In exemplary embodiments, the immunomodulatory bacterial cells are capable of reducing inflammation in a mammalian subject. Such immunomodulatory bacterial cells are referred to herein as “anti-inflammatory bacteria” or “anti-inflammatory bacterial cells”. Immunomodulatory bacterial cells can act to alter the immune activity of a subject directly or indirectly. For example, immunomodulatory bacteria can act directly on immune cells through receptors for bacterial components (e.g. Toll-like receptors) or by producing metabolites such as immunomodulatory short chain fatty acids (SCFAs). Such SCFAs can have many positive impacts on the health of the subject, by, for example, reducing inflammation, or improving intestinal barrier integrity. Immunomodulatory bacterial cells can also impact the immune activity of a subject by producing glutathione or gamma-glutamylcysteine.
Pharmaceutical compositions (e.g., probiotic compositions) containing immunomodulatory bacteria (i.e., bacterial cells) can additionally or alternatively impact the immune activity of a subject indirectly by modulating the activity of immune cells in the subject. For example, immunomodulatory bacteria may alter cytokine expression by host immune cells (e.g., macrophages, B lymphocytes, T lymphocytes, mast cells, peripheral blood mononuclear cells (PBMCs), etc.) or other types of host cells capable of cytokine secretion (e.g., endothelial cells, fibroblasts, stromal cells, etc.). In an exemplary embodiment, pharmaceutical compositions (e.g., probiotic compositions) contain a population of anti-inflammatory bacterial cells that are capable of inducing secretion of a anti-inflammatory cytokine by host cells (e.g., host immune cells). For example, anti-inflammatory bacterial cells can induce secretion of one or more anti-inflammatory cytokines such as, but not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGFβ, and combinations thereof, by host cells (e.g., host immune cells). In another exemplary embodiment, pharmaceutical compositions (e.g., probiotic compositions) contain anti-inflammatory bacterial cells that are capable of reducing secretion of one or more pro-inflammatory cytokines by a host cell (e.g., by a host immune cell). For example, anti-inflammatory bacterial cells can reduce secretion of one or more pro-inflammatory cytokines, such as, but not limited to, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof, by host cells (e.g., host immune cells). In some embodiments, the induction and/or secretion of said pro-inflammatory cytokines may be induced by (e.g., in response to, either directly or indirectly) a bacteria (e.g., Enterococcus faecalis). Other cytokines that may be modulated by immunomodulatory bacterial cells include, for example, IL-17A, IL-2, and IL-9.
In some embodiments, immunomodulatory bacteria (i.e., immunomodulatory bacterial cells) are selected for inclusion in a pharmaceutical composition (e.g., probiotic composition) of the invention based on the desired effect of the immunomodulatory bacteria on cytokine secretion by a host cell or a population of host cells (e.g., a host immune cell (e.g., a PBMC)). In some embodiments, said effect of the immunomodulatory bacteria is assessed in vitro using a population of host cells (e.g., a population of isolated host immune cells). For example, in one embodiment, a probiotic composition contains anti-inflammatory bacteria that increase secretion of a anti-inflammatory cytokine, for example, IL-10, IL-13, IL-9, IL-4, IL-5, TGFβ, and combinations thereof, by a host cell (e.g., a host immune cell (e.g., PBMCs, macrophages, B lymphocytes, T lymphocytes, mast cells). In some embodiments, the anti-inflammatory bacteria increase secretion of two or more anti-inflammatory cytokines. In some embodiments, the anti-inflammatory bacteria increase secretion of three or more anti-inflammatory cytokines. In some embodiments, the anti-inflammatory bacteria increase secretion of four or more anti-inflammatory cytokines. In some embodiments, the anti-inflammatory bacteria increase secretion of five or more anti-inflammatory cytokines. In exemplary embodiments, the increase is an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 80%, 100%, 200%, 300%, 500% or more. In other embodiments, a pharmaceutical composition contains anti-inflammatory bacteria that decrease secretion of a pro-inflammatory cytokine, for example, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof, by a host cell. In some embodiments, the anti-inflammatory bacteria decrease secretion of five or more pro-inflammatory cytokines. In exemplary embodiments, the decrease is a decrease of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 80%, 100%, 200%, 300%, 500% or more. In another embodiment, the pharmaceutical composition contains anti-inflammatory bacterial cells that increase secretion of one or more anti-inflammatory cytokines and reduce secretion of one or more pro-inflammatory cytokines by a host cell (e.g., a host immune cell). Alterations in cytokine expression may occur locally, e.g., in the gastrointestinal tract of a subject, or at a site distal to a microbial niche, e.g., distal to the gastrointestinal tract. In some embodiments, the induction and/or secretion of said pro-inflammatory cytokines may be induced by (e.g., in response to, either directly or indirectly) a bacteria (e.g., Enterococcus faecalis).
In some aspects, the pharmaceutical compositions described herein and/or a prebiotic (e.g., a carbohydrate) modulate the release of immune stimulatory cytokines by host cells (e.g., host immune cells). In preferred embodiments, the administered immunomodulatory bacterial cells (e.g., anti-inflammatory bacterial cells) and/or a prebiotic (e.g., a carbohydrate) inhibit or reduce the release of immune stimulatory cytokines. Non-limiting examples of immune modulating cytokines and ligands include B lymphocyte chemoattractant (“BLC”), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotactic protein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor (“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”), 1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon gamma (“IFN-γ”), Interlukin-1 alpha (“IL-1α”), Interlukin-1β (“IL-1β”), Interleukin 1 receptor antagonist (“IL-1 ra”), Interleukin-2 (“IL-2”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6 sR”), Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10 (“IL-10”), Interleukin-11 (“IL-11”), Subunit β of Interleukin-12 (“IL-12 p40” or “IL-12 p70”), Interleukin-13 (“IL-13”), Interleukin-15 (“IL-15”), Interleukin-16 (“IL-16”), Interleukin-17 (“IL-17”), Chemokine (C-C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor (“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-C motif) ligand 2 (“MIP-1 alpha”), Chemokine (C-C motif) ligand 4 (“MIP-1β”), Macrophage inflammatory protein-1-δ (“MIP-1δ”), Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-C motif) ligand 5, Regulated on Activation, Normal T cell Expressed and Secreted (“RANTES”), TIMP metallopeptidase inhibitor 1 (“TIMP-1”), TIMP metallopeptidase inhibitor 2 (“TIMP-2”), Tumor necrosis factor, lymphotoxin-a (“TNF-α”), Tumor necrosis factor, lymphotoxin-β (“TNF (3”), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR, Brain-derived neurotrophic factor (“BDNF”), Basic fibroblast growth factor (“bFGF”), Bone morphogenetic protein 4 (“BMP-4”), Bone morphogenetic protein 5 (“BMP-5”), Bone morphogenetic protein 7 (“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growth factor (“EGF”), Epidermal growth factor receptor (“EGFR”), Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”), Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor (“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glial cell-derived neurotrophic factor (“GDNF”), Growth Hormone, Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growth factor (“HGF”), Insulin-like growth factor binding protein 1 (“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”), Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-like growth factor binding protein 4 (“IGFBP-4”), Insulin-like growth factor binding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”), Insulin, Macrophage colony-stimulating factor (“M-CSF R”), Nerve growth factor receptor (“NGF R”), Neurotrophin-3 (“NT-3”), Neurotrophin-4 (“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”), Platelet-derived growth factor receptors (“PDGF-AA”), Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-box containing comples (“SCF”), Stem cell factor receptor (“SCF R”), Transforming growth factor α (“TGF-α”), Transforming growth factor β-1 (“TGF β1”), Transforming growth factor β-3 (“TGF (β3”), Vascular endothelial growth factor (“VEGF”), Vascular endothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelial growth factor receptor 3 (“VEGFR3”), VEGF-D6Ckine, Tyrosine-protein kinase receptor UFO (“Axl”), Betacellulin (“BTC”), Mucosae-associated epithelial chemokine (“CCL28”), Chemokine (C-C motif) ligand 27 (“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C-X-C motif chemokine 5 (“ENA-78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”), Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C-C motif) ligand 14 (“HCC-1”), Chemokine (C-C motif) ligand 16 (“HCC-4”), Interleukin-9 (“IL-9”), Interleukin-17 F (“IL-17F”), Interleukin-18-binding protein (“IL-18 BPa”), Interleukin-28 A (“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C-X-C motif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”), Leukemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand (“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocyte chemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4 (“MCP-4”), Macrophage-derived chemokine (“MDC”), Macrophage migration inhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3α”), C-C motif chemokine 19 (“MIP-3 (3”), Chemokine (C-C motif) ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain (“MSP-α”), Nucleosome assembly protein 1-like 4 (“NAP-2”), Secreted phosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulated cytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derived factor-1 a (“SDF-1α”), Chemokine (C-C motif) ligand 17 (“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromal lymphopoietin (“TSLP 4-IBB”), CD 166 antigen (“ALCAM”), Cluster of Differentiation 80 (“B7-1”), Tumor necrosis factor receptor superfamily member 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster of Differentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40 Ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (“CEACAM-1”), Death Receptor 6 (“DR6”), Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein (“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”), Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosis antigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosis factor receptor superfamily member 1 (“GITR”), Tumor necrosis factor receptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule 3 (“ICAM-3”), IL-1 R4, IL-1 RI, IL-10 Rβ, IL-17R, IL-2Rγ, IL-21R, Lysosome membrane protein 2 (“LIMPII”), Neutrophil gelatinase-associated lipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium (“LYVE-1”), MHC class I polypeptide-related sequence A (“MICA”), MHC class I polypeptide-related sequence B (“MICB”), NRG1-β1, Beta-type platelet-derived growth factor receptor (“PDGF Rβ”), Platelet endothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A virus cellular receptor 1 (“TIM-1”), Tumor necrosis factor receptor superfamily member IOC (“TRAIL R3”), Trappin protein transglutaminase binding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular cell adhesion protein 1 (“VCAM-1”), XEDARActivin A, Agouti-related protein (“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin, Catheprin S, CD40, Cryptic family protein IB (“Cripto-1”), DAN, Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial cell adhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB/C, FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”), IL-13 R1, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal cell adhesion molecule (“NrCAM”), Plasminogen activator inhibitor-1 (“PAI-1”), Platelet derived growth factor receptors (“PDGF-AB”), Resistin, stromal cell-derived factor 1 (“SDF-1β”), sgp130, Secreted frizzled-related protein 2 (“ShhN”), Sialic acid-binding immunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growth factor-β2 (“TGF β2”), Tie-2, Thrombopoietin (“TPO”), Tumor necrosis factor receptor superfamily member 10D (“TRAIL R4”), Triggering receptor expressed on myeloid cells 1 (“TREM-1”), Vascular endothelial growth factor C (“VEGF-C”), VEGFR1Adiponectin, Adipsin (“AND”), α-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”), β-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”), Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”), Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), Human Epidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin, Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1 (“GRO α”), human chorionic gonadotropin (“β HCG”), Insulin-like growth factor 1 receptor (“IGF-1 sR”), IL-1 sRII, IL-3, IL-18 Rb, IL-21, Leptin, Matrix metalloproteinase-1 (“MMP-1”), Matrix metalloproteinase-2 (“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrix metalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”), Matrix metalloproteinase-10 (“MMP-10”), Matrix metalloproteinase-13 (“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-1”), Entactin (“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”), Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialic acid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase (“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”), TSH2B4, Disintegrin and metalloproteinase domain-containing protein 9 (“ADAM-9”), Angiopoietin 2, Tumor necrosis factor ligand superfamily member 13/Acidic leucine-rich nuclear phosphoprotein 32 family member B (“APRIL”), Bone morphogenetic protein 2 (“BMP-2”), Bone morphogenetic protein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L, CD200, CD97, Chemerin, Tumor necrosis factor receptor superfamily member 6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblast activation protein, alpha (“FAP”), Fibroblast growth factor 19 (“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGF R”), IFN-γα/β R2, Insulin-like growth factor 2 (“IGF-2”), Insulin-like growth factor 2 receptor (“IGF-2 R”), Interleukin-1 receptor 6 (“IL-1R6”), Interleukin 24 (“IL-24”), Interleukin 33 (“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”), Oxidized low-density lipoprotein receptor 1 (“LOX-1”), Mannose-binding lectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1, translocation-associated (Drosophila) (“Notch-1”), Nephroblastoma overexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1 (“PD-1”), N-acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4, Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Toll-like receptor 2 (“TLR2”), Tumor necrosis factor receptor superfamily member 10A (“TRAIL R1”), Transferrin (“TRF”), WIF-lACE-2, Albumin, AMICA, Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen 19-9 (“CA19-9”), CD 163, Clusterin, CRT AM, Chemokine (C-X-C motif) ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-related protein 3 (“Dkk-3”), Delta-like protein 1 (“DLL1”), Fetuin A, Heparin-binding growth factor 1 (“aFGF”), Folate receptor a (“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-1”), GPCR-associated sorting protein 2 (“GASP-2”), Granulocyte colony-stimulating factor receptor (“GCSF R”), Serine protease hepsin (“HAI-2”), Interleukin-17B Receptor (“IL-17B R”), Interleukin 27 (“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V (“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST, Heparan sulfate proteoglycan (“Syndecan-1”), Tumor necrosis factor receptor superfamily member 13B (“TACI”), Tissue factor pathway inhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily, member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase Plasminogen Activator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascular endothelial) also known as CD144 (“VE-Cadherin”), WNT1-inducible-signaling pathway protein 1 (“WISP-1”), and Receptor Activator of Nuclear Factor κ B (“RANK”).
In other embodiments, pharmaceutical compositions (e.g., probiotic compositions) containing immunomodulatory bacteria that impact the immune activity of a subject by promoting the differentiation and/or expansion of particular subpopulations of immune cells. For example, immunomodulatory bacteria can increase or decrease the proportion of Treg cells, Th17 cells, Th1 cells, or Th2 cells in a subject. The increase or decrease in the proportion of immune cell subpopulations may be systemic, or it may be localized to a site of action of the probiotic, e.g., in the gastrointestinal tract or at a site of distal dysbiosis. In some embodiments, immunomodulatory bacteria (i.e., anti-inflammatory bacterial cells) are selected for inclusion in a pharmaceutical composition (e.g., a probiotic composition) of the invention based on the desired effect of the pharmaceutical composition on the differentiation and/or expansion of subpopulations of immune cells in the subject.
In one embodiment, a pharmaceutical composition (e.g., a probiotic composition) contains immunomodulatory bacteria (i.e., immunomodulatory bacterial cells) that increase the proportion of Treg cells in a subject (e.g., by inducing expansion of Treg cells in the subject). In another embodiment, a pharmaceutical composition contains immunomodulatory bacteria that decrease the proportion of Treg cells in a subject. In one embodiment, a pharmaceutical composition contains immunomodulatory bacteria that increase the proportion of Th17 cells in a subject (e.g., by inducing expansion of Th17 cells in the subject). In another embodiment, a pharmaceutical composition contains immunomodulatory bacteria that decrease the proportion of Th17 cells in a subject. In one embodiment, a pharmaceutical composition contains immunomodulatory bacteria that increase the proportion of Th1 cells in a subject (e.g., by inducing expansion of Th1 cells in the subject). In another embodiment, a pharmaceutical composition contains immunomodulatory bacteria that decrease the proportion of Th1 cells in a subject. In one embodiment, a pharmaceutical composition contains immunomodulatory bacteria that increase the proportion of Th2 cells in a subject (e.g., by inducing expansion of Th2 cells in the subject). In another embodiment, a pharmaceutical composition contains immunomodulatory bacteria that decrease the proportion of Th2 cells in a subject. The increase or decrease in the proportion of immune cell subpopulations may be systemic, or it may be localized to a site of action of the probiotic, e.g., in the gastrointestinal tract or at a site of distal dysbiosis.
In one embodiment, a pharmaceutical composition (e.g., a probiotic composition) contains immunomodulatory bacteria capable of modulating the proportion of one or more populations of Treg cells, Th17 cells, Th1 cells, Th2 cells, and combinations thereof, in a subject. Certain immune cell profiles may be particularly desirable to treat or prevent particular disorders associated with a dysbiosis. For example, treatment or prevention of an autoimmune or inflammatory disorder (e.g., GVHD) can be promoted by increasing the quantity of Treg cells and Th2 cells, and decreasing the quantity of Th17 cells and Th1 cells. Accordingly, pharmaceutical compositions (e.g., probiotic compositions) for the treatment or prevention of an autoimmune or inflammatory disorder (e.g., GVHD) contain immunomodulatory bacteria capable of promoting the differentiation and/or expansion of Treg cells and Th2 cells, and reducing Th17 and Th1 cells in the subject.
In one embodiment, pharmaceutical compositions (e.g., a therapeutic probiotic compositions) containing a purified population of immunomodulatory microbes, e.g., immunomodulatory bacterial cells, are provided, with or without one or more prebiotics, in an amount effective to: i) treat or prevent dysbiosis, e.g., gastrointestinal or distal dysbiosis, inflammation, or an autoimmune or inflammatory disorder; and/or ii) augment at least one type of microbe, e.g., a bacterium, not present in the therapeutic composition in a mammalian recipient subject to whom the pharmaceutical composition is administered; and/or iii) engraft at least one type of microbe, e.g., an anti-inflammatory bacterial cell, present in the therapeutic composition but not present in a mammalian subject prior to treatment.
In another embodiment, pharmaceutical compositions containing a purified population of immunomodulatory bacteria (e.g., anti-inflammatory bacterial cells) are provided, in an amount effective to i) augment the microbiota diversity present in the mammalian recipient and/or ii) treat or prevent dysbiosis, e.g., gastrointestinal or distal dysbiosis, inflammation, or an autoimmune or inflammatory disorder in a mammalian recipient subject to whom the therapeutic composition is administered, wherein the purified population of immunomodulatory bacteria is obtained by separating the population from at least one residual habitat product in a fecal material obtained from one or a plurality of mammalian donor subjects. In some embodiments, individual bacterial strains can be cultured from fecal material. These strains can then be purified or otherwise isolated and used singly or in combination. In one embodiment, the probiotic composition does not contain a fecal extract.
In one embodiment, the pharmaceutical compositions described herein may be used to treat or correct a dysbiosis in a subject. The dysbiosis may be, for example, a local dysbiosis, or a distal dysbiosis. In another embodiment, the probiotic compositions described herein may be used to prevent a dysbiosis in a subject at risk for developing a dysbiosis.
In another embodiment, pharmaceutical compositions containing a purified population of immunomodulatory bacteria (e.g., anti-inflammatory bacterial cells) are provided, in an amount effective to i) augment the microbiota diversity present in the mammalian recipient and/or ii) treat or prevent dysbiosis, e.g., gastrointestinal or distal dysbiosis, inflammation, or an autoimmune or inflammatory disorder in a mammalian recipient subject to whom the therapeutic composition is administered, wherein the purified population of immunomodulatory bacteria is obtained by separating the population from a non-fecal material source.
In some embodiments, a pharmaceutical composition containing a purified population of immunomodulatory bacterial cells (e.g., anti-inflammatory bacterial cells) described above is co-administered or co-formulated with one or more prebiotics, e.g., carbohydrates. In some embodiments, a pharmaceutical composition is administered before one or more prebiotics is administered to a subject. In some embodiments, the pharmaceutical composition is administered after one or more prebiotics is administered to a subject. In some embodiments, a pharmaceutical composition containing a purified population of immunomodulatory bacterial cells is administered concurrently with one or more prebiotics. In other embodiments, a pharmaceutical composition containing a purified population of immunomodulatory bacterial cells is administered sequentially with one or more prebiotics. In some embodiments, a purified population of immunomodulatory bacterial cells is administered in a pharmaceutical composition formulated to contain one or more pharmaceutical excipients, and optionally one or more prebiotics.
Immunomodulatory bacterial cells (e.g., anti-inflammatory bacterial cells) involved in modulation of the host immune system i) may be human commensals; ii) may be part of an organ's healthy-state microbiome; ii) may be part of a distal organ's healthy-state microbiome; iv) may be exogenous microbes; v) may be innocuous; vi) may be pathobionts; vii) may be pathogens; viii) may be opportunistic pathogens; or ix) any combination thereof. In some aspects, microbes are not required to be actively proliferating (e.g., spores, dormant cells, cells with reduced metabolic rate, or heat-killed cells) to have an immunomodulatory effect. In certain aspects, microbial cell components, rather than whole microbial cells, may have immunomodulatory effects. Non-limiting examples of microbial components are lipids, carbohydrates, proteins, nucleic acids, and small molecules.
The pharmaceutical compositions provided herein, may optionally further comprise a prebiotic, a non-microbial immunomodulatory carbohydrates, or a microbial immunomodulatory cell component, that are effective for the prevention or treatment of an autoimmune or inflammatory disorder such as graft-versus-host disease (GVHD), an inflammatory bowel disease (IBD) including, but not limited to, ulterative colitis and Crohn's disease, multiple sclerosis (MS), systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, Sjögren's syndrome, and Celiac disease, or dysbiosis.
In certain embodiments, the pharmaceutical compositions comprise at least one type of immunomodulatory bacterial cells (e.g., at least one type of anti-inflammatory bacterial cell) and, optionally, at least one prebiotic (e.g., a carbohydrate), and optionally further comprise a microbial immunomodulatory cell component or substrate for the production of immunomodulatory metabolites, that are effective for the prevention or treatment of an autoimmune or inflammatory disorder. Methods for the prevention and/or treatment of autoimmune and inflammatory diseases in human subjects are also disclosed herein.
In some embodiments, the pharmaceutical compositions, e.g., probiotic compositions, of the invention comprise purified spore populations of anti-inflammatory bacterial cells. In one embodiment, the purified spore populations can engraft in the host and remain present for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 25 days, 30 days, 60 days, 90 days, or longer than 90 days. Additionally, the purified spore populations can induce other healthy commensal bacteria found in a healthy gut to engraft in the host that are not present in the purified spore populations or present at lesser levels. Therefore, these species are considered to “augment” the delivered spore populations. In this manner, commensal species augmentation of the purified spore population in the recipient's gut leads to a more diverse population of gut microbiota than present initially.
Preferred bacterial cells for use in the present invention include bacterial cells of the genera Acetanaerobacterium, Acetivibrio, Alicyclobacillus, Alkaliphilus, Anaerofustis, Anaerosporobacter, Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Bacteroides, Blautia, Brachyspira, Brevibacillus, Bryantella, Bulleidia, Butyricicoccus, Butyrivibrio, Catenibacterium, Chlamydiales, Clostridiaceae, Clostridiales, Clostridium, Collinsella, Coprobacillus, Coprococcus, Coxiella, Deferribacteres, Desulfitobacterium, Desulfotomaculum, Dorea, Eggerthella, Erysipelothrix, Erysipelotrichaceae, Ethanoligenens, Eubacterium, Faecalibacterium, Filifactor, Flavonifractor, Flexistipes, Fulvimonas, Fusobacterium, Gemmiger, Geobacillus, Gloeobacter, Holdemania, Hydrogenoanaerobacterium, Kocuria, Lachnobacterium, Lachnospira, Lachnospiraceae, Lactobacillus, Lactonifactor, Leptospira, Lutispora, Lysinibacillus, Mollicutes, Moorella, Nocardia, Oscillibacter, Oscillospira, Paenibacillus, Papillibacter, Pseudoflavonifractor, Robinsoniella, Roseburia, Ruminococcaceae, Ruminococcus, Saccharomonospora, Sarcina, Solobacterium, Sporobacter, Sporolactobacillus, Streptomyces, Subdoligranulum, Sutterella, Syntrophococcus, Thermoanaerobacter, Thermobifida, and Turicibacter. 
Preferred bacterial cells also include bacterial cells of the genera Acetonema, Alkaliphilus, Amphibacillus, Ammonifex, Anaerobacter, Caldicellulosiruptor, Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Cohnella, Dendrosporobacter Desulfitobacterium, Desulfosporosinus, Halobacteroides, Heliobacterium, Heliophilum, Heliorestis, Lachnoanaerobaculum, Lysinibacillus, Oceanobacillus, Orenia (S.), Oxalophagus, Oxobacter, Pelospora, Pelotomaculum, Propionispora, Sporohalobacter, Sporomusa, Sporosarcina, Sporotomaculum, Symbiobacterium, Syntrophobotulus, Syntrophospora, Terribacillus, Thermoanaerobacter, and Thermosinus. 
In another embodiment, a probiotic composition of the invention consists essentially of Blautia. 
In one embodiment, a probiotic composition of the invention does not comprise Blautia alone.
As provided herein, the pharmaceutical compositions comprise, or in the alternative, modulate, the colonization and/or engraftment, of the following exemplary bacterial entities (e.g., bacterial cells belonging to particular bacterial strains, bacterial species, or bacterial genera): Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecalis, Enterococcus durans, Enterococcus villorum, Blautia luti, Blautia coccoides, Blautia hydrogenotrophica, Blautia hansenii, Blautia wexlerae, Lactobacillus plantarum, Pediococcus acidilactici, Staphylococcus pasteuri, Staphylococcus cohnii, Streptococcus sanguinis, Streptococcus sinensis, Streptococcus mitis, Streptococcus sp. SCA22, Streptococcus sp. CR-3145, Streptococcus anginosus, Streptococcus mutans, Coprobacillus cateniformis, Clostridium saccharogumia, Eubacterium dolichum DSM 3991, Clostridium sp. PPf35E6, Clostridium sordelli ATCC 9714, Ruminococcus torques, Ruminococcus gnavus, Clostridium clostridioforme, Ruminococcus obeum, Blautia producta, Clostridium sp. ID5, Megasphaera micronuciformis, Veillonella parvula, Clostridium methylpentosum, Clostridium islandicum, Faecalibacterium prausnitzii, Bacteroides uniformmis, Eubacterium rectale, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides ovatus, Bacteroides fragilis, Parabacteroides distasonis, Propinionibacteirum propionicum, Actinomycs hyovaginalis, Rothia mucilaginosa, Rothia aeria, Bifidobacterium breve, Scardovia inopinata and Eggerthella lenta. 
Preferred bacterial strains are provided in Table 1, Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, and Table 1F. Optionally, in some embodiments, preferred bacterial species are spore formers. The bacterial cells may be in the vegetative form and/or in the spore form. Thus, in some embodiments, the bacterial cell is present in the pharmaceutical composition solely in spore form. In other embodiments, the bacterial cell is present in the pharmaceutical composition solely in vegetative form. Yet, in other embodiments, the bacterial cell may be present in the pharmaceutical composition in a combination of vegetative form and spore form. Where specific strains of a species are provided, one of skill in the art will recognize that other strains of the species can be substituted for the named strain.
In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Acidaminococcus intestine. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Acinetobacter baumannii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Acinetobacter lwoffii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Akkermansia muciniphila. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Alistipes putredinis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Alistipes shahii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Anaerostipes hadrus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Anaerotruncus colihominis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides caccae. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides cellulosilyticus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides dorei. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides eggerthii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides finegoldii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides fragilis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides massiliensis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides ovatus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides salanitronis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides salyersiae. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides sp. 1_1_6. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides sp. 3_1_23. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides sp. D20. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides thetaiotaomicrond. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides uniformis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bacteroides vulgatus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bifidobacterium adolescentis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bifidobacterium bifidum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bifidobacterium breve. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bifidobacterium faecale. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bifidobacterium kashiwanohense. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bifidobacterium longum subsp. Longum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bifidobacterium pseudocatenulatum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Bifidobacterium stercoris. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia (Ruminococcus) coccoides. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia faecis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia glucerasea. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia (Ruminococcus) hansenii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia (Ruminococcus) luti. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia (Ruminococcus) obeum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia producta (Ruminococcus productus). In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia (Ruminococcus) schinkii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia stercoris. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia uncultured bacterium clone BKLE_a03_2 (GenBank: EU469501.1). In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank: EF402926.1). In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank: EF404657.1). In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia uncultured bacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia uncultured PAC000178_s (www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Blautia wexlerae. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Candidatus Arthromitus sp. SFB-mouse-Yit. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Catenibacterium mitsuokai. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridiaceae bacterium (Dielma fastidiosa) JC13. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridiales bacterium 1_7_47FAA. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium asparagiforme. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium bolteae. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium clostridioforme. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium glycyrrhizinilyticum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium (Hungatella) hathewayi. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium histolyticum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium indolis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium leptum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium (Tyzzerella) nexile. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium perfringens. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium (Erysipelatoclostridium) ramosum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium scindens. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium septum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium sp. 14774. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium sp. 7_3_54FAA. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium sp. HGF2. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium symbiosum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Collinsella aerofaciens. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Collinsella intestinalis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Coprobacillus sp. D7. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Coprococcus catus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Coprococcus comes. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Dorea formicigenerans. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Dorea longicatena. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Enterococcus faecalis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Enterococcus faecium. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Erysipelotrichaceae bacterium 3_1_53. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Escherichia coli. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Escherichia coli S88. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Eubacterium eligens. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Eubacterium fissicatena. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Eubacterium ramulus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Eubacterium rectale. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Faecalibacterium prausnitzii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Flavonifractor plautii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Fusobacterium mortiferum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Fusobacterium nucleatum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Holdemania filiformis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Hydrogenoanaerobacterium saccharovorans. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Klebsiella oxytoca. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Lachnospiraceae bacterium 3_1_57FAA_CT1. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Lachnospiraceae bacterium 7_1_58FAA. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Lachnospiraceae bacterium 5_1_57FAA. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Lactobacillus casei. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Lactobacillus rhamnosus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Lactobacillus ruminis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Lactococcus casei. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Odoribacter splanchnicus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Oscillibacter valericigenes. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Parabacteroides gordonii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Parabacteroides johnsonii. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Parabacteroides merdae. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Pediococcus acidilactici. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Peptostreptococcus asaccharolyticus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Propionibacterium granulosum. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Roseburia intestinalis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Roseburia inulinivorans. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Ruminococcus faecis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Ruminococcus gnavus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Ruminococcus sp. ID8. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Ruminococcus torques. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Slackia piriformis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Staphylococcus epidermidis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Staphylococcus saprophyticus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Streptococcus cristatus. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Streptococcus dysgalactiae subsp. Equisimilis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Streptococcus infantis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Streptococcus oralis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Streptococcus sanguinis. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Streptococcus viridans. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Streptococcus thermophiles. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Veillonella dispar. 
In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Acidaminococcus intestine. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Acinetobacter baumannii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Acinetobacter lwoffii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Akkermansia muciniphila. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Alistipes putredinis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Alistipes shahii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Anaerostipes hadrus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Anaerotruncus colihominis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides caccae. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides cellulosilyticus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides dorei. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides eggerthii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides finegoldii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides fragilis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides massiliensis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides ovatus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides salanitronis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides salyersiae. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides sp. 1_1_6. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides sp. 3_1_23. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides sp. D20. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides thetaiotaomicrond. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides uniformis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bacteroides vulgatus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bifidobacterium adolescentis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bifidobacterium bifidum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bifidobacterium breve. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bifidobacterium faecale. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bifidobacterium kashiwanohense. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bifidobacterium longum subsp. Longum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bifidobacterium pseudocatenulatum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Bifidobacterium stercoris. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia (Ruminococcus) coccoides. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia faecis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia glucerasea. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia (Ruminococcus) hansenii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia (Ruminococcus) luti. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia (Ruminococcus) obeum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia producta (Ruminococcus productus). In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia (Ruminococcus) schinkii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia stercoris. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia uncultured bacterium clone BKLE_a03_2 (GenBank: EU469501.1). In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank: EF402926.1). In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank: EF404657.1). In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia uncultured bacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia uncultured PAC000178_s (www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Blautia wexlerae. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Candidatus Arthromitus sp. SFB-mouse-Yit. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Catenibacterium mitsuokai. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridiaceae bacterium (Dielma fastidiosa) JC13. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridiales bacterium 1_7_47FAA. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium asparagiforme. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium bolteae. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium clostridioforme. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium glycyrrhizinilyticum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium (Hungatella) hathewayi. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium histolyticum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium indolis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium leptum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium (Tyzzerella) nexile. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium perfringens. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium (Erysipelatoclostridium) ramosum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium scindens. In one embodiment, the bacterial entity, e.g., species or strain, useful in the compositions and methods of the invention is Clostridium septum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium sp. 14774. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium sp. 7_3_54FAA. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium sp. HGF2. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Clostridium symbiosum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Collinsella aerofaciens. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Collinsella intestinalis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Coprobacillus sp. D7. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Coprococcus catus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Coprococcus comes. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Dorea formicigenerans. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Dorea longicatena. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Enterococcus faecalis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Enterococcus faecium. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Erysipelotrichaceae bacterium 3_1_53. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Escherichia coli. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Escherichia coli S88. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Eubacterium eligens. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Eubacterium fissicatena. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Eubacterium ramulus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Eubacterium rectale. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Faecalibacterium prausnitzii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Flavonifractor plautii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Fusobacterium mortiferum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Fusobacterium nucleatum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Holdemania filiformis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Hydrogenoanaerobacterium saccharovorans. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Klebsiella oxytoca. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Lachnospiraceae bacterium 3_1_57FAA_CT1. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Lachnospiraceae bacterium 7_1_58FAA. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Lachnospiraceae bacterium 5_1_57FAA. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Lactobacillus casei. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Lactobacillus rhamnosus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Lactobacillus ruminis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Lactococcus casei. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Odoribacter splanchnicus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Oscillibacter valericigenes. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Parabacteroides gordonii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Parabacteroides johnsonii. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Parabacteroides merdae. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Pediococcus acidilactici. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Peptostreptococcus asaccharolyticus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Propionibacterium granulosum. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Roseburia intestinalis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Roseburia inulinivorans. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Ruminococcus faecis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Ruminococcus gnavus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Ruminococcus sp. ID8. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Ruminococcus torques. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Slackia piriformis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Staphylococcus epidermidis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Staphylococcus saprophyticus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Streptococcus cristatus. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Streptococcus dysgalactiae subsp. Equisimilis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Streptococcus infantis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Streptococcus oralis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Streptococcus sanguinis. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Streptococcus viridans. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Streptococcus thermophiles. In one embodiment, the bacterial population useful in the compositions and methods of the invention comprises Veillonella dispar. 
In some embodiments, the pharmaceutical composition comprises engineered bacteria. For example, engineered bacteria include bacteria harboring i) one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or on an endogenous plasmid, wherein the genetic change may result in the alteration, disruption, removal, or addition of one or more protein-coding genes, non-protein-coding genes, gene regulatory regions, or any combination thereof, and wherein such change may be a fusion of two or more separate genomic regions or may be synthetically derived; ii) one or more foreign plasmids containing a mutant copy of an endogenous gene, such mutation being an insertion, deletion, or substitution, or any combination thereof, of one or more nucleotides; and iii) one or more foreign plasmids containing a mutant or non-mutant exogenous gene or a fusion of two or more endogenous, exogenous, or mixed genes. The engineered bacteria may be produced using techniques including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, or any combination thereof. Suitable bacteria for engineering are known in the art. For example, as described in PCT Publications Nos. WO 93/18163, DELIVERY AND EXPRESSION OF A HYBRID SURFACE PROTEIN ON THE SURFACE OF GRAM POSITIVE BACTERIA; WO 03/06593, METHODS FOR TREATING CANCER BY ADMINISTERING TUMOR-TARGETED BACTERIA AND AN IMMUNOMODULATORY AGENT; and WO 2010/141143, ENGINEERED AVIRULENT BACTERIA STRAINS AND USE IN MEDICAL TREATMENTS.
In some embodiments, the engineered bacteria are natural human commensals. In other embodiments, the engineered bacteria are attenuated strains of pathogens, and may include, but are not limited to, Pseudomonas aeruginosa, Salmonella species, Listeria monocytogenes, Mycoplasma hominis, Escherichia coli, Shigella species, and Streptococcus species, see, e.g. PCT Publications No. WO 03/06593, METHODS FOR TREATING CANCER BY ADMINISTERING TUMOR-TARGETTED BACTERIA AND AN IMMUNOMODULATORY AGENT. Attenuated strains of pathogens will lack all or parts of virulence operons, may lack immune-stimulatory surface moieties (e.g., lipopolysaccharide for Gram-negative bacteria), or may contain one or more nutrient auxotrophies. In specific embodiments, the engineered bacteria are attenuated intracellular pathogens, such as avirulent strains of Listeria monocytogenes. 
In some embodiments, the composition of the invention comprises one or more types of bacteria (e.g., one or more bacterial species or more than one strain of a particular bacterial species) capable of producing butyrate in a mammalian subject. Butyrate-producing bacteria may be identified experimentally, such as by NMR or gas chromatography analyses of microbial products or colorimetric assays (Rose (1955) METHODS ENZYMOL. 591-5). Butyrate-producing bacteria may also be identified computationally, such as by the identification of one or more enzymes involved in butyrate synthesis. Non-limiting examples of enzymes found in butyrate-producing bacteria include butyrate kinase, phosphotransbutyrylase, and butyryl CoA:acetate CoA transferase (Louis et al. (2004) J. BACT. 186(7): 2099-2106). Butyrate-producing strains include, but are not limited to, Faecalibacterium prausnitzii, Eubacterium spp., Butyrivibrio fibrisolvens, Roseburia intestinalis, Clostridium spp., Anaerostipes caccae, and Ruminococcus spp. In some embodiments, a pharmaceutical composition comprises two or more types of bacteria (e.g., two or more bacterial species or two or more strains of a particular bacterial species), wherein at least two types of bacteria are capable of producing butyrate in a mammalian subject. In other embodiments, the pharmaceutical composition comprises two or more types of bacteria, wherein two or more types of bacteria cooperate (i.e., cross-feed) to produce an immunomodulatory SCFA (e.g., butyrate) in a mammalian subject. In a preferred embodiment, the pharmaceutical composition comprises at least one type of bacteria (e.g., Bifidobacterium spp.) capable of metabolizing a prebiotic, including but not limited to, inulin, inulin-type fructans, or oligofructose, such that the resulting metabolic product may be converted by a second type of bacteria (e.g., a butyrate-producing bacteria such as Roseburia spp.) to an immunomodulatory SCFA such as butyrate (see, e.g., Falony et al. (2006) APPL. ENVIRON. MICROBIOL. 72(12): 7835-7841). In other aspects, the composition comprises at least one acetate-producing bacteria (e.g., Bacteroides thetaiotaomicron) and at least one acetate-consuming, butyrate-producing bacteria (e.g., Faecalibacterium prausnitzii).
In some embodiments, the pharmaceutical composition comprises one or more types of bacteria (e.g., one or more bacterial species or more than one strain of a particular bacterial species) capable of producing propionate in a mammalian subject, optionally further comprising a prebiotic or substrate appropriate for proprionate biosynthesis. Examples of prebiotics or substrates used for the production of propionate include, but are not limited to, L-rhamnose, D-tagalose, resistant starch, inulin, polydextrose, arabinoxylans, arabinoxylan oligosaccharides, mannooligosaccharides, and laminarans (Hosseini et al. (2011) NUTRITION REVIEWS 69(5): 245-258). Propionate-producing bacteria may be identified experimentally, such as by NMR or gas chromatography analyses of microbial products or colorimetric assays (Rose (1955)). Propionate-producing bacteria may also be identified computationally, such as by the identification of one or more enzymes involved in propionate biosynthesis. Non-limiting examples of enzymes found in propionate-producing bacteria include enzymes of the succinate pathway, including but not limited to phophoenylpyrvate carboxykinase, pyruvate kinase, pyruvate carboxylase, malate dehydrogenase, fumarate hydratase, succinate dehydrogenase, succinyl CoA synthetase, methylmalonyl Coa decarboxylase, and propionate CoA transferase, as well as enzymes of the acrylate pathway, including but not limited to L-lactate dehydrogenase, propionate CoA transferase, lactoyl CoA dehydratase, acyl CoA dehydrogenase, phosphate acetyltransferase, and propionate kinase. Non-limiting examples of bacteria that utilize the succinate pathway are Bacteroides fragilis and other species (including Bacteroides vulgatus), Propionibacterium spp. (including freudenrichii and acidipropionici), Veillonella spp. (including gazogenes), Micrococcus lactilyticus, Selenomonas ruminantium, Escherichia coli, and Prevotella ruminocola. Non-limiting examples of bacteria that utilize the acrylate pathway are Clostridium neopropionicum X4, and Megasphaera elsdenii. 
In preferred embodiments, the combination of a bacteria and a prebiotic is selected based on the fermentation or metabolic preferences of one or more bacteria capable of producing immunomodulatory SCFAs (e.g., preference for complex versus simple sugar or preference for a fermentation product versus a prebiotic). For example, M. eldsenii prefers lactate fermentation to glucose fermentation, and maximization of propionate production by M. eldsenii in a mammalian subject may therefore be achieved by administering along with M. eldsenii a favored substrate (e.g., lactate) or one or more bacteria capable of fermenting glucose into lactate (e.g., Streptococcus bovis) (see, e.g., Hosseini et al. (2011)). Thus, in some embodiments, the composition comprises at least one type of SCFA-producing bacteria and a sugar fermentation product (e.g., lactate). In other embodiments, the composition comprises at least one type of SCFA-producing bacteria and at least one type of sugar-fermenting bacteria, wherein the fermentation product of the second, sugar-fermenting bacteria is the preferred substrate of the SCFA-producing bacteria.
Immunomodulation can also be achieved by the bacterial production of glutathione or gamma-glutamylcysteine. Thus, in certain embodiments, the pharmaceutical composition, dosage form, or kit comprises at least one type of bacteria (e.g., one or more bacterial species or more than one strain of a particular bacterial species) capable of producing glutathione and/or gamma-glutamylcysteine in a mammalian subject. In some aspects, the composition comprises one or more bacteria selected for the presence of glutamate cysteine ligase (e.g., Lactobacillus fermentum) and/or L-proline biosynthesis enzymes (e.g., E. coli) (Peran et al. (2006) INT. J. COLORECTAL DIS. 21(8): 737-746; Veeravalli et al. (2011) NAT. CHEM. BIO. 7(2): 101-105). In a preferred embodiment, at least one bacteria in the composition is L. fermentum. 
Para-cresol (p-cresol) is a microbial product, via the fermentation of tyrosine or phenylalanine. Sulfated in the liver or colon to p-cresyl sulfate, this molecule reduces Th1-mediated responses (Shiba et al. (2014) TOXICOL. APPL. PHARMACOL. 274(2): 191-9). In some embodiments, the composition comprises at least one type of bacteria (e.g., one or more bacterial species or more than one strain of a particular bacterial species) capable of fermenting tyrosine and/or phenylalanine to p-cresol in a mammalian subject. Non-limiting examples of such bacteria include Bacteroides fragilis, Clostridium difficile, and Lactobacillus sp. Strain #11198-11201 (see, e.g., Yokoyama and Carlson (1981) APPL. ENVIRON. MICROBIOL. 41(1): 71-76), and other bacteria with p-hydroxylphenyl acetate decarboxylase activity.
In one aspect, provided herein are therapeutic compositions (e.g., pharmaceutical compositions) containing a purified population of bacterial cells. As used herein, the terms “purify”, “purified” and “purifying” refer to the state of a population (e.g., a plurality of known or unknown amount and/or concentration) of desired bacterial cells, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired bacteria, or alternatively a removal or reduction of residual habitat products as described herein. In some embodiments, a purified population has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other embodiments, a purified population has an amount and/or concentration of desired bacterial cells at or above an acceptable amount and/or concentration. In other embodiments, the purified population of bacterial cells is enriched as compared to the starting material (e.g., a fecal material) from which the population is obtained. This enrichment may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.9999%, or greater than 99.999999%, as compared to the starting material.
In certain embodiments, the purified populations of bacterial cells have reduced or undetectable levels of one or more pathogenic activities, such as toxicity, an ability to cause infection of the mammalian recipient subject, an undesired immunomodulatory activity, an autoimmune response, a metabolic response, or an inflammatory response or a neurological response. Such a reduction in a pathogenic activity may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%, as compared to the starting material. In other embodiments, the purified populations of bacterial cells have reduced sensory components as compared to fecal material, such as reduced odor, taste, appearance, and umami.
In another embodiment, the invention provides purified populations of bacterial cells that are substantially free of residual habitat products. In certain embodiments, this means that the bacterial composition no longer contains a substantial amount of the biological matter associated with the microbial community while living on or in the human or animal subject, and the purified population of bacterial cells (e.g., bacterial spores or vegetative cells) may be 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, 90% free, 85% free, 80% free, 75% free, 70% free, 60% free, or 50% free, of any contamination of the biological matter associated with the microbial community. Substantially free of residual habitat products may also mean that the bacterial composition contains no detectable cells from a human or animal, and that only microbial cells are detectable, in particular, only desired microbial cells are detectable. In another embodiment, it means that fewer than 1×10−2%, 1×10−3%, 1×104%, 1×10−5%, 1×10−6%, 1×10−7%, 1×10−8% of the cells in the bacterial composition are human or animal, as compared to microbial cells. In another embodiment, the residual habitat product present in the purified population is reduced at least a certain level from the fecal material obtained from the mammalian donor subject, e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%.
In one embodiment, substantially free of residual habitat products or substantially free of a detectable level of a pathogenic material means that the bacterial composition contains no detectable viral (including bacterial viruses (i.e., phage)), fungal, or mycoplasmal or toxoplasmal contaminants, or a eukaryotic parasite such as a helminth. Alternatively, the purified population of bacterial cells (e.g., bacterial spores and/or vegetative cells) is substantially free of an acellular material, e.g., DNA, viral coat material, or non-viable bacterial material. Alternatively, the purified population of bacterial cells may processed by a method that kills, inactivates, or removes one or more specific undesirable viruses, such as an enteric virus, including norovirus, poliovirus or hepatitis A virus.
As described herein, purified populations of bacterial cells can be demonstrated by, for example, genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, microscopic analysis, microbial analysis including germination and culturing, or methods using instrumentation such as flow cytometry with reagents that distinguish desired bacterial entities and/or fungal entities from non-desired, contaminating materials. In yet another embodiment, the spores in a purified population of bacterial cells undergo partial germination during processing and formulation such that the final composition comprises spores and vegetative bacteria.
In another embodiment, provided herein are methods for production of a pharmaceutical composition, e.g., a probiotic composition, comprising a population of bacterial cells (e.g., a population of anti-inflammatory bacterial cells), with or without one or more prebiotics, suitable for therapeutic administration to a mammalian subject in need thereof. In one embodiment, the composition can be produced by generally following the steps of: (a) providing a fecal material obtained from a mammalian donor subject; and (b) subjecting the fecal material to at least one purification treatment or step under conditions such that a population of bacterial cells is produced from the fecal material.
Individual bacterial strains can also be isolated from stool samples using culture methods. For example, 5 mls of phosphate-buffered saline (PBS) is added to 1 mg of frozen stool sample and homogenized by vortexing in an anaerobic chamber for isolation of anaerobic bacteria. The suspension is then serially diluted ten-fold (e.g., 10−1 to 10−9 dilutions) and 100 μl aliquots of each dilution are spread evenly over the surface of agar plates containing different formulations e.g., anaerobic blood agar plates, Bacteroides bile esculin plates, laked kanamycin vancomycin plates, egg yolk agar plates and de Man Rogosa and Sharpe agar plates. Inverted plates are incubated in an anaerobic chamber for 48 hr+/−4 hours. Colonies with different morphologies are picked and replated on anaerobic blood agar plates for further testing, PCR analysis and 16 S sequencing. Selected bacterial strains can be grown for therapeutic use singly or in combination.
In one embodiment, a probiotic composition of the invention is not a fecal transplant. In some embodiments all or essentially all of the bacterial entities present in a purified population are originally obtained from a fecal material and subsequently, e.g., for production of pharmaceutical compositions, are grown in culture as described herein or otherwise known in the art. In some embodiments all or essentially all of the bacterial entities and/or fungal entities present in a purified population are obtained from a fecal material and subsequently are grown in culture as described herein or otherwise known in the art. In one embodiment, the bacterial cells are cultured from a bacterial stock and purified as described herein. In one embodiment, each of the populations of bacterial cells are independently cultured and purified, e.g., each population is cultured separately and subsequently mixed together. In one embodiment, one or more of the populations of bacterial cells in the composition are co-cultured.
Identification of Immunomodulatory Bacteria.
In some embodiments, immunomodulatory bacteria are identified by screening bacteria to determine whether the bacteria induce secretion of a pro-inflammatory or a anti-inflammatory cytokines by a host cell (e.g., a host immune cell). In some embodiments, the immunomodulatory bacteria are screened in vitro. For example, human or mammalian cells capable of cytokine secretion, such as immune cells (e.g., PBMCs, macrophages, T cells, etc.) can be exposed to candidate immunomodulatory bacteria, or supernatants obtained from cultures of candidate immunomodulatory bacteria, and changes in cytokine expression or secretion can be measured using standard techniques, such as ELISA, immunoblot, Luminex, antibody array, quantitative PCR, microarray, etc. Bacteria for inclusion in a pharmaceutical composition (e.g., a probiotic composition) can be selected based on the ability to induce a desired cytokine profile in human or mammalian cells (e.g., immune cells). For example, anti-inflammatory bacteria can be selected for inclusion in a pharmaceutical composition based on the ability to induce secretion of one or more anti-inflammatory cytokines, and/or the ability to reduce secretion of one or more pro-inflammatory cytokines. Anti-inflammatory cytokines include, for example, IL-10, IL-13, IL-9, IL-4, IL-5, TGFβ, and combinations thereof. Pro-inflammatory cytokines include, for example, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof. In some embodiments, anti-inflammatory bacteria may be selected for inclusion in a pharmaceutical compositions based on the ability to modulate the secretion of one or more anti-inflammatory cytokines and/or that ability to reduce secretion of one or pro-inflammatory cytokines that have been induced by a bacterial cell of a different bacteria type. In some embodiments, the different bacterial cell is of a different bacterial genus. In some embodiments, the different bacterial cell is of a different bacterial species. In some embodiments, the different bacterial cell is of a different bacterial strain.
In other embodiments, immunomodulatory bacteria are identified by screening bacteria to determine whether the bacteria impact the differentiation and/or expansion of particular subpopulations of immune cells. For example, candidate bacteria can be screened for the ability to promote differentiation and/or expansion of Treg cells, Th17 cells, Th1 cells and/or Th2 cells from precursor cells, e.g., naive T cells. By way of example, naïve T cells can be cultured in the presence of candidate bacteria or supernatants obtained from cultures of candidate bacteria, and the quantity of Treg cells, Th17 cells, Th1 cells and/or Th2 cells can be determined using standard techniques, such as FACS analysis. Markers indicative of Treg cells include, for example, CD25+CD127lo. Markers indicative of Th17 cells include, for example, CXCR3−CCR6+. Markers indicative of Th1 cells include, for example, CXCR3+CCR6−. Markers indicative of Th2 cells include, for example, CXCR3−CCR6−. Other markers indicative of particular T cell subpopulations are known in the art, and may be used in the assays described herein, e.g., to identify populations of immune cells impacted by candidate immunomodulatory bacteria. Bacteria can be selected for inclusion in a pharmaceutical composition based on the ability to promote differentiation and/or expansion of a desired immune cell subpopulation.
In other embodiments, immunomodulatory bacteria are identified by screening bacteria to determine whether the bacteria secrete short chain fatty acids (SCFA), such as, for example, butyrate, acetate, propionate, or valerate, or combinations thereof. For example, secretion of short chain fatty acids into bacterial supernatants can be measured using standard techniques. In one embodiment, bacterial supernatants can be screened to measure the level of one or more short chain fatty acids using NMR, mass spectrometry (e.g., GC-MS, tandem mass spectrometry, matrix-assisted laser desorption/ionization, etc.), ELISA, or immunoblot. Expression of bacterial genes responsible for production of short chain fatty acids can also be determined by standard techniques, such as Northern blot, microarray, or quantitative PCR.
In some embodiments, provided herein are pharmaceutical compositions comprising a population of bacterial cells (e.g., bacterial cells of the order Clostridiales) containing one type of bacteria. In some embodiments, provided herein are pharmaceutical compositions comprising a population of bacterial cells (e.g., bacterial cells of the Order Clostridiales) containing more than one type of bacteria. As used herein, a “type” or more than one “types” of bacteria may be differentiated at the genus level, the species level, the sub-species level, the strain level or by any other taxonomic method, as described herein and otherwise known in the art.
In some embodiment, the pharmaceutical composition may contain one or more types of bacteria, including bacterial strains of the same species or of different species. For instance, a pharmaceutical composition may comprise bacterial cells of 1, at least 2, at least 3, or at least 4 types of bacteria. In another embodiment, a bacterial composition may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20, at least 30, at least 40, at least 50, or more than 50 types of bacteria, as defined by species or operational taxonomic unit (OTU) encompassing such species. In a preferred embodiment, a pharmaceutical composition comprises from 2 to no more than 40, from 2 to no more than 30, from 2 to no more than 20, from 2 to no more than 15, from 2 to no more than 10, from 2 to no more than 5, types of bacteria. In another preferred embodiment, a bacterial composition comprises a single type of bacteria.
In a preferred embodiment, the composition comprises about 20 or fewer isolated populations of bacterial cells. In another embodiment, the composition comprises about 15 or fewer isolated populations of bacterial cells. In another embodiment, the composition comprises about 10 or fewer isolated populations of bacterial cells. In another embodiment, the composition comprises about 5 or fewer isolated populations of bacterial cells. In another embodiment, the composition comprises about 4 or fewer isolated populations of bacterial cells. In another embodiment, the composition comprises about 3 or fewer isolated populations of bacterial cells. In another embodiment, the composition comprises about 2 isolated populations of bacterial cells. In another embodiment, the composition comprises between about 12 and 20 isolated populations of bacterial cells. In another embodiment, the composition comprises a single isolated population of bacterial cells. In another embodiment, the composition comprises at least two isolated populations of bacterial cells. In yet another embodiment, the composition comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated populations of bacterial cells.
In some embodiments, the pharmaceutical composition contains a defined mixture of isolated bacteria. For example, in some embodiments, the pharmaceutical composition contains no more than 100 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 75 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 60 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 50 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 45 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 40 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 35 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 30 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 25 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 20 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 15 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 14 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 13 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 12 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 11 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 10 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 9 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 8 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 7 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 6 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 5 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 4 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 3 types of bacteria. In other embodiments, the pharmaceutical composition contains no more than 2 types of bacteria. In some embodiments, the pharmaceutical composition contains no more than 1 type of bacteria. In some embodiments, the pharmaceutical composition contains defined quantities of each bacterial species. In an exemplary embodiment, the bacteria incorporated into the pharmaceutical composition are not isolated from fecal matter
Provided herein are pharmaceutical compositions comprising at least one, at least two or at least three types of bacteria that are not identical and that are capable of decreasing the risk and/or severity of an autoimmune or inflammatory disease, symptom, condition, or disorder, or dysbiosis. In an embodiment, the pharmaceutical composition comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 types of isolated bacteria. In one embodiment, the pharmaceutical composition comprises at least about 4 types of isolated bacteria or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more types of isolated bacteria. In some embodiments, the above invention relates to pharmaceutical compositions further comprising one or more prebiotics.
In some embodiments, the pharmaceutical composition of the invention includes at least one type of bacteria, wherein said bacteria is a bacterial strain (e.g., strain of anti-inflammatory bacterial cells), and the composition includes at least 1×103 colony forming units (CFU) per dose of said bacterial strain. In other embodiments, the pharmaceutical composition of the invention includes at least one type of bacteria, wherein said bacteria is a bacterial strain, and the composition includes at least about 1×103, 1×104, 1×105, 1×106, 1×107, 1×108, 1×109, 1×1010, 1×1011, 1×1012, 1×1013, 1×1014, 1×1015, or greater than 1×1015 CFU per dose of each bacterial strain present in the composition.
In some embodiments, the pharmaceutical compositions of the invention are formulated for oral or gastric administration, typically to a mammalian subject (e.g., a human). In some embodiments, the composition is formulated for oral administration as a solid, semi-solid, gel, or liquid form, such as in the form of a pill, tablet, capsule, or lozenge. In another embodiment, the pharmaceutical composition is formulated as a skin patch. In another embodiment, the pharmaceutical composition is formulated for topical administration. In one embodiment, the pharmaceutical composition is formulated as a food product. In some embodiments, such formulations contain or are coated by an enteric coating to protect the bacterial strain through the stomach and small intestine, although spores are generally resistant to the stomach and small intestines. In other embodiments, the pharmaceutical compositions may be formulated with a germinant to enhance engraftment, or efficacy. In yet other embodiments, the pharmaceutical compositions may be co-formulated or co-administered with prebiotic substances, to enhance engraftment or efficacy.
The composition(s) may include different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration such as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intlrapericardially, intraumbilically, intraocularally, orally, topically, locally, as an injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), as an aerosol, or by other method or any combination of the fore going as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
In some embodiments, the composition comprises at least one lipid. As used herein, a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans). In some embodiments, the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0). In other embodiments, the composition comprises at least one modified lipid, for example, a lipid that has been modified by cooking.
In some embodiments, the composition comprises at least one supplemental mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
In certain embodiments, the composition comprises at least one supplemental vitamin. The at least one vitamin can be fat-soluble or water soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
In other embodiments, the pharmaceutical composition comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
In another embodiment, the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
In cases where a pharmaceutical composition contains a anaerobic bacterial strain, the pharmaceutical formulation and excipients can be selected to prevent exposure of the bacterial strains to oxygen.
In other embodiments, the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
In another embodiment, the composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
In other embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.
In some embodiments, the pharmaceutical composition comprises a disintegrant as an excipient. In other embodiments, the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth. In another embodiment, the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
In another embodiment, the excipient comprises a flavoring agent. Flavoring agents can be chosen from synthetic flavor oils and flavoring aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof. In some embodiments the flavoring agent is selected from cinnamon oils; oil of wintergreen; peppermint oils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oil such as lemon oil, orange oil, grape and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot.
In other embodiments, the excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as the sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like. Also contemplated are hydrogenated starch hydrolysates and the synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularly the potassium salt (acesulfame-K), and sodium and calcium salts thereof.
In yet other embodiments, the composition comprises a coloring agent. Non-limiting examples of suitable color agents include food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), and external drug and cosmetic colors (Ext. D&C). The coloring agents can be used as dyes or their corresponding lakes.
The weight fraction of the excipient or combination of excipients in the formulation of the pharmaceutical composition is usually about 99% or less, such as about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, or about 1% or less of the total weight of the composition.
The compositions disclosed herein can be formulated into a variety of forms and administered by a number of different means. The compositions can be administered orally, rectally, or parenterally, in formulations containing conventionally acceptable carriers, adjuvants, and vehicles as desired. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, or intrasternal injection and infusion techniques. In an exemplary embodiment, the composition is administered orally.
Solid dosage forms for oral administration include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules. A capsule typically comprises a core material comprising a bacterial composition and a shell wall that encapsulates the core material. In some embodiments, the core material comprises at least one of a solid, a liquid, and an emulsion. In other embodiments, the shell wall material comprises at least one of a soft gelatin, a 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, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose succinate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, such as those formed from acrylic acid, methacrylic acid, methyl acrylate, ammonio methylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate (e.g., those copolymers sold under the trade name “Eudragit”); vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; and shellac (purified lac). In yet other embodiments, at least one polymer functions as taste-masking agents.
Tablets, pills, and the like can be compressed, multiply compressed, multiply layered, and/or coated. The coating can be single or multiple. In one embodiment, the coating material comprises at least one of a saccharide, a polysaccharide, and glycoproteins extracted from at least one of a plant, a fungus, and a microbe. Non-limiting examples include corn starch, wheat starch, potato starch, tapioca starch, cellulose, hemicellulose, dextrans, maltodextrin, cyclodextrins, inulins, pectin, mannans, gum arabic, locust bean gum, mesquite gum, guar gum, gum karaya, gum ghatti, tragacanth gum, funori, carrageenans, agar, alginates, chitosans, or gellan gum. In some embodiments the coating material comprises a protein. In another embodiment, the coating material comprises at least one of a fat and an oil. In other embodiments, the at least one of a fat and an oil is high temperature melting. In yet another embodiment, the at least one of a fat and an oil is hydrogenated or partially hydrogenated. In one embodiment, the at least one of a fat and an oil is derived from a plant. In other embodiments, the at least one of a fat and an oil comprises at least one of glycerides, free fatty acids, and fatty acid esters. In some embodiments, the coating material comprises at least one edible wax. The edible wax 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 additionally be prepared with enteric coatings.
Alternatively, powders or granules embodying the bacterial compositions disclosed herein can be incorporated into a food product. In some embodiments, the food product is a drink for oral administration. Non-limiting examples of a suitable drink include fruit juice, a fruit drink, an artificially flavored drink, an artificially sweetened drink, a carbonated beverage, a sports drink, a liquid diary product, a shake, an alcoholic beverage, a caffeinated beverage, infant formula and so forth. Other suitable means for oral administration include aqueous and nonaqueous solutions, emulsions, suspensions and solutions and/or suspensions reconstituted from non-effervescent granules, containing at least one of suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, coloring agents, and flavoring agents.
In some embodiments, the food product can be a solid foodstuff. Suitable examples of a solid foodstuff include without limitation a food bar, a snack bar, a cookie, a brownie, a muffin, a cracker, an ice cream bar, a frozen yogurt bar, and the like.
In other embodiments, the pharmaceutical compositions disclosed herein are incorporated into a therapeutic food. In some embodiments, the therapeutic food is a ready-to-use food that optionally contains some or all essential macronutrients and micronutrients. In another embodiment, the compositions disclosed herein are incorporated into a supplementary food that is designed to be blended into an existing meal. In one embodiment, the supplemental food contains some or all essential macronutrients and micronutrients. In another embodiment, the bacterial compositions disclosed herein are blended with or added to an existing food to fortify the food's protein nutrition. Examples include food staples (grain, salt, sugar, cooking oil, margarine), beverages (coffee, tea, soda, beer, liquor, sports drinks), snacks, sweets and other foods.
In one embodiment, the formulations are filled into gelatin capsules for oral administration. An example of an appropriate capsule is a 250 mg gelatin capsule containing from 10 mg (up to 100 mg) of lyophilized powder (108 to 1011 CFUs), 160 mg microcrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesium stearate. In an alternative embodiment, from 105 to 1012, 105 to 107, 106 to 107, or 108 to 1010 CFUs may be used, with attendant adjustments of the excipients if necessary. In an alternative embodiment, an enteric-coated capsule or tablet or with a buffering or protective composition can be used.
The pharmaceutical compositions, with or without one or more prebiotics, are generally formulated for oral or gastric administration, typically to a mammalian subject. In particular embodiments, the composition is formulated for oral administration as a solid, semi-solid, gel, or liquid form, such as in the form of a pill, tablet, capsule, or lozenge. In some embodiments, such formulations contain or are coated by an enteric coating to protect the bacteria through the stomach and small intestine, although spores are generally resistant to the stomach and small intestines. In other embodiments, the pharmaceutical compositions, with or without one or more prebiotics, may be formulated with a germinant to enhance engraftment, or efficacy. In yet other embodiments, the pharmaceutical compositions may be co-formulated or co-administered with prebiotic substances, to enhance engraftment or efficacy. In some embodiments, bacterial compositions may be co-formulated or co-administered with prebiotic substances, to enhance engraftment or efficacy.
In some formulations, the pharmaceutical composition contains at least about 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% spores on a mass basis. In some formulations, the administered dose does not exceed 200, 300, 400, 500, 600, 700, 800, 900 milligrams or 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 grams in mass.
The pharmaceutical compositions of the invention may include live microbes, dead microbes, microbes that are lyophilized, freeze-dried, and/or substantially dehydrated, or the composition may include bacterial or fungal spores or virions.
Bacterial compositions for use in the pharmaceutical compositions can be described by operational taxonomic units (OTUs). Bacterial compositions may be prepared comprising one or at least two types of isolated bacteria, wherein a first type and a second type are independently chosen from the species or OTUs listed in Table 1. Additionally, a bacterial composition may be prepared comprising at least two types of isolated bacteria, wherein a first OTU and a second OTU are independently characterized by, i.e., at least 95%, 96%, 97%, 98%, 99% or including 100% sequence identity to, sequences listed.
Pharmaceutical compositions may be prepared comprising one or at least two types of isolated bacteria, chosen from the species in Table 1, Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, or Table 1F. Generally, the first bacteria and the second bacteria are not the same. The sequences provided in the sequencing listing file for OTUs in Table 1 are full 16S sequences. Therefore, in one embodiment, the first and/or second OTUs may be characterized by the full 16S sequences of OTUs listed in Table 1. In another embodiment, the first and/or second OTUs may be characterized by one or more of the variable regions of the 16S sequence (V1-V9). In some embodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU. In one embodiment, the V1, V2, and V3 regions are used to characterize an OTU. In another embodiment, the V3, V4, and V5 regions are used to characterize an OTU. In another embodiment, the V4 region is used to characterize an OTU.
Using well known techniques, in order to determine the full 16S sequence or the sequence of any hypervariable region of the 16S sequence, genomic DNA is extracted from a bacterial sample, the 16S rDNA (full region or specific hypervariable regions) amplified using polymerase chain reaction (PCR), the PCR products cleaned, and nucleotide sequences delineated to determine the genetic composition of 16S gene or subdomain of the gene. If full 16S sequencing is performed, the sequencing method used may be, but is not limited to, Sanger sequencing. If one or more hypervariable regions are used, such as the V4 region, the sequencing may be, but is not limited to being, performed using the Sanger method or using a next-generation sequencing method, such as an Illumina (sequencing by synthesis) method using barcoded primers allowing for multiplex reactions.
OTUs can be defined by a combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof, full-genome sequence, or partial genome sequence generated using amplified genetic products, or whole genome sequence (WGS). Using well defined methods DNA extracted from a bacterial sample will have specific genomic regions amplified using PCR and sequenced to determine the nucleotide sequence of the amplified products. In the whole genome shotgun (WGS) method, extracted DNA will be directly sequenced without amplification. Sequence data can be generated using any sequencing technology including, but not limited to Sanger, Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences, and/or Oxford Nanopore.
Prebiotics
In one aspect, the pharmaceutical compositions described herein contain a prebiotic. In another aspect, the pharmaceutical compositions are co-administered with a prebiotic (e.g., sequentially or concurrently). A prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota, that confers benefits upon host well-being and health. Prebiotics can include complex carbohydrates, amino acids, peptides, or other nutritional components useful for the survival of the bacterial composition. Prebiotics include, but are not limited to, amino acids, biotin, fructooligosaccharide, galactooligosaccharides, inulin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, oligofructose, oligodextrose, tagatose, trans-galactooligosaccharide, and xylooligosaccharides.
Suitable prebiotics are usually plant-derived complex carbohydrates, oligosaccharides or polysaccharides. Generally, prebiotics are indigestible or poorly digested by humans and serve as a food source for bacteria. Prebiotics which can be used in the pharmaceutical dosage forms, pharmaceutical compositions, and kits provided herein include, without limitation, galactooligosaccharides (GOS), trans-galactooligosaccharides, fructooligosaccharides or oligofructose (FOS), inulin, oligofructose-enriched inulin, lactulose, arabinoxylan, xylooligosaccharides (XOS), mannooligosaccharides, gum guar, gum arabic, tagatose, amylose, amylopectin, xylan, pectin, and the like and combinations of thereof. Prebiotics can be found in certain foods, e.g. chicory root, Jerusalem artichoke, Dandelion greens, garlic, leek, onion, asparagus, wheat bran, wheat flour, banana, milk, yogurt, sorghum, burdock, broccoli, Brussels sprouts, cabbage, cauliflower, collard greens, kale, radish and rutabaga, and miso. Alternatively, prebiotics can be purified or chemically or enzymatically synthesized.
In some embodiments, the composition comprises at least one prebiotic. In some embodiment, the prebiotic is a carbohydrate. In some embodiments, the composition of the present invention comprises a prebiotic mixture, which comprises at least one carbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula (CH2O)n. A carbohydrate can be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates can contain modified saccharide units, such as 2′-deoxyribose wherein a hydroxyl group is removed, 2′-fluororibose wherein a hydroxyl group is replace with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2′-fluororibose, deoxyribose, and hexose). Carbohydrates can exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers. Carbohydrates may be purified from natural (e.g., plant or microbial) sources (i.e., they are enzymatically synthetized), or they may be chemically synthesized or modified.
Suitable prebiotic carbohydrates can include one or more of a carbohydrate, carbohydrate monomer, carbohydrate oligomer, or carbohydrate polymer. In certain embodiments, the pharmaceutical composition, dosage form, or kit comprises at least one type of microbe and at least one type of non-digestible saccharide, which includes non-digestible monosaccharides, non-digestible oligosaccharides, or non-digestible polysaccharides. In one embodiment, the sugar units of an oligosaccharide or polysaccharide can be linked in a single straight chain or can be a chain with one or more side branches. The length of the oligosaccharide or polysaccharide can vary from source to source. In one embodiment, small amounts of glucose can also be contained in the chain. In another embodiment, the prebiotic composition can be partially hydrolyzed or contain individual sugar moieties that are components of the primary oligosaccharide (see, e.g., U.S. Pat. No. 8,486,668, PREBIOTIC FORMULATIONS AND METHODS OF USE).
Prebiotic carbohydrates may include, but are not limited to monosaccharaides (e.g., trioses, tetroses, pentoses, aldopentoses, ketopentoses, hexoses, cyclic hemiacetals, ketohexoses, heptoses) and multimers thereof, as well as epimers, cyclic isomers, stereoisomers, and anomers thereof. Nonlimiting examples of monosaccharides include (in either the L- or D-conformation) glyceraldehyde, threose, ribose, altrose, glucose, mannose, talose, galactose, gulose, idose, lyxose, arabinose, xylose, allose, erythrose, erythrulose, tagalose, sorbose, ribulose, psicose, xylulose, fructose, dihydroxyacetone, and cyclic (alpha or beta) forms thereof. Multimers (disaccharides, trisaccharides, oligosaccharides, polysaccharides) thereof include but are not limited to sucrose, lactose, maltose, lactulose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, sophorose, laminaribiose, gentioboise, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiulose, rutinose, rutinulose, xylobiose, primeverose, amylose, amylopectin, starch (including resistant starch), chitin, cellulose, agar, agarose, xylan, glycogen, bacterial polysaccharides such as capsular polysaccharides, LPS, and peptodglycan, and biofilm exopolysaccharide (e.g., alginate, EPS), N-linked glycans, and O-linked glycans. Prebiotic sugars may be modified and carbohydrate derivatives include amino sugars (e.g., sialic acid, N-acetylglucosamine, galactosamine), deoxy sugars (e.g., rhamnose, fucose, deoxyribose), sugar phosphates, glycosylamines, sugar alcohols, and acidic sugars (e.g., glucuronic acid, ascorbic acid).
In some embodiments, the prebiotic carbohydrate component of the pharmaceutical composition, dosage form, or kit consists essentially of one or more non-digestible saccharides. In one embodiment, non-digestible oligosaccharides the non-digestible oligosaccharides are galactooligosaccharides (GOS). In another embodiment, the non-digestible oligosaccharides are fructooligosaccharides (FOS).
In some embodiments, the prebiotic composition of the invention comprises one or more of GOS, lactulose, raffinose, stachyose, lactosucrose, FOS (i.e., oligofructose or oligofructan), inulin, isomalto-oligosaccharide, xylo-oligosaccharide, paratinose oligosaccharide, transgalactosylated oligosaccharides (i.e., transgalacto-oligosaccharides), transgalactosylate disaccharides, soybean oligosaccharides (i.e., soyoligosaccharides), gentiooligosaccharides, glucooligosaccharides, pecticoligosaccharides, palatinose polycondensates, difructose anhydride III, sorbitol, maltitol, lactitol, polyols, polydextrose, reduced paratinose, cellulose, β-glucose, β-galactose, β-fructose, verbascose, galactinol, and β-glucan, guar gum, pectin, high, sodium alginate, and lambda carrageenan, or mixtures thereof. The GOS may be a short-chain GOS, a long-chain GOS, or any combination thereof. The FOS may be a short-chain FOS, a long-chain FOS, or any combination thereof.
In some embodiments, the prebiotic composition comprises two carbohydrate species (nonlimiting examples being a GOS and FOS) in a mixture of at least 1:1, at least 2:1, at least 5:1, at least 9:1, at least 10:1, about 20:1, or at least 20:1.
In some embodiments, the prebiotic composition of the invention comprises a mixture of one or more non-digestible oligosaccharides, non-digestible polysaccharides, free monosaccharides, non-digestible saccharides, starch, or non-starch polysaccharides. In one embodiment, a prebiotic component of a prebiotic composition is a GOS composition. In one embodiment, a prebiotic composition is a pharmaceutical composition. In one embodiment, a pharmaceutical composition is a GOS composition.
Oligosaccharides are generally considered to have a reducing end and a non-reducing end, whether or not the saccharide at the reducing end is in fact a reducing sugar. Most oligosaccharides described herein are described with the name or abbreviation for the non-reducing saccharide (e.g., Gal or D-Gal), preceded or followed by the configuration of the glycosidic bond (α or β), the ring bond, the ring position of the reducing saccharide involved in the bond, and then the name or abbreviation of the reducing saccharide (e.g., Glc or D-Glc). The linkage (e.g., glycosidic linkage, galactosidic linkage, glucosidic linkage) between two sugar units can be expressed, for example, as 1,4, 1->4, or (1-4).
Both FOS and GOS are non-digestible saccharides. (3 glycosidic linkages of saccharides, such as those found in, but not limited to, FOS and GOS, make these prebiotics mainly non-digestible and unabsorbable in the stomach and small intestine α-linked GOS (a-GOS) is also not hydrolyzed by human salivary amylase, but can be used by Bifidobacterium bifidum and Clostridium butyricum (Yamashita et al. (2004) J. APPL. GLYCOSCI. 51: 115-122). FOS and GOS can pass through the small intestine and into the large intestine (colon) mostly intact, except where commensal microbes and microbes administered as part of a pharmaceutical composition are able to metabolize the oligosaccharides.
GOS (also known as galacto-oligosaccharides, galactooligosaccharides, trans-oligosaccharide (TOS), trans-galacto-oligosaccharide (TGOS), and trans-galactooligosaccharide) are oligomers or polymers of galactose molecules ending mainly with a glucose or sometimes ending with a galactose molecule and have varying degree of polymerization (generally the DP is between 2-20) and type of linkages. In one embodiment, GOS comprises galactose and glucose molecules. In another embodiment, GOS comprises only galactose molecules. In a further embodiment, GOS are galactose-containing oligosaccharides of the form of [β-D-Gal-(1-6)]n-β-D-Gal-(1-4)-D-Glc wherein n is 2-20. In another embodiment, GOS are galactose-containing oligosaccharides of the form Glc α1-4-[β Gal 1-6)]n where n=2-20. In another embodiment, GOS are in the form of α-D-Glc (1-4)-[β-D-Gal-(1-6)-]n where n=2-20. Gal is a galactopyranose unit and Glc (or Glu) is a glucopyranose unit.
In one embodiment, a prebiotic composition comprises a GOS-related compound. A GOS-related compound can have the following properties: a) a “lactose” moiety; e.g., GOS with a gal-glu moiety and any polymerization value or type of linkage; or b) be stimulatory to “lactose fermenting” microbes in the human GI tract; for example, raffinose (gal-fru-glu) is a “related” GOS compound that is stimulatory to both lactobacilli and bifidobacteria.
In one embodiment, a prebiotic composition comprises GOS with a low degree of polymerization. In one embodiment a prebiotic composition comprising GOS with a low degree of polymerization increases growth of probiotic and select commensal bacteria to a greater extent than an equivalent amount of a prebiotic composition comprising GOS with a high degree of polymerization. In one embodiment, a prebiotic composition comprising a high percentage of GOS with a low degree of polymerization increases growth of probiotic and beneficial commensal bacteria to a greater extent than an equivalent amount of a prebiotic composition comprising a low percentage of GOS with a low degree of polymerization (DP). In one embodiment a prebiotic composition comprises GOS with a DP less than 20, such as less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3. In another embodiment a prebiotic composition comprising GOS with a low DP increases growth of co-formulated or co-administered microbes and/or beneficial commensal microbes in the GI tract of a subject.
Linkages between the individual sugar units found in GOS and other oligosaccharides include β-(1-6), β-(1-4), β-(1-3) and β-(1-2) linkages. In one embodiment, the administered oligosaccharides (e.g., GOS) are branched saccharides. In another embodiment, the administered oligosacchardies (e.g, GOS) are linear saccharides.
In some embodiments, the GOS comprises a disaccharide Gal α (1-6) Gal, at least one trisaccharide selected from Gal β(1-6)-Gal β(1-4)-Glc and Gal β(1-3)-Gal β(1-4)-Glc, the tetrasaccharide Gal β(1-6)-Gal β(1-6)-Gal β(1-4)-Glc and the pentasaccharide Gal β(1-6)-Gal β (1-6)-Gal β(1-6)-Gal β(1-4)-Glc.
In one embodiment, a GOS composition is a mixture of 10 to 45% w/v disaccharide, 10 to 45% w/v trisaccharide, 10 to 45% w/v tetrasaccharide and 10 to 45% w/v pentasaccharide. In another embodiment, a GOS composition is a mixture of oligosaccharides comprising 20-28% by weight of β(1-3) linkages, 20-25% by weight of β(1-4) linkages, and 45-55% by weight of β (1-6) linkages. In one embodiment, a GOS composition is a mixture of oligosaccharides comprising 26% by weight of β(1-3) linkages, 23% by weight of β(1-4) linkages, and 51% by weight of β(1-6) linkages.
Alpha-GOS (also called alpha-bond GOS or alpha-linked GOS) are oligosaccharides having an alpha-galactopyranosyl group. Alpha-GOS comprises at least one alpha glycosidic linkage between the saccharide units. Alpha-GOS are generally represented by α-(Gal)n (n usually represents an integer of 2 to 10) or α-(Gal)n Glc (n usually represents an integer of 1 to 9). Examples include a mixture of α-galactosylglucose, α-galactobiose, α-galactotriose, α-galactotetraose, and higher oligosaccharides. Additional non-limiting examples include melibiose, manninootriose, raffinose, stachyose, and the like, which can be produced from beat, soybean oligosaccharide, and the like.
Commercially available and enzyme synthesized alpha-GOS products are also useful for the compositions described herein. Synthesis of alpha-GOS with an enzyme is conducted utilizing the dehydration condensation reaction of α-galactosidase with the use of galactose, galactose-containing substance, or glucose as a substrate. The galactose-containing substance includes hydrolysates of galactose-containing substances, for example, a mixture of galactose and glucose obtained by allowing beta-galactosidase to act on lactose, and the like. Glucose can be mixed separately with galactose and be used as a substrate with α-galactosidase (see e.g., WO 02/18614). Methods of preparing alpha-GOS have been described (see, e.g., EP 1514551 and EP 2027863).
In one embodiment, a GOS composition comprises a mixture of saccharides that are alpha-GOS and saccharides that are produced by transgalactosylation using β-galactosidase. In another embodiment, GOS comprises alpha-GOS. In another embodiment, alpha-GOS comprises α-(Gal)2 from 10% to 100% by weight. In one embodiment, GOS comprises only saccharides that are produced by transgalactosylation using β-galactosidase.
In one embodiment, a GOS composition can comprise GOS with alpha linkages and beta linkages.
In one embodiment, the pharmaceutical composition, dosage form, or kit comprises, in addition to one or more microbes, an oligosaccharide composition that is a mixture of oligosaccharides comprising 1-20% by weight of di-saccharides, 1-20% by weight tri-saccharides, 1-20% by weight tetra-saccharides, and 1-20% by weight penta-saccharides. In another embodiment, an oligosaccharide composition is a mixture of oligosaccharides consisting essentially of 1-20% by weight of di-saccharides, 1-20% by weight tri-saccharides, 1-20% by weight tetra-saccharides, and 1-20% by weight penta-saccharides.
In one embodiment, a prebiotic composition is a mixture of oligosaccharides comprising 1-20% by weight of saccharides with a degree of polymerization (DP) of 1-3, 1-20% by weight of saccharides with DP of 4-6, 1-20% by weight of saccharides with DP of 7-9, and 1-20% by weight of saccharides with DP of 10-12, 1-20% by weight of saccharides with DP of 13-15.
In another embodiment, a prebiotic composition comprises a mixture of oligosaccharides comprising 50-55% by weight of di-saccharides, 20-30% by weight tri-saccharides, 10-20% by weight tetra-saccharide, and 1-10% by weight penta-saccharides. In one embodiment, a GOS composition is a mixture of oligosaccharides comprising 52% by weight of di-saccharides, 26% by weight tri-saccharides, 14% by weight tetra-saccharide, and 5% by weight penta-saccharides. In another embodiment, a prebiotic composition comprises a mixture of oligosaccharides comprising 45-55% by weight tri-saccharides, 15-25% by weight tetra-saccharides, 1-10% by weight penta-saccharides.
In certain embodiments, the composition according to the invention comprises a mixture of neutral and acid oligosaccharides as disclosed in PCT Application WO 2005/039597 (N. V. Nutricia) and US Patent Application 2015/0004130, which are hereby incorporated by reference. In one embodiment, the acid oligosaccharide has a degree of polymerization (DP) between 1 and 5000. In another embodiment, the DP is between 1 and 1000. In another embodiment, the DP is between 2 and 250. If a mixture of acid oligosaccharides with different degrees of polymerization is used, the average DP of the acid oligosaccharide mixture is preferably between 2 and 1000. The acid oligosaccharide may be a homogeneous or heterogeneous carbohydrate. The acid oligosaccharides may be prepared from pectin, pectate, alginate, chondroitine, hyaluronic acids, heparin, heparane, bacterial carbohydrates, sialoglycans, fucoidan, fucooligosaccharides or carrageenan, and are preferably prepared from pectin or alginate. The acid oligosaccharides may be prepared by the methods described in PCT Application WO 01/60378, which is hereby incorporated by reference. The acid oligosaccharide is preferably prepared from high methoxylated pectin, which is characterized by a degree of methoxylation above 50%. As used herein, “degree of methoxylation” (also referred to as DE or “degree of esterification”) is intended to mean the extent to which free carboxylic acid groups contained in the polygalacturonic acid chain have been esterified (e.g. by methylation). In some embodiments, the acid oligosaccharides have a degree of methoxylation above about 10%, above about 20%, above about 50%, above about 70%. In some embodiments, the acid oligosaccharides have a degree of methylation above about 10%, above about 20%, above about 50%, above about 70%.
The term neutral oligosaccharides as used in the present invention refers to saccharides which have a degree of polymerization of monose units exceeding 2, exceeding 3, exceeding 4, or exceeding 10, which are not or only partially digested in the intestine by the action of acids or digestive enzymes present in the human upper digestive tract (small intestine and stomach) but which are fermented by the human intestinal flora and preferably lack acidic groups. The neutral oligosaccharide is structurally (chemically) different from the acid oligosaccharide. The term “neutral oligosaccharides”, as used herein, preferably refers to saccharides which have a degree of polymerization of the oligosaccharide below 60 monose units. The term “monose units” refers to units having a closed ring structure, e.g., the pyranose or furanose forms. In some embodiments, the neutral oligosaccharide comprises at least 90% or at least 95% monose units selected from the group consisting of mannose, arabinose, fructose, fucose, rhamnose, galactose, -D-galactopyranose, ribose, glucose, xylose and derivatives thereof, calculated on the total number of monose units contained therein. Suitable neutral oligosaccharides are preferably fermented by the gut flora. Nonlimiting examples of suitable neutral oligosaccharides are cellobiose (4-O-β-D-glucopyranosyl-D-glucose), cellodextrins ((4-O-β-D-glucopyranosyl)n-D-glucose), B-cyclo-dextrins (cyclic molecules of α-1-4-linked D-glucose; a-cyclodextrin-hexamer, β-cyclodextrin-heptamer and γ-cyclodextrin-octamer), indigestible dextrin, gentiooligosaccharides (mixture of β-1-6 linked glucose residues, some 1-4 linkages), glucooligosaccharides (mixture of α-D-glucose), isomaltooligosaccharides (linear α-1-6 linked glucose residues with some 1-4 linkages), isomaltose (6-O-α-D-glucopyranosyl-D-glucose); isomaltriose (6-O-α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyl-D-glucose), panose (6-O-α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyl-(1-4)-D-glucose), leucrose (5-O-α-D-glucopyranosyl-D-fructopyranoside), palatinose or isomaltulose (6-O-α-D-glucopyranosyl-D-fructose), theanderose (O-α-D-glucopyranosyl-(1-6)-O-α-D-glucopyranosyl-(1-2)-B-D-fructo furanoside), D-agatose, D-lyxo-hexylose, lactosucrose (O-β-D-galactopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-2)-β-D-fructofuranoside), α-galactooligosaccharides including raffinose, stachyose and other soy oligosaccharides (O-α-D-galactopyranosyl-(1-6)-α-D-glucopyranosyl-β-D-fructofuranoside), β-galactooligosaccharides or transgalacto-oligosaccharides (β-D-galactopyranosyl-(1-6)-[β-D-glucopyranosyl]n-(1-4) α-D glucose), lactulose (4-O-β-D-galactopyranosyl-D-fructose), 4′-galatosyllactose (O-D-galactopyranosyl-(1-4)-O-β-D-glucopyranosyl-(1-4)-D-glucopyranose), synthetic galactooligosaccharide (neogalactobiose, isogalactobiose, galsucrose, isolactose I, II and III), fructans-Levan-type (β-D-(2→6)-fructofuranosyl)nα-D-glucopyranoside), fructans-Inulin-type (β-D-((2→1)-fructofuranosyl)nα-D-glucopyranoside), 1 f-β-fructofuranosylnystose (β-D-((2→1)-fructofuranosyl)n B-D-fructofuranoside), xylooligo-saccharides (B-D-((1→4)-xylose)n, lafinose, lactosucrose and arabinooligosaccharides.
In some embodiments, the neutral oligosaccharide is selected from the group consisting of fructans, fructooligosaccharides, indigestible dextrins galactooligo-saccharides (including transgalactooligosaccharides), xylooligosaccharides, arabinooligo-saccharides, glucooligosaccharides, mannooligosaccharides, fucooligosaccharides and mixtures thereof.
Suitable oligosaccharides and their production methods are further described in Laere K. J. M. (Laere, K. J. M., Degradation of structurally different non-digestible oligosaccharides by intestinal bacteria: glycosylhydrolases of Bi. adolescentis. PhD-thesis (2000), Wageningen Agricultural University, Wageningen, The Netherlands), the entire content of which is hereby incorporated by reference. Transgalactooligosaccharides (TOS) are for example sold under the trademark VivinalTM (Borculo Domo Ingredients, Netherlands). Indigestible dextrin, which may be produced by pyrolysis of corn starch, comprises α(1→4) and α(1→6) glucosidic bonds, as are present in the native starch, and contains 1→2 and 1→3 linkages and levoglucosan. Due to these structural characteristics, indigestible dextrin contains well-developed, branched particles that are partially hydrolysed by human digestive enzymes. Numerous other commercial sources of indigestible oligosaccharides are readily available and known to skilled persons in the art. For example, transgalactooligosaccharide is available from Yakult Honsha Co., Tokyo, Japan. Soybean oligosaccharide is available from Calpis Corporation distributed by Ajinomoto U.S.A. Inc., Teaneck, N.J.
In a further preferred embodiment, the pharmaceutical composition contains a prebiotic mixture of an acid oligosaccharide with a DP between 1 and 5000, prepared from pectin, alginate, and mixtures thereof; and a neutral oligosaccharide, selected from the group of fructans, fructooligosaccharides, indigestible dextrins, galactooligosaccharides including transgalacto-oligosaccharides, xylooligosaccharides, arabinooligosaccharides, glucooligosaccharides, manno-oligosaccharides, fucooligosaccharides, and mixtures thereof.
In certain embodiments, the prebiotic mixture comprises xylose. In other embodiments, the prebiotic mixture comprises a xylose polymer (i.e. xylan). In some embodiments, the prebiotic comprises xylose derivatives, such as xylitol, a sugar alcohol generated by reduction of xylose by catalytic hydrogenation of xylose, and also xylose oligomers (e.g., xylooligosaccharide). While xylose can be digested by humans, via xylosyltransferase activity, most xylose ingested by humans is excreted in urine. In contrast, some microorganisms are efficient at xylose metabolism or may be selected for enhanced xylose metabolism. Microbial xylose metabolism may occur by at least four pathways, including the isomerase pathway, the Weimburg pathway, the Dahms pathway, and, for eukaryotic microorganisms, the oxido-reductase pathway.
The xylose isomerase pathway involves the direct conversion of D-xylose into D-xylulose by xylose isomerase, after which D-xylulose is phosphorylated by xylulose kinase to yield D-xylolose-5-phosphate, an intermediate of the pentose phosphate pathway.
In the Weimberg pathway, D-xylose is oxidized to D-xylono-lactone by a D-xylose dehydrogenase. Then D-xylose dehydrogenase is hydrolyzed by a lactonase to yield D-xylonic acid, and xylonate dehydratase activity then yields 2-keto-3-deoxy-xylonate. The final steps of the Weimberg pathway are a dehydratase reaction to form 2-keto glutarate semialdehyde and an oxidizing reaction to form 2-ketoglutarate, an intermediate of the Krebs cycle.
The Dahms pathway follows the same mechanism as the Weimberg pathway but diverges once it has yielded 2-keto-3-deoxy-xylonate. In the Dahms pathway, an aldolase splits 2-keto-3-deoxy-xylonate into pyruvate and glycolaldehyde.
The xylose oxido-reductase pathway, also known as the xylose reductase-xylitol dehydrogenase pathway, begins by the reduction of D-xylose to xylitol by xylose reductase followed by the oxidation of xylitol to D-xylulose by xylitol dehydrogenase. As in the isomerase pathway, the next step in the oxido-reductase pathway is the phosphorylation of D-xylulose by xylulose kinase to yield D-xylolose-5-phosphate.
Xylose is present in foods like fruits and vegetables and other plants such as trees for wood and pulp production. Thus, xylose can be obtained in the extracts of such plants. Xylose can be obtained from various plant sources using known processes including acid hydrolysis followed by various types of chromatography. Examples of such methods to produce xylose include those described in Maurelli, L. et al. (2013), Appl. Biochem. Biotechnol. 170:1104-1118; Hooi H. T et al. (2013), Appl. Biochem. Biotechnol. 170:1602-1613; Zhang H-J. et al. (2014), Bioprocess Biosyst. Eng. 37:2425-2436.
Preferably, the metabolism of xylose and/or the shift in microbiota due to the metabolism of the xylose provided in a pharmaceutical composition of the invention confers a benefit to a host, e.g. immunological tolerance. For example, in aspects in which the patient is at risk or suffering from GVHD, the immunological tolerance may reduce graft-versus-host activity while maintaining graft-versus-leukemia activity. In another example, in aspects in which the patient suffers from Celiac disease, the immunological tolerance prevents an inappropriate immune response to gluten. The xylose may be, e.g. i) cytotoxic for an autoimmune disease- and/or inflammatory disease-associated associated pathogen or pathobiont, ii) cytostatic for an autoimmune disease- and/or inflammatory disease-associated pathogen or pathobiont, iii) capable of decreasing the growth of autoimmune disease- and/or inflammatory disease-associated pathogen or pathobiont, iv) capable of inhibiting the growth of an autoimmune disease- and/or inflammatory disease-associated pathogen or pathobiont, v) capable of decreasing the colonization of an autoimmune disease- and/or inflammatory disease-associated pathogen or pathobiont, vi) capable of inhibiting the colonization of an autoimmune disease- and/or inflammatory disease-associated pathogen or pathobiont, vii) capable of eliciting an immunomodulatory response in the host that reduces the risk of an autoimmune and/or inflammatory disorder, viii), capable of eliciting an immunomodulatory response in the host that reduces the severity of an autoimmune and/or inflammatory disorder, ix) capable of promoting barrier integrity directly or indirectly through its impact on microbiota, or x) any combination of i)-ix).
In some embodiments, the pharmaceutical composition or dosage form comprises a bacterial population and xylose in an amount effective to promote the growth of select bacteria of the family Clostridiacea, including members of the genus Clostridium, Ruminococcus, or Blautia or relatives thereof in a host. In some embodiments, the pharmaceutical composition or dosage form is further effective to promote the proliferation of select bacteria of the family Clostridiacea, including members of the genus Clostridium, Ruminococcus, or Blautia or relatives thereof in a host. In certain embodiments, the pharmaceutical composition or dosage form comprises a bacterial population and xylose in an amount effective to promote the colonization and/or engraftment of select bacteria of the family Clostridiacea, including members of the genus Clostridium, Ruminococcus, or Blautia or relatives thereof in a host. In preferred embodiments, the pharmaceutical composition or dosage form is further capable of altering a dysbiotic state such that the growth, proliferation, colonization, and/or engraftment of a host by a pathogen, pathobiont, disease-associated microbe, or a combination thereof such that the population of at least one pathogen, pathobiont, or disease-associated microbe is decreased 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 10000-fold, or over 10000-fold. In one embodiment, the pharmaceutical composition or dosage form is capable of locally or systemically eliminating at least one pathogen, pathobiont, or disease-associated microbe from a host.
In some embodiments, the prebiotic comprises a carbohydrate monomer or polymer that has been modified i.e., substituted with other substituents (e.g., acetyl group, glucuronic acid residue, arabinose residue, or the like) (see US Patent Application 20090148573, hereby incorporated by reference). The term “modified”, as used herein, refers to a molecule modified from a reference molecule, and includes not only artificially produced molecules but also naturally occurring molecules. In preferred embodiments, the modification occurs at one or more hydroxyl groups of the reference carbohydrate. In some embodiments, the modification occurs at carbon-2 (C2), the modification occurs at carbon-6 (C6), or a combination thereof.
In some embodiments, a carbohydrate (a monomer or, preferably, a polymer) is modified with one or more hydrophilic groups. Nonlimiting examples of the hydrophilic groups include an acetyl group, a 4-O-methyl-α-D-glucuronic acid residue, an L-arabinofuranose residue, an L-arabinose residue, and an a-D-glucuronic acid residue. In some embodiments, the modification is the replacement of one or more hydroxyl groups with —H, —CH2OH, —CH3, or —NH2.
In some embodiments, the composition comprises at least one carbohydrate that elicits an immunomodulatory response. Exemplary immunomodulary carbohydrates include (but are not limited to) fructo-oligosaccharides, glycosaminoglycans (e.g., heparin sulfate, chondroitin sulfate A, hyaluronan), 0-glycans, and carrageenan oligosaccharides, and galacto-oligosaccharides. Immunomodulatory carbohydrates may be purified from plants or microbes or may be synthetically derived. Immunomodulatory carbohydrates may be effective to, for example, prevent disease, suppress symptoms, treat disease, or any combination thereof.
In some embodiments, immunomodulatory carbohydrates are C-type lectin receptor ligands. In preferred embodiments, the C-type lectin receptor ligands are produced by one or more fungal species. In other embodiments, the immunomodulatory carbohydrates are bacterial exopolysaccharides, such as (but not limited to) the exopolysaccharides (EPS) produced by Bacillus subtilis, Bifidobacterium breve, or Bacteroides fragilis. In some aspects, immunomodulatory carbohydrates are zwitterionic polysaccharides. In some aspects, immunomodulatory carbohydrates modulate toll-like receptor 2 (TLR2) and/or toll-like receptor 4 (TLR4) responses in a host. For example, autoimmune or inflammatory diseases characterized by intestinal inflammation may be prevented by a TLR4 agonist such as but not limited to B. subtilis EPS (see, e.g., Jones et al. (2014) J. IMMUNOL. 192: 4813-4820). Immunomodulatory carbohydrates may also activate CD4+ T cells and/or lead to an upregulation of the anti-inflammatory cytokine interleukin-10 (Mazmanian and Kasper (2006) NAT. REV. IMMUNOL. 6: 849-858). Immunomodulatory carbohydrates may be selected for administration to a patient based on the presence, abundance, distribution, modification and/or linkages of sugar residues. For example, immunomodulatory carbohydrates used in the prevention of intestinal disorders or autoimmune conditions that manifest in the gut (non-limiting examples being IBD and GVHD) may be selected based on i) a high abundance of mannose residues; ii) the presence of terminal mannopyransosyl (t-Man) residues and/or 2,6 linked mannopyranosyl residues (2,6-Man), iii) a ratio of mannose to glucose residues in the approximate range of 8:2 to 9:1, iv) the presence of galactose residues, v) areas of positive charge, or vi) a combination thereof.
Carbohydrates may be selected according to the fermentation or metabolic preferences of a microbe (e.g., an anti-inflammatory bacterial cell) selected for administration to a mammalian subject. Selection criteria include but are not limited to sugar complexity (e.g., monosaccharides, including but not limited to glucose, versus oligosaccharides or starches) as well as by desired end-product. Non-liming examples include the fermentation products ethanol and carbon dioxide (CO2) (e.g., via ethanol fermentation by Saccharomyces sp. Zymomonas sp.), lactate (e.g., via homolactic acid fermentation by Lactococcus sp., Streptococcus sp., Enterococcus sp., Pediococcus sp. and some species Lactobacillus), lactate, ethanol, and CO2 (e.g., via heterolactic acid fermentation (which includes the phosphoketolase pathway) by some species of Lactobacillus as well as Leuconostoc sp., Oenococcus sp., and Weissella sp.), butanol, acetone, CO2 and H2 (via acetone-butanol fermentation by some Clostridium sp.), and short chain fatty acids (with or without the production of other products) (see, e.g., Muller (2011) Bacterial Fermentation. Encyclopedia of Life Sciences). Examples of fermentation leading to short chain fatty acid production include homoacetic acid fermentation (e.g., by Acetobacterium sp., and resulting in acetate), propionic acid fermentation (e.g., by Propionibacterium sp., and resulting in propionate, acetate and CO2) mixed acid fermentation (e.g., by Escherichia sp., and resulting in ethanol, lactate, acetate, succinate, formate, CO2, and H2), butyrate fermentation (e.g., by some Clostridium sp., resulting in butyrate, CO2, and H2), and 2,3-butanediol fermentation (e.g., by Enterobacter sp., resulting in ethanol, butanediol, lactate, formate, CO2, and H2). In some embodiments, selection of carbohydrates for co-formulation of co-administration with a type of microbe or types of microbe may be achieved by computational analysis of microbial enzymatic pathways, including but not limited to the presence of metabolic/fermentation pathway enzymes.
Other prebiotics include molecules capable of selective or semi-selective utilization by microbes (e.g., bacterial cells) of the compositions contained herein. The ability of a microbe to utilize a metabolite of interest is determined by the genomic capacity of that microbe. Public databases have characterized many microbes and automate the annotation of the genome to allow a computational analysis of the metabolites a microbe is potentially able to utilize. Databases such as the Cluster of Orthologous Groups (COGs) database characterize genomes from a variety of species in this manner and are capable of characterizing newly sequenced genomes as well (e.g. see in this fashion (Tatusov et al. (2000) NUCL. ACID RES. 28(1): 33-36). Furthermore, pathway analysis classifies COGs into different categories with associated one letter codes including J, translation; L replication, recombination, and repair, K transcription; 0 molecular chaperones and related functions, M, cell wall structure and biogenesis and outer membrane, N secretion motility and chemotaxis; T signal Transduction; P inorganic ion transport and metabolism; C energy production and conversion; G, carbohydrate metabolism and transport; E amino acid metabolism and transport; F, nueclotide metabolism and transport; D cell Division and chromosome partitioning; R general functional prediction. In preferred embodiments, COGs of the categories, N, M, P, C, G, E, and F are selected as preferred COGs to both provide enhanced growth on specific substrates and modified behaviors relevant for anti-tumor properties.
COGs are selected to be specific or semi enriched in the host or other microbes within a host by searching for specific functions present in the microbe of interest but absent from a large set of other competition organisms. Tissue specific analysis of the host for enzymes expressed within a tissue is performed to identify tissue specific enzymatic activities in the host. Specific functions are absent from at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30% at least 20% or at least 10% of the other organisms selected from the group of the host, the host tissue, the disease-associated microbiota, the host gut microbiota, the host niche specific to the engraftment of the microbial composition (e.g., GI tract, skin).
Once these COGs are identified, databases like KEGG are used to link the enzymatic functions to identify the metabolites that are substrates for these selective COGs. Furthermore, the selective analysis to generate selective metabolites is repeated on the set of substrate of COGs to validate that the pathways and metabolites are selective to the desired microbial composition.
Methods of the Invention
In one aspect, the invention provides methods for modulating an immune response in a subject in need thereof, the method comprising administering a pharmaceutical composition of the invention to thereby modulate the immune response in the subject. In some embodiments, the immune response is against a microorganism. In some embodiments, the immune response is against self (e.g., an auto-immune response). In some embodiments, the immune response is a pro-inflammatory immune response.
In another aspect, the invention provides methods for reducing inflammation in a subject in need thereof, the method comprising administering a pharmaceutical composition of the invention to thereby reduce inflammation in the subject. In some embodiments, the immune response is against a microorganism. In some embodiments, the subject has an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from the group consisting of graft-versus-host disease (GVHD), an inflammatory bowel disease (IBD), ulterative colitis, Crohn's disease, multiple sclerosis (MS), systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, Sjögren's syndrome, and Celiac disease. In an exemplary embodiments, the autoimmune or inflammatory disorder is GVHD. In another exemplary embodiment, the autoimmune or inflammatory disorder is IBD. In yet another exemplary embodiment, the autoimmune or inflammatory disorder is ulcerative colitis. In an exemplary embodiment, the autoimmune or inflammatory disorder is Crohn's disease. In another exemplary embodiment, the autoimmune or inflammatory disorder is multiple sclerosis (MS). In yet another embodiment, the autoimmune or inflammatory disorder is systemic lupus erythematosus. In an exemplary embodiment, the autoimmune or inflammatory disorder is type I diabetes. In another exemplary embodiment, the autoimmune or inflammatory disorder is rheumatoid arthritis. In yet another exemplary embodiment, the autoimmune or inflammatory disorder is rheumatoid arthritis. In an exemplary embodiment, the autoimmune or inflammatory disorder is Sjögren's syndrome. In another exemplary embodiment, the autoimmune or inflammatory disorder is Celiac disease.
Autoimmune and inflammatory diseases that may be treated with the pharmaceutical compositions of the present invention, include, but are not limited to: Acute Disseminated Encephalomyelitis, Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, adhesive capsulitis, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM nephritis, Anti-TBM nephritis, Antiphospholipid syndrome, arthofibrosis, atrial fibrosis, autoimmune angioedema, autoimmune aplastic anemia, autoimmune dusautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathies, Balo disease, Behçet's disease, benign mucosal pemphigold, Bullous pemphigold, cardiomyopathy, Castleman disease, Celiac Disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy, chronic Lyme disease, chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigold, cirrhosis, Cogans syndrome, cold agglutinin disease, congenital heart block, Coxsackle myocarditis, CREST disease, Crohn's disease, Cystic Fibrosis, essential mixed cryoglobulinemia, deficiency of the interleukin-1 receptor antagonist, demyelinating neuropathies, dermatitis herpetiformis, dermatomyosis, Devic's disease, discoid lupus, Dressler's syndrome, Dupuytren's contracture, endometriosis, endomyocardial fibrosis, eosinophilic esophagitis, eosinophilic facsciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, Familial Mediterranean Fever, fibromyalgia, fibrosing alveolitis, giant cell arteritis, giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, Graft-versus-host disease (GVHD), granulomatosus with polyanglitis, Graves' disease, Guillain-Bare syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, hepatitis, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, inflammatory bowel disorders, interstitial cystitis, juvenile arthritis, juvenile myositis, Kawasaki syndrome, keloid, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease, mediastinal fibrosis, Meniere's disease, microscopic polyanglitis, mixed connective tissue disease, Mooren's ulcer, Mucha-Hamermann disease, Multiple Sclerosis (MS), Myasthenia gravis, myelofibrosis, Myositis, narcolepsy, Neonatal Onset Multisystem Inflammatory Disease, nephrogenic systemic fibrosis, neutropenia, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis (NASH), ocular-cicatricial pemphigold, optic neuritis, palindromic rheumatism, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS), paraneoplastic cerebellar degeneration, paroxysmal nocturnal nemoglobinuria, Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus, Peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, Peyronie's disease, POEMS syndrome, polyarteritis nodosa, progressive massive fibrosis, Tumor Necrosis Factor Receptor-associated Periodic Syndrome, Type I autoimmune polyglandular syndrome, Type II autoimmune polyglandular syndrome, Type III autoimmune polyglandular syndrome, polymyalgia rhematica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynauds phenomenon, reactic arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis, Susac's syndrome, sympathetic ophthalmia, systemic lupus erythematosus (SLE), Takayasu's arthritis, temporal arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, Type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vesiculobullous dermatosis, and Vitiligo.
The microbes described herein may additively or synergistically reduce the number of types of autoimmune disease- or inflammatory disease-associated pathogens or pathobionts either distally—e.g., orally-administered microbes reduce the total microbial burden in an organ not in the gastrointestinal tract, or intravaginally-administered microbes reduce the total microbial burden in an organ that is not the vagina—or locally, e.g., the intestines or vagina, respectively. Distal sites include but are not limited to the liver, spleen, fallopian tubes and uterus.
Similarly, the microbes described herein may additively or synergistically elicit an immunomodulatory response either distally, e.g., in which enteral administration of microbes results in altering the immune response at the skin or liver, or locally, e.g., the enteral administration of microbes results in altering the immune response in the intestines.
In some situations, the recipient subject is immunocompromised or immunosuppressed, or is at risk of developing an immune or inflammatory disorder.
In embodiments, the microbial composition is administered enterically, with or without prebiotics. This preferentially includes oral administration, or by an oral or nasal tube (including nasogastric, nasojejunal, oral gastric, or oral jejunal). In other embodiments, administration includes rectal administration (including enema, suppository, or colonoscopy). The pharmaceutical composition may be administered to at least one region of the gastrointestinal tract, including the mouth, esophagus, stomach, small intestine, large intestine, and rectum. In some embodiments, the pharmaceutical composition is administered to all regions of the gastrointestinal tract. The pharmaceutical compositions may be administered orally in the form of medicaments such as powders, capsules, tablets, gels or liquids. The pharmaceutical compositions may also be administered in gel or liquid form by the oral route or through a nasogastric tube, or by the rectal route in a gel or liquid form, by enema or instillation through a colonoscope or by a suppository. In some embodiments, the pharmaceutical composition of the invention is administered enterically with one ore more prebiotics.
If the composition is administered colonoscopically and, optionally, if the microbial composition, with or without one or more prebiotics, is administered by other rectal routes (such as an enema or suppository) or even if the subject has an oral administration, the subject may have a colonic-cleansing preparation. The colon-cleansing preparation can facilitate proper use of the colonoscope or other administration devices, but even when it does not serve a mechanical purpose it can also maximize the proportion of bacteria from the pharmaceutical composition relative to the other organisms previously residing in the gastrointestinal tract of the subject. Any ordinarily acceptable colonic-cleansing preparation may be used such as those typically provided when a subject undergoes a colonoscopy.
Pretreatment Protocols. Prior to administration of the pharmaceutical composition, with or without one or more prebiotics, the subject can optionally have a pretreatment protocol to prepare the gastrointestinal tract or vagina to receive the pharmaceutical composition. In these instances, the pretreatment protocol can enhance the ability of the pharmaceutical composition to affect the patient's microbiome.
As one way of preparing the patient for administration of the microbial ecosystem, at least one antibiotic can be administered to alter the bacteria in the patient. As another way of preparing the patient for administration of the microbial ecosystem, a standard colon-cleansing preparation can be administered to the patient to substantially empty the contents of the colon, such as used to prepare a patient for a colonscopy. By “substantially emptying the contents of the colon,” this application means removing at least 75%, at least 80%, at least 90%, at least 95%, or about 100% of the contents of the ordinary volume of colon contents. Antibiotic treatment can precede the colon-cleansing protocol.
If a patient has received an antibiotic for treatment of an infection, or if a patient has received an antibiotic as part of a specific pretreatment protocol, in one embodiment, the antibiotic can be stopped in sufficient time to allow the antibiotic to be substantially reduced in concentration in the gut or vagina before the pharmaceutical composition is administered. In one embodiment, the antibiotic can be discontinued 1, 2, or 3 days before the administration of the pharmaceutical composition. In another embodiment, the antibiotic can be discontinued 3, 4, 5, 6, or 7 antibiotic half-lives before administration of the pharmaceutical composition. In another embodiment, the antibiotic can be chosen so the bacterial constituents in the pharmaceutical composition have an MIC50 that is higher than the concentration of the antibiotic in the gut or vagina.
MIC50 of the bacterial constituents in the pharmaceutical composition can be determined by methods well known in the art (see, e.g., Reller et al. (2009) CLINICAL INFECTIOUS DISEASES 49(11):1749-1755). In such an embodiment, the additional time between antibiotic administration and administration of the pharmaceutical composition is not necessary. If the pretreatment protocol is part of treatment of an acute infection, the antibiotic can be chosen so that the infection is sensitive to the antibiotic, but the bacterial constituents in the pharmaceutical composition are not sensitive to the antibiotic.
Routes of Administration. Compositions can be administered by any route suitable for the delivery of disclosed compositions for treating, inhibiting, or preventing a dysbiosis, or an diseases and disorders associated with a dysbiosis, include, but are not limited to orally, sublingually, rectally, parentally (e.g., intravenous injection (i.v.), intracranial injection (i.e.); intramuscular injection (i.m.), intraperitoneal injection (i.p.), and subcutaneous injection (s.c.) and intraosseous infusion (i.o.)), transdermally, extracorporeally, inhalation, topically or the like, including topical intranasal administration or administration by inhalant.
In some embodiments, the subject is fed a meal within one hour of administration of the pharmaceutical composition. In another embodiment, the subject is fed a meal concurrently with administration of the pharmaceutical composition.
In some embodiments, the therapeutic composition is administered at intervals greater than two days, such as once every three, four, five or six days, or every week or less frequently than every week. In other embodiments, the preparation is administered intermittently according to a set schedule, e.g., once a day, once weekly, or once monthly, or when the subject relapses from the primary illness.
In certain embodiments, the pharmaceutical composition is administered enterically. This preferentially includes oral administration, or by an oral or nasal tube (including nasogastric, nasojejunal, oral gastric, or oral jejunal). In other embodiments, administration includes rectal administration (including enema, suppository, or colonoscopy). The pharmaceutical composition can be administered to at least one region of the gastrointestinal tract, including the mouth, esophagus, stomach, small intestine, large intestine, and rectum. In some embodiments, it is administered to all regions of the gastrointestinal tract. The pharmaceutical compositions can be administered orally in the form of medicaments such as powders, capsules, tablets, gels or liquids. The bacterial compositions can also be administered in gel or liquid form by the oral route or through a nasogastric tube, or by the rectal route in a gel or liquid form, by enema or instillation through a colonoscope or by a suppository. In certain embodiments of the above invention, the microbial composition is administered enterically with one or more prebiotics.
If the composition is administered colonoscopically and, optionally, if the composition is administered by other rectal routes (such as an enema or suppository) or even if the subject has an oral administration, the subject can have a colon-cleansing preparation. The colon-cleansing preparation can facilitate proper use of the colonoscope or other administration devices, but even when it does not serve a mechanical purpose, it can also maximize the proportion of the bacterial cells in the pharmaceutical composition relative to the other organisms previously residing in the gastrointestinal tract of the subject. For example, the colon cleansing preparation may maximize the amount of bacterial entities of the bacterial composition that reach and/or engraft in the gastrointestinal tract of the subject.
Dosages and Schedule for Administration. In some embodiments, the pharmaceutical compositions are provided in a dosage form. In certain embodiments, the dosage form is designed for administration of at least one OTU or combinations thereof disclosed herein, wherein the total amount of pharmaceutical composition administered is selected from 0.1 ng to 10 g, 10 ng to 1 g, 100 ng to 0.1 g, 0.1 mg to 500 mg, 1 mg to 100 mg, or from 10-15 mg. In other embodiments, the pharmaceutical composition is consumed at a rate of from 0.1 ng to 10 g a day, 10 ng to 1 g a day, 100 ng to 0.1 g a day, 0.1 mg to 500 mg a day, 1 mg to 100 mg a day, or from 10-15 mg a day, or more.
In certain embodiments, the treatment period is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year. In some embodiments the treatment period is from 1 day to 1 week, from 1 week to 4 weeks, from 1 month, to 3 months, from 3 months to 6 months, from 6 months to 1 year, or for over a year.
In one embodiment, between about 105 and about 1012 CFUs total can be administered to the patient in a given dosage form. In another embodiment, an effective amount can be provided in from 1 to 500 ml or from 1 to 500 grams of the pharmaceutical composition having from 107 to 1011 bacteria per ml or per gram, or, for example, a capsule, tablet or suppository may contain from 1 mg to 1000 mg lyophilized powder having from 107 to 1011 CFUs. Those receiving acute treatment can receive higher doses than those who are receiving chronic administration (such as hospital workers or those admitted into long-term care facilities).
Any of the pharmaceutical compositions described herein can be administered once on a single occasion or on multiple occasions, such as once a day for several days or more than once a day on the day of administration (including twice daily, three times daily, or up to five times daily). In another embodiment, the preparation can be administered intermittently according to a set schedule, e.g., once weekly, once monthly, or when the patient relapses from the primary illness.
Combination Therapy. The pharmaceutical compositions, with or without one or more prebiotics, can be administered with other agents in a combination therapy mode, including anti-microbial agents. Administration can be sequential, over a period of hours or days, or simultaneous.
In one embodiment, the microbial compositions, with or without one or more prebiotics, are included in combination therapy with one or more anti-microbial agents, which include anti-bacterial agents, anti-fungal agents, anti-viral agents and anti-parasitic agents.
Anti-bacterial agents can include cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).
Anti-viral agents can include Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, Foscarnet, Fomivirsen, Ganciclovir, Indinavir, Idoxuridine, Lamivudine, Lopinavir Maraviroc, MK-2048, Nelfinavir, Nevirapine, Penciclovir, Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine, Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine, Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir, Zalcitabine, Zanamivir and Zidovudine.
Anti-fungal agents include, but are not limited, to polyene antifungals such as natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, and hamycin; imidazole antifungals such as miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole; triazole antifungals such as fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, and albaconazole; thiazole antifungals such as abafungin; allylamine antifungals such as terbinafine, naftifine, and butenafine; and echinocandin antifungals such as anidulafungin, caspofungin, and micafungin. Other compounds that have antifungal properties include, but are not limited to polygodial, benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine or 5-fluorocytosine, griseofulvin, and haloprogin.
In one embodiment, the pharmaceutical compositions are administered in combination with one or more corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines, and combinations thereof.
The pharmaceutical compositions described herein have beneficial effects for the subject locally, at the site of administration (e.g., in the gastrointestinal tract for compositions administered orally, or in the vagina for compositions administered vaginally), as previously described. Surprisingly, the pharmaceutical compositions described herein may also be used to correct or prevent a dysbiosis at a site distal to the site of administration, intended engraftment, or intended colonization of a composition, e.g., a probiotic composition, of the invention. For example, if a probiotic composition is administered vaginally, a distal effect of the composition would occur outside the vagina. Similarly, if a probiotic composition is administered to the skin, e.g., through a skin patch, transdermal lotion, etc., a distal effect of the composition would occur in a niche other than the skin. If a probiotic composition is administered to the lungs, e.g., in an inhalable formulation, a distal effect of the composition would occur outside the lungs. If a probiotic composition is administered to the ear, eye, nose, etc., a distal effect of the composition would occur at a site other than the site of administration, engraftment, or colonization of the composition (i.e., distal to the ear, distal to the eye, distal to the nose, etc.).
Distal sites include but are not limited to the liver, spleen, fallopian tubes and uterus. Other distal sites include skin, blood and lymph nodes. In other embodiments, the distal site is placenta, spleen, liver, uterus, blood, eyes, ears, lungs, liver, pancreas, brain, embryonic sac, or vagina. In another embodiment, the distal site is vagina, skin, lungs, brain, nose, ear, eyes/conjunctiva, mouth, circulatory system, e.g., blood, placenta, reproductive tract, cardiovascular system, and/or nervous system. A probiotic composition may have an effect on the microbiota of more than one distal site in a subject. For example, in some embodiments, a probiotic composition modulates the microbiota of one or more sites distal to the site of administration, engraftment, or colonization, e.g., one or more of placenta, spleen, liver, uterus, blood, eyes, ears, lungs, liver, pancreas, brain, embryonic sac, vagina, skin, brain, nose, mouth, reproductive tract, cardiovascular system, and/or nervous system. In preferred embodiments, the probiotic composition contains a immunomodulatory bacteria, e.g., a anti-inflammatory bacteria.
Without wishing to be bound by theory, the probiotic compositions of the invention may impact sites distal sites in several ways.
Pharmaceutical compositions described herein can correct or treat a distal dysbiosis by correcting the imbalance in microbial diversity that is present at the distal site. Bacteria contained in the pharmaceutical composition can correct the distal dysbiosis directly, by translocating to the distal site. Bacteria contained in the pharmaceutical composition can also correct the distal dysbiosis indirectly, by promoting translocation of other gut commensals to the distal site, or by modifying the microenvironment of the distal site to create conditions that restore a healthy microbiome, e.g., by reducing inflammation.
A distal dysbiosis includes disruptions in the normal diversity and/or function of the microbial network in a subject at a site other than the gastrointestinal tract, which is generally the site of administration of probiotics provided orally. In cases where a probiotic composition is administered vaginally to a subject, a distal dysbiosis can include disruptions in the normal diversity and/or function of the microbial network in a subject at a site other than the vagina.
In order to characterize a distal dysbiosis, provided are methods of detecting, quantifying and characterizing 16S, 18S and ITS signatures in immune organs, such as the lymph nodes, spleen, etc. Moreover, provided are methods of detecting bacterial and fungal components typically associated with one microbiota in a distal site, often associating (in a physiological or pathological manner) with the microbiota of that distal site. For example, bacteria normally detected in the GI tract or vagina are detected in distal sites, for example, the blood.
In one embodiment, a bacterial strain present in the pharmaceutical composition engrafts in the gastrointestinal tract of a subject, and translocates to a distal site, thereby augmenting the bacterial strain present in the pharmaceutical composition at the distal site. In one embodiment, the bacterial strain present in the pharmaceutical composition is not detectably present at the distal site prior to administration of the pharmaceutical composition.
In another embodiment, a bacterial strain present in the pharmaceutical composition is augmented in the gastrointestinal tract of a subject without engraftment, and translocates to a distal site, thereby augmenting the bacterial strain present in the pharmaceutical composition at the distal site. In one embodiment, the bacterial strain present in the pharmaceutical composition is not detectably present at the distal site prior to administration of the pharmaceutical composition.
In another embodiment, a bacterial strain present in the pharmaceutical composition modulates the microenvironment of the gut, augmenting a second bacterial strain present within the gut microbiota. The second bacterial strain augmented in the gut translocates to a distal site, thereby augmenting the second bacterial strain at the distal site. In embodiments, the second bacterial strain is not present in the pharmaceutical composition. In some embodiments, the bacterial strain present in the pharmaceutical composition is an immunomodulatory bacteria, e.g., an anti-inflammatory bacteria. Modulation of the microenvironment of the gut may include, for example, alteration of cytokines secreted by host cells in and around the gut, reducing inflammation in the gut, increasing secretion of short chain fatty acids in the gut, or altering the proportion of immune cell subpopulations in the gut, each of which impacts the gut microbiome. Modulation of the microenvironment of the gut can include increasing or decreasing overall microbial diversity.
In another embodiment, a bacterial strain present in the pharmaceutical composition modulates the microenvironment at a distal site in a subject, thereby augmenting a second bacterial strain at the distal site. In embodiments, the second bacterial strain is not present in the pharmaceutical composition. In some embodiments, the bacterial strain present in the pharmaceutical composition is an immunomodulatory bacteria, e.g., an anti-inflammatory bacteria. Immunomodulatory bacteria can modulate the microenvironment at a distal site in a subject by, for example, reducing systemic inflammation. This can be achieved by altering the profile of cytokine expression by immune cells which circulate throughout the body, or altering the proportion of immune cell subpopulations which circulate throughout the body. Bacterial strains present in the pharmaceutical composition can also modulate intestinal permeability, e.g., by secretion of short chain fatty acids, which impacts the microenvironment of distal sites. In addition or alternatively, bacterial strains present in the pharmaceutical composition can increase or decrease overall microbial diversity.
Accordingly, the pharmaceutical compositions described herein may additively or synergistically elicit an immunomodulatory response either distally, e.g., in which enteral administration of microbes results in altering the immune response at a site outside the gastrointestinal tract such as the skin or liver, or locally, e.g. the enteral administration of microbes results in altering the immune response in the gastrointestinal tract, e.g., in the intestines.
The immune system of a subject and the microbiome of the subject are closely linked, and interact systemically. Disruptions to the microbiome, both in the gastrointestinal tract and at distal sites, can have profound effects throughout the body of the subject. In particular, disruptions to the microbiome increase systemic inflammation and intestinal barrier dysfunction in a subject. Increased inflammation and intestinal barrier dysfunction negatively impact the health of the subject in many ways, by contributing to a wide range of inflammatory and autoimmune conditions distal to the gastrointestinal tract. Conversely, increased inflammation in a subject leads to disruptions in the subject's microbiome, and disruptions to the microbiome lead in turn to further increases in inflammation. Administration of a pharmaceutical composition containing immunomodulatory bacteria can reduce inflammation in the gastrointestinal tract and restore intestinal barrier integrity, resulting in a reduction in inflammation at sites distal to the gastrointestinal tract, and improvement in the symptoms of autoimmune or inflammatory disorders associated with systemic inflammation. Administration of a pharmaceutical composition containing bacterial strains that secrete short chain fatty acids are also capable of reducing inflammation restoring intestinal barrier integrity.
The pharmaceutical compositions and methods described herein can prevent or treat the loss or reduction of barrier function recognized to occur during dysbiosis or in the shift in one or more microbiotal populations that give rise to the dysbiosis. The loss of barrier function results in systemic seeding of bacterial populations resulting in dysbiotic activity, and in some events, the loss of barrier function results in a local reseeding of the bacterial populations. In both situations, the resulting immune activation leads to pathogenic inflammatory and immune responses. In response, provided are compositions that are capable of restoring barrier function, restoring the normal microbiotal components, and reducing (e.g., suppressing) immune/inflammatory response. In some compositions, provided are antibiotic agents that remove the existing microflora in a target niche, while newly administered or recruited bacteria populate (or re-populate) the target niche. Co-administration or co-formulation with a carbohydrate may synergistically affect this population/repopulation technique.
Disorders associated with a dysbiosis, i.e., a gastrointestinal dysbiosis or a distal dysbiosis, which increases systemic inflammation and/or reduces intestinal barrier integrity include, for example, autoimmune or inflammatory disorders, Crohn's Disease, vaginal dysbiosis, and transplant disorders such as graft-versus-host disease. These disorders can be treated by administration (e.g., oral administration) of pharmaceutical compositions containing immunomodulatory (e.g., anti-inflammatory) bacterial strains.
The pharmaceutical compositions described herein may additively or synergistically reduce the number of types of autoimmune disease- or inflammatory disease-associated pathogens or pathobionts either distally—e.g., orally-administered microbes reduce the total microbial burden in an organ not in the gastrointestinal tract, or intravaginally-administered microbes reduce the total microbial burden in an organ that is not the vagina—or locally, e.g., the intestines or vagina, respectively.
Accordingly, in one aspect, the invention provides a method of reducing inflammation in a subject, comprising administering to the subject a probiotic composition comprising an isolated, anti-inflammatory bacterial population, such that inflammation in the subject is reduced. A systemic reduction in inflammation can modulate the microbiome of niches distal to the site of administration, intended engraftment, or intended colonization of the bacterial population. The probiotic composition can contain an excipient useful for formulation as a pharmaceutical composition. In instances where the bacterial population includes anaerobic bacteria, the excipient can, in one embodiment, reduce exposure of the bacterial population to oxygen.
In a preferred embodiment, administration of the probiotic composition can reduce inflammation at a site distal to the site of administration, engraftment, or colonization, such as, for example, vagina, skin, lungs, brain, nose, ear, eyes/conjunctiva, mouth, circulatory system, e.g., blood, placenta, embryonic sac, reproductive tract, cardiovascular system, and/or nervous system. In one embodiment, administration of the probiotic composition can reduce inflammation at a site selected from blood, skin, vagina, liver, spleen, fallopian tubes, uterus, or a combination thereof. In one embodiment, administration of the probiotic composition modulates the microbiome at a distal site.
The anti-inflammatory bacterial population can induce a decrease in secretion of pro-inflammatory cytokines and/or an increase in secretion of anti-inflammatory cytokines by host cells. The anti-inflammatory properties of the bacterial population can be determined by methods described herein or known in the art, for example, by measuring alterations in cytokine secretion by peripheral blood mononuclear cells (PBMCs) exposed to the bacterial population. Anti-inflammatory bacteria can be selected for inclusion in the probiotic formulation based on modulation of particular cytokines of interest. For example, anti-inflammatory bacteria can be selected based on the ability to decrease secretion of one or more pro-inflammatory cytokines, e.g., IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof, and/or the ability to increase secretion of one or more anti-inflammatory cytokines, e.g., IL-10, IL-13, IL-4, IL-5, TGFβ, and combinations thereof.
In another aspect, the invention provides methods of treating or preventing a distal dysbiosis in a subject, by administering to the subject a probiotic composition comprising an isolated bacterial population in an amount sufficient to alter the microbiome at a site distal to the site of administration, engraftment, or colonization of the bacterial population, such that the distal dysbiosis is treated. For example, administration of the probiotic composition may modulate a first microbiome at the site of administration, engraftment or colonization of the bacterial population, causing subsequent modulation of a second microbiome at a site that is distinct from the first microbiome, e.g., a distal site.
In one embodiment, the invention provides methods of treating or preventing a distal dysbiosis, by orally administering a probiotic composition which alters the microbiome at a site distal to the gastrointestinal tract.
In another aspect, the invention provides a method of treating or preventing a disorder associated with a distal dysbiosis in a subject in need thereof, comprising administering to the subject a probiotic composition comprising an isolated bacterial population in an amount sufficient to alter the microbiome at a site of the distal dysbiosis, such that the disorder associated with the distal dysbiosis is treated. Disorders associated with distal dysbiosis, including disruptions to the systemic microbiome, are described herein and include, for example, autoimmune or inflammatory disorders such as graft-versus-host disease (GVHD), an inflammatory bowel disease (IBD), ulterative colitis, Crohn's disease, multiple sclerosis (MS), systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, Sjögren's syndrome, and Celiac disease; transplant disorders such as graft-versus-host disease; and vaginal dysbiosis. In one embodiment, the disorder associated with distal dysbiosis occurs in the respiratory tract (e.g., lung), including but not limited to Cystic Fibrosis and chronic obstructive pulmonary disorder (COPD).
In one embodiment, the probiotic composition contains a species of bacteria that is deficient at the site of the distal dysbiosis. Administration of the probiotic composition can increase the quantity of the deficient species in the distal microbiome. In one embodiment, the deficient species is not detectably present at the site of the distal dysbiosis prior to administration of the probiotic composition. In one embodiment, the species of bacteria in the probiotic composition translocates to the site of the distal dysbiosis.
In another embodiment, the probiotic composition results in augmentation of a species of bacteria not present in the probiotic composition at a distal site. This augmentation can result from, for example, translocation of a species of bacteria not present in the probiotic composition to the distal site, and/or modulation of the microenvironment of the distal site in a manner that alters the microbiome.
In preferred embodiments, the probiotic composition contains immunomodulatory bacteria, e.g., anti-inflammatory bacteria.
In another aspect, the invention provides a method of reducing intestinal permeability in a subject, by administering a probiotic composition comprising an isolated bacterial population, wherein administration of the probiotic composition augments a species of bacteria that produces short chain fatty acids, such that the intestinal permeability of the subject is reduced. In other embodiments, intestinal permeability and disorders associated therewith is improved by administering a probiotic composition containing mucin-containing bacteria, and/or anti-inflammatory bacteria.
Pharmaceutical compositions useful for correcting or treating a distal dysbiosis, or for treating a disorder distal to the site of administration (e.g., the gastrointestinal tract) associated with a dysbiosis, can include any of the pharmaceutical compositions described herein. In exemplary embodiments, a pharmaceutical composition useful for correcting or treating a distal dysbiosis includes one or more bacterial strains from Table 1. In other embodiments, the pharmaceutical composition useful for correcting or treating a distal dysbiosis includes one or more bacterial strains from Table 1A. In other embodiments, the pharmaceutical composition useful for correcting or treating a distal dysbiosis includes one or more bacterial strains from Table 1B. In other embodiments, the pharmaceutical composition useful for correcting or treating a distal dysbiosis includes one or more bacterial strains from Table 1C. In other embodiments, the pharmaceutical composition useful for correcting or treating a distal dysbiosis includes one or more bacterial strains from Table 1D. In other embodiments, the pharmaceutical composition useful for correcting or treating a distal dysbiosis includes one or more bacterial strains from Table 1E. In other embodiments, the pharmaceutical composition useful for correcting or treating a distal dysbiosis includes one or more bacterial strains from Table 1F. In some embodiments, the pharmaceutical composition contains a single strain of bacteria. In other embodiments, the pharmaceutical composition contains two or more strains of bacteria, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more strains of bacteria. In other embodiments, the pharmaceutical composition contains or is administered in conjunction with a prebiotic, as described herein.
Exemplary pharmaceutical compositions useful for treatment of disorders associated with a dysbiosis distal to the site of administration (e.g., the gastrointestinal tract) contain bacterial strains capable of reducing inflammation in a subject. As described herein, such immunomodulatory (anti-inflammatory) bacteria can modulate cytokine expression by host immune cells, resulting in an overall increase in secretion of anti-inflammatory cytokines and/or an overall decrease in secretion of pro-inflammatory cytokines, systemically reducing inflammation in the subject. In exemplary embodiments, pharmaceutical compositions useful for treatment of disorders associated with a distal dysbiosis stimulate secretion of one or more anti-inflammatory cytokines by host immune cells, such as PBMCs. Anti-inflammatory cytokines include, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGFβ, and combinations thereof. In other exemplary embodiments, pharmaceutical compositions useful for treatment of disorders associated with a distal dysbiosis inhibit secretion of one or more pro-inflammatory cytokines by host immune cells, such as PBMCs. Pro-inflammatory cytokines include, but are not limited to, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof. Other exemplary cytokines are known in the art and are described herein. Pharmaceutical compositions containing anti-inflammatory bacteria reduce inflammation at the site of administration, e.g., in the gastrointestinal tract, as well as at distal sites throughout the body of the subject.
Other exemplary pharmaceutical compositions useful for treatment of disorders associated with a dysbiosis distal to the site of administration (e.g., the gastrointestinal tract) contain bacterial strains capable of altering the proportion of immune subpopulations, e.g., T cell subpopulations, in the subject.
For example, immunomodulatory bacteria can increase or decrease the proportion of Treg cells, Th17 cells, Th1 cells, or Th2 cells in a subject. The increase or decrease in the proportion of immune cell subpopulations may be systemic, or it may be localized to a site of action of the pharmaceutical, e.g., in the gastrointestinal tract or at the site of a distal dysbiosis. In some embodiments, a pharmaceutical composition comprising immunomodulatory bacteria is used for treatment of disorders associated with a dysbiosis distal to the site of administration (e.g., the gastrointestinal tract) based on the desired effect of the pharmaceutical composition on the differentiation and/or expansion of subpopulations of immune cells in the subject.
In one embodiment, a pharmaceutical composition contains immunomodulatory bacteria that increase the proportion of Treg cells in a subject. In another embodiment, a pharmaceutical composition contains immunomodulatory bacteria that decrease the proportion of Treg cells in a subject. In one embodiment, a pharmaceutical composition contains immunomodulatory bacteria that increase the proportion of Th17 cells in a subject. In another embodiment, a pharmaceutical composition contains immunomodulatory bacteria that decrease the proportion of Th17 cells in a subject. In one embodiment, a pharmaceutical composition contains immunomodulatory bacteria that increase the proportion of Th1 cells in a subject. In another embodiment, a pharmaceutical composition contains immunomodulatory bacteria that decrease the proportion of Th1 cells in a subject. In one embodiment, a pharmaceutical composition contains immunomodulatory bacteria that increase the proportion of Th2 cells in a subject. In another embodiment, a pharmaceutical composition contains immunomodulatory bacteria that decrease the proportion of Th2 cells in a subject.
In one embodiment, a pharmaceutical composition contains immunomodulatory bacteria capable of modulating the proportion of one or more of Treg cells, Th17 cells, Th1 cells, and combinations thereof in a subject. Certain immune cell profiles may be particularly desirable to treat or prevent particular disorders associated with a dysbiosis. For example, treatment or prevention of autoimmune or inflammatory disorders can be promoted by increasing numbers of Treg cells and Th2 cells, and decreasing numbers of Th17 cells and Th1 cells. Accordingly, pharmaceutical compositions for the treatment or prevention of autoimmune or inflammatory disorders may contain pharmaceuticals capable of promoting Treg cells and Th2 cells, and reducing Th17 and Th1 cells.
Distal disorders associated with loss of intestinal barrier function can be treated or improved by administration of pharmaceutical compositions containing bacterial strains that produce short chain fatty acids (SCFAs), such as, for example, butyrate, acetate, propionate, or valerate, or combinations thereof. Distal disorders associated with loss of intestinal barrier function can be treated or improved by administration of probiotic compositions containing bacterial strains that reduce inflammation, as described herein.
In other embodiments, the distal dysbiosis is caused by a deficiency in microbes that produce lactic acid. Accordingly, in one embodiment, the probiotic composition can contain a species of bacteria that produce lactic acid.
EXAMPLES
The invention is further illustrated by the following examples. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. The entire contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference in their entirety.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press, Vols A and B, 1992). Enzyme Linked Immunosorbent Assays (ELISAs) and Western blots described below are performed using kits according to the manufacturers' (e.g., Life Technologies, Thermo Fisher Scientific, New York, USA) instructions.
Examples
The invention is further illustrated by the following examples. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. The entire contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference in their entirety.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press, Vols A and B, 1992). Enzyme Linked Immunosorbent Assays (ELISAs) and Western blots described below are performed using kits according to the manufacturers' (e.g., Life Technologies, Thermo Fisher Scientific, New York, USA) instructions.
Example 1
Assessment of Intestinal Permeability after Administration of Bacteria, Prebiotic or Combinations Thereof
The main function of the gastrointestinal (GI) tract is to digest and absorb nutrients from food. The mucosa of the GI tract forms a selective barrier between the host and the environment of the gut lumen. The mucosa allows transport of nutrients while restricting passage of larger molecules and bacteria. Impaired barrier integrity is believed to contribute to the pathogenesis of many disorders including autoimmune diseases, including transplant disorders such as graft-versus-host-disease (GVHD), and neurological disorders. Disruption of the intestinal barrier due to toxins, dysbiosis, inflammation or other factors is believed to result in the passage and presentation of environmental antigens to the immune system leading to aberrant immune responses. Similarly, the leakage of bacterial endotoxin or other toxic metabolites into the circulation can lead to systemic inflammation promoting the development of autoimmunity and neuroinflammation.
Restoration of GI barrier integrity through the administration of selected prebiotics and/or probiotics represents an approach to correct a basic defect underlying multiple pathological conditions.
In a first set of experiments, intestinal permeability was assessed using serum endotoxin levels as a marker of gut permeability in mice treated with xylose and/or antibiotics. Basal levels of intestinal permeability can be measured under disease or normal conditions. Intestinal permeability can be induced in mice through administration of inflammatory stimuli such as cholera toxin (3 oral gavages of 10 μg cholera toxin, 5 days apart), Poly I:C (3 intraperioneal injections of 1 mg/kg, 3 days apart) or dextran sulfate (3% dextran sulfate sodium salt in drinking water for 7 days). Quantitation of intestinal permeability was carried out by quantitatively measuring plasma levels of endotoxin originating from gut bacteria using a commercially available chromogenic assay (Lonza, Rockland, Me.). The results of these experiments are shown in FIG. 1.
Quantitation of intestinal permeability can also be conducted using a number of alternative methods (reviewed in Bischoff et al, 2014) for example, by quantifying leakage of fluorescently-labeled high molecular weight dextran (FITC-dextran) into the plasma following oral administration (oral gavage with 0.6 g/kg 4 kDa FITC-dextran, serum samples collected 4 hours later and read for fluorescence intensity at 521 nm; Hsiao et al, 2013). To study the effect of bacterial strains on intestinal permeability, mice are gavaged orally with 107-1010 bacterial cells for an average of 5 administrations, typically daily or 2 days apart. Bacteria can be administred as single strains or combinations of strains. The bacteria can be administered alone or in combination with a pre-biotic(s). The pre-biotic can be xylose or xylose-containing molecules as a preferred carbon source for anaerobic bacteria. Other prebiotics that can be used include, for example, those described in Table 4. After administration of bacteria+/−pre-biotic, intestinal permability is assessed using the preferred method at the desired time point(s) starting on day 1 post-treatment.
As shown in FIG. 1, C57BL/6 mice were either left untreated or were treated with xylose at 10 g/L in drinking water from day −7 to day 14; ciprofloxacin (cipro) at 0.25 g/L in drinking water from day −7 to day −2; enrofloxacin (enro) at 0.25 g/L in drinking water from day −7 to day −2; xylose+cipro or xylose+enro. Analysis of serum samples collected on days 0 and 14 showed that basal levels of serum endotoxin are present in normal mice that remained unchanged in untreated mice. Xylose treatment reduced these basal levels over time, suggesting an increase in gut barrier integrity even in normal animals. Antibiotic treatment with cipro, a broad spectrum quinolone antibiotic, or enro, an anaerobe-sparing antibiotic, led to an increase in serum endotoxin levels (measured 2 days after a 5 day course), likely due to disruption of the microbiota. Serum endotoxin levels returned to baseline over time. As shown in FIG. 1, xylose appeared to counteract the increase in serum endotoxin level caused by cipro, but not enro. The differential effect of xylose on these 2 antibiotics may relate to its ability to preserve/promote expansion of anaerobic bacteria, which are killed by cipro but not enro.
Example 2
Immunomodulatory Properties of Different Human Commensal Bacteria on Human Peripheral Blood Mononuclear Cells
The microbiota of mammalian hosts is composed of bacterial species that possess both pro- and anti-inflammatory properties. In healthy individuals, a balance or state of eubiosis is maintained that supports gut barrier integrity, immune containment of commensal bacteria and promotion of a tolerogenic environment. Under disease conditions, dysbiosis characterized by an imbalance in pro- and anti-inflammatory bacteria results in local inflammation and compromised gut barrier integrity, leading to systemic inflammation and aberrant immune responses. Administration of selected probiotic bacterial strains (+/−prebiotics) that possess anti-inflammatory activity and promote immune tolerance represents an approach to correct a basic defect underlying multiple pathological conditions.
An in vitro system was developed to efficiently test the inflammatory and immunomodulatory properties of different human commensal bacteria on human peripheral blood mononuclear cells (PBMCs). Experiments were carried out with 21 bacterial candidates to profile their anti-inflammatory properties against human PBMCs. The innate properties of bacteria alone on human PBMCs were tested as well as their ability to counteract the pro-inflammatory activity of Enterococcus feacalis. 
Human PBMCs were isolated from fresh blood by density-gradient centrifugation using Ficoll (1-4). Freshly isolated PBMCs were plated at 1.5×106 cells per ml per well of a 24-well plate in a total volume of 2 mls RPMI-1640 medium+5% human serum, and incubated at 37° C./5% CO2 with the following:

    
    
        (1) 500 μl of different commensal bacteria suspensions at OD 0.8
        (2) E. faecalis at 107 colony-forming units (cfu)
        (3) A combination of commensal bacteria (OD 0.8)+E. faecalis (107 cfu)
        (4) Complete medium alone as a negative control
        (5) Bacterial lipopolysaccharide (LPS; 100 ng/ml) as an immunomodulatory “positive” control

Culture supernatants were collected at 24, 48 and 72 h, and the cytokine profile was analyzed by Luminex technology according to manufacturer's instruction (EMD Millipore, Danvers, Mass.). Cytokine production was detectable in culture supernatants by 24 h with levels increasing over 48-72 h and sometimes exceeding the range of quantitation. The results are presented in FIGS. 2-5 for all time points. The 24 h time point was chosen as the optimal time point for further analysis. The 24 h results are shown as a composite in FIG. 6 and with statistical analysis on individual cytokines in FIGS. 7-10. The results represent the properties of each bacterial species against human PBMCs and their ability to counteract inflammatory stimulation with E. faecalis in vitro. It was found that the commensal bacteria tested have distinct immunomodulatory properties, and most appear to counteract the inflammatory activity of E. Faecalis for at least one cytokine.

    
    


FIG. 2 shows the time course of Th1 related cytokines that were released by human PBMCs incubated with Ruminococcus gnavus (Epv 1), Eubacterium rectale (Epv 2), Blautia luti (Epv 3), Blautia wexlerae (Epv 5) and Enterococcus faecalis (Epv 8), or combinations of each bacterium with E. faecalis. Amounts of Th1-related pro-inflammatory cytokines interferon gamma (IFN-γ), interleukin-12 p70 (IL-12p70), interleukin-6 (IL-6), interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFα) released by PBMCs were measured after 24, 48 and 72 hours. As shown in FIG. 2, all commensals have unique immunomodulatory properties. As expected, E. faecalis induced high levels of these pro-inflammatory cytokines. By comparison, most of the other bacterial candidates induced lower levels of Th1-related cytokines and were able to counteract the induction of one or more inflammatory cytokines by E. faecalis. In particular, Blautia luti (Epv 3), showed minimal induction of Th1-related cytokines on its own and was most effective in counteracting induction of these cytokines by E. faecalis (Epv 8). This profile is desirable for disease indications which are primarily driven by Th1 immune responses, such as GVHD.
FIG. 3 shows the time course of Th2 related cytokines that were released in cells treated with R. gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv 5) and E. faecalis (Epv 8), or combinations thereof. Amounts of anti-inflammatory cytokines interleukin-13 (IL-13), interleukin-4 (IL-4) and interleukin-5 (IL-5) released by PBMCs were measured after 24, 48 and 72 hours. Each bacterium displayed detectable pattern of cytokine induction and ability to modulate the effect of E. faecalis. Th2-related cytokines are beneficial in counteracting Th1 responses. Bacteria capable of promoting Th2 cytokine release are therefore of interest in Th1-driven diseases. R. gnavus appeared the most active in terms of elicitng Th2 cytokine on its own or in the presence of E. faecalis. 
FIG. 4 shows the time course of Th9, Th17 and Treg cytokines that were released in cells treated with R. gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv 5) and E. faecalis (Epv 8), or combinations thereof. Amounts of interleukin-9 (IL-9), interleukin-17 (IL-17) and interleukin-10 (IL-10) released by PBMCs were measured after 24, 48 and 72 hours. The activity of IL-9 and IL-17 is context-dependent in that these cytokines can be beneficial under some conditions but detrimental under other conditions depending on the mechanisms responsible for disease pathogenesis. For example, IL-17 is expected to contribute to disease pathogenesis in GVHD but could provide a benefit in Th2-driven disorders. IL-10 produced by regulatory T cells (Treg) is generally immunosuppressive and is expected to provide a benefit in most inflammatory disorders whether Th1- or Th2-driven. As shown in FIG. 4, all bacterial candidates elicited IL-9 and IL-17 to varying degrees and B. wexlerae (Epv 5) was the most potent in inducing IL-10.
FIG. 5 shows the time course of monocyte, macrophage and neutrophil-related inflammatory cytokines that were released by PBMCs treated with R. gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv 5) and E. faecalis (Epv 8), or combinations thereof. Amounts of monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP1β), macrophage inflammatory protein 1α (MIP1a), regulated on activation, normal T expressed and secreted protein (RANTES), interleukin-1α (IL-1α), interleukin-1β (IL1β), interferon α2 (IFN-α2) and interleukin-8 (IL-8) that were released were measured after 24, 48 and 72 hours. In general, these cytokines contribute to inflammation by innate immune effector cells. The bacteria tested showed different degrees of induction and effects on E. faecalis. Overall, E. rectale (Epv 2) and B. luti (Epv 3) were the least inflammatory and the most effective at countering the effect of E. faecalis (Epv 8).
A composite illustration of the secretion of each of the pro-inflammatory and anti-inflammatory cytokines described above in the presence of each commensal alone or in combination with EPV8 is graphed relative to the pro-inflammatory bacterial strain E. faecalis (Epv 8) in FIG. 6. In the context of GVHD, IFNγ(IFNg), IL-12p70, IL-1α(IL-1a), IL-6, IL-8, MCP1, MIP1α(MIP1a), MIP1β (MIP1b) and TNFα(TNFa) are considered pro-inflammatory cytokines. IL-10, IL-13, IL-9, IL-4 and IL-5 are considered anti-inflammatory cytokines. IL-17 (IL-17A), IL-9 and IL-2 have context dependent activity. The results are shown as a percentage of Epv 8, where cytokine levels in the presence of E. faecalis after 24 hours is set at 100%. Each commensal has a unique signature and each one added alone to human PBMCs appeared to be less inflammatory than E. faecalis (below 100% for pro-inflammatory cytokines), except for B. wexlerae (Epv 5). When added to PBMCs in combination with E. faecalis, most commensals tested (except for Epv 5) also counteracted the pro-inflammatory activity of E. faecalis (below 100% for pro-inflammatory cytokines).
FIGS. 7-10 detail individual cytokine profiles of PBMCs following exposure to various commensals, alone or in combination with the pro-inflammatory bacterium E. faecalis (Epv8). In particular, FIG. 7 shows the effect of R. gnavus (EPV1) on cytokine concentration (pg/ml) either alone or in combination with Epv 8 (E. faecalis).
FIG. 8 shows the effect of E. rectale (EPV 2) on cytokine concentration (pg/ml) either alone or in combination with Epv 8 (E. faecalis). FIG. 9 shows the effect of B. luti (EPV 3) on cytokine concentration (pg/ml) either alone or in combination with Epv 8 (E. faecalis). FIG. 10 shows the effect of B. wexlerae (EPV 5) on cytokine concentration (pg/ml) either alone or in combination with Epv 8 (E. faecalis).
Overall, the foregoing data indicate that, among the bacteria tested, EPV3 has a significantly desirable anti-inflammatory profile for a Th-1-driven condition, such as GVHD while EPV5 has a suboptimal anti-inflammatory profile for GVHD. As shown in FIG. 11, EPV3 has relatively low intrinsic inflammatory activity compared to EPV 8 and is able to reduce the induction of pro-inflammatory cytokines by EPV 8, including IL-6, MCP-1, IL-12p70, and IFNγ which are believed to contribute to the pathogenesis of GVHD. By comparison, EPV 5 is similar to EPV 8 in terms of induction of pro-inflammatory cytokines and shows little ability to counteract the induction of pro-inflammatory cytokines by EPV 8.
Additional bacteria were profiled using this methodology including: Clostridium leptum (EPV 6), Blautia faecis (EPV15), Blautia/Ruminococcus obeum ATCC 29174 (EPV 20), Blautia product ATCC 27340 (EPV 21), Blautia coccoides ATCC 29236 (EPV 22), Blautia hydrogenotrophica ATCC BAA-2371 (EPV-23) and Blautia Hansenii ATCC27752 (EPV 24). Strains freshly isolated by Epiva from the stool of a normal healthy volunteer were also profiled and included: Eubacterium rectale (EPV 35), a previously uncultured Blautia, similar to GQ898099_s S1-5 (EPV 47), a previously uncultured Blautia, similar to SJTU_C_14_16 (EPV 51), Blautia wexlerae (SJTU_B_09_77) (EPV 52), Blautia luti ELU0087-T13-S-NI_000247 (EPV 54), Blautia wexlerae WAL 14507 (EPV 64), Blautia obeum (EPV 78), Ruminococcus gnavus (EPV 102) and Blautia luti (BlnIX) (EPV 114). Results focusing on key pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1 RA) and anti-inflammatory (IL-10, IL-4, IL-13) cytokines are shown in FIGS. 12-27. As observed with the initial set of bacterial candidates, each isolate displayed a defined signature. Candidates for treatment of autoimmune or inflammatory disorders, such as GVHD, displayed low induction of pro-inflammatory cytokines and/or positive induction of anti-inflammatory cytokines, and had ability to counteract the inflammatory activity of E. faecalis. Bacterial candidates meeting these criteria include, for example, EPV 35, 51, 78 and 114.
Taken together, these results show that commensals have distinct immunomodulatory properties and display a definable signature in terms of their ability to induce cytokines in human host cells, or counteract the pro-inflammatory activity of another bacterium (E. faecalis). Accordingly, bacterial compositions may be selected in order to achieve a desired modulation of pro- and anti-inflammatory cytokines. For example, anti-inflammatory bacterial strains may be selected based on their ability to reduce key pro-inflammatory cytokines such as interferon gamma, IL-12p70, IP-10 and IL-1RA and/or increase anti-inflammatory cytokines such as IL-13, IL-10 and IL-4.
Example 3
Effect of Commensal Human Bacteria on T-Cell Polarization
In order to determine whether exposure to commensal bacteria may polarize T cells toward a particular phenotype, flow cytometry analysis was performed on human PBMCs cultured with various commensal bacteria as described above. The cells recovered from culture were washed in phosphate-buffered saline and stained with a cocktail of fluorescently labeled antibodies against specific cell surface protein markers to allow for the detection of Th1 cells (CXCR3+CCR6−), Th2 cells (CXCR3−CCR6−), Th17 cells (CXCR3−CCR6+) and Tregs) (CD25+CD127lo. Negative control wells contained PBMCs in culture medium alone and positive control wells contained PBMCs+LPS (100 ng/ml) as a known immune stimulus. The commensal bacteria examined included: Epv 1: R. gnavus; Epv 3: B. luti; Epv 2: E. rectale; Epv 5: B. wexlerae; Epv. 8: E. faecalis; Epv 20: B. obeum, ATCC 29174; Epv 21: B. product, ATCC 27340; Epv 24: B. hansenii, ATCC 27752. As shown in FIG. 28, exposure of human PBMCs to bacteria did result in a shift in the relative proportion of T cell populations compared to the PBMCs alone (control) although statistical significance was not achieved in every case. Overall, most bacteria tested caused an increase in the proportion of T cells with a regulatory phenotype (Tregs) with EPV 21 and EPV 24 having the greatest impact and EPV8 (E. faecalis) causing little or no increase in Tregs. Most bacteria also caused a decrease in the proportion of Th17 cells, an increase in Th2 cells and had little or no effect on the proportion of Th1 cells. This type of analysis indicates that commensal bacteria can modulate the proportions of effector T cell types and can be used to select the desired phenotype for a given disease application. For example, the optimal T cell profile to address pro-inflammatory disorders such as GVHD would consist of ⇑Treg, ⇓Th17, ⇓ or unchanged Th1, and ⇑Th2. This phenotype was induced by many of the bacteria tested.
Example 4
Pattern of Carbon Source Utilization by Commensal Bacteria
Modulation of the microbiota to correct a dysbiosis associated with pathological conditions can potentially be achieved through administration of bacteria (or bacterial combinations) and prebiotic(s) as a carbon source to promote endogenous expansion of beneficial bacteria. Alternatively, prebiotics can be administered in combination with bacteria to promote their growth or create a favorable environment for their growth. Profiling of carbon source usage by bacterial isolates can be used to customize and optimize selection of prebiotics for particular bacterial strains. Profiling of carbon source usage was conducted on 21 anaerobic commensal bacteria (Table 3) using 96 well plates from Biolog (Hayward, Calif.) where each well contains a different carbon source for a total of 192 different carbon sources (PM01 and PM02A plates). The carbon sources tested are listed in Table 4. The assay was conducted according to manufacturer's instructions. Briefly, pre-cultured bacteria were suspended in Biolog assay medium at a 750 nm optical density (OD) of 0.3-0.4 and 100 μl of the suspension was transferred to each well of the 96 well PM01 and PM02 assay plates. The plates were then incubated at 37° C. in an anaerobic chamber for 24 hr or longer. The amount of growth on each carbon source was evaluated by measuring the optical density (OD) of each well at 750 nm. The results are summarized in FIG. 29, and indicate that each individual strain displays a unique pattern of carbon source usage. Interestingly, different isolates of the same species (e.g. B. luti and B. wexlerae) show related (albeit distinct) patterns. Overall, these results indicate that characterization of carbon source usage for profiling of bacterial candidates allows optimal selection of prebiotics. Preferred prebiotics can be selected which increase the growth (indicated by an increase in optical density) of bacterial species contained in probiotic compositions.
Example 5
Normal Human Volunteer Study of a Prebiotic Formulation Containing Xylose
D-xylose is a carbon source generally preferred by anaerobic bacteria. Preliminary results in the mouse indicate that it may act to promote gut barrier integrity (FIG. 1). It is also used as a carbon source by several bacterial strains (FIG. 29) that were determined to possess a desirable immunological profile for target indications such as GVHD (FIG. 19, 25, 27). A parallel, double-blind, 5 cohort escalation food safety study was conducted to examine D-xylose in normal human volunteers. The study was a double-blind, single-center, parallel group study designed to evaluate the tolerability and potential microbiome changes induced by ingestion of D-xylose at 5 different amounts in healthy, adult volunteers enrolled at 1 study center in the United States (US).
Subjects were screened for eligibility within 21 days prior to the first planned ingestion of study sweetener on Day 1 (Baseline). Within each of 5 cohorts, eligible subjects were randomly assigned in a double-blinded, 6:2 ratio to ingest either D-xylose or the GRAS sweetener Splenda® (control), dissolved into 2 to 6 oz of sterile water and ingested TID with meals for a total of 82 ingestions taken over 28 consecutive days. D-xylose ingestion amounts ranged from 1 to 15 g TID (total daily amount of 3 to 45 g), and all subjects randomized to Splenda® ingested 1 dissolved, commercially available packet TID (3 packets total per day).
Subjects returned to the study center weekly on Days 8, 15, 22, and 28 for ingestion, tolerability, and compliance evaluations. Safety was evaluated on a continual basis through adverse events (AE) monitoring, clinical laboratory measurements, vital sign monitoring, physical examinations, electrocardiograms (ECGs), telephone follow-up, and electronic subject ingestion diaries. Stool was collected pre-ingestion and at pre-specified time points, and post-ingestion samples were evaluated for changes in the gut microbiome compared with Baseline for all subjects. For subjects who consented to further sampling, additional stool specimens were used to potentially isolate living bacteria that could be categorized for research and potential commercialization purposes. Serum and urine were collected for measurement of D-xylose levels and pharmacokinetic (PK) assessments and PK/pharmacodynamics (PD) correlations. Telephone follow-up was conducted as needed, but minimally once per week. The total duration for each participant was up to 60 days, including the Screening period (Day −21 to 0), the ingestion period (Day 1 to 28), and an End-of-Study (EOS) follow-up visit conducted 7 (±3) days after the last ingestion of study sweetener.
Criteria for Evaluation
Safety
Safety was evaluated on a continual basis through AE monitoring, clinical laboratory measurements, vital sign monitoring, physical examinations, ECGs, telephone follow-up, and electronic subject ingestion diaries.
Immunology and Other Assessments
Stool was collected at pre-specified pre- and post-ingestion time points and post-ingestion samples were evaluated for changes in the gut microbiome compared with Baseline. Additional optional specimens were collected to potentially isolate living bacteria that could be categorized for research and potential commercialization purposes.
Blood was collected at pre-specified pre- and post-ingestion time points to evaluate C-reactive protein (CRP), serum cytokines (tumor necrosis factor alpha [TNF-α], interleukin [IL]-2, IL-6, interferon gamma [IFN-γ], and IL-10), and T-cell markers CD3, CD4, CD8, CD25, and FOXP3. Plasma was also stored and may be tested for biomarkers and/or metabolic markers for up to 7 years.
Pharmacokinetics
Blood and urine were collected at pre-specified pre- and post-ingestion time points to measure D-xylose levels and to characterize the systemic absorption profiles of D-xylose.
Statistical Methods
Statistical analyses were conducted using SAS®, Version 9.2 (SAS Institute, Inc., Cary, N.C., USA). The sample size calculations were empiric and based on an estimation of normal healthy volunteer variability in reported symptoms and side effects and not on a statistical method. A weighted randomization scheme was implemented such that more subjects were enrolled at the higher D-xylose ingestion amounts to account for potential toxicity-related effects that could have resulted in withdrawal and/or analysis ineligibility, and to enable collection of more data at ingestion amounts for which limited data were available.
Analysis Populations
The safety population comprised all subjects who ingested any amount of study sweetener.
Safety
AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA), Version 18.0 (Northrup Grumman Corporation, Chantilly, Va., USA), and summarized by cohort. Laboratory, vital sign, and physical examination data were summarized by cohort using descriptive statistics over time, including statistics for changes from Baseline. ECG findings were also summarized by cohort over time as well as using frequency counts and percentages, as normal or abnormal, with the relevance of abnormalities categorized by clinical significance.
Immunology and Other Assessments
Stool sample compliance was summarized by cohort, using the following calculation for each subject:




  
    Percentage
    ⁢
    
      
    
    ⁢
    compliance
  
  =
  
    
      
        Total
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        ⁢
        number
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        of
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        stool
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        samples
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        ⁢
        collected
      
      
        Total
        ⁢
        
          
        
        ⁢
        number
        ⁢
        
          
        
        ⁢
        of
        ⁢
        
          
        
        ⁢
        stool
        ⁢
        
          
        
        ⁢
        samples
        ⁢
        
          
        
        ⁢
        expected
      
    
    ×
    100
  




A total of 7 stool samples were expected to be collected for each subject. Evaluation of changes in the gut microbiome were evaluated in stool samples through taxonomic classification, relative and statistical differential abundance analyses by cohort and time point, an alpha diversity analysis calculated using the Shannon diversity index by cohort and time point, a beta diversity analysis using Bray-Curtis dissimilarity and Unifrac distance by subject and time point, and a principal coordinates analysis using the beta diversity data.
Summary statistics (n, mean, standard deviation, median, minimum, and maximum) were presented for serum concentrations of CRP, flow cytometry T-cell markers (CD3, CD4, CD8, CD25, and FOXP3), and cytokines (TNF-α, IL-2, IL-6, IFN-γ, and IL-10) as per their nominal time points.
Pharmacokinetics
Phoenix® WinNonLin®, Version 6.2.1, was used for PK analyses.
Serum D-xylose concentrations were summarized by cohort using nominal sample times according to actual amount received using summary statistics (n, coefficient of variation [CV], mean, standard deviation [SD], median, minimum, and maximum). Evidence for the occurrence of steady-state was assessed graphically by comparing the time course of either trough or 2-hour post-ingestion serum concentrations of D-xylose as different levels of D-xylose. Accumulation was assessed by comparing the 2-hour post-first-ingestion serum levels with those observed at Week 2 (Day 15) and Week 4 (Day 28).
The total amount of D-xylose excreted in urine was analyzed for all subjects over 5 hours post-ingestion and pooled for analysis; the pooling for analysis reflected the subject mean within a given time of collection (e.g., Day 15 and then Day 28) sorted by ingested amount. Urine PK parameters for D-xylose levels included Ae(0-t) (cumulative amount of sweetener recovered in urine) and percent sweetener amount excreted over a 5-hour period.
Summary of Results
Forty-eight subjects were randomized to ingest either 1 packet of commercially-available Splenda® TID (n=12) or D-xylose TID at the following ingestion amounts (n=36 total):

    1 g: 6 subjects
    2 g: 6 subjects
    8 g: 7 subjects
    12.5 g: 8 subjects
    15 g: 9 subjects


Over the 28-day ingestion period, study sweetener ingestion compliance was >90% for all subjects. Two subjects (4.2%) discontinued from the study prematurely; primary reasons for discontinuation were a protocol violation (positive urine drug screen) and withdrawal of consent. The proportion of males (47.9%) and females (52.1%) was balanced, and the majority of subjects were White (89.6%) and not Hispanic or Latino (77.1%). Subject ages spanned a wide range, with a median of 38.3 (range 22.5 to 60.5) years for the combined D-xylose cohorts and 43.6 (range 24.9 to 64.3) years for the Splenda® cohort.
Safety
D-xylose and Splenda® were both well tolerated, with no new safety concerns identified. One subject required a D-xylose reduction from 15 g to 12.5 g TID at the Week 1 (Day 8) visit due to AEs of moderate abdominal distension, diarrhea, and GI pain; no other modifications to sweetener ingestion amounts were implemented.
Overall, 17 subjects (35.4%) experienced at least 1 AE, including a higher proportion of subjects who ingested any amount of D-xylose (14 subjects [38.9%]) than Splenda® (3 subjects [25.0%]). Reported AE rates increased with increasing D-xylose ingestion amounts, with incidences ranging from 16.7% in subjects who ingested the 2 lowest amounts (1 and 2 g TID) to 66.7% in subjects who ingested the highest amount (15 g TID). AEs reported for more than 1 subject in the D-xylose cohorts included diarrhea (3 subjects [8.3%]) and flatulence and GI pain (2 subjects [5.6%] each). AEs in the Splenda® cohort included abdominal distension, flatulence, increased blood creatinine, infrequent bowel movements, and rhinitis. The incidence of AEs was highest during Weeks 1 and 2 (Days 2 through 15), regardless of sweetener type or ingestion amount. During this 2-week period, 18 subjects overall (37.5%) experienced AEs, compared with 7 subjects (14.6%) overall who experienced AEs either on Day 1 or after Week 2.
All AEs were mild in severity with the exception of moderate AEs reported for 4 subjects (11.1%) in the D-xylose cohorts. These moderate AEs included abdominal distension, concussion/post-concussion syndrome, diarrhea, GI pain, increased blood bilirubin, and neutropenia.
No SAEs, severe AEs, or subject deaths were reported. One subject in the 8 g TID D-xylose cohort experienced non-serious, moderate AEs of concussion and post-concussion syndrome that were noted to have contributed to study discontinuation; however, this subject's primary reason for discontinuation was withdrawal of consent.
GI-related AEs, which were of special interest, were reported for 7 subjects (19.4%) in the D-xylose cohorts and 2 subjects (16.7%) in the Splenda® cohort. GI-related events were mild for all but 1 subject in the 15 g TID D-xylose cohort who experienced moderate GI-related AEs of abdominal distension, diarrhea, and GI pain that required reduction of the D-xylose ingestion amount to 12.5 g TID.
Eleven subjects (22.9%) experienced at least 1 AE that was considered by the Investigator to be related to study sweetener, including 9 subjects (25.0%) in the D-xylose cohorts and 2 subjects (16.7%) in the Splenda® cohort. The incidence of sweetener-related AEs appeared to increase with increasing D-xylose ingestion amounts. Sweetener-related AEs reported for more than 1 subject in the D-xylose cohorts included diarrhea (3 subjects [8.3%]) and flatulence and GI pain (2 subjects [5.6%] each). Sweetener-related AEs reported in the Splenda® cohort were abdominal distension, flatulence, and infrequent bowel movements.
No fluctuations in clinical laboratory measurements over time were considered to be clinically meaningful. Categorical shifts from Baseline that occurred in >10% of subjects in either the combined D-xylose or Splenda® cohorts included decreased or increased glucose (27.7% D-xylose and 16.7% Splenda®) and decreased absolute neutrophil count (ANC) (13.9% and 8.3%); these shifts were not associated with sweetener type or ingestion amount.
Immunology and Other Assessments
To assess the effect of D-xylose on the gut microbiome, this study incorporated an analysis of alpha diversity, beta diversity, and differentially abundant taxa. These factors were assessed both across cohorts and over time. Regardless of sweetener ingestion amount, no apparent significant impact on the intra-sample alpha diversity of the gut microbiome was observed, and no significant changes in community composition were observed over time on study. Numerous taxa were identified as differentially abundant, but these findings may reflect the relatively small sample sizes in each cohort.
Across all D-xylose cohorts, 8.3% of subjects with normal serum CRP at Baseline experienced at least 1 post-ingestion CRP value >2.9 mg/L. A substantially higher proportion of subjects in the Splenda® cohort (41.7%) had normal serum CRP at Baseline and experienced at least 1 post-ingestion CRP value >2.9 mg/L. None of the post-ingestion CRP values for any subject were deemed clinically significant.
Because most individual cytokine data points were below the limit of quantitation (BLQ) and therefore set to zero, cytokine summary statistics were limited and did not indicate any consistent or clinically meaningful changes over time for either sweetener or any D-xylose ingestion amount. There was a trend for reduced levels of serum interferon gamma over time in the 2 g and 15 g D-xylose cohorts (FIG. 30). No consistent or clinically meaningful changes over time in total T-cells or any T-cell subsets were observed for either sweetener or any D-xylose ingestion amount.
Pharmacokinetics
Serum D-xylose concentrations increased linearly with increasing ingestion amounts. Little to no accumulation of serum D-xylose occurred at Day 15 following 1 g to 12.5 g TID ingestion, while an approximately 1.9-fold accumulation ratio was observed in the 15 mg TID cohort (although variability was high). On Day 28, the accumulation ratio ranged from 1.08 to 1.31 following 1 g to 12.5 g TID ingestion and 1.68 following 15 g TID ingestion, although variability was moderate to high in all but the 8 g TID cohort.
In the 1 g TID cohort, approximately 40% of the ingested amount of D-xylose was recovered in urine within 5 hours post-ingestion on Days 1, 15, and 28. In the 2 g through 15 g TID cohorts, between 23% and 32% of the ingested amount of D-xylose was recovered in urine within 5 hours post-ingestion on Days 1, 15, and 28. The fraction excreted in urine was similar among Days 1, 15, and 28.
A review of the time course of serum D-xylose concentrations and the corresponding urinary excretion profiles indicated high ingestion compliance.
Changes in the Gut Microbiome
A total of 344 stool samples were collected in OMNIgene●GUT collection kits and shipped to the GenoFIND laboratory for DNA extraction and V3-V4 16S amplicon sequencing. There were no major shifts in the microbiome alpha diversity between the different treatment groups (absolute number of OTUs, abundance of OTUs) or over time on study. There was an overall decrease in the Chao diversity index over time (indicator of community richness—# of singleton, doubleton OTUs), as shown in FIG. 31. Numerous taxa were identified as differentially abundant, but this finding may be attributable to the relatively small sample sizes of each cohort. Similar observations were made in the mouse study, e.g., xylose treatment did not cause major shifts in the gut microbiome but showed some differences at the family level. Overall, these results suggest that, under the conditions tested in normal individuals and normal mice, ingestion of xylose exerts subtle changes in the gut microbiome. The impact of xylose on the microbiome under disease conditions remains to be determined.
Taken together, the results of this trial show that D-xylose is safe and well-tolerated, and indicate that prebiotic formulations containing xylose may reduce inflammation in a subject, resulting in reduction of serum levels of pro-inflammatory cytokines.
Example 6
Distal Augmentation
The trillions of organisms forming the microbiome function as an organ system interconnected throughout the body. The possibility that modification of the microbiome in a given physical location may influence the microbiome at other sites in the body (distal augmentation) was investigated. Seven week old C57Bl/6 female mice were acclimatized for 7 days prior to the start of the study by daily handling and shuffling between cages. All mice were housed at three mice per cage in individually vented cages (Thoren, Hazleton, Pa.). At day 0, baseline fresh fecal pellets, and vaginal lavages with 100 μL of sterile double-distilled water were collected and immediately frozen at −80° C. for microbiome analysis. After baseline collection, mice were given to drink either autoclaved water (N=6) or 0.5 mg/L of the antibiotic vancomycin in autoclaved water (N=6) ad libitum. Water alone is not expected to influence the microbiome and acted as a negative control. Oral vancomycin is poorly absorbed from the gut and its ingestion does not result in significant levels of drug in the body (Rao et al, 2011). The impact of oral vancomycin is therefore expected to be limited to the gastrointestinal tract such that microbiome changes elsewhere in the body (e.g. vagina) would be attributable to distal augmentation. At day 6, fresh fecal pellets and vaginal lavages with 100 μL of sterile double-distilled water were collected and immediately stored at −80° C. for microbiome analysis.
Isolation and sequencing of microbial DNA from the stool and vaginal samples was performed by DNA Genotek (Ottawa, ON, Canada). The V3-V4 region of the 16S ribosomal subunit was amplified with custom PCR primers and sequenced on an Illumina MiSeq to a minimum acceptable read depth of 25,000 sequences per sample. The widely accepted read depth requirement for accurate taxonomic profiling is 15,000-100,000 reads (Illumina, 2014). A closed-reference taxonomic classification was performed, where each sequence was aligned to the SILVA reference database, version 123. Sequences were aligned using the UCLUST algorithm included in QIIME version 1.9.1 (Caporaso et al., 2010). A minimum threshold of 97% sequence identity was used to classify sequences according to representative sequences in the database. At 97% sequence identity, each OTU represents a genetically unique group of biological organisms. These OTU's were then assigned a curated taxonomic label based on the seven level SILVA taxonomy.
As expected, oral vancomycin treatment had a strong impact on the microbiome of the gut. As shown by principal component analysis (PCA) at the family level, the day 0 to day 6 pattern in fecal samples was clearly different in the control vs oral vancomycin group (FIG. 32). Interestingly, the day 0 to day 6 pattern in the vaginal samples also showed an overall difference between the PBS and oral vancomycin groups even though the vaginal environment is not exposed to vancomycin following oral administration of the antibiotic (FIG. 32). In addition, some bacterial species were detected at low frequency in vaginal samples of the vancomycin-treated group at day 6 (median abundance of approximately 0.00002%) that were not present at day 0 (Table 5). These results support the concept of distal augmentation whereby modification of the microbiome at one site also has an impact at a distal site(s). This finding opens the possibility of modulating the microbiome, for example at the level of the gut, to effect therapeutic changes in the microbiome at other sites, for example the lung.
Example 7
Co-Culture of Bacteria Plus Prebiotic and Host-Cells and Analysis of Host Cell Cytokine Response
The following work is done in the presence and absence (as a control) of one or more selected prebiotic carbohydrates. This assay may be used to test or confirm the ability of a prebiotic-bacterium pair to elicit an immunomodulatory response such that the production or release of proinflammatory cytokines decreases and/or the production or release of anti-inflammatory cytokines increases, may be used to evaluate the difference in cytokine response in the presence or absence of a prebiotic mixture, and/or may be used to evaluate an array of prebiotic candidates. Clostridales bacteria are obtained from the ATCC or purified from a human donor and cultured in brain-heart infusion broth at 37° C. The bacteria are harvested by centrifugation (3000 g, 15 minutes) after 24 hours of stationary growth. To test the effects of spores on human intestinal cells and/or human peripheral blood mononuclear cells (huPBMC), bacteria are first heat killed (95° C., 30 minutes) before the centrifugation step. Bacteria (or spores) are washed three times with lx PBS (pH 7.2, Gibco BRL) and subsequently diluted to obtain final cell densities of 106 and 107 colony forming units (cfu)/ml in RPMI 1640 medium (Gibco BRL).
Human enterocyte-like CaCO-2 cells (passage 60-65) are seeded at a density of 2.5×105 cells/ml on 25 mm cell culture inserts (0.4 μm nucleopore size; Becton Dickinson). The inserts are placed into six well tissue culture plates (Nunc) and cultured 18-22 days at 37° C./10% CO2in DMEM (glutamine, high glucose; Amimed) supplemented with 20% decomplemented fetal calf serum (56° C., 30 minutes; Amimed), 1% MEM non-essential amino acids (Gibco BRL), 10 μm/ml gentamycin (Gibco BRL), and 0.1% penicillin/streptomycin (10 000 IU/ml/10 000 UG/ml; Gibco BRL). The cell culture medium is changed every second day until the cells are fully differentiated. Transepithelial electrical resistance (TEER) is determined continuously in confluent CaCO-2 monolayers using a MultiCell-ERS voltmeter/ohmmeter or as described in Example 44.
Tissue culture inserts covered with CaCO-2 cell monolayers are washed twice with prewarmed RPMI 1640 medium and transferred to six well tissue culture plates. 2 mL culture medium is added to the apical and basolateral compartments of the transwell cell culture system.
Next, the apical surface of CaCO-2 monolayers is challenged by addition of 106 or 107 cfu/ml of Clostridiales bacteria or spores, in the absence of gentamicin. After four hours, gentamicin is added (at 150 μg/mL) to stop bacterial growth and metabolite secretion. CaCO-2 cells are stimulated with the bacteria or spores for 6-36 hours in a 37° C., 10% CO2 incubator. Then the CaCO-2 cells are collected, washed once with cold 1×PBS (pH 7.2), and lysed in denaturation solution for RNA extraction (Micro RNA Isolation Kit, Stratagene). Cellular lysates are stored at −20° C. and cell culture supernatants are collected from the apical compartment and frozen at −20° C. The immune response of CaCO-2 cells is monitored by analysis of cytokine gene transcription (TNF-α, IL-8, monocyte chemoattracting protein 1 (MCP-1), TGF-β, IL-12, IFN-γ, IL-4, IL-10) using a reverse transcription-polymerase chain reaction (RT-PCR) technique and determination of cytokine secretion in cell culture supernatants using an ELISA (Haller D, Bode C, Hammes W P, Pfeifer A M A, Schiffrin E J, Blum S, 2000. Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures. Gut. 47:79-97).
REFERENCES


    1. Bischoff, S C, Giovanni, B, Buuman, W, Ockhuizen, T, Schulzke, J-D, Serino, M, Tilg, H, Watson, A and Wells, J M. (2014) Intestinal permeability—a new target for disease prevention and therapy. BMC GASTROENTEROLOGY 14: 189.
    2. Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. CLIN. LAB. INVEST. 21, Suppl 97 (Paper IV), 77-89.
    3. Boyum A. (1976) Isolation of lymphocytes, granulocytes and macrophages. SCAND J IMMUNOL. (Suppl 5):9-15.
    4. Bach M K, Brashler J R. (1970) Isolation of subpopulations of lymphocytic cells by the use of isotonically balanced solutions of Ficoll. I. Development of methods and demonstration of the existence of a large but finite number of subpopulations. EXP CELL RES. 61:387-96.
    5. Fotino, M., Merson, E. J. and Allen, F. H. (1971) Micromethod for rapid separation of lymphocytes from peripheral blood. ANN. CLIN. LAB. SCI. 1:131-133.
    6. Hsiao, E Y, McBride, S W, Hsien, S, Sharon G, Hyde, E R, McCue T, Codelli, J A, Chow, J, Reisman, S E, Petrosino, J F, Patterson, P H and Mazmanian, S K (2013) Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. CELL 155: 1451-1463.
    7. Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., Knight, R. (2010). QIIME allows analysis of high-throughput community sequencing data. NATURE METHODS 7 (5): 335-336. doi:10.1038/nmeth.f.303
    8. Illumina. (2014). Frequently Asked Questions: 16S Metagenomic Sequencing. Retrieved from http://www.illumina.com/content/dam/illuminamarketing/documents/products/other/16smetagen omics-faq-1270-2014-003.pdf
    9. Rao S, Kupfer Y, Pagala M, Chapnick E and Tessler S. (2011) Systemic absorption of oral vancomycin in patients with Clostridium difficile infection. SCAND J INFECT DIS 5: 386-388.









TABLE 1







See, e.g., WO 2014/121304













SEQ ID
Public DB

Spore
Pathogen


OTU
Number
Accession
Clade
Former
Status















Eubacterium saburreum
858
AB525414
clade_178
Y
N


Eubacterium sp. oral clone
866
AY349376
clade_178
Y
N


IR009


Lachnospiraceae bacterium
1061
HQ616401
clade_178
Y
N


ICM62


Lachnospiraceae bacterium
1062
HQ616384
clade_178
Y
N


MSX33


Lachnospiraceae bacterium
1063
ADDS01000069
clade_178
Y
N


oral taxon 107


Alicyclobacillus
122
NR_074721
clade_179
Y
N


acidocaldarius


Clostridium baratii
555
NR_029229
clade_223
Y
N


Clostridium colicanis
576
FJ957863
clade_223
Y
N


Clostridium paraputrificum
611
AB536771
clade_223
Y
N


Clostridium sardiniense
621
NR_041006
clade_223
Y
N


Eubacterium budayi
837
NR_024682
clade_223
Y
N


Eubacterium moniliforme
851
HF558373
clade_223
Y
N


Eubacterium multiforme
852
NR_024683
clade_223
Y
N


Eubacterium nitritogenes
853
NR_024684
clade_223
Y
N


Anoxybacillus flavithermus
173
NR_074667
clade_238
Y
N


Bacillus aerophilus
196
NR_042339
clade_238
Y
N


Bacillus aestuarii
197
GQ980243
clade_238
Y
N


Bacillus amyloliquefaciens
199
NR_075005
clade_238
Y
N


Bacillus anthracis
200
AAEN01000020
clade_238
Y
Category-A


Bacillus atrophaeus
201
NR_075016
clade_238
Y
OP


Bacillus badius
202
NR_036893
clade_238
Y
OP


Bacillus cereus
203
ABDJ01000015
clade_238
Y
OP


Bacillus circulans
204
AB271747
clade_238
Y
OP


Bacillus firmus
207
NR_025842
clade_238
Y
OP


Bacillus flexus
208
NR_024691
clade_238
Y
OP


Bacillus fordii
209
NR_025786
clade_238
Y
OP


Bacillus halmapalus
211
NR_026144
clade_238
Y
OP


Bacillus herbersteinensis
213
NR_042286
clade_238
Y
OP


Bacillus idriensis
215
NR_043268
clade_238
Y
OP


Bacillus lentus
216
NR_040792
clade_238
Y
OP


Bacillus licheniformis
217
NC_006270
clade_238
Y
OP


Bacillus megaterium
218
GU252124
clade_238
Y
OP


Bacillus nealsonii
219
NR_044546
clade_238
Y
OP


Bacillus niabensis
220
NR_043334
clade_238
Y
OP


Bacillus niacini
221
NR_024695
clade_238
Y
OP


Bacillus pocheonensis
222
NR_041377
clade_238
Y
OP


Bacillus pumilus
223
NR_074977
clade_238
Y
OP


Bacillus safensis
224
JQ624766
clade_238
Y
OP


Bacillus simplex
225
NR_042136
clade_238
Y
OP


Bacillus sonorensis
226
NR_025130
clade_238
Y
OP


Bacillus sp. 10403023
227
CAET01000089
clade_238
Y
OP


MM10403188


Bacillus sp. 2_A_57_CT2
230
ACWD01000095
clade_238
Y
OP


Bacillus sp. 2008724126
228
GU252108
clade_238
Y
OP


Bacillus sp. 2008724139
229
GU252111
clade_238
Y
OP


Bacillus sp. 7_16AIA
231
FN397518
clade_238
Y
OP


Bacillus sp. AP8
233
JX101689
clade_238
Y
OP


Bacillus sp. B27(2008)
234
EU362173
clade_238
Y
OP


Bacillus sp. BT1B_CT2
235
ACWC01000034
clade_238
Y
OP


Bacillus sp. GB1.1
236
FJ897765
clade_238
Y
OP


Bacillus sp. GB9
237
FJ897766
clade_238
Y
OP


Bacillus sp. HU19.1
238
FJ897769
clade_238
Y
OP


Bacillus sp. HU29
239
FJ897771
clade_238
Y
OP


Bacillus sp. HU33.1
240
FJ897772
clade_238
Y
OP


Bacillus sp. JC6
241
JF824800
clade_238
Y
OP


Bacillus sp. oral taxon F79
248
HM099654
clade_238
Y
OP


Bacillus sp. SRC_DSF1
243
GU797283
clade_238
Y
OP


Bacillus sp. SRC_DSF10
242
GU797292
clade_238
Y
OP


Bacillus sp. SRC_DSF2
244
GU797284
clade_238
Y
OP


Bacillus sp. SRC_DSF6
245
GU797288
clade_238
Y
OP


Bacillus sp. tc09
249
HQ844242
clade_238
Y
OP


Bacillus sp. zh168
250
FJ851424
clade_238
Y
OP


Bacillus sphaericus
251
DQ286318
clade_238
Y
OP


Bacillus sporothermodurans
252
NR_026010
clade_238
Y
OP


Bacillus subtilis
253
EU627588
clade_238
Y
OP


Bacillus thermoamylovorans
254
NR_029151
clade_238
Y
OP


Bacillus thuringiensis
255
NC_008600
clade_238
Y
OP


Bacillus weihenstephanensis
256
NR_074926
clade_238
Y
OP


Geobacillus kaustophilus
933
NR_074989
clade_238
Y
N


Geobacillus
936
NR_040794
clade_238
Y
N


stearothermophilus


Geobacillus
938
NR_074976
clade_238
Y
N


thermodenitrificans


Geobacillus
939
NR_043022
clade_238
Y
N


thermoglucosidasius


Lysinibacillus sphaericus
1193
NR_074883
clade_238
Y
N


Clostridiales sp. SS3_4
543
AY305316
clade_246
Y
N


Clostridium beijerinckii
557
NR_074434
clade_252
Y
N


Clostridium botulinum
560
NC_010723
clade_252
Y
Category-A


Clostridium butyricum
561
ABDT01000017
clade_252
Y
N


Clostridium chauvoei
568
EU106372
clade_252
Y
N


Clostridium favososporum
582
X76749
clade_252
Y
N


Clostridium histolyticum
592
HF558362
clade_252
Y
N


Clostridium isatidis
597
NR_026347
clade_252
Y
N


Clostridium limosum
602
FR870444
clade_252
Y
N


Clostridium sartagoforme
622
NR_026490
clade_252
Y
N


Clostridium septicum
624
NR_026020
clade_252
Y
N


Clostridium sp. 7_2_43FAA
626
ACDK01000101
clade_252
Y
N


Clostridium sporogenes
645
ABKW02000003
clade_252
Y
N


Clostridium tertium
653
Y18174
clade_252
Y
N


Clostridium carnis
564
NR_044716
clade_253
Y
N


Clostridium celatum
565
X77844
clade_253
Y
N


Clostridium disporicum
579
NR_026491
clade_253
Y
N


Clostridium gasigenes
585
NR_024945
clade_253
Y
N


Clostridium quinii
616
NR_026149
clade_253
Y
N


Clostridium hylemonae
593
AB023973
clade_260
Y
N


Clostridium scindens
623
AF262238
clade_260
Y
N


Lachnospiraceae bacterium
1054
ACTR01000020
clade_260
Y
N


5_1_57FAA


Clostridium
588
AB233029
clade_262
Y
N


glycyrrhizinilyticum


Clostridium nexile
607
X73443
clade_262
Y
N


Coprococcus comes
674
ABVR01000038
clade_262
Y
N


Lachnospiraceae bacterium
1048
ACTM01000065
clade_262
Y
N


1_1_57FAA


Lachnospiraceae bacterium
1049
ACTN01000028
clade_262
Y
N


1_4_56FAA


Lachnospiraceae bacterium
1057
ACWQ01000079
clade_262
Y
N


8_1_57FAA


Ruminococcus lactaris
1663
ABOU02000049
clade_262
Y
N


Ruminococcus torques
1670
AAVP02000002
clade_262
Y
N


Paenibacillus lautus
1397
NR_040882
clade_270
Y
N


Paenibacillus polymyxa
1399
NR_037006
clade_270
Y
N


Paenibacillus sp. HGF5
1402
AEXS01000095
clade_270
Y
N


Paenibacillus sp. HGF7
1403
AFDH01000147
clade_270
Y
N


Eubacterium sp. oral clone
868
AY349379
clade_298
Y
N


JI012


Alicyclobacillus
124
NR_041475
clade_301
Y
N


contaminans


Alicyclobacillus herbarius
126
NR_024753
clade_301
Y
N


Alicyclobacillus pomorum
127
NR_024801
clade_301
Y
N


Blautia coccoides
373
AB571656
clade_309
Y
N


Blautia glucerasea
374
AB588023
clade_309
Y
N


Blautia glucerasei
375
AB439724
clade_309
Y
N


Blautia hansenii
376
ABYU02000037
clade_309
Y
N


Blautia luti
378
AB691576
clade_309
Y
N


Blautia producta
379
AB600998
clade_309
Y
N


Blautia schinkii
380
NR_026312
clade_309
Y
N


Blautia sp. M25
381
HM626178
clade_309
Y
N


Blautia stercoris
382
HM626177
clade_309
Y
N


Blautia wexlerae
383
EF036467
clade_309
Y
N


Bryantella formatexigens
439
ACCL02000018
clade_309
Y
N


Clostridium coccoides
573
EF025906
clade_309
Y
N


Eubacterium cellulosolvens
839
AY178842
clade_309
Y
N


Lachnospiraceae bacterium
1056
ACTV01000014
clade_309
Y
N


6_1_63FAA


Ruminococcus hansenii
1662
M59114
clade_309
Y
N


Ruminococcus obeum
1664
AY169419
clade_309
Y
N


Ruminococcus sp.
1666
ACII01000172
clade_309
Y
N


5_1_39BFAA


Ruminococcus sp. K_1
1669
AB222208
clade_309
Y
N


Syntrophococcus
1911
NR_036869
clade_309
Y
N


sucromutans


Bacillus alcalophilus
198
X76436
clade_327
Y
N


Bacillus clausii
205
FN397477
clade_327
Y
OP


Bacillus gelatini
210
NR_025595
clade_327
Y
OP


Bacillus halodurans
212
AY144582
clade_327
Y
OP


Bacillus sp. oral taxon F26
246
HM099642
clade_327
Y
OP


Clostridium innocuum
595
M23732
clade_351
Y
N


Clostridium sp. HGF2
628
AENW01000022
clade_351
Y
N


Clostridium perfringens
612
ABDW01000023
clade_353
Y
Category-B


Sarcina ventriculi
1687
NR_026146
clade_353
Y
N


Clostridium bartlettii
556
ABEZ02000012
clade_354
Y
N


Clostridium bifermentans
558
X73437
clade_354
Y
N


Clostridium ghonii
586
AB542933
clade_354
Y
N


Clostridium glycolicum
587
FJ384385
clade_354
Y
N


Clostridium mayombei
605
FR733682
clade_354
Y
N


Clostridium sordellii
625
AB448946
clade_354
Y
N


Clostridium sp. MT4 E
635
FJ159523
clade_354
Y
N


Eubacterium tenue
872
M59118
clade_354
Y
N


Clostridium argentinense
553
NR_029232
clade_355
Y
N


Clostridium sp. JC122
630
CAEV01000127
clade_355
Y
N


Clostridium sp. NMBHI_1
636
JN093130
clade_355
Y
N


Clostridium subterminale
650
NR_041795
clade_355
Y
N


Clostridium sulfidigenes
651
NR_044161
clade_355
Y
N


Dorea formicigenerans
773
AAXA02000006
clade_360
Y
N


Dorea longicatena
774
AJ132842
clade_360
Y
N


Lachnospiraceae bacterium
1050
ADLB01000035
clade_360
Y
N


2_1_46FAA


Lachnospiraceae bacterium
1051
ACTO01000052
clade_360
Y
N


2_1_58FAA


Lachnospiraceae bacterium
1053
ADCR01000030
clade_360
Y
N


4_1_37FAA


Lachnospiraceae bacterium
1058
ACTX01000023
clade_360
Y
N


9_1_43BFAA


Ruminococcus gnavus
1661
X94967
clade_360
Y
N


Ruminococcus sp. ID8
1668
AY960564
clade_360
Y
N


Blautia hydrogenotrophica
377
ACBZ01000217
clade_368
Y
N


Lactonifactor longoviformis
1147
DQ100449
clade_368
Y
N


Robinsoniella peoriensis
1633
AF445258
clade_368
Y
N


Eubacterium infirmum
849
U13039
clade_384
Y
N


Eubacterium sp. WAL
864
FJ687606
clade_384
Y
N


14571


Erysipelotrichaceae
823
ACZW01000054
clade_385
Y
N


bacterium 5_2_54FAA


Eubacterium biforme
835
ABYT01000002
clade_385
Y
N


Eubacterium cylindroides
842
FP929041
clade_385
Y
N


Eubacterium dolichum
844
L34682
clade_385
Y
N


Eubacterium sp. 3_1_31
861
ACTL01000045
clade_385
Y
N


Eubacterium tortuosum
873
NR_044648
clade_385
Y
N


Bulleidia extructa
441
ADFR01000011
clade_388
Y
N


Solobacterium moorei
1739
AECQ01000039
clade_388
Y
N


Coprococcus catus
673
EU266552
clade_393
Y
N


Lachnospiraceae bacterium
1064
HM099641
clade_393
Y
N


oral taxon F15


Clostridium cochlearium
574
NR_044717
clade_395
Y
N


Clostridium malenominatum
604
FR749893
clade_395
Y
N


Clostridium tetani
654
NC_004557
clade_395
Y
N


Acetivibrio ethanolgignens
6
FR749897
clade_396
Y
N


Anaerosporobacter mobilis
161
NR_042953
clade_396
Y
N


Bacteroides pectinophilus
288
ABVQ01000036
clade_396
Y
N


Clostridium aminovalericum
551
NR_029245
clade_396
Y
N


Clostridium
613
NR_074652
clade_396
Y
N


phytofermentans


Eubacterium hallii
848
L34621
clade_396
Y
N


Eubacterium xylanophilum
875
L34628
clade_396
Y
N


Ruminococcus callidus
1658
NR_029160
clade_406
Y
N


Ruminococcus
1659
FP929052
clade_406
Y
N


champanellensis


Ruminococcus sp. 18P13
1665
AJ515913
clade_406
Y
N


Ruminococcus sp. 9SE51
1667
FM954974
clade_406
Y
N


Anaerostipes caccae
162
ABAX03000023
clade_408
Y
N


Anaerostipes sp.
163
ACWB01000002
clade_408
Y
N


3_2_56FAA


Clostridiales bacterium
541
ABQR01000074
clade_408
Y
N


1_7_47FAA


Clostridiales sp. SM4_1
542
FP929060
clade_408
Y
N


Clostridiales sp. SSC_2
544
FP929061
clade_408
Y
N


Clostridium aerotolerans
546
X76163
clade_408
Y
N


Clostridium aldenense
547
NR_043680
clade_408
Y
N


Clostridium
550
NR_028726
clade_408
Y
N


algidixylanolyticum


Clostridium amygdalinum
552
AY353957
clade_408
Y
N


Clostridium asparagiforme
554
ACCJ01000522
clade_408
Y
N


Clostridium bolteae
559
ABCC02000039
clade_408
Y
N


Clostridium celerecrescens
566
JQ246092
clade_408
Y
N


Clostridium citroniae
569
ADLJ01000059
clade_408
Y
N


Clostridium clostridiiformes
571
M59089
clade_408
Y
N


Clostridium clostridioforme
572
NR_044715
clade_408
Y
N


Clostridium hathewayi
590
AY552788
clade_408
Y
N


Clostridium indolis
594
AF028351
clade_408
Y
N


Clostridium lavalense
600
EF564277
clade_408
Y
N


Clostridium
620
CP002109
clade_408
Y
N


saccharolyticum


Clostridium sp. M62_1
633
ACFX02000046
clade_408
Y
N


Clostridium sp. SS2_1
638
ABGC03000041
clade_408
Y
N


Clostridium sphenoides
643
X73449
clade_408
Y
N


Clostridium symbiosum
652
ADLQ01000114
clade_408
Y
N


Clostridium xylanolyticum
658
NR_037068
clade_408
Y
N


Eubacterium hadrum
847
FR749933
clade_408
Y
N


Lachnospiraceae bacterium
1052
ACTP01000124
clade_408
Y
N


3_1_57FAA_CT1


Lachnospiraceae bacterium
1055
ACTS01000081
clade_408
Y
N


5_1_63FAA


Lachnospiraceae bacterium
1059
DQ789118
clade_408
Y
N


A4


Lachnospiraceae bacterium
1060
EU728771
clade_408
Y
N


DJF VP30


Lachnospiraceae genomosp.
1065
AY278618
clade_408
Y
N


C1


Clostridium difficile
578
NC_013315
clade_409
Y
OP


Eubacterium sp. AS15b
862
HQ616364
clade_428
Y
N


Eubacterium sp. OBRC9
863
HQ616354
clade_428
Y
N


Eubacterium sp. oral clone
871
AY947497
clade_428
Y
N


OH3A


Eubacterium yurii
876
AEES01000073
clade_428
Y
N


Clostridium acetobutylicum
545
NR_074511
clade_430
Y
N


Clostridium algidicarnis
549
NR_041746
clade_430
Y
N


Clostridium cadaveris
562
AB542932
clade_430
Y
N


Clostridium carboxidivorans
563
FR733710
clade_430
Y
N


Clostridium estertheticum
580
NR_042153
clade_430
Y
N


Clostridium fallax
581
NR_044714
clade_430
Y
N


Clostridium felsineum
583
AF270502
clade_430
Y
N


Clostridium frigidicarnis
584
NR_024919
clade_430
Y
N


Clostridium kluyveri
598
NR_074165
clade_430
Y
N


Clostridium magnum
603
X77835
clade_430
Y
N


Clostridium putrefaciens
615
NR_024995
clade_430
Y
N


Clostridium sp. HPB_46
629
AY862516
clade_430
Y
N


Clostridium tyrobutyricum
656
NR_044718
clade_430
Y
N


Sutterella parvirubra
1899
AB300989
clade_432
Y
N


Acetanaerobacterium
4
NR_042930
clade_439
Y
N


elongatum


Clostridium cellulosi
567
NR_044624
clade_439
Y
N


Ethanoligenens harbinense
832
AY675965
clade_439
Y
N


Eubacterium rectale
856
FP929042
clade_444
Y
N


Eubacterium sp. oral clone
865
AY349374
clade_444
Y
N


GI038


Lachnobacterium bovis
1045
GU324407
clade_444
Y
N


Roseburia cecicola
1634
GU233441
clade_444
Y
N


Roseburia faecalis
1635
AY804149
clade_444
Y
N


Roseburia faecis
1636
AY305310
clade_444
Y
N


Roseburia hominis
1637
AJ270482
clade_444
Y
N


Roseburia intestinalis
1638
FP929050
clade_444
Y
N


Roseburia inulinivorans
1639
AJ270473
clade_444
Y
N


Brevibacillus brevis
410
NR_041524
clade_448
Y
N


Brevibacillus laterosporus
414
NR_037005
clade_448
Y
N


Bacillus coagulans
206
DQ297928
clade_451
Y
OP


Sporolactobacillus inulinus
1752
NR_040962
clade_451
Y
N


Kocuria palustris
1041
EU333884
clade_453
Y
N


Nocardia farcinica
1353
NC_006361
clade_455
Y
N


Bacillus sp. oral taxon F28
247
HM099650
clade_456
Y
OP


Catenibacterium mitsuokai
495
AB030224
clade_469
Y
N


Clostridium sp. TM_40
640
AB249652
clade_469
Y
N


Coprobacillus cateniformis
670
AB030218
clade_469
Y
N


Coprobacillus sp. 29_1
671
ADKX01000057
clade_469
Y
N


Clostridium rectum
618
NR_029271
clade_470
Y
N


Eubacterium nodatum
854
U13041
clade_476
Y
N


Eubacterium saphenum
859
NR_026031
clade_476
Y
N


Eubacterium sp. oral clone
867
AY349373
clade_476
Y
N


JH012


Eubacterium sp. oral clone
870
AY349378
clade_476
Y
N


JS001


Faecalibacterium prausnitzii
880
ACOP02000011
clade_478
Y
N


Gemmiger formicilis
932
GU562446
clade_478
Y
N


Subdoligranulum variabile
1896
AJ518869
clade_478
Y
N


Clostridiaceae bacterium
532
JF824807
clade_479
Y
N


JC13


Clostridium sp. MLG055
634
AF304435
clade_479
Y
N


Erysipelotrichaceae
822
ACTJ01000113
clade_479
Y
N


bacterium 3_1_53


Clostridium cocleatum
575
NR_026495
clade_481
Y
N


Clostridium ramosum
617
M23731
clade_481
Y
N


Clostridium saccharogumia
619
DQ100445
clade_481
Y
N


Clostridium spiroforme
644
X73441
clade_481
Y
N


Coprobacillus sp. D7
672
ACDT01000199
clade_481
Y
N


Clostridiales bacterium
535
AB477431
clade_482
Y
N


SY8519


Clostridium sp. SY8519
639
AP012212
clade_482
Y
N


Eubacterium ramulus
855
AJ011522
clade_482
Y
N


Erysipelothrix inopinata
819
NR_025594
clade_485
Y
N


Erysipelothrix rhusiopathiae
820
ACLK01000021
clade_485
Y
N


Erysipelothrix tonsillarum
821
NR_040871
clade_485
Y
N


Holdemania filiformis
1004
Y11466
clade_485
Y
N


Mollicutes bacterium
1258
AY297808
clade_485
Y
N


pACH93


Coxiella burnetii
736
CP000890
clade_486
Y
Category-B


Clostridium hiranonis
591
AB023970
clade_487
Y
N


Clostridium irregulare
596
NR_029249
clade_487
Y
N


Clostridium orbiscindens
609
Y18187
clade_494
Y
N


Clostridium sp. NML
637
EU815224
clade_494
Y
N


04A032


Flavonifractor plautii
886
AY724678
clade_494
Y
N


Pseudoflavonifractor
1591
AY136666
clade_494
Y
N


capillosus


Ruminococcaceae bacterium
1655
ADDX01000083
clade_494
Y
N


D16


Acetivibrio cellulolyticus
5
NR_025917
clade_495
Y
N


Clostridium aldrichii
548
NR_026099
clade_495
Y
N


Clostridium clariflavum
570
NR_041235
clade_495
Y
N


Clostridium stercorarium
647
NR_025100
clade_495
Y
N


Clostridium straminisolvens
649
NR_024829
clade_495
Y
N


Clostridium thermocellum
655
NR_074629
clade_495
Y
N


Fusobacterium nucleatum
901
ADVK01000034
clade_497
Y
N


Eubacterium barkeri
834
NR_044661
clade_512
Y
N


Eubacterium callanderi
838
NR_026330
clade_512
Y
N


Eubacterium limosum
850
CP002273
clade_512
Y
N


Anaerotruncus colihominis
164
ABGD02000021
clade_516
Y
N


Clostridium
606
ACEC01000059
clade_516
Y
N


methylpentosum


Clostridium sp. YIT 12070
642
AB491208
clade_516
Y
N


Hydrogenoanaerobacterium
1005
NR_044425
clade_516
Y
N


saccharovorans


Ruminococcus albus
1656
AY445600
clade_516
Y
N


Ruminococcus flavefaciens
1660
NR_025931
clade_516
Y
N


Clostridium haemolyticum
589
NR_024749
clade_517
Y
N


Clostridium novyi
608
NR_074343
clade_517
Y
N


Clostridium sp. LMG 16094
632
X95274
clade_517
Y
N


Eubacterium ventriosum
874
L34421
clade_519
Y
N


Bacteroides galacturonicus
280
DQ497994
clade_522
Y
N


Eubacterium eligens
845
CP001104
clade_522
Y
N


Lachnospira multipara
1046
FR733699
clade_522
Y
N


Lachnospira pectinoschiza
1047
L14675
clade_522
Y
N


Lactobacillus rogosae
1114
GU269544
clade_522
Y
N


Bacillus horti
214
NR_036860
clade_527
Y
OP


Bacillus sp. 9_3AIA
232
FN397519
clade_527
Y
OP


Eubacterium brachy
836
U13038
clade_533
Y
N


Filifactor alocis
881
CP002390
clade_533
Y
N


Filifactor villosus
882
NR_041928
clade_533
Y
N


Clostridium leptum
601
AJ305238
clade_537
Y
N


Clostridium sp. YIT 12069
641
AB491207
clade_537
Y
N


Clostridium
646
NR_044835
clade_537
Y
N


sporosphaeroides


Eubacterium
841
HM037995
clade_537
Y
N


coprostanoligenes


Ruminococcus bromii
1657
EU266549
clade_537
Y
N


Eubacterium siraeum
860
ABCA03000054
clade_538
Y
N


Clostridium viride
657
NR_026204
clade_540
Y
N


Oscillibacter sp. G2
1386
HM626173
clade_540
Y
N


Oscillibacter valericigenes
1387
NR_074793
clade_540
Y
N


Oscillospira guilliermondii
1388
AB040495
clade_540
Y
N


Butyrivibrio crossotus
455
ABWN01000012
clade_543
Y
N


Clostridium sp. L2_50
631
AAYW02000018
clade_543
Y
N


Coprococcus eutactus
675
EF031543
clade_543
Y
N


Coprococcus sp. ART55_1
676
AY350746
clade_543
Y
N


Eubacterium ruminantium
857
NR_024661
clade_543
Y
N


Collinsella aerofaciens
659
AAVN02000007
clade_553
Y
N


Alkaliphilus
137
AY137848
clade_554
Y
N


metalliredigenes


Alkaliphilus oremlandii
138
NR_043674
clade_554
Y
N


Clostridium sticklandii
648
L04167
clade_554
Y
N


Turicibacter sanguinis
1965
AF349724
clade_555
Y
N


Fulvimonas sp. NML
892
EF589680
clade_557
Y
N


060897


Desulfitobacterium frappieri
753
AJ276701
clade_560
Y
N


Desulfitobacterium
754
NR_074996
clade_560
Y
N


hafniense


Desulfotomaculum
756
NR_044832
clade_560
Y
N


nigrificans


Lutispora thermophila
1191
NR_041236
clade_564
Y
N


Brachyspira pilosicoli
405
NR_075069
clade_565
Y
N


Eggerthella lenta
778
AF292375
clade_566
Y
N


Streptomyces albus
1888
AJ697941
clade_566
Y
N


Chlamydiales bacterium
505
JN606074
clade_567
Y
N


NS11


Anaerofustis
159
ABIL02000005
clade_570
Y
N


stercorihominis


Butyricicoccus
453
HH793440
clade_572
Y
N


pullicaecorum


Eubacterium desmolans
843
NR_044644
clade_572
Y
N


Papillibacter cinnamivorans
1415
NR_025025
clade_572
Y
N


Sporobacter termitidis
1751
NR_044972
clade_572
Y
N


Deferribacteres sp. oral
744
AY349371
clade_575
Y
N


clone JV006


Clostridium colinum
577
NR_026151
clade_576
Y
N


Clostridium
599
NR_025651
clade_576
Y
N


lactatifermentans


Clostridium piliforme
614
D14639
clade_576
Y
N


Saccharomonospora viridis
1671
X54286
clade_579
Y
N


Thermobifida fusca
1921
NC_007333
clade_579
Y
N


Leptospira licerasiae
1164
EF612284
clade_585
Y
OP


Moorella thermoacetica
1259
NR_075001
clade_590
Y
N


Thermoanaerobacter
1920
CP000924
clade_590
Y
N


pseudethanolicus


Flexistipes sinusarabici
888
NR_074881
clade_591
Y
N


Gloeobacter violaceus
942
NR_074282
clade_596
Y
N


Eubacterium sp. oral clone
869
AY349377
clade_90
Y
N


JN088


Clostridium oroticum
610
FR749922
clade_96
Y
N


Clostridium sp. D5
627
ADBG01000142
clade_96
Y
N


Eubacterium contortum
840
FR749946
clade_96
Y
N


Eubacterium fissicatena
846
FR749935
clade_96
Y
N


Corynebacterium coyleae
692
X96497
clade_100
N
N


Corynebacterium
711
NR_026396
clade_100
N
N


mucifaciens


Corynebacterium
733
AM397636
clade_100
N
N


ureicelerivorans


Corynebacterium appendicis
684
NR_028951
clade_102
N
N


Corynebacterium genitalium
698
ACLJ01000031
clade_102
N
N


Corynebacterium glaucum
699
NR_028971
clade_102
N
N


Corynebacterium imitans
703
AF537597
clade_102
N
N


Corynebacterium riegelii
719
EU848548
clade_102
N
N


Corynebacterium sp.
723
HE575405
clade_102
N
N


L_2012475


Corynebacterium sp. NML
724
GU238409
clade_102
N
N


93_0481


Corynebacterium
728
Y09655
clade_102
N
N


sundsvallense


Corynebacterium tuscaniae
730
AY677186
clade_102
N
N


Prevotella maculosa
1504
AGEK01000035
clade_104
N
N


Prevotella oris
1513
ADDV01000091
clade_104
N
N


Prevotella salivae
1517
AB108826
clade_104
N
N


Prevotella sp. ICM55
1521
HQ616399
clade_104
N
N


Prevotella sp. oral clone
1528
AY005057
clade_104
N
N


AA020


Prevotella sp. oral clone
1538
AY349396
clade_104
N
N


GI032


Prevotella sp. oral taxon
1558
GU432179
clade_104
N
N


G70


Prevotella corporis
1491
L16465
clade_105
N
N


Bacteroides sp. 4_1_36
312
ACTC01000133
clade_110
N
N


Bacteroides sp. AR20
315
AF139524
clade_110
N
N


Bacteroides sp. D20
319
ACPT01000052
clade_110
N
N


Bacteroides sp. F_4
322
AB470322
clade_110
N
N


Bacteroides uniformis
329
AB050110
clade_110
N
N


Prevotella nanceiensis
1510
JN867228
clade_127
N
N


Prevotella sp. oral taxon 299
1548
ACWZ01000026
clade_127
N
N


Prevotella bergensis
1485
ACKS01000100
clade_128
N
N


Prevotella buccalis
1489
JN867261
clade_129
N
N


Prevotella timonensis
1564
ADEF01000012
clade_129
N
N


Prevotella oralis
1512
AEPE01000021
clade_130
N
N


Prevotella sp. SEQ072
1525
JN867238
clade_130
N
N


Leuconostoc carnosum
1177
NR_040811
clade_135
N
N


Leuconostoc gasicomitatum
1179
FN822744
clade_135
N
N


Leuconostoc inhae
1180
NR_025204
clade_135
N
N


Leuconostoc kimchii
1181
NR_075014
clade_135
N
N


Edwardsiella tarda
777
CP002154
clade_139
N
N


Photorhabdus asymbiotica
1466
Z76752
clade_139
N
N


Psychrobacter arcticus
1607
CP000082
clade_141
N
N


Psychrobacter cibarius
1608
HQ698586
clade_141
N
N


Psychrobacter
1609
CP000323
clade_141
N
N


cryohalolentis


Psychrobacter faecalis
1610
HQ698566
clade_141
N
N


Psychrobacter nivimaris
1611
HQ698587
clade_141
N
N


Psychrobacter pulmonis
1612
HQ698582
clade_141
N
N


Pseudomonas aeruginosa
1592
AABQ07000001
clade_154
N
N


Pseudomonas sp. 2_1_26
1600
ACWU01000257
clade_154
N
N


Corynebacterium confusum
691
Y15886
clade_158
N
N


Corynebacterium
712
NR_037038
clade_158
N
N


propinquum


Corynebacterium
713
X84258
clade_158
N
N


pseudodiphtheriticum


Bartonella bacilliformis
338
NC_008783
clade_159
N
N


Bartonella grahamii
339
CP001562
clade_159
N
N


Bartonella henselae
340
NC_005956
clade_159
N
N


Bartonella quintana
341
BX897700
clade_159
N
N


Bartonella tamiae
342
EF672728
clade_159
N
N


Bartonella washoensis
343
FJ719017
clade_159
N
N


Brucella abortus
430
ACBJ01000075
clade_159
N
Category-B


Brucella canis
431
NR_044652
clade_159
N
Category-B


Brucella ceti
432
ACJD01000006
clade_159
N
Category-B


Brucella melitensis
433
AE009462
clade_159
N
Category-B


Brucella microti
434
NR_042549
clade_159
N
Category-B


Brucella ovis
435
NC_009504
clade_159
N
Category-B


Brucella sp. 83_13
436
ACBQ01000040
clade_159
N
Category-B


Brucella sp. BO1
437
EU053207
clade_159
N
Category-B


Brucella suis
438
ACBK01000034
clade_159
N
Category-B


Ochrobactrum anthropi
1360
NC_009667
clade_159
N
N


Ochrobactrum intermedium
1361
ACQA01000001
clade_159
N
N


Ochrobactrum
1362
DQ365921
clade_159
N
N


pseudintermedium


Prevotella genomosp. C2
1496
AY278625
clade_164
N
N


Prevotella
1509
AFJE01000016
clade_164
N
N


multisaccharivorax


Prevotella sp. oral clone
1543
AY550997
clade_164
N
N


IDR_CEC_0055


Prevotella sp. oral taxon 292
1547
GQ422735
clade_164
N
N


Prevotella sp. oral taxon 300
1549
GU409549
clade_164
N
N


Prevotella marshii
1505
AEEI01000070
clade_166
N
N


Prevotella sp. oral clone
1544
AY349401
clade_166
N
N


IK053


Prevotella sp. oral taxon 781
1554
GQ422744
clade_166
N
N


Prevotella stercorea
1562
AB244774
clade_166
N
N


Prevotella brevis
1487
NR_041954
clade_167
N
N


Prevotella ruminicola
1516
CP002006
clade_167
N
N


Prevotella sp. sp24
1560
AB003384
clade_167
N
N


Prevotella sp. sp34
1561
AB003385
clade_167
N
N


Prevotella albensis
1483
NR_025300
clade_168
N
N


Prevotella copri
1490
ACBX02000014
clade_168
N
N


Prevotella oulorum
1514
L16472
clade_168
N
N


Prevotella sp. BI_42
1518
AJ581354
clade_168
N
N


Prevotella sp. oral clone
1546
AY207050
clade_168
N
N


P4PB_83 P2


Prevotella sp. oral taxon
1557
GU432133
clade_168
N
N


G60


Prevotella amnii
1484
AB547670
clade_169
N
N


Bacteroides caccae
268
EU136686
clade_170
N
N


Bacteroides finegoldii
277
AB222699
clade_170
N
N


Bacteroides intestinalis
283
ABJL02000006
clade_171
N
N


Bacteroides sp. XB44A
326
AM230649
clade_171
N
N


Bifidobacteriaceae
345
AY278612
clade_172
N
N


genomosp. C1


Bifidobacterium
346
AAXD02000018
clade_172
N
N


adolescentis


Bifidobacterium angulatum
347
ABYS02000004
clade_172
N
N


Bifidobacterium animalis
348
CP001606
clade_172
N
N


Bifidobacterium breve
350
CP002743
clade_172
N
N


Bifidobacterium
351
ABXY01000019
clade_172
N
N


catenulatum


Bifidobacterium dentium
352
CP001750
clade_172
N
OP


Bifidobacterium gallicum
353
ABXB03000004
clade_172
N
N


Bifidobacterium infantis
354
AY151398
clade_172
N
N


Bifidobacterium
355
AB491757
clade_172
N
N


kashiwanohense


Bifidobacterium longum
356
ABQQ01000041
clade_172
N
N


Bifidobacterium
357
ABXX02000002
clade_172
N
N


pseudocatenulatum


Bifidobacterium
358
NR_043442
clade_172
N
N


pseudolongum


Bifidobacterium scardovii
359
AJ307005
clade_172
N
N


Bifidobacterium sp. HM2
360
AB425276
clade_172
N
N


Bifidobacterium sp.
361
JF519685
clade_172
N
N


HMLN12


Bifidobacterium sp. M45
362
HM626176
clade_172
N
N


Bifidobacterium sp. MSX5B
363
HQ616382
clade_172
N
N


Bifidobacterium sp. TM_7
364
AB218972
clade_172
N
N


Bifidobacterium
365
DQ340557
clade_172
N
N


thermophilum


Leuconostoc citreum
1178
AM157444
clade_175
N
N


Leuconostoc lactis
1182
NR_040823
clade_175
N
N


Alicyclobacillus
123
NR_040844
clade_179
N
N


acidoterrestris


Alicyclobacillus
125
NR_024754
clade_179
N
N


cycloheptanicus


Acinetobacter baumannii
27
ACYQ01000014
clade_181
N
N


Acinetobacter calcoaceticus
28
AM157426
clade_181
N
N


Acinetobacter genomosp.
29
AY278636
clade_181
N
N


C1


Acinetobacter haemolyticus
30
ADMT01000017
clade_181
N
N


Acinetobacter johnsonii
31
ACPL01000162
clade_181
N
N


Acinetobacter junii
32
ACPM01000135
clade_181
N
N


Acinetobacter lwoffii
33
ACPN01000204
clade_181
N
N


Acinetobacter parvus
34
AIEB01000124
clade_181
N
N


Acinetobacter schindleri
36
NR_025412
clade_181
N
N


Acinetobacter sp. 56A1
37
GQ178049
clade_181
N
N


Acinetobacter sp. CIP
38
JQ638573
clade_181
N
N


101934


Acinetobacter sp. CIP
39
JQ638578
clade_181
N
N


102143


Acinetobacter sp. M16_22
41
HM366447
clade_181
N
N


Acinetobacter sp. RUH2624
42
ACQF01000094
clade_181
N
N


Acinetobacter sp. SH024
43
ADCH01000068
clade_181
N
N


Lactobacillus jensenii
1092
ACQD01000066
clade_182
N
N


Alcaligenes faecalis
119
AB680368
clade_183
N
N


Alcaligenes sp. CO14
120
DQ643040
clade_183
N
N


Alcaligenes sp. S3
121
HQ262549
clade_183
N
N


Oligella ureolytica
1366
NR_041998
clade_183
N
N


Oligella urethralis
1367
NR_041753
clade_183
N
N


Eikenella corrodens
784
ACEA01000028
clade_185
N
N


Kingella denitrificans
1019
AEWV01000047
clade_185
N
N


Kingella genomosp. P1 oral
1020
DQ003616
clade_185
N
N


cone MB2_C20


Kingella kingae
1021
AFHS01000073
clade_185
N
N


Kingella oralis
1022
ACJW02000005
clade_185
N
N


Kingella sp. oral clone
1023
AY349381
clade_185
N
N


ID059


Neisseria elongata
1330
ADBF01000003
clade_185
N
N


Neisseria genomosp. P2 oral
1332
DQ003630
clade_185
N
N


clone MB5_P15


Neisseria sp. oral clone
1345
AY349388
clade_185
N
N


JC012


Neisseria sp. SMC_A9199
1342
FJ763637
clade_185
N
N


Simonsiella muelleri
1731
ADCY01000105
clade_185
N
N


Corynebacterium
700
ABYP01000081
clade_193
N
N


glucuronolyticum


Corynebacterium
716
FJ185225
clade_193
N
N


pyruviciproducens


Rothia aeria
1649
DQ673320
clade_194
N
N


Rothia dentocariosa
1650
ADDW01000024
clade_194
N
N


Rothia sp. oral taxon 188
1653
GU470892
clade_194
N
N


Corynebacterium accolens
681
ACGD01000048
clade_195
N
N


Corynebacterium
707
AB359393
clade_195
N
N


macginleyi


Corynebacterium
714
ABYQ01000237
clade_195
N
N


pseudogenitalium


Corynebacterium
729
ACVP01000009
clade_195
N
N


tuberculostearicum


Lactobacillus casei
1074
CP000423
clade_198
N
N


Lactobacillus paracasei
1106
ABQV01000067
clade_198
N
N


Lactobacillus zeae
1143
NR_037122
clade_198
N
N


Prevotella dentalis
1492
AB547678
clade_205
N
N


Prevotella sp. oral clone
1529
AY923148
clade_206
N
N


ASCG10


Prevotella sp. oral clone
1541
AY349399
clade_206
N
N


HF050


Prevotella sp. oral clone
1542
AY349400
clade_206
N
N


ID019


Prevotella sp. oral clone
1545
AY349402
clade_206
N
N


IK062


Prevotella genomosp. P9
1499
DQ003633
clade_207
N
N


oral clone MB7_G16


Prevotella sp. oral clone
1531
AY005062
clade_207
N
N


AU069


Prevotella sp. oral clone
1532
AY005063
clade_207
N
N


CY006


Prevotella sp. oral clone
1534
AY349392
clade_207
N
N


FL019


Actinomyces genomosp. C1
56
AY278610
clade_212
N
N


Actinomyces genomosp. C2
57
AY278611
clade_212
N
N


Actinomyces genomosp. P1
58
DQ003632
clade_212
N
N


oral clone MB6_C03


Actinomyces georgiae
59
GU561319
clade_212
N
N


Actinomyces israelii
60
AF479270
clade_212
N
N


Actinomyces massiliensis
61
AB545934
clade_212
N
N


Actinomyces meyeri
62
GU561321
clade_212
N
N


Actinomyces odontolyticus
66
ACYT01000123
clade_212
N
N


Actinomyces orihominis
68
AJ575186
clade_212
N
N


Actinomyces sp. CCUG
71
AJ234058
clade_212
N
N


37290


Actinomyces sp. ICM34
75
HQ616391
clade_212
N
N


Actinomyces sp. ICM41
76
HQ616392
clade_212
N
N


Actinomyces sp. ICM47
77
HQ616395
clade_212
N
N


Actinomyces sp. ICM54
78
HQ616398
clade_212
N
N


Actinomyces sp. oral clone
87
AY349366
clade_212
N
N


IP081


Actinomyces sp. oral taxon
91
AEUH01000060
clade_212
N
N


178


Actinomyces sp. oral taxon
92
AEPP01000041
clade_212
N
N


180


Actinomyces sp. TeJ5
80
GU561315
clade_212
N
N


Haematobacter sp. BC14248
968
GU396991
clade_213
N
N


Paracoccus denitrificans
1424
CP000490
clade_213
N
N


Paracoccus marcusii
1425
NR_044922
clade_213
N
N


Grimontia hollisae
967
ADAQ01000013
clade_216
N
N


Shewanella putrefaciens
1723
CP002457
clade_216
N
N


Afipia genomosp. 4
111
EU117385
clade_217
N
N


Rhodopseudomonas
1626
CP000301
clade_217
N
N


palustris


Methylobacterium
1223
NC_010172
clade_218
N
N


extorquens


Methylobacterium podarium
1224
AY468363
clade_218
N
N


Methylobacterium
1225
GU294320
clade_218
N
N


radiotolerans


Methylobacterium sp. 1sub
1226
AY468371
clade_218
N
N


Methylobacterium sp. MM4
1227
AY468370
clade_218
N
N


Achromobacter denitrificans
18
NR_042021
clade_224
N
N


Achromobacter piechaudii
19
ADMS01000149
clade_224
N
N


Achromobacter
20
ACRC01000072
clade_224
N
N


xylosoxidans


Bordetella bronchiseptica
384
NR_025949
clade_224
N
OP


Bordetella holmesii
385
AB683187
clade_224
N
OP


Bordetella parapertussis
386
NR_025950
clade_224
N
OP


Bordetella pertussis
387
BX640418
clade_224
N
OP


Microbacterium chocolatum
1230
NR_037045
clade_225
N
N


Microbacterium flavescens
1231
EU714363
clade_225
N
N


Microbacterium lacticum
1233
EU714351
clade_225
N
N


Microbacterium oleivorans
1234
EU714381
clade_225
N
N


Microbacterium oxydans
1235
EU714348
clade_225
N
N


Microbacterium
1236
AJ491806
clade_225
N
N


paraoxydans


Microbacterium
1237
EU714359
clade_225
N
N


phyllosphaerae


Microbacterium schleiferi
1238
NR_044936
clade_225
N
N


Microbacterium sp. 768
1239
EU714378
clade_225
N
N


Microbacterium sp. oral
1240
AF287752
clade_225
N
N


strain C24KA


Microbacterium testaceum
1241
EU714365
clade_225
N
N


Corynebacterium atypicum
686
NR_025540
clade_229
N
N


Corynebacterium mastitidis
708
AB359395
clade_229
N
N


Corynebacterium sp. NML
725
GU238411
clade_229
N
N


97_0186


Mycobacterium elephantis
1275
AF385898
clade_237
N
OP


Mycobacterium paraterrae
1288
EU919229
clade_237
N
OP


Mycobacterium phlei
1289
GU142920
clade_237
N
OP


Mycobacterium sp. 1776
1293
EU703152
clade_237
N
N


Mycobacterium sp. 1781
1294
EU703147
clade_237
N
N


Mycobacterium sp.
1297
HM210417
clade_237
N
N


AQ1GA4


Mycobacterium sp.
1299
FJ497243
clade_237
N
N


GN_10546


Mycobacterium sp.
1300
FJ497247
clade_237
N
N


GN_10827


Mycobacterium sp.
1301
FJ652846
clade_237
N
N


GN_11124


Mycobacterium sp.
1302
FJ497240
clade_237
N
N


GN_9188


Mycobacterium sp.
1303
FJ555538
clade_237
N
N


GR_2007_210


Anoxybacillus contaminans
172
NR_029006
clade_238
N
N


Bacillus aeolius
195
NR_025557
clade_238
N
N


Brevibacterium
422
NR_042639
clade_238
N
N


frigoritolerans


Geobacillus sp. E263
934
DQ647387
clade_238
N
N


Geobacillus sp. WCH70
935
CP001638
clade_238
N
N


Geobacillus
937
NR_043020
clade_238
N
N


thermocatenulatus


Geobacillus
940
NR_074931
clade_238
N
N


thermoleovorans


Lysinibacillus fusiformis
1192
FN397522
clade_238
N
N


Planomicrobium koreense
1468
NR_025011
clade_238
N
N


Sporosarcina newyorkensis
1754
AFPZ01000142
clade_238
N
N


Sporosarcina sp. 2681
1755
GU994081
clade_238
N
N


Ureibacillus composti
1968
NR_043746
clade_238
N
N


Ureibacillus suwonensis
1969
NR_043232
clade_238
N
N


Ureibacillus terrenus
1970
NR_025394
clade_238
N
N


Ureibacillus thermophilus
1971
NR_043747
clade_238
N
N


Ureibacillus
1972
NR_040961
clade_238
N
N


thermosphaericus


Prevotella micans
1507
AGWK01000061
clade_239
N
N


Prevotella sp. oral clone
1533
AY005065
clade_239
N
N


DA058


Prevotella sp. SEQ053
1523
JN867222
clade_239
N
N


Treponema socranskii
1937
NR_024868
clade_240
N
OP


Treponema sp. 6:H:D15A_4
1938
AY005083
clade_240
N
N


Treponema sp. oral taxon
1953
GU408850
clade_240
N
N


265


Treponema sp. oral taxon
1958
GU432215
clade_240
N
N


G85


Porphyromonas
1472
ACNN01000021
clade_241
N
N


endodontalis


Porphyromonas sp. oral
1478
AY005068
clade_241
N
N


clone BB134


Porphyromonas sp. oral
1479
AY005069
clade_241
N
N


clone F016


Porphyromonas sp. oral
1480
AY207054
clade_241
N
N


clone P2PB_52 P1


Porphyromonas sp. oral
1481
AY207057
clade_241
N
N


clone P4GB_100 P2


Acidovorax sp. 98_63833
26
AY258065
clade_245
N
N


Comamonadaceae
663
JN585335
clade_245
N
N


bacterium NML000135


Comamonadaceae
664
JN585331
clade_245
N
N


bacterium NML790751


Comamonadaceae
665
JN585332
clade_245
N
N


bacterium NML910035


Comamonadaceae
666
JN585333
clade_245
N
N


bacterium NML910036


Comamonas sp. NSP5
668
AB076850
clade_245
N
N


Delftia acidovorans
748
CP000884
clade_245
N
N


Xenophilus aerolatus
2018
JN585329
clade_245
N
N


Oribacterium sp. oral taxon
1380
ACIQ02000009
clade_246
N
N


078


Oribacterium sp. oral taxon
1381
GQ422713
clade_246
N
N


102


Weissella cibaria
2007
NR_036924
clade_247
N
N


Weissella confusa
2008
NR_040816
clade_247
N
N


Weissella hellenica
2009
AB680902
clade_247
N
N


Weissella kandleri
2010
NR_044659
clade_247
N
N


Weissella koreensis
2011
NR_075058
clade_247
N
N


Weissella
2012
ACKU01000017
clade_247
N
N


paramesenteroides


Weissella sp. KLDS 7.0701
2013
EU600924
clade_247
N
N


Mobiluncus curtisii
1251
AEPZ01000013
clade_249
N
N


Enhydrobacter aerosaccus
785
ACYI01000081
clade_256
N
N


Moraxella osloensis
1262
JN175341
clade_256
N
N


Moraxella sp. GM2
1264
JF837191
clade_256
N
N


Brevibacterium casei
420
JF951998
clade_257
N
N


Brevibacterium epidermidis
421
NR_029262
clade_257
N
N


Brevibacterium sanguinis
426
NR_028016
clade_257
N
N


Brevibacterium sp. H15
427
AB177640
clade_257
N
N


Acinetobacter radioresistens
35
ACVR01000010
clade_261
N
N


Lactobacillus alimentarius
1068
NR_044701
clade_263
N
N


Lactobacillus farciminis
1082
NR_044707
clade_263
N
N


Lactobacillus kimchii
1097
NR_025045
clade_263
N
N


Lactobacillus nodensis
1101
NR_041629
clade_263
N
N


Lactobacillus tucceti
1138
NR_042194
clade_263
N
N


Pseudomonas mendocina
1595
AAUL01000021
clade_265
N
N


Pseudomonas
1598
NR_037000
clade_265
N
N


pseudoalcaligenes


Pseudomonas sp. NP522b
1602
EU723211
clade_265
N
N


Pseudomonas stutzeri
1603
AM905854
clade_265
N
N


Paenibacillus barcinonensis
1390
NR_042272
clade_270
N
N


Paenibacillus barengoltzii
1391
NR_042756
clade_270
N
N


Paenibacillus chibensis
1392
NR_040885
clade_270
N
N


Paenibacillus cookii
1393
NR_025372
clade_270
N
N


Paenibacillus durus
1394
NR_037017
clade_270
N
N


Paenibacillus glucanolyticus
1395
D78470
clade_270
N
N


Paenibacillus lactis
1396
NR_025739
clade_270
N
N


Paenibacillus pabuli
1398
NR_040853
clade_270
N
N


Paenibacillus popilliae
1400
NR_040888
clade_270
N
N


Paenibacillus sp. CIP
1401
HM212646
clade_270
N
N


101062


Paenibacillus sp. JC66
1404
JF824808
clade_270
N
N


Paenibacillus sp. R_27413
1405
HE586333
clade_270
N
N


Paenibacillus sp. R_27422
1406
HE586338
clade_270
N
N


Paenibacillus timonensis
1408
NR_042844
clade_270
N
N


Rothia mucilaginosa
1651
ACVO01000020
clade_271
N
N


Rothia nasimurium
1652
NR_025310
clade_271
N
N


Prevotella sp. oral taxon 302
1550
ACZK01000043
clade_280
N
N


Prevotella sp. oral taxon F68
1556
HM099652
clade_280
N
N


Prevotella tannerae
1563
ACIJ02000018
clade_280
N
N


Prevotellaceae bacterium
1566
AY207061
clade_280
N
N


P4P_62 P1


Porphyromonas
1471
AENO01000048
clade_281
N
N


asaccharolytica


Porphyromonas gingivalis
1473
AE015924
clade_281
N
N


Porphyromonas macacae
1475
NR_025908
clade_281
N
N


Porphyromonas sp. UQD
1477
EU012301
clade_281
N
N


301


Porphyromonas uenonis
1482
ACLR01000152
clade_281
N
N


Leptotrichia buccalis
1165
CP001685
clade_282
N
N


Leptotrichia hofstadii
1168
ACVB02000032
clade_282
N
N


Leptotrichia sp. oral clone
1173
AY349386
clade_282
N
N


HE012


Leptotrichia sp. oral taxon
1176
GU408547
clade_282
N
N


223


Bacteroides fluxus
278
AFBN01000029
clade_285
N
N


Bacteroides helcogenes
281
CP002352
clade_285
N
N


Parabacteroides johnsonii
1419
ABYH01000014
clade_286
N
N


Parabacteroides merdae
1420
EU136685
clade_286
N
N


Treponema denticola
1926
ADEC01000002
clade_288
N
OP


Treponema genomosp. P5
1929
DQ003624
clade_288
N
N


oral clone MB3_P23


Treponema putidum
1935
AJ543428
clade_288
N
OP


Treponema sp. oral clone
1942
AY207055
clade_288
N
N


P2PB_53 P3


Treponema sp. oral taxon
1949
GU408748
clade_288
N
N


247


Treponema sp. oral taxon
1950
GU408776
clade_288
N
N


250


Treponema sp. oral taxon
1951
GU408781
clade_288
N
N


251


Anaerococcus hydrogenalis
144
ABXA01000039
clade_289
N
N


Anaerococcus sp. 8404299
148
HM587318
clade_289
N
N


Anaerococcus sp. gpac215
156
AM176540
clade_289
N
N


Anaerococcus vaginalis
158
ACXU01000016
clade_289
N
N


Propionibacterium
1569
NC_019395
clade_290
N
N


acidipropionici


Propionibacterium avidum
1571
AJ003055
clade_290
N
N


Propionibacterium
1573
FJ785716
clade_290
N
N


granulosum


Propionibacterium jensenii
1574
NR_042269
clade_290
N
N


Propionibacterium
1575
NR_025277
clade_290
N
N


propionicum


Propionibacterium sp. H456
1577
AB177643
clade_290
N
N


Propionibacterium thoenii
1581
NR_042270
clade_290
N
N


Bifidobacterium bifidum
349
ABQP01000027
clade_293
N
N


Leuconostoc mesenteroides
1183
ACKV01000113
clade_295
N
N


Leuconostoc
1184
NR_040814
clade_295
N
N


pseudomesenteroides


Johnsonella ignava
1016
X87152
clade_298
N
N


Propionibacterium acnes
1570
ADJM01000010
clade_299
N
N


Propionibacterium sp.
1576
AFIL01000035
clade_299
N
N


434_HC2


Propionibacterium sp. LG
1578
AY354921
clade_299
N
N


Propionibacterium sp.
1579
AB264622
clade_299
N
N


S555a


Alicyclobacillus sp. CCUG
128
HE613268
clade_301
N
N


53762


Actinomyces cardiffensis
53
GU470888
clade_303
N
N


Actinomyces funkei
55
HQ906497
clade_303
N
N


Actinomyces sp. HKU31
74
HQ335393
clade_303
N
N


Actinomyces sp. oral taxon
94
HM099646
clade_303
N
N


C55


Kerstersia gyiorum
1018
NR_025669
clade_307
N
N


Pigmentiphaga daeguensis
1467
JN585327
clade_307
N
N


Aeromonas
104
S39232
clade_308
N
N


allosaccharophila


Aeromonas enteropelogenes
105
X71121
clade_308
N
N


Aeromonas hydrophila
106
NC_008570
clade_308
N
N


Aeromonas jandaei
107
X60413
clade_308
N
N


Aeromonas salmonicida
108
NC_009348
clade_308
N
N


Aeromonas trota
109
X60415
clade_308
N
N


Aeromonas veronii
110
NR_044845
clade_308
N
N


Marvinbryantia
1196
AJ505973
clade_309
N
N


formatexigens


Rhodobacter sp. oral taxon
1620
HM099648
clade_310
N
N


C30


Rhodobacter sphaeroides
1621
CP000144
clade_310
N
N


Lactobacillus antri
1071
ACLL01000037
clade_313
N
N


Lactobacillus coleohominis
1076
ACOH01000030
clade_313
N
N


Lactobacillus fermentum
1083
CP002033
clade_313
N
N


Lactobacillus gastricus
1085
AICN01000060
clade_313
N
N


Lactobacillus mucosae
1099
FR693800
clade_313
N
N


Lactobacillus oris
1103
AEKL01000077
clade_313
N
N


Lactobacillus pontis
1111
HM218420
clade_313
N
N


Lactobacillus reuteri
1112
ACGW02000012
clade_313
N
N


Lactobacillus sp. KLDS
1127
EU600911
clade_313
N
N


1.0707


Lactobacillus sp. KLDS
1128
EU600913
clade_313
N
N


1.0709


Lactobacillus sp. KLDS
1129
EU600915
clade_313
N
N


1.0711


Lactobacillus sp. KLDS
1131
EU600917
clade_313
N
N


1.0713


Lactobacillus sp. KLDS
1132
EU600921
clade_313
N
N


1.0716


Lactobacillus sp. KLDS
1133
EU600922
clade_313
N
N


1.0718


Lactobacillus sp. oral taxon
1137
GQ422710
clade_313
N
N


052


Lactobacillus vaginalis
1140
ACGV01000168
clade_313
N
N


Brevibacterium aurantiacum
419
NR_044854
clade_314
N
N


Brevibacterium linens
423
AJ315491
clade_314
N
N


Lactobacillus pentosus
1108
JN813103
clade_315
N
N


Lactobacillus plantarum
1110
ACGZ02000033
clade_315
N
N


Lactobacillus sp. KLDS
1123
EU600906
clade_315
N
N


1.0702


Lactobacillus sp. KLDS
1124
EU600907
clade_315
N
N


1.0703


Lactobacillus sp. KLDS
1125
EU600908
clade_315
N
N


1.0704


Lactobacillus sp. KLDS
1126
EU600909
clade_315
N
N


1.0705


Agrobacterium radiobacter
115
CP000628
clade_316
N
N


Agrobacterium tumefaciens
116
AJ389893
clade_316
N
N


Corynebacterium
685
EF463055
clade_317
N
N


argentoratense


Corynebacterium
693
NC_002935
clade_317
N
OP


diphtheriae


Corynebacterium
715
NR_037070
clade_317
N
N


pseudotuberculosis


Corynebacterium renale
717
NR_037069
clade_317
N
N


Corynebacterium ulcerans
731
NR_074467
clade_317
N
N


Aurantimonas coralicida
191
AY065627
clade_318
N
N


Aureimonas altamirensis
192
FN658986
clade_318
N
N


Lactobacillus acidipiscis
1066
NR_024718
clade_320
N
N


Lactobacillus salivarius
1117
AEBA01000145
clade_320
N
N


Lactobacillus sp. KLDS
1134
EU600923
clade_320
N
N


1.0719


Lactobacillus buchneri
1073
ACGH01000101
clade_321
N
N


Lactobacillus genomosp. C1
1086
AY278619
clade_321
N
N


Lactobacillus genomosp. C2
1087
AY278620
clade_321
N
N


Lactobacillus hilgardii
1089
ACGP01000200
clade_321
N
N


Lactobacillus kefiri
1096
NR_042230
clade_321
N
N


Lactobacillus parabuchneri
1105
NR_041294
clade_321
N
N


Lactobacillus parakefiri
1107
NR_029039
clade_321
N
N


Lactobacillus curvatus
1079
NR_042437
clade_322
N
N


Lactobacillus sakei
1116
DQ989236
clade_322
N
N


Aneurinibacillus
167
AB101592
clade_323
N
N


aneurinilyticus


Aneurinibacillus danicus
168
NR_028657
clade_323
N
N


Aneurinibacillus migulanus
169
NR_036799
clade_323
N
N


Aneurinibacillus
170
NR_042271
clade_323
N
N


terranovensis


Staphylococcus aureus
1757
CP002643
clade_325
N
Category-B


Staphylococcus auricularis
1758
JQ624774
clade_325
N
N


Staphylococcus capitis
1759
ACFR01000029
clade_325
N
N


Staphylococcus caprae
1760
ACRH01000033
clade_325
N
N


Staphylococcus carnosus
1761
NR_075003
clade_325
N
N


Staphylococcus cohnii
1762
JN175375
clade_325
N
N


Staphylococcus condimenti
1763
NR_029345
clade_325
N
N


Staphylococcus epidermidis
1764
ACHE01000056
clade_325
N
N


Staphylococcus equorum
1765
NR_027520
clade_325
N
N


Staphylococcus
1767
NC_007168
clade_325
N
N


haemolyticus


Staphylococcus hominis
1768
AM157418
clade_325
N
N


Staphylococcus lugdunensis
1769
AEQA01000024
clade_325
N
N


Staphylococcus pasteuri
1770
FJ189773
clade_325
N
N


Staphylococcus
1771
CP002439
clade_325
N
N


pseudintermedius


Staphylococcus
1772
NR_029158
clade_325
N
N


saccharolyticus


Staphylococcus
1773
NC_007350
clade_325
N
N


saprophyticus


Staphylococcus sp. clone
1777
AF467424
clade_325
N
N


bottae7


Staphylococcus sp. H292
1775
AB177642
clade_325
N
N


Staphylococcus sp. H780
1776
AB177644
clade_325
N
N


Staphylococcus succinus
1778
NR_028667
clade_325
N
N


Staphylococcus warneri
1780
ACPZ01000009
clade_325
N
N


Staphylococcus xylosus
1781
AY395016
clade_325
N
N


Cardiobacterium hominis
490
ACKY01000036
clade_326
N
N


Cardiobacterium valvarum
491
NR_028847
clade_326
N
N


Pseudomonas fluorescens
1593
AY622220
clade_326
N
N


Pseudomonas gessardii
1594
FJ943496
clade_326
N
N


Pseudomonas monteilii
1596
NR_024910
clade_326
N
N


Pseudomonas poae
1597
GU188951
clade_326
N
N


Pseudomonas putida
1599
AF094741
clade_326
N
N


Pseudomonas sp. G1229
1601
DQ910482
clade_326
N
N


Pseudomonas tolaasii
1604
AF320988
clade_326
N
N


Pseudomonas viridiflava
1605
NR_042764
clade_326
N
N


Listeria grayi
1185
ACCR02000003
clade_328
N
OP


Listeria innocua
1186
JF967625
clade_328
N
N


Listeria ivanovii
1187
X56151
clade_328
N
N


Listeria monocytogenes
1188
CP002003
clade_328
N
Category-B


Listeria welshimeri
1189
AM263198
clade_328
N
OP


Capnocytophaga sp. oral
484
AY923149
clade_333
N
N


clone ASCH05


Capnocytophaga sputigena
489
ABZV01000054
clade_333
N
N


Leptotrichia genomosp. C1
1166
AY278621
clade_334
N
N


Leptotrichia shahii
1169
AY029806
clade_334
N
N


Leptotrichia sp.
1170
AF189244
clade_334
N
N


neutropenic Patient


Leptotrichia sp. oral clone
1171
AY349384
clade_334
N
N


GT018


Leptotrichia sp. oral clone
1172
AY349385
clade_334
N
N


GT020


Bacteroides sp. 20_3
296
ACRQ01000064
clade_335
N
N


Bacteroides sp. 3_1_19
307
ADCJ01000062
clade_335
N
N


Bacteroides sp. 3_2_5
311
ACIB01000079
clade_335
N
N


Parabacteroides distasonis
1416
CP000140
clade_335
N
N


Parabacteroides goldsteinii
1417
AY974070
clade_335
N
N


Parabacteroides gordonii
1418
AB470344
clade_335
N
N


Parabacteroides sp. D13
1421
ACPW01000017
clade_335
N
N


Capnocytophaga genomosp.
477
AY278613
clade_336
N
N


C1


Capnocytophaga ochracea
480
AEOH01000054
clade_336
N
N


Capnocytophaga sp. GEJ8
481
GU561335
clade_336
N
N


Capnocytophaga sp. oral
486
AY005077
clade_336
N
N


strain A47ROY


Capnocytophaga sp. S1b
482
U42009
clade_336
N
N


Paraprevotella clara
1426
AFFY01000068
clade_336
N
N


Bacteroides heparinolyticus
282
JN867284
clade_338
N
N


Prevotella heparinolytica
1500
GQ422742
clade_338
N
N


Treponema genomosp. P4
1928
DQ003618
clade_339
N
N


oral clone MB2_G19


Treponema genomosp. P6
1930
DQ003625
clade_339
N
N


oral clone MB4_G11


Treponema sp. oral taxon
1952
GU408803
clade_339
N
N


254


Treponema sp. oral taxon
1956
GU413616
clade_339
N
N


508


Treponema sp. oral taxon
1957
GU413640
clade_339
N
N


518


Chlamydia muridarum
502
AE002160
clade_341
N
OP


Chlamydia trachomatis
504
U68443
clade_341
N
OP


Chlamydia psittaci
503
NR_036864
clade_342
N
Category-B


Chlamydophila pneumoniae
509
NC_002179
clade_342
N
OP


Chlamydophila psittaci
510
D85712
clade_342
N
OP


Anaerococcus octavius
146
NR_026360
clade_343
N
N


Anaerococcus sp. 8405254
149
HM587319
clade_343
N
N


Anaerococcus sp. 9401487
150
HM587322
clade_343
N
N


Anaerococcus sp. 9403502
151
HM587325
clade_343
N
N


Gardnerella vaginalis
923
CP001849
clade_344
N
N


Campylobacter lari
466
CP000932
clade_346
N
OP


Anaerobiospirillum
142
NR_026075
clade_347
N
N


succiniciproducens


Anaerobiospirillum thomasii
143
AJ420985
clade_347
N
N


Ruminobacter amylophilus
1654
NR_026450
clade_347
N
N


Succinatimonas hippei
1897
AEVO01000027
clade_347
N
N


Actinomyces europaeus
54
NR_026363
clade_348
N
N


Actinomyces sp. oral clone
82
AY349361
clade_348
N
N


GU009


Moraxella catarrhalis
1260
CP002005
clade_349
N
N


Moraxella lincolnii
1261
FR822735
clade_349
N
N


Moraxella sp. 16285
1263
JF682466
clade_349
N
N


Psychrobacter sp. 13983
1613
HM212668
clade_349
N
N


Actinobaculum massiliae
49
AF487679
clade_350
N
N


Actinobaculum schaalii
50
AY957507
clade_350
N
N


Actinobaculum sp.
51
AY282578
clade_350
N
N


BM#101342


Actinobaculum sp. P2P_19
52
AY207066
clade_350
N
N


P1


Actinomyces sp. oral clone
84
AY349363
clade_350
N
N


IO076


Actinomyces sp. oral taxon
93
ACUY01000072
clade_350
N
N


848


Actinomyces neuii
65
X71862
clade_352
N
N


Mobiluncus mulieris
1252
ACKW01000035
clade_352
N
N


Blastomonas natatoria
372
NR_040824
clade_356
N
N


Novosphingobium
1357
AAAV03000008
clade_356
N
N


aromaticivorans


Sphingomonas sp. oral
1745
AY349411
clade_356
N
N


clone FI012


Sphingopyxis alaskensis
1749
CP000356
clade_356
N
N


Oxalobacter formigenes
1389
ACDQ01000020
clade_357
N
N


Veillonella atypica
1974
AEDS01000059
clade_358
N
N


Veillonella dispar
1975
ACIK02000021
clade_358
N
N


Veillonella genomosp. P1
1976
DQ003631
clade_358
N
N


oral clone MB5_P17


Veillonella parvula
1978
ADFU01000009
clade_358
N
N


Veillonella sp. 3_1_44
1979
ADCV01000019
clade_358
N
N


Veillonella sp. 6_1_27
1980
ADCW01000016
clade_358
N
N


Veillonella sp. ACP1
1981
HQ616359
clade_358
N
N


Veillonella sp. AS16
1982
HQ616365
clade_358
N
N


Veillonella sp. BS32b
1983
HQ616368
clade_358
N
N


Veillonella sp. ICM51a
1984
HQ616396
clade_358
N
N


Veillonella sp. MSA12
1985
HQ616381
clade_358
N
N


Veillonella sp. NVG 100cf
1986
EF108443
clade_358
N
N


Veillonella sp. OK11
1987
JN695650
clade_358
N
N


Veillonella sp. oral clone
1990
AY923144
clade_358
N
N


ASCG01


Veillonella sp. oral clone
1991
AY953257
clade_358
N
N


ASCG02


Veillonella sp. oral clone
1992
AY947495
clade_358
N
N


OH1A


Veillonella sp. oral taxon
1993
AENU01000007
clade_358
N
N


158


Kocuria marina
1040
GQ260086
clade_365
N
N


Kocuria rhizophila
1042
AY030315
clade_365
N
N


Kocuria rosea
1043
X87756
clade_365
N
N


Kocuria varians
1044
AF542074
clade_365
N
N


Clostridiaceae bacterium
531
EF451053
clade_368
N
N


END_2


Micrococcus antarcticus
1242
NR_025285
clade_371
N
N


Micrococcus luteus
1243
NR_075062
clade_371
N
N


Micrococcus lylae
1244
NR_026200
clade_371
N
N


Micrococcus sp. 185
1245
EU714334
clade_371
N
N


Lactobacillus brevis
1072
EU194349
clade_372
N
N


Lactobacillus parabrevis
1104
NR_042456
clade_372
N
N


Pediococcus acidilactici
1436
ACXB01000026
clade_372
N
N


Pediococcus pentosaceus
1437
NR_075052
clade_372
N
N


Lactobacillus dextrinicus
1081
NR_036861
clade_373
N
N


Lactobacillus perolens
1109
NR_029360
clade_373
N
N


Lactobacillus rhamnosus
1113
ABWJ01000068
clade_373
N
N


Lactobacillus saniviri
1118
AB602569
clade_373
N
N


Lactobacillus sp. BT6
1121
HQ616370
clade_373
N
N


Mycobacterium mageritense
1282
FR798914
clade_374
N
OP


Mycobacterium neoaurum
1286
AF268445
clade_374
N
OP


Mycobacterium smegmatis
1291
CP000480
clade_374
N
OP


Mycobacterium sp. HE5
1304
AJ012738
clade_374
N
N


Dysgonomonas gadei
775
ADLV01000001
clade_377
N
N


Dysgonomonas mossii
776
ADLW01000023
clade_377
N
N


Porphyromonas levii
1474
NR_025907
clade_377
N
N


Porphyromonas somerae
1476
AB547667
clade_377
N
N


Bacteroides barnesiae
267
NR_041446
clade_378
N
N


Bacteroides coprocola
272
ABIY02000050
clade_378
N
N


Bacteroides coprophilus
273
ACBW01000012
clade_378
N
N


Bacteroides dorei
274
ABWZ01000093
clade_378
N
N


Bacteroides massiliensis
284
AB200226
clade_378
N
N


Bacteroides plebeius
289
AB200218
clade_378
N
N


Bacteroides sp. 3_1_33FAA
309
ACPS01000085
clade_378
N
N


Bacteroides sp. 3_1_40A
310
ACRT01000136
clade_378
N
N


Bacteroides sp. 4_3_47FAA
313
ACDR02000029
clade_378
N
N


Bacteroides sp. 9_1_42FAA
314
ACAA01000096
clade_378
N
N


Bacteroides sp. NB_8
323
AB117565
clade_378
N
N


Bacteroides vulgatus
331
CP000139
clade_378
N
N


Bacteroides ovatus
287
ACWH01000036
clade_38
N
N


Bacteroides sp. 1_1_30
294
ADCL01000128
clade_38
N
N


Bacteroides sp. 2_1_22
297
ACPQ01000117
clade_38
N
N


Bacteroides sp. 2_2_4
299
ABZZ01000168
clade_38
N
N


Bacteroides sp. 3_1_23
308
ACRS01000081
clade_38
N
N


Bacteroides sp. D1
318
ACAB02000030
clade_38
N
N


Bacteroides sp. D2
321
ACGA01000077
clade_38
N
N


Bacteroides sp. D22
320
ADCK01000151
clade_38
N
N


Bacteroides xylanisolvens
332
ADKP01000087
clade_38
N
N


Treponema lecithinolyticum
1931
NR_026247
clade_380
N
OP


Treponema parvum
1933
AF302937
clade_380
N
OP


Treponema sp. oral clone
1940
AY349417
clade_380
N
N


JU025


Treponema sp. oral taxon
1954
GQ422733
clade_380
N
N


270


Parascardovia denticolens
1428
ADEB01000020
clade_381
N
N


Scardovia inopinata
1688
AB029087
clade_381
N
N


Scardovia wiggsiae
1689
AY278626
clade_381
N
N


Clostridiales bacterium
533
HM587320
clade_384
N
N


9400853


Mogibacterium diversum
1254
NR_027191
clade_384
N
N


Mogibacterium neglectum
1255
NR_027203
clade_384
N
N


Mogibacterium pumilum
1256
NR_028608
clade_384
N
N


Mogibacterium timidum
1257
Z36296
clade_384
N
N


Borrelia burgdorferi
389
ABGI01000001
clade_386
N
OP


Borrelia garinii
392
ABJV01000001
clade_386
N
OP


Borrelia sp. NE49
397
AJ224142
clade_386
N
OP


Caldimonas manganoxidans
457
NR_040787
clade_387
N
N


Comamonadaceae
667
HM099651
clade_387
N
N


bacterium oral taxon F47


Lautropia mirabilis
1149
AEQP01000026
clade_387
N
N


Lautropia sp. oral clone
1150
AY005030
clade_387
N
N


AP009


Peptoniphilus
1441
D14145
clade_389
N
N


asaccharolyticus


Peptoniphilus duerdenii
1442
EU526290
clade_389
N
N


Peptoniphilus harei
1443
NR_026358
clade_389
N
N


Peptoniphilus indolicus
1444
AY153431
clade_389
N
N


Peptoniphilus lacrimalis
1446
ADDO01000050
clade_389
N
N


Peptoniphilus sp. gpac077
1450
AM176527
clade_389
N
N


Peptoniphilus sp. JC140
1447
JF824803
clade_389
N
N


Peptoniphilus sp. oral taxon
1452
ADCS01000031
clade_389
N
N


386


Peptoniphilus sp. oral taxon
1453
AEAA01000090
clade_389
N
N


836


Peptostreptococcaceae
1454
JN837495
clade_389
N
N


bacterium ph1


Dialister pneumosintes
765
HM596297
clade_390
N
N


Dialister sp. oral taxon 502
767
GQ422739
clade_390
N
N


Cupriavidus metallidurans
741
GU230889
clade_391
N
N


Herbaspirillum seropedicae
1001
CP002039
clade_391
N
N


Herbaspirillum sp. JC206
1002
JN657219
clade_391
N
N


Janthinobacterium sp. SY12
1015
EF455530
clade_391
N
N


Massilia sp. CCUG 43427A
1197
FR773700
clade_391
N
N


Ralstonia pickettii
1615
NC_010682
clade_391
N
N


Ralstonia sp. 5_7_47FAA
1616
ACUF01000076
clade_391
N
N


Francisella novicida
889
ABSS01000002
clade_392
N
N


Francisella philomiragia
890
AY928394
clade_392
N
N


Francisella tularensis
891
ABAZ01000082
clade_392
N
Category-A


Ignatzschineria indica
1009
HQ823562
clade_392
N
N


Ignatzschineria sp. NML
1010
HQ823559
clade_392
N
N


95_0260


Streptococcus mutans
1814
AP010655
clade_394
N
N


Lactobacillus gasseri
1084
ACOZ01000018
clade_398
N
N


Lactobacillus hominis
1090
FR681902
clade_398
N
N


Lactobacillus iners
1091
AEKJ01000002
clade_398
N
N


Lactobacillus johnsonii
1093
AE017198
clade_398
N
N


Lactobacillus senioris
1119
AB602570
clade_398
N
N


Lactobacillus sp. oral clone
1135
AY349382
clade_398
N
N


HT002


Weissella beninensis
2006
EU439435
clade_398
N
N


Sphingomonas echinoides
1744
NR_024700
clade_399
N
N


Sphingomonas sp. oral
1747
HM099639
clade_399
N
N


taxon A09


Sphingomonas sp. oral
1748
HM099645
clade_399
N
N


taxon F71


Zymomonas mobilis
2032
NR_074274
clade_399
N
N


Arcanobacterium
174
NR_025347
clade_400
N
N


haemolyticum


Arcanobacterium pyogenes
175
GU585578
clade_400
N
N


Trueperella pyogenes
1962
NR_044858
clade_400
N
N


Lactococcus garvieae
1144
AF061005
clade_401
N
N


Lactococcus lactis
1145
CP002365
clade_401
N
N


Brevibacterium mcbrellneri
424
ADNU01000076
clade_402
N
N


Brevibacterium paucivorans
425
EU086796
clade_402
N
N


Brevibacterium sp. JC43
428
JF824806
clade_402
N
N


Selenomonas artemidis
1692
HM596274
clade_403
N
N


Selenomonas sp. FOBRC9
1704
HQ616378
clade_403
N
N


Selenomonas sp. oral taxon
1715
AENV01000007
clade_403
N
N


137


Desmospora activa
751
AM940019
clade_404
N
N


Desmospora sp. 8437
752
AFHT01000143
clade_404
N
N


Paenibacillus sp. oral taxon
1407
HM099647
clade_404
N
N


F45


Corynebacterium
682
ADNS01000011
clade_405
N
N


ammoniagenes


Corynebacterium
687
ACLH01000041
clade_405
N
N


aurimucosum


Corynebacterium bovis
688
AF537590
clade_405
N
N


Corynebacterium canis
689
GQ871934
clade_405
N
N


Corynebacterium casei
690
NR_025101
clade_405
N
N


Corynebacterium durum
694
Z97069
clade_405
N
N


Corynebacterium efficiens
695
ACLI01000121
clade_405
N
N


Corynebacterium falsenii
696
Y13024
clade_405
N
N


Corynebacterium flavescens
697
NR_037040
clade_405
N
N


Corynebacterium
701
BA000036
clade_405
N
N


glutamicum


Corynebacterium jeikeium
704
ACYW01000001
clade_405
N
OP


Corynebacterium
705
NR_026380
clade_405
N
N


kroppenstedtii


Corynebacterium
706
ACHJ01000075
clade_405
N
N


lipophiloflavum


Corynebacterium
709
ACSH02000003
clade_405
N
N


matruchotii


Corynebacterium
710
X82064
clade_405
N
N


minutissimum


Corynebacterium resistens
718
ADGN01000058
clade_405
N
N


Corynebacterium simulans
720
AF537604
clade_405
N
N


Corynebacterium singulare
721
NR_026394
clade_405
N
N


Corynebacterium sp. 1 ex
722
Y13427
clade_405
N
N


sheep


Corynebacterium sp. NML
726
GU238413
clade_405
N
N


99_0018


Corynebacterium striatum
727
ACGE01000001
clade_405
N
OP


Corynebacterium
732
X81913
clade_405
N
OP


urealyticum


Corynebacterium variabile
734
NR_025314
clade_405
N
N


Aerococcus sanguinicola
98
AY837833
clade_407
N
N


Aerococcus urinae
99
CP002512
clade_407
N
N


Aerococcus urinaeequi
100
NR_043443
clade_407
N
N


Aerococcus viridans
101
ADNT01000041
clade_407
N
N


Fusobacterium naviforme
898
HQ223106
clade_408
N
N


Moryella indoligenes
1268
AF527773
clade_408
N
N


Selenomonas genomosp. P5
1697
AY341820
clade_410
N
N


Selenomonas sp. oral clone
1710
AY349408
clade_410
N
N


IQ048


Selenomonas sputigena
1717
ACKP02000033
clade_410
N
N


Hyphomicrobium
1007
AY468372
clade_411
N
N


sulfonivorans


Methylocella silvestris
1228
NR_074237
clade_411
N
N


Legionella pneumophila
1153
NC_002942
clade_412
N
OP


Lactobacillus coryniformis
1077
NR_044705
clade_413
N
N


Arthrobacter agilis
178
NR_026198
clade_414
N
N


Arthrobacter arilaitensis
179
NR_074608
clade_414
N
N


Arthrobacter bergerei
180
NR_025612
clade_414
N
N


Arthrobacter globiformis
181
NR_026187
clade_414
N
N


Arthrobacter nicotianae
182
NR_026190
clade_414
N
N


Mycobacterium abscessus
1269
AGQU01000002
clade_418
N
OP


Mycobacterium chelonae
1273
AB548610
clade_418
N
OP


Bacteroides salanitronis
291
CP002530
clade_419
N
N


Paraprevotella xylaniphila
1427
AFBR01000011
clade_419
N
N


Barnesiella intestinihominis
336
AB370251
clade_420
N
N


Barnesiella viscericola
337
NR_041508
clade_420
N
N


Parabacteroides sp. NS31_3
1422
JN029805
clade_420
N
N


Porphyromonadaceae
1470
EF184292
clade_420
N
N


bacterium NML 060648


Tannerella forsythia
1913
CP003191
clade_420
N
N


Tannerella sp.
1914
ACWX01000068
clade_420
N
N


6_1_58FAA_CT1


Mycoplasma amphoriforme
1311
AY531656
clade_421
N
N


Mycoplasma genitalium
1317
L43967
clade_421
N
N


Mycoplasma pneumoniae
1322
NC_000912
clade_421
N
N


Mycoplasma penetrans
1321
NC_004432
clade_422
N
N


Ureaplasma parvum
1966
AE002127
clade_422
N
N


Ureaplasma urealyticum
1967
AAYN01000002
clade_422
N
N


Treponema genomosp. P1
1927
AY341822
clade_425
N
N


Treponema sp. oral taxon
1943
GU408580
clade_425
N
N


228


Treponema sp. oral taxon
1944
GU408603
clade_425
N
N


230


Treponema sp. oral taxon
1945
GU408631
clade_425
N
N


231


Treponema sp. oral taxon
1946
GU408646
clade_425
N
N


232


Treponema sp. oral taxon
1947
GU408673
clade_425
N
N


235


Treponema sp. ovine footrot
1959
AJ010951
clade_425
N
N


Treponema vincentii
1960
ACYH01000036
clade_425
N
OP


Burkholderiales bacterium
452
ADCQ01000066
clade_432
N
OP


1_1_47


Parasutterella
1429
AFBP01000029
clade_432
N
N


excrementihominis


Parasutterella secunda
1430
AB491209
clade_432
N
N


Sutterella morbirenis
1898
AJ832129
clade_432
N
N


Sutterella sanguinus
1900
AJ748647
clade_432
N
N


Sutterella sp. YIT 12072
1901
AB491210
clade_432
N
N


Sutterella stercoricanis
1902
NR_025600
clade_432
N
N


Sutterella wadsworthensis
1903
ADMF01000048
clade_432
N
N


Propionibacterium
1572
NR_036972
clade_433
N
N


freudenreichii


Propionibacterium sp. oral
1580
GQ422728
clade_433
N
N


taxon 192


Tessaracoccus sp. oral taxon
1917
HM099640
clade_433
N
N


F04


Peptoniphilus ivorii
1445
Y07840
clade_434
N
N


Peptoniphilus sp. gpac007
1448
AM176517
clade_434
N
N


Peptoniphilus sp. gpac018A
1449
AM176519
clade_434
N
N


Peptoniphilus sp. gpac148
1451
AM176535
clade_434
N
N


Flexispira rappini
887
AY126479
clade_436
N
N


Helicobacter bilis
993
ACDN01000023
clade_436
N
N


Helicobacter cinaedi
995
ABQT01000054
clade_436
N
N


Helicobacter sp. None
998
U44756
clade_436
N
N


Brevundimonas
429
CP002102
clade_438
N
N


subvibrioides


Hyphomonas neptunium
1008
NR_074092
clade_438
N
N


Phenylobacterium zucineum
1465
AY628697
clade_438
N
N


Streptococcus downei
1793
AEKN01000002
clade_441
N
N


Streptococcus sp. SHV515
1848
Y07601
clade_441
N
N


Acinetobacter sp. CIP 53.82
40
JQ638584
clade_443
N
N


Halomonas elongata
990
NR_074782
clade_443
N
N


Halomonas johnsoniae
991
FR775979
clade_443
N
N


Butyrivibrio fibrisolvens
456
U41172
clade_444
N
N


Roseburia sp. 11SE37
1640
FM954975
clade_444
N
N


Roseburia sp. 11SE38
1641
FM954976
clade_444
N
N


Shuttleworthia satelles
1728
ACIP02000004
clade_444
N
N


Shuttleworthia sp. MSX8B
1729
HQ616383
clade_444
N
N


Shuttleworthia sp. oral
1730
GU432167
clade_444
N
N


taxon G69


Bdellovibrio sp. MPA
344
AY294215
clade_445
N
N


Desulfobulbus sp. oral clone
755
AY005036
clade_445
N
N


CH031


Desulfovibrio desulfuricans
757
DQ092636
clade_445
N
N


Desulfovibrio fairfieldensis
758
U42221
clade_445
N
N


Desulfovibrio piger
759
AF192152
clade_445
N
N


Desulfovibrio sp. 3_1_syn3
760
ADDR01000239
clade_445
N
N


Geobacter bemidjiensis
941
CP001124
clade_445
N
N


Brachybacterium
401
NR_026269
clade_446
N
N


alimentarium


Brachybacterium
402
AB537169
clade_446
N
N


conglomeratum


Brachybacterium
403
NR_026272
clade_446
N
N


tyrofermentans


Dermabacter hominis
749
FJ263375
clade_446
N
N


Aneurinibacillus
171
NR_029303
clade_448
N
N


thermoaerophilus


Brevibacillus agri
409
NR_040983
clade_448
N
N


Brevibacillus centrosporus
411
NR_043414
clade_448
N
N


Brevibacillus choshinensis
412
NR_040980
clade_448
N
N


Brevibacillus invocatus
413
NR_041836
clade_448
N
N


Brevibacillus parabrevis
415
NR_040981
clade_448
N
N


Brevibacillus reuszeri
416
NR_040982
clade_448
N
N


Brevibacillus sp. phR
417
JN837488
clade_448
N
N


Brevibacillus thermoruber
418
NR_026514
clade_448
N
N


Lactobacillus murinus
1100
NR_042231
clade_449
N
N


Lactobacillus oeni
1102
NR_043095
clade_449
N
N


Lactobacillus ruminis
1115
ACGS02000043
clade_449
N
N


Lactobacillus vini
1141
NR_042196
clade_449
N
N


Gemella haemolysans
924
ACDZ02000012
clade_450
N
N


Gemella morbillorum
925
NR_025904
clade_450
N
N


Gemella morbillorum
926
ACRX01000010
clade_450
N
N


Gemella sanguinis
927
ACRY01000057
clade_450
N
N


Gemella sp. oral clone
929
AY923133
clade_450
N
N


ASCE02


Gemella sp. oral clone
930
AY923139
clade_450
N
N


ASCF04


Gemella sp. oral clone
931
AY923143
clade_450
N
N


ASCF12


Gemella sp. WAL 1945J
928
EU427463
clade_450
N
N


Sporolactobacillus
1753
NR_042247
clade_451
N
N


nakayamae


Gluconacetobacter entanii
945
NR_028909
clade_452
N
N


Gluconacetobacter
946
NR_026513
clade_452
N
N


europaeus


Gluconacetobacter hansenii
947
NR_026133
clade_452
N
N


Gluconacetobacter
949
NR_041295
clade_452
N
N


oboediens


Gluconacetobacter xylinus
950
NR_074338
clade_452
N
N


Auritibacter ignavus
193
FN554542
clade_453
N
N


Dermacoccus sp. Ellin185
750
AEIQ01000090
clade_453
N
N


Janibacter limosus
1013
NR_026362
clade_453
N
N


Janibacter melonis
1014
EF063716
clade_453
N
N


Acetobacter aceti
7
NR_026121
clade_454
N
N


Acetobacter fabarum
8
NR_042678
clade_454
N
N


Acetobacter lovaniensis
9
NR_040832
clade_454
N
N


Acetobacter malorum
10
NR_025513
clade_454
N
N


Acetobacter orientalis
11
NR_028625
clade_454
N
N


Acetobacter pasteurianus
12
NR_026107
clade_454
N
N


Acetobacter pomorum
13
NR_042112
clade_454
N
N


Acetobacter syzygii
14
NR_040868
clade_454
N
N


Acetobacter tropicalis
15
NR_036881
clade_454
N
N


Gluconacetobacter
943
NR_028767
clade_454
N
N


azotocaptans


Gluconacetobacter
944
NR_074292
clade_454
N
N


diazotrophicus


Gluconacetobacter johannae
948
NR_024959
clade_454
N
N


Nocardia brasiliensis
1351
AIHV01000038
clade_455
N
N


Nocardia cyriacigeorgica
1352
HQ009486
clade_455
N
N


Nocardia puris
1354
NR_028994
clade_455
N
N


Nocardia sp. 01_Je_025
1355
GU574059
clade_455
N
N


Rhodococcus equi
1623
ADNW01000058
clade_455
N
N


Oceanobacillus caeni
1358
NR_041533
clade_456
N
N


Oceanobacillus sp. Ndiop
1359
CAER01000083
clade_456
N
N


Ornithinibacillus bavariensis
1384
NR_044923
clade_456
N
N


Ornithinibacillus sp.
1385
FN397526
clade_456
N
N


7_10AIA


Virgibacillus proomii
2005
NR_025308
clade_456
N
N


Corynebacterium
683
ABZU01000033
clade_457
N
OP


amycolatum


Corynebacterium hansenii
702
AM946639
clade_457
N
N


Corynebacterium xerosis
735
FN179330
clade_457
N
OP


Staphylococcaceae
1756
AY841362
clade_458
N
N


bacterium NML 92_0017


Staphylococcus fleurettii
1766
NR_041326
clade_458
N
N


Staphylococcus sciuri
1774
NR_025520
clade_458
N
N


Staphylococcus vitulinus
1779
NR_024670
clade_458
N
N


Stenotrophomonas
1782
AAVZ01000005
clade_459
N
N


maltophilia


Stenotrophomonas sp. FG_6
1783
EF017810
clade_459
N
N


Mycobacterium africanum
1270
AF480605
clade_46
N
OP


Mycobacterium alsiensis
1271
AJ938169
clade_46
N
OP


Mycobacterium avium
1272
CP000479
clade_46
N
OP


Mycobacterium
1274
AM062764
clade_46
N
OP


colombiense


Mycobacterium gordonae
1276
GU142930
clade_46
N
OP


Mycobacterium
1277
GQ153276
clade_46
N
OP


intracellulare


Mycobacterium kansasii
1278
AF480601
clade_46
N
OP


Mycobacterium lacus
1279
NR_025175
clade_46
N
OP


Mycobacterium leprae
1280
FM211192
clade_46
N
OP


Mycobacterium
1281
EU203590
clade_46
N
OP


lepromatosis


Mycobacterium mantenii
1283
FJ042897
clade_46
N
OP


Mycobacterium marinum
1284
NC_010612
clade_46
N
OP


Mycobacterium microti
1285
NR_025234
clade_46
N
OP


Mycobacterium
1287
ADNV01000350
clade_46
N
OP


parascrofulaceum


Mycobacterium seoulense
1290
DQ536403
clade_46
N
OP


Mycobacterium sp. 1761
1292
EU703150
clade_46
N
N


Mycobacterium sp. 1791
1295
EU703148
clade_46
N
N


Mycobacterium sp. 1797
1296
EU703149
clade_46
N
N


Mycobacterium sp.
1298
HQ174245
clade_46
N
N


B10_07.09.0206


Mycobacterium sp.
1305
HM627011
clade_46
N
N


NLA001000736


Mycobacterium sp. W
1306
DQ437715
clade_46
N
N


Mycobacterium tuberculosis
1307
CP001658
clade_46
N
Category-C


Mycobacterium ulcerans
1308
AB548725
clade_46
N
OP


Mycobacterium vulneris
1309
EU834055
clade_46
N
OP


Xanthomonas campestris
2016
EF101975
clade_461
N
N


Xanthomonas sp. kmd_489
2017
EU723184
clade_461
N
N


Dietzia natronolimnaea
769
GQ870426
clade_462
N
N


Dietzia sp. BBDP51
770
DQ337512
clade_462
N
N


Dietzia sp. CA149
771
GQ870422
clade_462
N
N


Dietzia timorensis
772
GQ870424
clade_462
N
N


Gordonia bronchialis
951
NR_027594
clade_463
N
N


Gordonia
952
DQ385609
clade_463
N
N


polyisoprenivorans


Gordonia sp. KTR9
953
DQ068383
clade_463
N
N


Gordonia sputi
954
FJ536304
clade_463
N
N


Gordonia terrae
955
GQ848239
clade_463
N
N


Leptotrichia goodfellowii
1167
ADAD01000110
clade_465
N
N


Leptotrichia sp. oral clone
1174
AY349387
clade_465
N
N


IK040


Leptotrichia sp. oral clone
1175
AY207053
clade_465
N
N


P2PB_51 P1


Bacteroidales genomosp. P7
264
DQ003623
clade_466
N
N


oral clone MB3_P19


Butyricimonas virosa
454
AB443949
clade_466
N
N


Odoribacter laneus
1363
AB490805
clade_466
N
N


Odoribacter splanchnicus
1364
CP002544
clade_466
N
N


Capnocytophaga gingivalis
478
ACLQ01000011
clade_467
N
N


Capnocytophaga granulosa
479
X97248
clade_467
N
N


Capnocytophaga sp. oral
483
AY005074
clade_467
N
N


clone AH015


Capnocytophaga sp. oral
487
AY005073
clade_467
N
N


strain S3


Capnocytophaga sp. oral
488
AEXX01000050
clade_467
N
N


taxon 338


Capnocytophaga canimorsus
476
CP002113
clade_468
N
N


Capnocytophaga sp. oral
485
AY349368
clade_468
N
N


clone ID062


Lactobacillus catenaformis
1075
M23729
clade_469
N
N


Lactobacillus vitulinus
1142
NR_041305
clade_469
N
N


Cetobacterium somerae
501
AJ438155
clade_470
N
N


Fusobacterium
896
ACET01000043
clade_470
N
N


gonidiaformans


Fusobacterium mortiferum
897
ACDB02000034
clade_470
N
N


Fusobacterium necrogenes
899
X55408
clade_470
N
N


Fusobacterium necrophorum
900
AM905356
clade_470
N
N


Fusobacterium sp. 12_1B
905
AGWJ01000070
clade_470
N
N


Fusobacterium sp. 3_1_5R
911
ACDD01000078
clade_470
N
N


Fusobacterium sp. D12
918
ACDG02000036
clade_470
N
N


Fusobacterium ulcerans
921
ACDH01000090
clade_470
N
N


Fusobacterium varium
922
ACIE01000009
clade_470
N
N


Mycoplasma arthritidis
1312
NC_011025
clade_473
N
N


Mycoplasma faucium
1314
NR_024983
clade_473
N
N


Mycoplasma hominis
1318
AF443616
clade_473
N
N


Mycoplasma orale
1319
AY796060
clade_473
N
N


Mycoplasma salivarium
1324
M24661
clade_473
N
N


Mitsuokella jalaludinii
1247
NR_028840
clade_474
N
N


Mitsuokella multacida
1248
ABWK02000005
clade_474
N
N


Mitsuokella sp. oral taxon
1249
GU413658
clade_474
N
N


521


Mitsuokella sp. oral taxon
1250
GU432166
clade_474
N
N


G68


Selenomonas genomosp. C1
1695
AY278627
clade_474
N
N


Selenomonas genomosp. P8
1700
DQ003628
clade_474
N
N


oral clone MB5_P06


Selenomonas ruminantium
1703
NR_075026
clade_474
N
N


Veillonellaceae bacterium
1994
GU402916
clade_474
N
N


oral taxon 131


Alloscardovia omnicolens
139
NR_042583
clade_475
N
N


Alloscardovia sp. OB7196
140
AB425070
clade_475
N
N


Bifidobacterium urinalis
366
AJ278695
clade_475
N
N


Prevotella loescheii
1503
JN867231
clade_48
N
N


Prevotella sp. oral clone
1530
DQ272511
clade_48
N
N


ASCG12


Prevotella sp. oral clone
1540
AY349398
clade_48
N
N


GU027


Prevotella sp. oral taxon 472
1553
ACZS01000106
clade_48
N
N


Selenomonas dianae
1693
GQ422719
clade_480
N
N


Selenomonas flueggei
1694
AF287803
clade_480
N
N


Selenomonas genomosp. C2
1696
AY278628
clade_480
N
N


Selenomonas genomosp. P6
1698
DQ003636
clade_480
N
N


oral clone MB3_C41


Selenomonas genomosp. P7
1699
DQ003627
clade_480
N
N


oral clone MB5_C08


Selenomonas infelix
1701
AF287802
clade_480
N
N


Selenomonas noxia
1702
GU470909
clade_480
N
N


Selenomonas sp. oral clone
1705
AY349403
clade_480
N
N


FT050


Selenomonas sp. oral clone
1706
AY349404
clade_480
N
N


GI064


Selenomonas sp. oral clone
1707
AY349405
clade_480
N
N


GT010


Selenomonas sp. oral clone
1708
AY349406
clade_480
N
N


HU051


Selenomonas sp. oral clone
1709
AY349407
clade_480
N
N


IK004


Selenomonas sp. oral clone
1711
AY349409
clade_480
N
N


JI021


Selenomonas sp. oral clone
1712
AY349410
clade_480
N
N


JS031


Selenomonas sp. oral clone
1713
AY947498
clade_480
N
N


OH4A


Selenomonas sp. oral clone
1714
AY207052
clade_480
N
N


P2PA_80 P4


Selenomonas sp. oral taxon
1716
AEEJ01000007
clade_480
N
N


149


Veillonellaceae bacterium
1995
GU470897
clade_480
N
N


oral taxon 155


Agrococcus jenensis
117
NR_026275
clade_484
N
N


Microbacterium
1232
NR_025098
clade_484
N
N


gubbeenense


Pseudoclavibacter sp.
1590
FJ375951
clade_484
N
N


Timone


Tropheryma whipplei
1961
BX251412
clade_484
N
N


Zimmermannella bifida
2031
AB012592
clade_484
N
N


Legionella hackeliae
1151
M36028
clade_486
N
OP


Legionella longbeachae
1152
M36029
clade_486
N
OP


Legionella sp. D3923
1154
JN380999
clade_486
N
OP


Legionella sp. D4088
1155
JN381012
clade_486
N
OP


Legionella sp. H63
1156
JF831047
clade_486
N
OP


Legionella sp. NML 93L054
1157
GU062706
clade_486
N
OP


Legionella steelei
1158
HQ398202
clade_486
N
OP


Tatlockia micdadei
1915
M36032
clade_486
N
N


Helicobacter pullorum
996
ABQU01000097
clade_489
N
N


Acetobacteraceae bacterium
16
AGEZ01000040
clade_490
N
N


AT_5844


Roseomonas cervicalis
1643
ADVL01000363
clade_490
N
N


Roseomonas mucosa
1644
NR_028857
clade_490
N
N


Roseomonas sp.
1645
AF533357
clade_490
N
N


NML94_0193


Roseomonas sp.
1646
AF533359
clade_490
N
N


NML97_0121


Roseomonas sp.
1647
AF533358
clade_490
N
N


NML98_0009


Roseomonas sp.
1648
AF533360
clade_490
N
N


NML98_0157


Rickettsia akari
1627
CP000847
clade_492
N
OP


Rickettsia conorii
1628
AE008647
clade_492
N
OP


Rickettsia prowazekii
1629
M21789
clade_492
N
Category-B


Rickettsia rickettsii
1630
NC_010263
clade_492
N
OP


Rickettsia slovaca
1631
L36224
clade_492
N
OP


Rickettsia typhi
1632
AE017197
clade_492
N
OP


Anaeroglobus geminatus
160
AGCJ01000054
clade_493
N
N


Megasphaera genomosp. C1
1201
AY278622
clade_493
N
N


Megasphaera
1203
AECS01000020
clade_493
N
N


micronuciformis


Clostridiales genomosp.
540
CP001850
clade_495
N
N


BVAB3


Tsukamurella
1963
X80628
clade_496
N
N


paurometabola


Tsukamurella
1964
AB478958
clade_496
N
N


tyrosinosolvens


Abiotrophia para_adiacens
2
AB022027
clade_497
N
N


Carnobacterium divergens
492
NR_044706
clade_497
N
N


Carnobacterium
493
NC_019425
clade_497
N
N


maltaromaticum


Enterococcus avium
800
AF133535
clade_497
N
N


Enterococcus caccae
801
AY943820
clade_497
N
N


Enterococcus casseliflavus
802
AEWT01000047
clade_497
N
N


Enterococcus durans
803
AJ276354
clade_497
N
N


Enterococcus faecalis
804
AE016830
clade_497
N
N


Enterococcus faecium
805
AM157434
clade_497
N
N


Enterococcus gallinarum
806
AB269767
clade_497
N
N


Enterococcus gilvus
807
AY033814
clade_497
N
N


Enterococcus hawaiiensis
808
AY321377
clade_497
N
N


Enterococcus hirae
809
AF061011
clade_497
N
N


Enterococcus italicus
810
AEPV01000109
clade_497
N
N


Enterococcus mundtii
811
NR_024906
clade_497
N
N


Enterococcus raffinosus
812
FN600541
clade_497
N
N


Enterococcus sp.
813
JN809766
clade_497
N
N


BV2CASA2


Enterococcus sp.
814
GU457263
clade_497
N
N


CCRI_16620


Enterococcus sp. F95
815
FJ463817
clade_497
N
N


Enterococcus sp. RfL6
816
AJ133478
clade_497
N
N


Enterococcus thailandicus
817
AY321376
clade_497
N
N


Fusobacterium canifelinum
893
AY162222
clade_497
N
N


Fusobacterium genomosp.
894
AY278616
clade_497
N
N


C1


Fusobacterium genomosp.
895
AY278617
clade_497
N
N


C2


Fusobacterium
902
ACJY01000002
clade_497
N
N


periodonticum


Fusobacterium sp.
906
ADGG01000053
clade_497
N
N


1_1_41FAA


Fusobacterium sp. 11_3_2
904
ACUO01000052
clade_497
N
N


Fusobacterium sp. 2_1_31
907
ACDC02000018
clade_497
N
N


Fusobacterium sp. 3_1_27
908
ADGF01000045
clade_497
N
N


Fusobacterium sp. 3_1_33
909
ACQE01000178
clade_497
N
N


Fusobacterium sp.
910
ACPU01000044
clade_497
N
N


3_1_36A2


Fusobacterium sp. AC18
912
HQ616357
clade_497
N
N


Fusobacterium sp. ACB2
913
HQ616358
clade_497
N
N


Fusobacterium sp. AS2
914
HQ616361
clade_497
N
N


Fusobacterium sp. CM1
915
HQ616371
clade_497
N
N


Fusobacterium sp. CM21
916
HQ616375
clade_497
N
N


Fusobacterium sp. CM22
917
HQ616376
clade_497
N
N


Fusobacterium sp. oral
919
AY923141
clade_497
N
N


clone ASCF06


Fusobacterium sp. oral
920
AY953256
clade_497
N
N


clone ASCF11


Granulicatella adiacens
959
ACKZ01000002
clade_497
N
N


Granulicatella elegans
960
AB252689
clade_497
N
N


Granulicatella paradiacens
961
AY879298
clade_497
N
N


Granulicatella sp. oral clone
963
AY923126
clade_497
N
N


ASC02


Granulicatella sp. oral clone
964
DQ341469
clade_497
N
N


ASCA05


Granulicatella sp. oral clone
965
AY953251
clade_497
N
N


ASCB09


Granulicatella sp. oral clone
966
AY923146
clade_497
N
N


ASCG05


Tetragenococcus halophilus
1918
NR_075020
clade_497
N
N


Tetragenococcus koreensis
1919
NR_043113
clade_497
N
N


Vagococcus fluvialis
1973
NR_026489
clade_497
N
N


Chryseobacterium anthropi
514
AM982793
clade_498
N
N


Chryseobacterium gleum
515
ACKQ02000003
clade_498
N
N


Chryseobacterium hominis
516
NR_042517
clade_498
N
N


Treponema refringens
1936
AF426101
clade_499
N
OP


Treponema sp. oral clone
1941
AY349416
clade_499
N
N


JU031


Treponema sp. oral taxon
1948
GU408738
clade_499
N
N


239


Treponema sp. oral taxon
1955
GU408871
clade_499
N
N


271


Alistipes finegoldii
129
NR_043064
clade_500
N
N


Alistipes onderdonkii
131
NR_043318
clade_500
N
N


Alistipes putredinis
132
ABFK02000017
clade_500
N
N


Alistipes shahii
133
FP929032
clade_500
N
N


Alistipes sp. HGB5
134
AENZ01000082
clade_500
N
N


Alistipes sp. JC50
135
JF824804
clade_500
N
N


Alistipes sp. RMA 9912
136
GQ140629
clade_500
N
N


Mycoplasma agalactiae
1310
AF010477
clade_501
N
N


Mycoplasma bovoculi
1313
NR_025987
clade_501
N
N


Mycoplasma fermentans
1315
CP002458
clade_501
N
N


Mycoplasma flocculare
1316
X62699
clade_501
N
N


Mycoplasma
1320
NR_025989
clade_501
N
N


ovipneumoniae


Arcobacter butzleri
176
AEPT01000071
clade_502
N
N


Arcobacter cryaerophilus
177
NR_025905
clade_502
N
N


Campylobacter curvus
461
NC_009715
clade_502
N
OP


Campylobacter rectus
467
ACFU01000050
clade_502
N
OP


Campylobacter showae
468
ACVQ01000030
clade_502
N
OP


Campylobacter sp.
469
HQ616379
clade_502
N
OP


FOBRC14


Campylobacter sp.
470
HQ616380
clade_502
N
OP


FOBRC15


Campylobacter sp. oral
471
AY005038
clade_502
N
OP


clone BB120


Campylobacter sputorum
472
NR_044839
clade_502
N
OP


Bacteroides ureolyticus
330
GQ167666
clade_504
N
N


Campylobacter gracilis
463
ACYG01000026
clade_504
N
OP


Campylobacter hominis
464
NC_009714
clade_504
N
OP


Dialister invisus
762
ACIM02000001
clade_506
N
N


Dialister micraerophilus
763
AFBB01000028
clade_506
N
N


Dialister microaerophilus
764
AENT01000008
clade_506
N
N


Dialister propionicifaciens
766
NR_043231
clade_506
N
N


Dialister succinatiphilus
768
AB370249
clade_506
N
N


Megasphaera elsdenii
1200
AY038996
clade_506
N
N


Megasphaera genomosp.
1202
ADGP01000010
clade_506
N
N


type_1


Megasphaera sp.
1204
HM990964
clade_506
N
N


BLPYG_07


Megasphaera sp. UPII
1205
AFIJ01000040
clade_506
N
N


199_6


Chromobacterium
513
NC_005085
clade_507
N
N


violaceum


Laribacter hongkongensis
1148
CP001154
clade_507
N
N


Methylophilus sp. ECd5
1229
AY436794
clade_507
N
N


Finegoldia magna
883
ACHM02000001
clade_509
N
N


Parvimonas micra
1431
AB729072
clade_509
N
N


Parvimonas sp. oral taxon
1432
AFII01000002
clade_509
N
N


110


Peptostreptococcus micros
1456
AM176538
clade_509
N
N


Peptostreptococcus sp. oral
1460
AY349390
clade_509
N
N


clone FJ023


Peptostreptococcus sp.
1458
AY207059
clade_509
N
N


P4P_31 P3


Helicobacter pylori
997
CP000012
clade_510
N
OP


Anaplasma marginale
165
ABOR01000019
clade_511
N
N


Anaplasma
166
NC_007797
clade_511
N
N


phagocytophilum


Ehrlichia chaffeensis
783
AAIF01000035
clade_511
N
OP


Neorickettsia risticii
1349
CP001431
clade_511
N
N


Neorickettsia sennetsu
1350
NC_007798
clade_511
N
N


Pseudoramibacter
1606
AB036759
clade_512
N
N


alactolyticus


Veillonella montpellierensis
1977
AF473836
clade_513
N
N


Veillonella sp. oral clone
1988
AY923118
clade_513
N
N


ASCA08


Veillonella sp. oral clone
1989
AY923122
clade_513
N
N


ASCB03


Inquilinus limosus
1012
NR_029046
clade_514
N
N


Sphingomonas sp. oral
1746
AY349412
clade_514
N
N


clone FZ016


Anaerococcus lactolyticus
145
ABYO01000217
clade_515
N
N


Anaerococcus prevotii
147
CP001708
clade_515
N
N


Anaerococcus sp. gpac104
152
AM176528
clade_515
N
N


Anaerococcus sp. gpac126
153
AM176530
clade_515
N
N


Anaerococcus sp. gpac155
154
AM176536
clade_515
N
N


Anaerococcus sp. gpac199
155
AM176539
clade_515
N
N


Anaerococcus tetradius
157
ACGC01000107
clade_515
N
N


Bacteroides coagulans
271
AB547639
clade_515
N
N


Clostridiales bacterium
534
HM587324
clade_515
N
N


9403326


Clostridiales bacterium ph2
539
JN837487
clade_515
N
N


Peptostreptococcus sp.
1457
X90471
clade_515
N
N


9succ1


Peptostreptococcus sp. oral
1459
AB175072
clade_515
N
N


clone AP24


Tissierella praeacuta
1924
NR_044860
clade_515
N
N


Helicobacter canadensis
994
ABQS01000108
clade_518
N
N


Peptostreptococcus
1455
AY326462
clade_520
N
N


anaerobius


Peptostreptococcus stomatis
1461
ADGQ01000048
clade_520
N
N


Bilophila wadsworthia
367
ADCP01000166
clade_521
N
N


Desulfovibrio vulgaris
761
NR_074897
clade_521
N
N


Actinomyces nasicola
64
AJ508455
clade_523
N
N


Cellulosimicrobium funkei
500
AY501364
clade_523
N
N


Lactococcus raffinolactis
1146
NR_044359
clade_524
N
N


Bacteroidales genomosp. P1
258
AY341819
clade_529
N
N


Bacteroidales genomosp. P2
259
DQ003613
clade_529
N
N


oral clone MB1_G13


Bacteroidales genomosp. P3
260
DQ003615
clade_529
N
N


oral clone MB1_G34


Bacteroidales genomosp. P4
261
DQ003617
clade_529
N
N


oral clone MB2_G17


Bacteroidales genomosp. P5
262
DQ003619
clade_529
N
N


oral clone MB2_P04


Bacteroidales genomosp. P6
263
DQ003634
clade_529
N
N


oral clone MB3_C19


Bacteroidales genomosp. P8
265
DQ003626
clade_529
N
N


oral clone MB4_G15


Bacteroidetes bacterium oral
333
HM099638
clade_530
N
N


taxon D27


Bacteroidetes bacterium oral
334
HM099643
clade_530
N
N


taxon F31


Bacteroidetes bacterium oral
335
HM099649
clade_530
N
N


taxon F44


Flavobacterium sp. NF2_1
885
FJ195988
clade_530
N
N


Myroides odoratimimus
1326
NR_042354
clade_530
N
N


Myroides sp. MY15
1327
GU253339
clade_530
N
N


Chlamydiales bacterium
507
JN606076
clade_531
N
N


NS16


Chlamydophila pecorum
508
D88317
clade_531
N
OP


Parachlamydia sp. UWE25
1423
BX908798
clade_531
N
N


Fusobacterium russii
903
NR_044687
clade_532
N
N


Streptobacillus moniliformis
1784
NR_027615
clade_532
N
N


Eubacteriaceae bacterium
833
AY207060
clade_533
N
N


P4P_50 P4


Abiotrophia defectiva
1
ACIN02000016
clade_534
N
N


Abiotrophia sp. oral clone
3
AY207063
clade_534
N
N


P4PA_155 P1


Catonella genomosp. P1
496
DQ003629
clade_534
N
N


oral clone MB5_P12


Catonella morbi
497
ACIL02000016
clade_534
N
N


Catonella sp. oral clone
498
AY349369
clade_534
N
N


FL037


Eremococcus coleocola
818
AENN01000008
clade_534
N
N


Facklamia hominis
879
Y10772
clade_534
N
N


Granulicatella sp.
962
AJ271861
clade_534
N
N


M658_99_3


Campylobacter coli
459
AAFL01000004
clade_535
N
OP


Campylobacter concisus
460
CP000792
clade_535
N
OP


Campylobacter fetus
462
ACLG01001177
clade_535
N
OP


Campylobacter jejuni
465
AL139074
clade_535
N
Category-B


Campylobacter upsaliensis
473
AEPU01000040
clade_535
N
OP


Atopobium minutum
183
HM007583
clade_539
N
N


Atopobium parvulum
184
CP001721
clade_539
N
N


Atopobium rimae
185
ACFE01000007
clade_539
N
N


Atopobium sp. BS2
186
HQ616367
clade_539
N
N


Atopobium sp. F0209
187
EU592966
clade_539
N
N


Atopobium sp. ICM42b10
188
HQ616393
clade_539
N
N


Atopobium sp. ICM57
189
HQ616400
clade_539
N
N


Atopobium vaginae
190
AEDQ01000024
clade_539
N
N


Coriobacteriaceae bacterium
677
JN809768
clade_539
N
N


BV3Ac1


Actinomyces naeslundii
63
X81062
clade_54
N
N


Actinomyces oricola
67
NR_025559
clade_54
N
N


Actinomyces oris
69
BABV01000070
clade_54
N
N


Actinomyces sp. 7400942
70
EU484334
clade_54
N
N


Actinomyces sp. ChDC
72
AF543275
clade_54
N
N


B197


Actinomyces sp. GEJ15
73
GU561313
clade_54
N
N


Actinomyces sp.
79
AJ234063
clade_54
N
N


M2231_94_1


Actinomyces sp. oral clone
83
AY349362
clade_54
N
N


GU067


Actinomyces sp. oral clone
85
AY349364
clade_54
N
N


IO077


Actinomyces sp. oral clone
86
AY349365
clade_54
N
N


IP073


Actinomyces sp. oral clone
88
AY349367
clade_54
N
N


JA063


Actinomyces sp. oral taxon
89
AFBL01000010
clade_54
N
N


170


Actinomyces sp. oral taxon
90
AECW01000034
clade_54
N
N


171


Actinomyces urogenitalis
95
ACFH01000038
clade_54
N
N


Actinomyces viscosus
96
ACRE01000096
clade_54
N
N


Orientia tsutsugamushi
1383
AP008981
clade_541
N
OP


Megamonas funiformis
1198
AB300988
clade_542
N
N


Megamonas hypermegale
1199
AJ420107
clade_542
N
N


Aeromicrobium marinum
102
NR_025681
clade_544
N
N


Aeromicrobium sp. JC14
103
JF824798
clade_544
N
N


Luteococcus sanguinis
1190
NR_025507
clade_544
N
N


Propionibacteriaceae
1568
EF599122
clade_544
N
N


bacterium NML 02_0265


Rhodococcus
1622
X80615
clade_546
N
N


corynebacterioides


Rhodococcus erythropolis
1624
ACNO01000030
clade_546
N
N


Rhodococcus fascians
1625
NR_037021
clade_546
N
N


Segniliparus rotundus
1690
CP001958
clade_546
N
N


Segniliparus rugosus
1691
ACZI01000025
clade_546
N
N


Exiguobacterium acetylicum
878
FJ970034
clade_547
N
N


Macrococcus caseolyticus
1194
NR_074941
clade_547
N
N


Streptomyces sp. 1
1890
FJ176782
clade_548
N
N


AIP_2009


Streptomyces sp. SD 524
1892
EU544234
clade_548
N
N


Streptomyces sp. SD 528
1893
EU544233
clade_548
N
N


Streptomyces
1895
NR_027616
clade_548
N
N


thermoviolaceus


Borrelia afzelii
388
ABCU01000001
clade_549
N
OP


Borrelia crocidurae
390
DQ057990
clade_549
N
OP


Borrelia duttonii
391
NC_011229
clade_549
N
OP


Borrelia hermsii
393
AY597657
clade_549
N
OP


Borrelia hispanica
394
DQ057988
clade_549
N
OP


Borrelia persica
395
HM161645
clade_549
N
OP


Borrelia recurrentis
396
AF107367
clade_549
N
OP


Borrelia spielmanii
398
ABKB01000002
clade_549
N
OP


Borrelia turicatae
399
NC_008710
clade_549
N
OP


Borrelia valaisiana
400
ABCY01000002
clade_549
N
OP


Providencia alcalifaciens
1586
ABXW01000071
clade_55
N
N


Providencia rettgeri
1587
AM040492
clade_55
N
N


Providencia rustigianii
1588
AM040489
clade_55
N
N


Providencia stuartii
1589
AF008581
clade_55
N
N


Treponema pallidum
1932
CP001752
clade_550
N
OP


Treponema phagedenis
1934
AEFH01000172
clade_550
N
N


Treponema sp. clone
1939
Y08894
clade_550
N
N


DDKL_4


Acholeplasma laidlawii
17
NR_074448
clade_551
N
N


Mycoplasma putrefaciens
1323
U26055
clade_551
N
N


Mycoplasmataceae
1325
DQ003614
clade_551
N
N


genomosp. P1 oral clone


MB1_G23


Spiroplasma insolitum
1750
NR_025705
clade_551
N
N


Collinsella intestinalis
660
ABXH02000037
clade_553
N
N


Collinsella stercoris
661
ABXJ01000150
clade_553
N
N


Collinsella tanakaei
662
AB490807
clade_553
N
N


Caminicella sporogenes
458
NR_025485
clade_554
N
N


Acidaminococcus
21
CP001859
clade_556
N
N


fermentans


Acidaminococcus intestini
22
CP003058
clade_556
N
N


Acidaminococcus sp. D21
23
ACGB01000071
clade_556
N
N


Phascolarctobacterium
1462
NR_026111
clade_556
N
N


faecium


Phascolarctobacterium sp.
1463
AB490812
clade_556
N
N


YIT 12068


Phascolarctobacterium
1464
AB490811
clade_556
N
N


succinatutens


Acidithiobacillus ferrivorans
25
NR_074660
clade_557
N
N


Xanthomonadaceae
2015
EU313791
clade_557
N
N


bacterium NML 03_0222


Catabacter hongkongensis
494
AB671763
clade_558
N
N


Christensenella minuta
512
AB490809
clade_558
N
N


Clostridiales bacterium oral
536
AY207065
clade_558
N
N


clone P4PA_66 P1


Clostridiales bacterium oral
537
GQ422712
clade_558
N
N


taxon 093


Heliobacterium
1000
NR_074517
clade_560
N
N


modesticaldum


Alistipes indistinctus
130
AB490804
clade_561
N
N


Bacteroidales bacterium ph8
257
JN837494
clade_561
N
N


Candidatus Sulcia muelleri
475
CP002163
clade_561
N
N


Cytophaga xylanolytica
742
FR733683
clade_561
N
N


Flavobacteriaceae
884
AY278614
clade_561
N
N


genomosp. C1


Gramella forsetii
958
NR_074707
clade_561
N
N


Sphingobacterium faecium
1740
NR_025537
clade_562
N
N


Sphingobacterium mizutaii
1741
JF708889
clade_562
N
N


Sphingobacterium
1742
NR_040953
clade_562
N
N


multivorum


Sphingobacterium
1743
ACHA02000013
clade_562
N
N


spiritivorum


Jonquetella anthropi
1017
ACOO02000004
clade_563
N
N


Pyramidobacter piscolens
1614
AY207056
clade_563
N
N


Synergistes genomosp. C1
1904
AY278615
clade_563
N
N


Synergistes sp. RMA 14551
1905
DQ412722
clade_563
N
N


Synergistetes bacterium
1906
GQ258968
clade_563
N
N


ADV897


Candidatus Arthromitus sp.
474
NR_074460
clade_564
N
N


SFB_mouse_Yit


Gracilibacter thermotolerans
957
NR_043559
clade_564
N
N


Brachyspira aalborgi
404
FM178386
clade_565
N
N


Brachyspira sp. HIS3
406
FM178387
clade_565
N
N


Brachyspira sp. HIS4
407
FM178388
clade_565
N
N


Brachyspira sp. HIS5
408
FM178389
clade_565
N
N


Adlercreutzia equolifaciens
97
AB306661
clade_566
N
N


Coriobacteriaceae bacterium
678
CAEM01000062
clade_566
N
N


JC110


Coriobacteriaceae bacterium
679
JN837493
clade_566
N
N


phI


Cryptobacterium curtum
740
GQ422741
clade_566
N
N


Eggerthella sinensis
779
AY321958
clade_566
N
N


Eggerthella sp. 1_3_56FAA
780
ACWN01000099
clade_566
N
N


Eggerthella sp. HGA1
781
AEXR01000021
clade_566
N
N


Eggerthella sp. YY7918
782
AP012211
clade_566
N
N


Gordonibacter pamelaeae
680
AM886059
clade_566
N
N


Gordonibacter pamelaeae
956
FP929047
clade_566
N
N


Slackia equolifaciens
1732
EU377663
clade_566
N
N


Slackia exigua
1733
ACUX01000029
clade_566
N
N


Slackia faecicanis
1734
NR_042220
clade_566
N
N


Slackia heliotrinireducens
1735
NR_074439
clade_566
N
N


Slackia isoflavoniconvertens
1736
AB566418
clade_566
N
N


Slackia piriformis
1737
AB490806
clade_566
N
N


Slackia sp. NATTS
1738
AB505075
clade_566
N
N


Chlamydiales bacterium
506
JN606075
clade_567
N
N


NS13


Victivallaceae bacterium
2003
FJ394915
clade_567
N
N


NML 080035


Victivallis vadensis
2004
ABDE02000010
clade_567
N
N


Streptomyces griseus
1889
NR_074787
clade_573
N
N


Streptomyces sp. SD 511
1891
EU544231
clade_573
N
N


Streptomyces sp. SD 534
1894
EU544232
clade_573
N
N


Cloacibacillus evryensis
530
GQ258966
clade_575
N
N


Deferribacteres sp. oral
743
AY349370
clade_575
N
N


clone JV001


Deferribacteres sp. oral
745
AY349372
clade_575
N
N


clone JV023


Synergistetes bacterium
1907
GQ258969
clade_575
N
N


LBVCM1157


Synergistetes bacterium oral
1909
GU410752
clade_575
N
N


taxon 362


Synergistetes bacterium oral
1910
GU430992
clade_575
N
N


taxon D48


Peptococcus sp. oral clone
1439
AY349389
clade_576
N
N


JM048


Helicobacter winghamensis
999
ACDO01000013
clade_577
N
N


Wolinella succinogenes
2014
BX571657
clade_577
N
N


Olsenella genomosp. C1
1368
AY278623
clade_578
N
N


Olsenella profusa
1369
FN178466
clade_578
N
N


Olsenella sp. F0004
1370
EU592964
clade_578
N
N


Olsenella sp. oral taxon 809
1371
ACVE01000002
clade_578
N
N


Olsenella uli
1372
CP002106
clade_578
N
N


Nocardiopsis dassonvillei
1356
CP002041
clade_579
N
N


Peptococcus niger
1438
NR_029221
clade_580
N
N


Peptococcus sp. oral taxon
1440
GQ422727
clade_580
N
N


167


Akkermansia muciniphila
118
CP001071
clade_583
N
N


Opitutus terrae
1373
NR_074978
clade_583
N
N


Clostridiales bacterium oral
538
HM099644
clade_584
N
N


taxon F32


Leptospira borgpetersenii
1161
NC_008508
clade_585
N
OP


Leptospira broomii
1162
NR_043200
clade_585
N
OP


Leptospira interrogans
1163
NC_005823
clade_585
N
OP


Methanobrevibacter
1213
NR_044789
clade_587
N
N


gottschalkii


Methanobrevibacter
1214
NR_042785
clade_587
N
N


millerae


Methanobrevibacter oralis
1216
HE654003
clade_587
N
N


Methanobrevibacter thaueri
1219
NR_044787
clade_587
N
N


Methanobrevibacter smithii
1218
ABYV02000002
clade_588
N
N


Deinococcus radiodurans
746
AE000513
clade_589
N
N


Deinococcus sp. R_43890
747
FR682752
clade_589
N
N


Thermus aquaticus
1923
NR_025900
clade_589
N
N


Actinomyces sp. c109
81
AB167239
clade_590
N
N


Syntrophomonadaceae
1912
AY341821
clade_590
N
N


genomosp. P1


Anaerobaculum
141
ACJX02000009
clade_591
N
N


hydrogeniformans


Microcystis aeruginosa
1246
NC_010296
clade_592
N
N


Prochlorococcus marinus
1567
CP000551
clade_592
N
N


Methanobrevibacter
1208
NR_028779
clade_593
N
N


acididurans


Methanobrevibacter
1209
NR_042783
clade_593
N
N


arboriphilus


Methanobrevibacter
1210
NR_044796
clade_593
N
N


curvatus


Methanobrevibacter
1211
NR_044776
clade_593
N
N


cuticularis


Methanobrevibacter
1212
NR_044801
clade_593
N
N


filiformis


Methanobrevibacter woesei
1220
NR_044788
clade_593
N
N


Roseiflexus castenholzii
1642
CP000804
clade_594
N
N


Methanobrevibacter olleyae
1215
NR_043024
clade_595
N
N


Methanobrevibacter
1217
NR_042784
clade_595
N
N


ruminantium


Methanobrevibacter wolinii
1221
NR_044790
clade_595
N
N


Methanosphaera stadtmanae
1222
AY196684
clade_595
N
N


Chloroflexi genomosp. P1
511
AY331414
clade_596
N
N


Halorubrum lipolyticum
992
AB477978
clade_597
N
N


Methanobacterium
1207
NR_025028
clade_597
N
N


formicicum


Acidilobus saccharovorans
24
AY350586
clade_598
N
N


Hyperthermus butylicus
1006
CP000493
clade_598
N
N


Ignicoccus islandicus
1011
X99562
clade_598
N
N


Metallosphaera sedula
1206
D26491
clade_598
N
N


Thermofilum pendens
1922
X14835
clade_598
N
N


Prevotella melaninogenica
1506
CP002122
clade_6
N
N


Prevotella sp. ICM1
1520
HQ616385
clade_6
N
N


Prevotella sp. oral clone
1535
AY349393
clade_6
N
N


FU048


Prevotella sp. oral clone
1537
AY349395
clade_6
N
N


GI030


Prevotella sp. SEQ116
1526
JN867246
clade_6
N
N


Streptococcus anginosus
1787
AECT01000011
clade_60
N
N


Streptococcus milleri
1812
X81023
clade_60
N
N


Streptococcus sp. 16362
1829
JN590019
clade_60
N
N


Streptococcus sp. 69130
1832
X78825
clade_60
N
N


Streptococcus sp. AC15
1833
HQ616356
clade_60
N
N


Streptococcus sp. CM7
1839
HQ616373
clade_60
N
N


Streptococcus sp. OBRC6
1847
HQ616352
clade_60
N
N


Burkholderia ambifaria
442
AAUZ01000009
clade_61
N
OP


Burkholderia cenocepacia
443
AAHI01000060
clade_61
N
OP


Burkholderia cepacia
444
NR_041719
clade_61
N
OP


Burkholderia mallei
445
CP000547
clade_61
N
Category-B


Burkholderia multivorans
446
NC_010086
clade_61
N
OP


Burkholderia oklahomensis
447
DQ108388
clade_61
N
OP


Burkholderia pseudomallei
448
CP001408
clade_61
N
Category-B


Burkholderia rhizoxinica
449
HQ005410
clade_61
N
OP


Burkholderia sp. 383
450
CP000151
clade_61
N
OP


Burkholderia xenovorans
451
U86373
clade_61
N
OP


Prevotella buccae
1488
ACRB01000001
clade_62
N
N


Prevotella genomosp. P8
1498
DQ003622
clade_62
N
N


oral clone MB3_P13


Prevotella sp. oral clone
1536
AY349394
clade_62
N
N


FW035


Prevotella bivia
1486
ADFO01000096
clade_63
N
N


Prevotella disiens
1494
AEDO01000026
clade_64
N
N


Bacteroides faecis
276
GQ496624
clade_65
N
N


Bacteroides fragilis
279
AP006841
clade_65
N
N


Bacteroides nordii
285
NR_043017
clade_65
N
N


Bacteroides salyersiae
292
EU136690
clade_65
N
N


Bacteroides sp. 1_1_14
293
ACRP01000155
clade_65
N
N


Bacteroides sp. 1_1_6
295
ACIC01000215
clade_65
N
N


Bacteroides sp. 2_1_56FAA
298
ACWI01000065
clade_65
N
N


Bacteroides sp. AR29
316
AF139525
clade_65
N
N


Bacteroides sp. B2
317
EU722733
clade_65
N
N


Bacteroides
328
NR_074277
clade_65
N
N


thetaiotaomicron


Actinobacillus minor
45
ACFT01000025
clade_69
N
N


Haemophilus parasuis
978
GU226366
clade_69
N
N


Vibrio cholerae
1996
AAUR01000095
clade_71
N
Category-B


Vibrio fluvialis
1997
X76335
clade_71
N
Category-B


Vibrio furnissii
1998
CP002377
clade_71
N
Category-B


Vibrio mimicus
1999
ADAF01000001
clade_71
N
Category-B


Vibrio parahaemolyticus
2000
AAWQ01000116
clade_71
N
Category-B


Vibrio sp. RC341
2001
ACZT01000024
clade_71
N
Category-B


Vibrio vulnificus
2002
AE016796
clade_71
N
Category-B


Lactobacillus acidophilus
1067
CP000033
clade_72
N
N


Lactobacillus amylolyticus
1069
ADNY01000006
clade_72
N
N


Lactobacillus amylovorus
1070
CP002338
clade_72
N
N


Lactobacillus crispatus
1078
ACOG01000151
clade_72
N
N


Lactobacillus delbrueckii
1080
CP002341
clade_72
N
N


Lactobacillus helveticus
1088
ACLM01000202
clade_72
N
N


Lactobacillus kalixensis
1094
NR_029083
clade_72
N
N


Lactobacillus
1095
NR_042440
clade_72
N
N


kefiranofaciens


Lactobacillus leichmannii
1098
JX986966
clade_72
N
N


Lactobacillus sp. 66c
1120
FR681900
clade_72
N
N


Lactobacillus sp. KLDS
1122
EU600905
clade_72
N
N


1.0701


Lactobacillus sp. KLDS
1130
EU600916
clade_72
N
N


1.0712


Lactobacillus sp. oral clone
1136
AY349383
clade_72
N
N


HT070


Lactobacillus ultunensis
1139
ACGU01000081
clade_72
N
N


Prevotella intermedia
1502
AF414829
clade_81
N
N


Prevotella nigrescens
1511
AFPX01000069
clade_81
N
N


Prevotella pallens
1515
AFPY01000135
clade_81
N
N


Prevotella sp. oral taxon 310
1551
GQ422737
clade_81
N
N


Prevotella genomosp. C1
1495
AY278624
clade_82
N
N


Prevotella sp. CM38
1519
HQ610181
clade_82
N
N


Prevotella sp. oral taxon 317
1552
ACQH01000158
clade_82
N
N


Prevotella sp. SG12
1527
GU561343
clade_82
N
N


Prevotella denticola
1493
CP002589
clade_83
N
N


Prevotella genomosp. P7
1497
DQ003620
clade_83
N
N


oral clone MB2_P31


Prevotella histicola
1501
JN867315
clade_83
N
N


Prevotella multiformis
1508
AEWX01000054
clade_83
N
N


Prevotella sp. JCM 6330
1522
AB547699
clade_83
N
N


Prevotella sp. oral clone
1539
AY349397
clade_83
N
N


GI059


Prevotella sp. oral taxon 782
1555
GQ422745
clade_83
N
N


Prevotella sp. oral taxon
1559
GU432180
clade_83
N
N


G71


Prevotella sp. SEQ065
1524
JN867234
clade_83
N
N


Prevotella veroralis
1565
ACVA01000027
clade_83
N
N


Bacteroides acidifaciens
266
NR_028607
clade_85
N
N


Bacteroides cellulosilyticus
269
ACCH01000108
clade_85
N
N


Bacteroides clarus
270
AFBM01000011
clade_85
N
N


Bacteroides eggerthii
275
ACWG01000065
clade_85
N
N


Bacteroides oleiciplenus
286
AB547644
clade_85
N
N


Bacteroides pyogenes
290
NR_041280
clade_85
N
N


Bacteroides sp. 315_5
300
FJ848547
clade_85
N
N


Bacteroides sp. 31SF15
301
AJ583248
clade_85
N
N


Bacteroides sp. 31SF18
302
AJ583249
clade_85
N
N


Bacteroides sp. 35AE31
303
AJ583244
clade_85
N
N


Bacteroides sp. 35AE37
304
AJ583245
clade_85
N
N


Bacteroides sp. 35BE34
305
AJ583246
clade_85
N
N


Bacteroides sp. 35BE35
306
AJ583247
clade_85
N
N


Bacteroides sp. WH2
324
AY895180
clade_85
N
N


Bacteroides sp. XB12B
325
AM230648
clade_85
N
N


Bacteroides stercoris
327
ABFZ02000022
clade_85
N
N


Actinobacillus
46
NR_074857
clade_88
N
N


pleuropneumoniae


Actinobacillus ureae
48
AEVG01000167
clade_88
N
N


Haemophilus aegyptius
969
AFBC01000053
clade_88
N
N


Haemophilus ducreyi
970
AE017143
clade_88
N
OP


Haemophilus haemolyticus
973
JN175335
clade_88
N
N


Haemophilus influenzae
974
AADP01000001
clade_88
N
OP


Haemophilus
975
GU561425
clade_88
N
N


parahaemolyticus


Haemophilus parainfluenzae
976
AEWU01000024
clade_88
N
N


Haemophilus
977
M75076
clade_88
N
N


paraphrophaemolyticus


Haemophilus somnus
979
NC_008309
clade_88
N
N


Haemophilus sp. 70334
980
HQ680854
clade_88
N
N


Haemophilus sp. HK445
981
FJ685624
clade_88
N
N


Haemophilus sp. oral clone
982
AY923117
clade_88
N
N


ASCA07


Haemophilus sp. oral clone
983
AY923147
clade_88
N
N


ASCG06


Haemophilus sp. oral clone
984
AY005034
clade_88
N
N


BJ021


Haemophilus sp. oral clone
985
AY005033
clade_88
N
N


BJ095


Haemophilus sp. oral taxon
987
AGRK01000004
clade_88
N
N


851


Haemophilus sputorum
988
AFNK01000005
clade_88
N
N


Histophilus somni
1003
AF549387
clade_88
N
N


Mannheimia haemolytica
1195
ACZX01000102
clade_88
N
N


Pasteurella bettyae
1433
L06088
clade_88
N
N


Moellerella wisconsensis
1253
JN175344
clade_89
N
N


Morganella morganii
1265
AJ301681
clade_89
N
N


Morganella sp. JB_T16
1266
AJ781005
clade_89
N
N


Proteus mirabilis
1582
ACLE01000013
clade_89
N
N


Proteus penneri
1583
ABVP01000020
clade_89
N
N


Proteus sp. HS7514
1584
DQ512963
clade_89
N
N


Proteus vulgaris
1585
AJ233425
clade_89
N
N


Oribacterium sinus
1374
ACKX01000142
clade_90
N
N


Oribacterium sp. ACB1
1375
HM120210
clade_90
N
N


Oribacterium sp. ACB7
1376
HM120211
clade_90
N
N


Oribacterium sp. CM12
1377
HQ616374
clade_90
N
N


Oribacterium sp. ICM51
1378
HQ616397
clade_90
N
N


Oribacterium sp. OBRC12
1379
HQ616355
clade_90
N
N


Oribacterium sp. oral taxon
1382
AFIH01000001
clade_90
N
N


108


Actinobacillus
44
AY362885
clade_92
N
N


actinomycetemcomitans


Actinobacillus succinogenes
47
CP000746
clade_92
N
N


Aggregatibacter
112
CP001733
clade_92
N
N


actinomycetemcomitans


Aggregatibacter aphrophilus
113
CP001607
clade_92
N
N


Aggregatibacter segnis
114
AEPS01000017
clade_92
N
N


Averyella dalhousiensis
194
DQ481464
clade_92
N
N


Bisgaard Taxon
368
AY683487
clade_92
N
N


Bisgaard Taxon
369
AY683489
clade_92
N
N


Bisgaard Taxon
370
AY683491
clade_92
N
N


Bisgaard Taxon
371
AY683492
clade_92
N
N


Buchnera aphidicola
440
NR_074609
clade_92
N
N


Cedecea davisae
499
AF493976
clade_92
N
N


Citrobacter amalonaticus
517
FR870441
clade_92
N
N


Citrobacter braakii
518
NR_028687
clade_92
N
N


Citrobacter farmeri
519
AF025371
clade_92
N
N


Citrobacter freundii
520
NR_028894
clade_92
N
N


Citrobacter gillenii
521
AF025367
clade_92
N
N


Citrobacter koseri
522
NC_009792
clade_92
N
N


Citrobacter murliniae
523
AF025369
clade_92
N
N


Citrobacter rodentium
524
NR_074903
clade_92
N
N


Citrobacter sedlakii
525
AF025364
clade_92
N
N


Citrobacter sp. 30_2
526
ACDJ01000053
clade_92
N
N


Citrobacter sp. KMSI_3
527
GQ468398
clade_92
N
N


Citrobacter werkmanii
528
AF025373
clade_92
N
N


Citrobacter youngae
529
ABWL02000011
clade_92
N
N


Cronobacter malonaticus
737
GU122174
clade_92
N
N


Cronobacter sakazakii
738
NC_009778
clade_92
N
N


Cronobacter turicensis
739
FN543093
clade_92
N
N


Enterobacter aerogenes
786
AJ251468
clade_92
N
N


Enterobacter asburiae
787
NR_024640
clade_92
N
N


Enterobacter cancerogenus
788
Z96078
clade_92
N
N


Enterobacter cloacae
789
FP929040
clade_92
N
N


Enterobacter cowanii
790
NR_025566
clade_92
N
N


Enterobacter hormaechei
791
AFHR01000079
clade_92
N
N


Enterobacter sp. 247BMC
792
HQ122932
clade_92
N
N


Enterobacter sp. 638
793
NR_074777
clade_92
N
N


Enterobacter sp. JC163
794
JN657217
clade_92
N
N


Enterobacter sp. SCSS
795
HM007811
clade_92
N
N


Enterobacter sp. TSE38
796
HM156134
clade_92
N
N


Enterobacteriaceae
797
ADCU01000033
clade_92
N
N


bacterium 9_2_54FAA


Enterobacteriaceae
798
AJ489826
clade_92
N
N


bacterium CF01Ent_1


Enterobacteriaceae
799
AY538694
clade_92
N
N


bacterium Smarlab 3302238


Escherichia albertii
824
ABKX01000012
clade_92
N
N


Escherichia coli
825
NC_008563
clade_92
N
Category-B


Escherichia fergusonii
826
CU928158
clade_92
N
N


Escherichia hermannii
827
HQ407266
clade_92
N
N


Escherichia sp. 1_1_43
828
ACID01000033
clade_92
N
N


Escherichia sp. 4_1_40B
829
ACDM02000056
clade_92
N
N


Escherichia sp. B4
830
EU722735
clade_92
N
N


Escherichia vulneris
831
NR_041927
clade_92
N
N


Ewingella americana
877
JN175329
clade_92
N
N


Haemophilus genomosp. P2
971
DQ003621
clade_92
N
N


oral clone MB3_C24


Haemophilus genomosp. P3
972
DQ003635
clade_92
N
N


oral clone MB3_C38


Haemophilus sp. oral clone
986
AY349380
clade_92
N
N


JM053


Hafnia alvei
989
DQ412565
clade_92
N
N


Klebsiella oxytoca
1024
AY292871
clade_92
N
OP


Klebsiella pneumoniae
1025
CP000647
clade_92
N
OP


Klebsiella sp. AS10
1026
HQ616362
clade_92
N
N


Klebsiella sp. Co9935
1027
DQ068764
clade_92
N
N


Klebsiella sp. enrichment
1036
HM195210
clade_92
N
N


culture clone SRC_DSD25


Klebsiella sp. OBRC7
1028
HQ616353
clade_92
N
N


Klebsiella sp. SP_BA
1029
FJ999767
clade_92
N
N


Klebsiella sp. SRC_DSD1
1033
GU797254
clade_92
N
N


Klebsiella sp. SRC_DSD11
1030
GU797263
clade_92
N
N


Klebsiella sp. SRC_DSD12
1031
GU797264
clade_92
N
N


Klebsiella sp. SRC_DSD15
1032
GU797267
clade_92
N
N


Klebsiella sp. SRC_DSD2
1034
GU797253
clade_92
N
N


Klebsiella sp. SRC_DSD6
1035
GU797258
clade_92
N
N


Klebsiella variicola
1037
CP001891
clade_92
N
N


Kluyvera ascorbata
1038
NR_028677
clade_92
N
N


Kluyvera cryocrescens
1039
NR_028803
clade_92
N
N


Leminorella grimontii
1159
AJ233421
clade_92
N
N


Leminorella richardii
1160
HF558368
clade_92
N
N


Pantoea agglomerans
1409
AY335552
clade_92
N
N


Pantoea ananatis
1410
CP001875
clade_92
N
N


Pantoea brenneri
1411
EU216735
clade_92
N
N


Pantoea citrea
1412
EF688008
clade_92
N
N


Pantoea conspicua
1413
EU216737
clade_92
N
N


Pantoea septica
1414
EU216734
clade_92
N
N


Pasteurella dagmatis
1434
ACZR01000003
clade_92
N
N


Pasteurella multocida
1435
NC_002663
clade_92
N
N


Plesiomonas shigelloides
1469
X60418
clade_92
N
N


Raoultella ornithinolytica
1617
AB364958
clade_92
N
N


Raoultella planticola
1618
AF129443
clade_92
N
N


Raoultella terrigena
1619
NR_037085
clade_92
N
N


Salmonella bongori
1683
NR_041699
clade_92
N
Category-B


Salmonella enterica
1672
NC_011149
clade_92
N
Category-B


Salmonella enterica
1673
NC_011205
clade_92
N
Category-B


Salmonella enterica
1674
DQ344532
clade_92
N
Category-B


Salmonella enterica
1675
ABEH02000004
clade_92
N
Category-B


Salmonella enterica
1676
ABAK02000001
clade_92
N
Category-B


Salmonella enterica
1677
NC_011080
clade_92
N
Category-B


Salmonella enterica
1678
EU118094
clade_92
N
Category-B


Salmonella enterica
1679
NC_011094
clade_92
N
Category-B


Salmonella enterica
1680
AE014613
clade_92
N
Category-B


Salmonella enterica
1682
ABFH02000001
clade_92
N
Category-B


Salmonella enterica
1684
ABEM01000001
clade_92
N
Category-B


Salmonella enterica
1685
ABAM02000001
clade_92
N
Category-B


Salmonella typhimurium
1681
DQ344533
clade_92
N
Category-B


Salmonella typhimurium
1686
AF170176
clade_92
N
Category-B


Serratia fonticola
1718
NR_025339
clade_92
N
N


Serratia liquefaciens
1719
NR_042062
clade_92
N
N


Serratia marcescens
1720
GU826157
clade_92
N
N


Serratia odorifera
1721
ADBY01000001
clade_92
N
N


Serratia proteamaculans
1722
AAUN01000015
clade_92
N
N


Shigella boydii
1724
AAKA01000007
clade_92
N
Category-B


Shigella dysenteriae
1725
NC_007606
clade_92
N
Category-B


Shigella flexneri
1726
AE005674
clade_92
N
Category-B


Shigella sonnei
1727
NC_007384
clade_92
N
Category-B


Tatumella ptyseos
1916
NR_025342
clade_92
N
N


Trabulsiella guamensis
1925
AY373830
clade_92
N
N


Yersinia aldovae
2019
AJ871363
clade_92
N
OP


Yersinia aleksiciae
2020
AJ627597
clade_92
N
OP


Yersinia bercovieri
2021
AF366377
clade_92
N
OP


Yersinia enterocolitica
2022
FR729477
clade_92
N
Category-B


Yersinia frederiksenii
2023
AF366379
clade_92
N
OP


Yersinia intermedia
2024
AF366380
clade_92
N
OP


Yersinia kristensenii
2025
ACCA01000078
clade_92
N
OP


Yersinia mollaretii
2026
NR_027546
clade_92
N
OP


Yersinia pestis
2027
AE013632
clade_92
N
Category-A


Yersinia pseudotuberculosis
2028
NC_009708
clade_92
N
OP


Yersinia rohdei
2029
ACCD01000071
clade_92
N
OP


Yokenella regensburgei
2030
AB273739
clade_92
N
N


Conchiformibius kuhniae
669
NR_041821
clade_94
N
N


Morococcus cerebrosus
1267
JN175352
clade_94
N
N


Neisseria bacilliformis
1328
AFAY01000058
clade_94
N
N


Neisseria cinerea
1329
ACDY01000037
clade_94
N
N


Neisseria flavescens
1331
ACQV01000025
clade_94
N
N


Neisseria gonorrhoeae
1333
CP002440
clade_94
N
OP


Neisseria lactamica
1334
ACEQ01000095
clade_94
N
N


Neisseria macacae
1335
AFQE01000146
clade_94
N
N


Neisseria meningitidis
1336
NC_003112
clade_94
N
OP


Neisseria mucosa
1337
ACDX01000110
clade_94
N
N


Neisseria pharyngis
1338
AJ239281
clade_94
N
N


Neisseria polysaccharea
1339
ADBE01000137
clade_94
N
N


Neisseria sicca
1340
ACKO02000016
clade_94
N
N


Neisseria sp. KEM232
1341
GQ203291
clade_94
N
N


Neisseria sp. oral clone
1344
AY005027
clade_94
N
N


AP132


Neisseria sp. oral strain
1346
AY005028
clade_94
N
N


B33KA


Neisseria sp. oral taxon 014
1347
ADEA01000039
clade_94
N
N


Neisseria sp. TM10_1
1343
DQ279352
clade_94
N
N


Neisseria subflava
1348
ACEO01000067
clade_94
N
N


Okadaella gastrococcus
1365
HQ699465
clade_98
N
N


Streptococcus agalactiae
1785
AAJO01000130
clade_98
N
N


Streptococcus alactolyticus
1786
NR_041781
clade_98
N
N


Streptococcus australis
1788
AEQR01000024
clade_98
N
N


Streptococcus bovis
1789
AEEL01000030
clade_98
N
N


Streptococcus canis
1790
AJ413203
clade_98
N
N


Streptococcus constellatus
1791
AY277942
clade_98
N
N


Streptococcus cristatus
1792
AEVC01000028
clade_98
N
N


Streptococcus dysgalactiae
1794
AP010935
clade_98
N
N


Streptococcus equi
1795
CP001129
clade_98
N
N


Streptococcus equinus
1796
AEVB01000043
clade_98
N
N


Streptococcus gallolyticus
1797
FR824043
clade_98
N
N


Streptococcus genomosp.
1798
AY278629
clade_98
N
N


C1


Streptococcus genomosp.
1799
AY278630
clade_98
N
N


C2


Streptococcus genomosp.
1800
AY278631
clade_98
N
N


C3


Streptococcus genomosp.
1801
AY278632
clade_98
N
N


C4


Streptococcus genomosp.
1802
AY278633
clade_98
N
N


C5


Streptococcus genomosp.
1803
AY278634
clade_98
N
N


C6


Streptococcus genomosp.
1804
AY278635
clade_98
N
N


C7


Streptococcus genomosp.
1805
AY278609
clade_98
N
N


C8


Streptococcus gordonii
1806
NC_009785
clade_98
N
N


Streptococcus infantarius
1807
ABJK02000017
clade_98
N
N


Streptococcus infantis
1808
AFNN01000024
clade_98
N
N


Streptococcus intermedius
1809
NR_028736
clade_98
N
N


Streptococcus lutetiensis
1810
NR_037096
clade_98
N
N


Streptococcus massiliensis
1811
AY769997
clade_98
N
N


Streptococcus mitis
1813
AM157420
clade_98
N
N


Streptococcus
1815
AY099095
clade_98
N
N


oligofermentans


Streptococcus oralis
1816
ADMV01000001
clade_98
N
N


Streptococcus parasanguinis
1817
AEKM01000012
clade_98
N
N


Streptococcus pasteurianus
1818
AP012054
clade_98
N
N


Streptococcus peroris
1819
AEVF01000016
clade_98
N
N


Streptococcus pneumoniae
1820
AE008537
clade_98
N
N


Streptococcus porcinus
1821
EF121439
clade_98
N
N


Streptococcus
1822
FJ827123
clade_98
N
N


pseudopneumoniae


Streptococcus
1823
AENS01000003
clade_98
N
N


pseudoporcinus


Streptococcus pyogenes
1824
AE006496
clade_98
N
OP


Streptococcus ratti
1825
X58304
clade_98
N
N


Streptococcus sanguinis
1827
NR_074974
clade_98
N
N


Streptococcus sinensis
1828
AF432857
clade_98
N
N


Streptococcus sp.
1831
ACOI01000028
clade_98
N
N


2_1_36FAA


Streptococcus sp. 2285_97
1830
AJ131965
clade_98
N
N


Streptococcus sp. ACS2
1834
HQ616360
clade_98
N
N


Streptococcus sp. AS20
1835
HQ616366
clade_98
N
N


Streptococcus sp. BS35a
1836
HQ616369
clade_98
N
N


Streptococcus sp. C150
1837
ACRI01000045
clade_98
N
N


Streptococcus sp. CM6
1838
HQ616372
clade_98
N
N


Streptococcus sp. ICM10
1840
HQ616389
clade_98
N
N


Streptococcus sp. ICM12
1841
HQ616390
clade_98
N
N


Streptococcus sp. ICM2
1842
HQ616386
clade_98
N
N


Streptococcus sp. ICM4
1844
HQ616387
clade_98
N
N


Streptococcus sp. ICM45
1843
HQ616394
clade_98
N
N


Streptococcus sp. M143
1845
ACRK01000025
clade_98
N
N


Streptococcus sp. M334
1846
ACRL01000052
clade_98
N
N


Streptococcus sp. oral clone
1849
AY923121
clade_98
N
N


ASB02


Streptococcus sp. oral clone
1850
DQ272504
clade_98
N
N


ASCA03


Streptococcus sp. oral clone
1851
AY923116
clade_98
N
N


ASCA04


Streptococcus sp. oral clone
1852
AY923119
clade_98
N
N


ASCA09


Streptococcus sp. oral clone
1853
AY923123
clade_98
N
N


ASCB04


Streptococcus sp. oral clone
1854
AY923124
clade_98
N
N


ASCB06


Streptococcus sp. oral clone
1855
AY923127
clade_98
N
N


ASCC04


Streptococcus sp. oral clone
1856
AY923128
clade_98
N
N


ASCC05


Streptococcus sp. oral clone
1857
DQ272507
clade_98
N
N


ASCC12


Streptococcus sp. oral clone
1858
AY923129
clade_98
N
N


ASCD01


Streptococcus sp. oral clone
1859
AY923130
clade_98
N
N


ASCD09


Streptococcus sp. oral clone
1860
DQ272509
clade_98
N
N


ASCD10


Streptococcus sp. oral clone
1861
AY923134
clade_98
N
N


ASCE03


Streptococcus sp. oral clone
1862
AY953253
clade_98
N
N


ASCE04


Streptococcus sp. oral clone
1863
DQ272510
clade_98
N
N


ASCE05


Streptococcus sp. oral clone
1864
AY923135
clade_98
N
N


ASCE06


Streptococcus sp. oral clone
1865
AY923136
clade_98
N
N


ASCE09


Streptococcus sp. oral clone
1866
AY923137
clade_98
N
N


ASCE10


Streptococcus sp. oral clone
1867
AY923138
clade_98
N
N


ASCE12


Streptococcus sp. oral clone
1868
AY923140
clade_98
N
N


ASCF05


Streptococcus sp. oral clone
1869
AY953255
clade_98
N
N


ASCF07


Streptococcus sp. oral clone
1870
AY923142
clade_98
N
N


ASCF09


Streptococcus sp. oral clone
1871
AY923145
clade_98
N
N


ASCG04


Streptococcus sp. oral clone
1872
AY005042
clade_98
N
N


BW009


Streptococcus sp. oral clone
1873
AY005044
clade_98
N
N


CH016


Streptococcus sp. oral clone
1874
AY349413
clade_98
N
N


GK051


Streptococcus sp. oral clone
1875
AY349414
clade_98
N
N


GM006


Streptococcus sp. oral clone
1876
AY207051
clade_98
N
N


P2PA_41 P2


Streptococcus sp. oral clone
1877
AY207064
clade_98
N
N


P4PA_30 P4


Streptococcus sp. oral taxon
1878
AEEP01000019
clade_98
N
N


071


Streptococcus sp. oral taxon
1879
GU432132
clade_98
N
N


G59


Streptococcus sp. oral taxon
1880
GU432146
clade_98
N
N


G62


Streptococcus sp. oral taxon
1881
GU432150
clade_98
N
N


G63


Streptococcus suis
1882
FM252032
clade_98
N
N


Streptococcus thermophilus
1883
CP000419
clade_98
N
N


Streptococcus salivarius
1826
AGBV01000001
clade_98
N
N


Streptococcus uberis
1884
HQ391900
clade_98
N
N


Streptococcus urinalis
1885
DQ303194
clade_98
N
N


Streptococcus vestibularis
1886
AEKO01000008
clade_98
N
N


Streptococcus viridans
1887
AF076036
clade_98
N
N


Synergistetes bacterium oral
1908
GU227192
clade_98
N
N


clone 03 5 D05
















TABLE 1A





Exemplary Immunomodulatory Bacterial Species

















Alkaliphilus metalliredigens



Ammonifex degensii



Anaerofustis stercorihominis



Anaerostipes caccae



Anaerotruncus colihominis



Bacillus amyloliquefaciens



Bacillus anthracis



Bacillus cellulosilyticus



Bacillus cereus



Bacillus clausii



Bacillus coagulans



Bacillus cytotoxicus



Bacillus halodurans



Bacillus licheniformis



Bacillus pumilus



Bacillus subtilis



Bacillus thuringiensis



Bacillus weihenstephanensis



Blautia (Ruminococcus) hansenii



Blautia (Ruminococcus) obeum



Brevibacillus brevis



Bryantella formatexigens



Caldicellulosiruptor saccharolyticus



Candidatus Desulforudis audaxviato



Carboxydibrachium pacificum



Carboxydothermus hydrogenoformans



Clostridium acetobutylicum



Clostridium asparagiforme



Clostridium bartlettii



Clostridium beijerinckii



Clostridium bolteae



Clostridium botulinum A str. ATCC 19397



Clostridium botulinum B str. Eklund 17B



Clostridium butyricum pathogenic E4 str. BoNT BL5262



Clostridium carboxidivorans



Clostridium cellulolyticum



Clostridium cellulovorans



Clostridium difficile



Clostridium (Hungatella) hathewayi



Clostridium hylemonae



Clostridium kluyveri



Clostridium leptum



Clostridium methylpentosum



Clostridium (Tyzzerella) nexile



Clostridium novyi NT



Clostridium papyrosolvens



Clostridium perfringens



Clostridium phytofermentans ISDg



Clostridium scindens



Clostridium sp. 7_2_43FAA



Clostridium sporogenes



Clostridium tetani



Clostridium thermocellum



Coprococcus comes



Desulfotomaculum reducens



Dorea longicatena



Eubacterium eligens



Eubacterium hallii



Eubacterium rectale



Eubacterium ventriosum



Faecalibacterium prausnitzii



Geobacillus kaustophilus



Geobacillus sp. G11MC16



Geobacillus thermodenitrificans



Heliobacterium modesticaldum



Lysinibacillus sphaericus



Oceanobacillus iheyensis



Paenibacillus sp. JDR-2



Pelotomaculum thermopropionicum



Roseburia intestinalis



Ruminococcus bromii



Ruminococcus gnavus



Ruminococcus torques



Subdoligranulum variabile



Symbiobacterium thermophilum



Thermoanaerobacter italicus



Thermoanaerobacter tengcongensis



Thermoanaerobacterium thermosaccharolyticum



Thermosinus carboxydivorans

















TABLE 1B





Exemplary Bacteria Useful in the Present Invention















Acidaminococcus intestini


Acinetobacter baumannii


Acinetobacter lwoffii


Akkermansia muciniphila


Alistipes putredinis


Alistipes shahii


Anaerostipes hadrus


Anaerotruncus colihominis


Bacteroides caccae


Bacteroides cellulosilyticus


Bacteroides dorei


Bacteroides eggerthii


Bacteroides finegoldii


Bacteroides fragilis


Bacteroides massiliensis


Bacteroides ovatus


Bacteroides salanitronis


Bacteroides salyersiae


Bacteroides sp. 1_1_6


Bacteroides sp. 3_1_23


Bacteroides sp. D20


Bacteroides thetaiotaomicron


Bacteroides uniformis


Bacteroides vulgatus


Bifidobacterium adolescentis


Bifidobacterium bifidum


Bifidobacterium breve


Bifidobacterium faecale


Bifidobacterium kashiwanohense


Bifidobacterium longum subsp. longum


Bifidobacterium pseudocatenulatum


Bifidobacterium stercoris


Blautia (Ruminococcus) coccoides


Blautia faecis


Blautia glucerasea


Blautia (Ruminococcus) hansenii


Blautia hydrogenotrophica (Ruminococcus hydrogenotrophicus)


Blautia (Ruminococcus) luti


Blautia (Ruminococcus) obeum


Blautia producta (Ruminococcus productus)


Blautia (Ruminococcus) schinkii


Blautia stercoris


Blautia uncultured bacterium clone BKLE_a03_2


(GenBank: EU469501.1)


Blautia uncultured bacterium clone SJTU_B_14_30


(GenBank: EF402926.1)


Blautia uncultured bacterium clone SJTU_C_14_16


(GenBank: EF404657.1)


Blautia uncultured bacterium clone S1-5 (GenBank: GQ898099.1)


Blautia uncultured PAC000178_s


(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2)


Blautia wexlerae


Candidatus Arthromitus sp. SFB-mouse-Yit


Catenibacterium mitsuokai


Clostridiaceae bacterium (Dielma fastidiosa) JC13


Clostridiales bacterium 1_7_47FAA


Clostridium asparagiforme


Clostridium bolteae


Clostridium clostridioforme


Clostridium glycyrrhizinilyticum


Clostridium (Hungatella) hathewayi


Clostridium histolyticum


Clostridium indolis


Clostridium leptum


Clostridium (Tyzzerella) nexile


Clostridium perfringens


Clostridium (Erysipelatoclostridium) ramosum


Clostridium scindens


Clostridium sp. 14774


Clostridium sp. 7_3_54FAA


Clostridium sp. HGF2


Clostridium symbiosum


Collinsella aerofaciens


Collinsella intestinalis


Coprobacillus sp. D7


Coprococcus catus


Coprococcus comes


Dorea formicigenerans


Dorea longicatena


Enterococcus faecalis


Enterococcus faecium


Erysipelotrichaceae bacterium 3_1_53


Escherichia coli


Escherichia coli S88


Eubacterium eligens


Eubacterium fissicatena


Eubacterium ramulus


Eubacterium rectale


Faecalibacterium prausnitzii


Flavonifractor plautii


Fusobacterium mortiferum


Fusobacterium nucleatum


Holdemania filiformis


Hydrogenoanaerobacterium saccharovorans


Klebsiella oxytoca


Lachnospiraceae bacterium 3_1_57FAA_CT1


Lachnospiraceae bacterium 7_1_58FAA


Lachnospiraceae bacterium 5_1_57FAA


Lactobacillus casei


Lactobacillus rhamnosus


Lactobacillus ruminis


Lactococcus casei


Odoribacter splanchnicus


Oscillibacter valericigenes


Parabacteroides gordonii


Parabacteroides johnsonii


Parabacteroides merdae


Pediococcus acidilactici


Peptostreptococcus asaccharolyticus


Propionibacterium granulosum


Roseburia intestinalis


Roseburia inulinivorans


Ruminococcus faecis


Ruminococcus gnavus


Ruminococcus sp. ID8


Ruminococcus torques


Slackia piriformis


Staphylococcus epidermidis


Staphylococcus saprophyticus


Streptococcus cristatus


Streptococcus dysgalactiae subsp. equisimilis


Streptococcus infantis


Streptococcus oralis


Streptococcus sanguinis


Streptococcus viridans


Streptococcus thermophilus


Veillonella dispar
















TABLE 1C





Exemplary Bacteria Useful in the Present Invention

















Anaerotruncus colihominis strain 13



Blautia producta strain 6



Clostridium bolteae strain 7



Clostridiaceae bacterium JC13 strain 8



Clostridiales bacterium 1_7_47FAA strain 28



Clostridium sp. 7_3_54FAA strain 16



Clostridium asparagiforme strain 15



Clostridium clostridioforme



Clostridium (Hungatella) hathewayi strain 4



Clostridium indolis strain 9



Clostridium (Erysipelatoclostridium) ramosum strain 18



Clostridium scindens strain 26



Clostridium sp. 14774 strain 1



Eubacterium fissicatena strain 21



Hydrogenoanaerobacterium saccharovorans



Lachnospiraceae bacterium 3_1_57FAA strain 27



Lachnospiraceae bacterium 3_1_57FAA strain 29



Lachnospiraceae bacterium 7_1_58FAA strain 3



Oscillibacter valericigenes



Ruminococcus sp. ID8 strain 14

















TABLE 1D





Exemplary Bacteria Useful in the Present Invention

















Bacteroides caccae



Bacteroides eggerthii



Bacteroides ovatus



Bacteroides sp. 1_1_6



Bacteroides sp. 3_1_23



Bacteroides sp. D20



Bacteroides vulgatus



Bifidobacterium adolescentis



Bifidobacterium pseudocatenulatum



Blautia (Ruminococcus) obeum



Blautia producta (Ruminococcus productus)



Blautia (Ruminococcus) schinkii



Clostridium (Hungatella) hathewayi



Clostridium (Tyzzerella) nexile



Clostridium sp. HGF2



Clostridium symbiosum



Collinsella aerofaciens



Coprobacillus sp. D7



Coprococcus catus



Coprococcus comes



Dorea formicigenerans



Dorea longicatena



Enterococcus faecalis



Erysipelotrichaceae bacterium 3_1_53



Escherichia coli



Escherichia coli S88



Eubacterium eligens



Eubacterium rectale



Faecalibacterium prausnitzii



Lachnospiraceae bacterium 5_1_57FAA



Odoribacter splanchnicus



Parabacteroides merdae



Roseburia intestinalis



Ruminococcus torques



Streptococcus thermophilus

















TABLE 1E





Exemplary Bacteria Useful in the Present Invention

















Akkermansia muciniphila



Enterococcus faecalis



Klebsiella oxytoca



Lactobacillus rhamnosus



Staphylococcus epidermidis



Streptococcus viridans



Veillonella dispar

















TABLE 1F





Exemplary Bacteria Useful in the Present Invention

















Acinetobacter baumannii



Acinetobacter lwoffii



Akkermansia muciniphila



Alistipes shahii



Anaerotruncus colihominis



Bacteroides caccae



Bacteroides dorei



Bacteroides eggerthii



Bacteroides finegoldii



Bacteroides fragilis



Bacteroides massiliensis



Bacteroides ovatus



Bacteroides salanitronis



Bacteroides sp. 1_1_6



Bacteroides sp. 3_1_23



Bacteroides sp. D20



Bacteroides thetaiotaomicron



Bacteroides uniformis



Bacteroides vulgatus



Bifidobacterium adolescentis



Bifidobacterium breve



Bifidobacterium pseudocatenulatum



Blautia (Ruminococcus) coccoides



Blautia faecis



Blautia glucerasea



Blautia (Ruminococcus) hansenii



Blautia hydrogenotrophica (Ruminococcus hydrogenotrophicus)



Blautia (Ruminococcus) luti



Blautia (Ruminococcus) obeum



Blautia producta (Ruminococcus productus)



Blautia (Ruminococcus) schinkii



Blautia stercoris



Blautia wexlerae



Candidatus Arthromitus sp. SFB-mouse-Yit



Clostridiaceae bacterium (Dielma fastidiosa) JC13



Clostridiales bacterium 1_7_47FAA



Clostridium asparagiforme



Clostridium bolteae



Clostridium clostridioforme



Clostridium (Hungatella) hathewayi



Clostridium histolyticum



Clostridium indolis



Clostridium leptum



Clostridium (Tyzzerella) nexile



Clostridium perfringens



Clostridium (Erysipelatoclostridium) ramosum



Clostridium scindens



Clostridium sp. 14774



Clostridium sp. 7_3_54FAA



Clostridium sp. HGF2



Clostridium symbiosum



Collinsella aerofaciens



Coprobacillus sp. D7



Coprococcus catus



Coprococcus comes



Dorea formicigenerans



Dorea longicatena



Enterococcus faecium



Erysipelotrichaceae bacterium 3_1_53



Escherichia coli



Escherichia coli S88



Eubacterium eligens



Eubacterium fissicatena



Eubacterium rectale



Faecalibacterium prausnitzii



Fusobacterium mortiferum



Fusobacterium nucleatum



Hydrogenoanaerobacterium saccharovorans



Lachnospiraceae bacterium 3_1_57FAA_CT1



Lachnospiraceae bacterium 7_1_58FAA



Lachnospiraceae bacterium 5_1_57FAA



Lactobacillus casei



Lactococcus casei



Odoribacter splanchnicus



Oscillibacter valericigenes



Parabacteroides johnsonii



Parabacteroides merdae



Pediococcus acidilactici



Peptostreptococcus asaccharolyticus



Propionibacterium granulosum



Roseburia intestinalis



Ruminococcus gnavus



Ruminococcus sp. ID8



Ruminococcus torques



Staphylococcus saprophyticus



Streptococcus thermophilus

















TABLE 2A







Species identified as germinable and sporulatable by colony picking















GAM +

Sweet B +
Sweet



OTU
BBA
FOS/inulin
M2GSC
FOS/Inulin
GAM
Total
















Blautia producta
1




1


Clostridium bartlettii
4

1


5


Clostridium bolteae
2


5
1
8


Clostridium botulinum



5

5


Clostridium butyricum
37
43
8
1
33
122


Clostridium celatum
4



1
5


Clostridium clostridioforme
1



1
2


Clostridium disporicum
26
26
22
33
50
157


Clostridium glycolicum
4
9
14


27


Clostridium mayombei
2
2



4


Clostridium paraputrificum
8
8
33
16
6
71


Clostridium sordellii


14


14


Clostridium sp. 7_2_43FAA

1



1


Clostridium symbiosum
3




3


Clostridium tertium

1

1

2


(blank)

2

31

33


Totals
92
92
92
92
92
460
















TABLE 2B





Species identified as germinable by 16S colony pick approach

















Clostridium paraputrificum



Clostridium disporicum



Clostridium glycolicum



Clostridium bartlettii



Clostridium butyricum



Ruminococcus bromii



Lachnospiraceae bacterium 2_1_58FAA



Eubacterium hadrum



Turicibacter sanguinis



Lachnospiraceae bacterium oral taxon F15



Clostridium perfringens



Clostridium bifermentans



Roseburia sp 11SE37



Clostridium quinii



Ruminococcus lactaris



Clostridium botulinum



Clostridium tyrobutyricum



Blautia hansenii



Clostridium kluyveri



Clostridium sp JC122



Clostridium hylemonae



Clostridium celatum



Clostridium straminisolvens



Clostridium orbiscindens



Roseburia cecicola



Eubacterium tenue



Clostridium sp. 7_2_43FAA



Lachnospiraceae bacterium 4_1_37FAA



Eubacterium rectale



Clostridium viride



Ruminococcus sp. K_1



Clostridium symbiosum



Ruminococcus torques



Clostridium algidicarnis

















TABLE 2C





Species identified as sporulatable by 16S NGS approach

















Clostridium paraputrificum



Clostridium bartlettii



Lachnospiraceae bacterium 2_1_58FAA



Clostridium disporicum



Ruminococcus bromii



Eubacterium hadrum



Clostridium butyricum



Roseburia sp. 11SE37



Clostridium perfringens



Clostridium glycolicum



Clostridium hylemonae



Clostridium orbiscindens



Ruminococcus lactaris



Clostridium symbiosum



Lachnospiraceae bacterium oral taxon F15



Blautia hansenii



Turicibacter sanguinis



Clostridium straminisolvens



Clostridium botulinum



Lachnospiraceae bacterium 4_1_37FAA



Roseburia cecicola



Ruminococcus sp. K_1



Clostridium bifermentans



Eubacterium rectale



Clostridium quinii



Clostridium viride



Clostridium kluyveri



Clostridium tyrobutyricum



Oscillibacter sp. G2



Clostridium sp. JC122



Lachnospiraceae bacterium 3_1_57FAA



Clostridium aldenense



Ruminococcus torques



Clostridium sp. 7_2_43FAA



Clostridium celatum



Eubacterium sp. WAL_14571



Eubacterium tenue



Lachnospiraceae bacterium 5_1_57FAA



Clostridium clostridioforme



Clostridium sp. YIT_12070



Blautia sp. M25



Anaerostipes caccae



Roseburia inulinivorans



Clostridium sp. D5



Clostridium asparagiforme



Coprobacillus sp. D7



Clostridium sp. HGF2



Clostridium citroniae



Clostridium difficile



Oscillibacter valericigenes



Clostridium algidicarnis

















TABLE 3







Anaerobic bacterial species tested for


carbon source usage (Biolog plates)








Species purchased:
Species Freshly Isolated:





R. gnavus (EPV1)
Blautia luti BlnIX (EPV114)


E. rectale (EPV2)
Blautia luti ELU (EPV54)


B. luti (EPV3)
Ruminococcus gnavus (EPV102)


B. wexlerae (EPV5)
Blautia faecis (EPV78)


C. leptum (EPV6)
Ruminococcus torques (EPV76)


B. faecis (EPV15)
Blautia wexlerae SJTU1416 (EPV52)


B. obeum (EPV20)
Blautia WAL14507 (EPV64)


B. producta (EPV21)
Uncultured bacterium



SJTU1416 (EPV51)


B. coccoides (EPV22)
Uncultured bacterium



GQ8980099 (EPV47)


B. hydrogenotrophica (EPV23)
Eubacterium rectale (EPV35)


B. hansenii (EPV24)
















TABLE 4







Exemplary Prebiotics/Carbon Sources








Chemical
MoA





L-Arabinose
C-Source, carbohydrate


N-Acetyl-D-Glucosamine
C-Source, carbohydrate


D-Saccharic acid
C-Source, carboxylic acid


Succinic acid
C-Source, carboxylic acid


D-Galactose
C-Source, carbohydrate


L-Aspartic acid
C-Source, amino acid


L-Proline
C-Source, amino acid


D-Alanine
C-Source, amino acid


D-Trehalose
C-Source, carbohydrate


D-Mannose
C-Source, carbohydrate


Dulcitol
C-Source, carbohydrate


D-Serine
C-Source, amino acid


D-Sorbitol
C-Source, carbohydrate


Glycerol
C-Source, carbohydrate


L-Fucose
C-Source, carbohydrate


D-Glucuronic acid
C-Source, carboxylic acid


D-Gluconic acid
C-Source, carboxylic acid


DL-a-Glycerol Phosphate
C-Source, carbohydrate


D-Xylose
C-Source, carbohydrate


L-Lactic acid
C-Source, carboxylic acid


Formic acid
C-Source, carboxylic acid


D-Mannitol
C-Source, carbohydrate


L-Glutamic acid
C-Source, amino acid


D-Glucose-6-Phosphate
C-Source, carbohydrate


D-Galactonic acid-g-Lactone
C-Source, carboxylic acid


DL-Malic acid
C-Source, carboxylic acid


D-Ribose
C-Source, carbohydrate


Tween 20
C-Source, fatty acid


L-Rhamnose
C-Source, carbohydrate


D-Fructose
C-Source, carbohydrate


Acetic acid
C-Source, carboxylic acid


a-D-Glucose
C-Source, carbohydrate


Maltose
C-Source, carbohydrate


D-Melibiose
C-Source, carbohydrate


Thymidine
C-Source, carbohydrate


L-Asparagine
C-Source, amino acid


D-Aspartic acid
C-Source, amino acid


D-Glucosaminic acid
C-Source, carboxylic acid


1,2-Propanediol
C-Source, alcohol


Tween 40
C-Source, fatty acid


a-Ketoglutaric acid
C-Source, carboxylic acid


a-Ketobutyric acid
C-Source, carboxylic acid


a-Methyl-D-Galactoside
C-Source, carbohydrate


a-D-Lactose
C-Source, carbohydrate


Lactulose
C-Source, carbohydrate


Sucrose
C-Source, carbohydrate


Uridine
C-Source, carbohydrate


L-Glutamine
C-Source, amino acid


m-Tartaric acid
C-Source, carboxylic acid


D-Glucose-1-Phosphate
C-Source, carbohydrate


D-Fructose-6-Phosphate
C-Source, carbohydrate


Tween 80
C-Source, fatty acid


a-Hydroxyglutaric acid-g-Lactone
C-Source, carboxylic acid


a-Hydroxybutyric acid
C-Source, carboxylic acid


b-Methyl-D-Glucoside
C-Source, carbohydrate


Adonitol
C-Source, carbohydrate


Maltotriose
C-Source, carbohydrate


2′-Deoxyadenosine
C-Source, carbohydrate


Adenosine
C-Source, carbohydrate


Gly-Asp
C-Source, amino acid


Citric acid
C-Source, carboxylic acid


m-Inositol
C-Source, carbohydrate


D-Threonine
C-Source, amino acid


Fumaric acid
C-Source, carboxylic acid


Bromosuccinic acid
C-Source, carboxylic acid


Propionic acid
C-Source, carboxylic acid


Mucic acid
C-Source, carboxylic acid


Glycolic acid
C-Source, carboxylic acid


Glyoxylic acid
C-Source, carboxylic acid


D-Cellobiose
C-Source, carbohydrate


Inosine
C-Source, carbohydrate


Gly-Glu
C-Source, amino acid


Tricarballylic acid
C-Source, carboxylic acid


L-Serine
C-Source, amino acid


L-Threonine
C-Source, amino acid


L-Alanine
C-Source, amino acid


Ala-Gly
C-Source, amino acid


Acetoacetic acid
C-Source, carboxylic acid


N-Acetyl-D-Mannosamine
C-Source, carbohydrate


Mono-Methylsuccinate
C-Source, carboxylic acid


Methylpyruvate
C-Source, ester


D-Malic acid
C-Source, carboxylic acid


L-Malic acid
C-Source, carboxylic acid


Gly-Pro
C-Source, amino acid


p-Hydroxyphenyl Acetic acid
C-Source, carboxylic acid


m-Hydroxyphenyl Acetic acid
C-Source, carboxylic acid


Tyramine
C-Source, amine


D-Psicose
C-Source, carbohydrate


L-Lyxose
C-Source, carbohydrate


Glucuronamide
C-Source, amide


Pyruvic acid
C-Source, carboxylic acid


L-Galactonic acid-g-Lactone
C-Source, carboxylic acid


D-Galacturonic acid
C-Source, carboxylic acid


Phenylethylamine
C-Source, amine


2-Aminoethanol
C-Source, alcohol


Negative Control
C-Source, negative control


Chondroitin Sulfate C
C-Source, polymer


a-Cyclodextrin
C-Source, polymer


b-Cyclodextrin
C-Source, polymer


g-Cyclodextrin
C-Source, polymer


Dextrin
C-Source, polymer


Gelatin
C-Source, polymer


Glycogen
C-Source, polymer


Inulin
C-Source, polymer


Laminarin
C-Source, polymer


Mannan
C-Source, polymer


Pectin
C-Source, polymer


N-Acetyl-D-Galactosamine
C-Source, carbohydrate


N-Acetyl-Neuraminic acid
C-Source, carboxylic acid


b-D-Allose
C-Source, carbohydrate


Amygdalin
C-Source, carbohydrate


D-Arabinose
C-Source, carbohydrate


D-Arabitol
C-Source, carbohydrate


L-Arabitol
C-Source, carbohydrate


Arbutin
C-Source, carbohydrate


2-Deoxy-D-Ribose
C-Source, carbohydrate


i-Erythritol
C-Source, carbohydrate


D-Fucose
C-Source, carbohydrate


3-O-b-D-Galactopyranosyl-D-Arabinose
C-Source, carbohydrate


Gentiobiose
C-Source, carbohydrate


L-Glucose
C-Source, carbohydrate


D-Lactitol
C-Source, carbohydrate


D-Melezitose
C-Source, carbohydrate


Maltitol
C-Source, carbohydrate


a-Methyl-D-Glucoside
C-Source, carbohydrate


b-Methyl-D-Galactoside
C-Source, carbohydrate


3-Methylglucose
C-Source, carbohydrate


b-Methyl-D-Glucuronic acid
C-Source, carboxylic acid


a-Methyl-D-Mannoside
C-Source, carbohydrate


b-Methyl-D-Xyloside
C-Source, carbohydrate


Palatinose
C-Source, carbohydrate


D-Raffinose
C-Source, carbohydrate


Salicin
C-Source, carbohydrate


Sedoheptulosan
C-Source, carbohydrate


L-Sorbose
C-Source, carbohydrate


Stachyose
C-Source, carbohydrate


D-Tagatose
C-Source, carbohydrate


Turanose
C-Source, carbohydrate


Xylitol
C-Source, carbohydrate


N-Acetyl-D-Glucosaminitol
C-Source, carbohydrate


g-Amino-N-Butyric acid
C-Source, carboxylic acid


d-Amino Valeric acid
C-Source, carboxylic acid


Butyric acid
C-Source, carboxylic acid


Capric acid
C-Source, carboxylic acid


Caproic acid
C-Source, carboxylic acid


Citraconic acid
C-Source, carboxylic acid


Citramalic acid
C-Source, carboxylic acid


D-Glucosamine
C-Source, carbohydrate


2-Hydroxybenzoic acid
C-Source, carboxylic acid


4-Hydroxybenzoic acid
C-Source, carboxylic acid


b-Hydroxybutyric acid
C-Source, carboxylic acid


g-Hydroxybutyric acid
C-Source, carboxylic acid


a-Keto-Valeric acid
C-Source, carboxylic acid


Itaconic acid
C-Source, carboxylic acid


5-Keto-D-Gluconic acid
C-Source, carboxylic acid


D-Lactic acid Methyl Ester
C-Source, ester


Malonic acid
C-Source, carboxylic acid


Melibionic acid
C-Source, carbohydrate


Oxalic acid
C-Source, carboxylic acid


Oxalomalic acid
C-Source, carboxylic acid


Quinic acid
C-Source, carboxylic acid


D-Ribono-1,4-Lactone
C-Source, carboxylic acid


Sebacic acid
C-Source, carboxylic acid


Sorbic acid
C-Source, carboxylic acid


Succinamic acid
C-Source, carboxylic acid


D-Tartaric acid
C-Source, carboxylic acid


L-Tartaric acid
C-Source, carboxylic acid


Acetamide
C-Source, amide


L-Alaninamide
C-Source, amide


N-Acetyl-L-Glutamic acid
C-Source, amino acid


L-Arginine
C-Source, amino acid


Glycine
C-Source, amino acid


L-Histidine
C-Source, amino acid


L-Homoserine
C-Source, amino acid


Hydroxy-L-Proline
C-Source, amino acid


L-Isoleucine
C-Source, amino acid


L-Leucine
C-Source, amino acid


L-Lysine
C-Source, amino acid


L-Methionine
C-Source, amino acid


L-Ornithine
C-Source, amino acid


L-Phenylalanine
C-Source, amino acid


L-Pyroglutamic acid
C-Source, amino acid


L-Valine
C-Source, amino acid


D,L-Carnitine
C-Source, carboxylic acid


sec-Butylamine
C-Source, amine


D,L-Octopamine
C-Source, amine


Putrescine
C-Source, amine


Dihydroxyacetone
C-Source, alcohol


2,3-Butanediol
C-Source, alcohol


2,3-Butanedione
C-Source, alcohol


3-Hydroxy-2-butanone
C-Source, alcohol
















TABLE 5







Bacterial Species Detected at Low Frequency in


Vaginal Samples from Vancomycin-Treated Mice














Mean
Median





abundance
abundance





day 6 (out
day 6 (out


Site
Group
Taxonomy
of 10,000)
of 10,000)














vaginal
Vancomycin
KF008552.1.1432 D_0——Bacteria;
0.291242675
0.024255713




D_1——Proteobacteria;




D_2——Gammaproteobacteria;




D_3——Enterobacteriales;




D_4——Enterobacteriaceae; D_5——Klebsiella;




D_6——Klebsiella pneumooiae


vaginal
Vancomycin
AB740357.1.1462 D_0——Bacteria;
1.436524722
0




D_1——Proteobacteria;




D_2——Gammaproteobacteria;




D_3——Enterobacteriales;




D_4——Enterobacteriaceae; D_5——Pantoea;




D_6——Pantoea sp. NCCP-532


vaginal
Vancomycin
DQ799428.1.1372 D_0——Bacteria;
0.348310693
0




D_1——Verrucomicrobia;




D_2——Verrucomicrobiae;




D_3——Verrucomicrobiales;




D_4——Verrucomicrobiaceae;




D_5——Akkermansia; D_6——uncultured




bacterium


vaginal
Vancomycin
JX094996.1.1390 D_0——Bacteria;
0.348310693
0




D_1——Firmicutes; D_2——Clostridia;




D_3——Clostridiales; D_4——Lachnospiraceae;




D_5——Blautia; D_6——uncultured bacterium


vaginal
Vancomycin
EU459716.1.1286 D_0——Bacteria;
0.348310693
0




D_1——Firmicutes; D_2——Clostridia;




D_3——Clostridiales; D_4——Lachnospiraceae;




D_5——uncultured; D_6——uncultured




bacterium


vaginal
Vancomycin
EU457230.1.1391 D_0——Bacteria;
0.696621386
0




D_1——Firmicutes; D_2——Clostridia;




D_3——Clostridiales; D_4——Lachnospiraceae;




D_5——Incertae Sedis; D_6——uncultured




bacterium


vaginal
Vancomycin
EU459317.1.1373 D_0——Bacteria;
0.348310693
0




D_1——Firmicutes; D_2——Clostridia;




D_3——Clostridiales; D_4——Clostridiaceae 1;




D_5——Clostridium sensu stricto 1;




D_6——uncultured bacterium


vaginal
Vancomycin
HM817954.1.1353 D_0——Bacteria;
0.348310693
0




D_1——Firmicutes; D_2——Clostridia;




D_3——Clostridiales; D_4——Lachnospiraceae;




D_5——Roseburia; D_6——uncultured




bacterium


vaginal
Vancomycin
GQ134873.1.1373 D_0——Bacteria;
0.348310693
0




D_1——Firmicutes; D_2——Clostridia;




D_3——Clostridiales; D_4——Clostridiaceae 1;




D_5——Clostridium sensu stricto 1;




D_6——uncultured bacterium


vaginal
Vancomycin
FJ879074.1.1494 D_0——Bacteria;
0.348310693
0




D_1——Firmicutes; D_2——Clostridia;




D_3——Clostridiales; D_4——Lachnospiraceae;




D_5——uncultured; D_6——uncultured




bacterium


vaginal
Vancomycin
EU774816.1.1381 D_0——Bacteria;
0.348310693
0




D_1——Firmicutes; D_2——Clostridia;




D_3——Clostridiales; D_4——Clostridiaceae 1;




D_5——Clostridium sensu stricto 1;




D_6——uncultured bacterium


vaginal
Vancomycin
EU775614.1.1398 D_0——Bacteria;
0.417063419
0




D_1——Proteobacteria;




D_2——Gammaproteobacteria;




D_3——Enterobacteriales;




D_4——Enterobacteriaceae;




D_5——Enterobacter; D_6——uncultured




bacterium









Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series.
While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.
The foregoing description of the embodiments of the invention has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure.
Finally, the language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims.

## Claims

1. A pharmaceutical composition comprising:
an isolated population of anti-inflammatory bacterial cells consisting essentially of 3 or more strains of anti-inflammatory bacteria, wherein the 3 or more strains comprise Blautia producta, Ruminococcus torques, and one or more strains selected from Eubacterium rectale, Ruminococcus obeum, and Eubacterium ventriosum, wherein each strain of anti-inflammatory bacteria selected for inclusion in the composition is capable of increasing secretion of Interleukin 10 (IL-10) by a population of human peripheral blood mononuclear cells (PBMCs) in vitro, and wherein the isolated population of anti-inflammatory bacterial cells increases IL-10 secretion by PBMCs by at least 80% or more relative to each strain individually; and
a pharmaceutically acceptable excipient.

2. The pharmaceutical composition of claim 1, wherein the isolated population of anti-inflammatory bacterial cells counteracts secretion of a pro-inflammatory cytokine induced by Enterococcus faecalis in a population of human PBMCs.

3. The pharmaceutical composition of claim 1, wherein the 3 or more strains of anti-inflammatory bacteria comprise Blautia producta, Ruminococcus torques, and Ruminococcus obeum.

4. The pharmaceutical composition of claim 1, wherein the 3 or more strains of anti-inflammatory bacteria comprise Blautia producta, Ruminococcus torques, and Eubacterium rectale.

5. The pharmaceutical composition of claim 1, wherein the 3or more strains of anti-inflammatory bacteria compromise Blautia producta, Ruminococcus torques, and Eubacterium ventriosum.

6. The pharmaceutical composition of claim 1, wherein the isolated population of anti-inflammatory bacterial cells comprises a bacterial cell in vegetative form.

7. The pharmaceutical composition of claim 1, wherein the isolated population of anti-inflammatory bacterial cells comprises a bacterial cell in spore form.

8. The pharmaceutical composition of claim 1, wherein the isolated population of anti-inflammatory bacterial cells increases IL-10 secretion by PBMCs by at least 100% or more relative to each strain individually.

9. The pharmaceutical composition of claim 1, further comprising a prebiotic.

10. The pharmaceutical composition of claim 9, wherein the prebiotic comprises a monomer or polymer selected from the group consisting of arabinoxylan, xylose, soluble fiber dextran, soluble corn fiber, polydextrose, lactose, N-acetyl-lactosamine, glucose, galactose, fructose, rhamnose, mannose, uronic acids, 3′-fucosyllactose, 3′-sialylactose, 6′-sialyllactose, lacto-N-neotetraose, 2′-2′-fucosyllactose, and combinations thereof.

11. The pharmaceutical composition of claim 9, wherein the prebiotic comprises a sugar selected from the group consisting of arabinose, fructose, fucose, lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose, xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose, xylooligosaccharide, and combinations thereof.

12. The pharmaceutical composition of claim 1, wherein the isolated population of anti-inflammatory bacterial cells reduces the secretion of one or more pro-inflammatory cytokines selected from the group consisting of IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof, by a population of human PBMCs.

13. The pharmaceutical composition of claim 1, wherein the isolated population of anti-inflammatory bacterial cells increases the secretion of one or more anti-inflammatory cytokines selected from the group consisting of IL-13, IL-4, IL-5, TGFβ, and combinations thereof, by a population of human PBMCs.

14. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for oral administration.

15. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for rectal administration.

16. A method for reducing inflammation in a subject having an autoimmune or inflammatory disorder, the method comprising administering the pharmaceutical composition of claim 1 to the subject, thereby reducing inflammation in the subject.

17. The method of claim 16, wherein the autoimmune or inflammatory disorder is selected from the group consisting of graft-versus-host disease (GVHD), an inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, multiple sclerosis (MS), systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, Sjogren's syndrome, and Celiac disease.

18. The method of claim 17, wherein the autoimmune or inflammatory disorder is GVHD.

19. The method of claim 16, wherein the pharmaceutical composition is administered orally.

20. The method of claim 16, wherein administration of the pharmaceutical composition reduces inflammation in the gastrointestinal tract of the subject.

21. The method of claim 16, wherein administration of the pharmaceutical composition reduces inflammation at a site distal to the gastrointestinal tract of the subject.

22. The method of claim 16, wherein the subject has a dysbiosis.

23. The method of claim 22, wherein the dysbiosis is a gastrointestinal dysbiosis.

24. The method of claim 22, wherein the dysbiosis is a distal dysbiosis.

25. The method of claim 16, wherein the anti-inflammatory bacterial cells of the pharmaceutical composition engraft in the gastrointestinal tract of the subject.

26. The method of claim 16, further comprising administering a prebiotic to the subject.

27. A pharmaceutical composition comprising: an isolated population of anti-inflammatory bacterial cells consisting essentially of 3 or more strains of anti-inflammatory bacteria, wherein the 3 or more strains comprise Blautia producta, Eubacterium ventriosum and Coprococcus comes, wherein each strain of anti-inflammatory bacteria selected for inclusion in the composition is capable of increasing secretion of Interleukin 10 (IL-10) by a population of peripheral blood mononuclear cells (PBMCs) in vitro, and wherein the isolated population of anti-inflammatory bacterial cells increases IL-10 secretion by PBMCs by at least 80% or more relative to each strain individually; and a pharmaceutically acceptable carrier.

28. The pharmaceutical composition of claim 27, wherein the 3or more strains of anti-inflammatory bacteria comprise Blautia producta, Eubacterium ventriosum, and Coprococcus comes.

29. A pharmaceutical composition comprising: an isolated population of anti-inflammatory bacterial cells consisting essentially of 3 or more strains of anti-inflammatory bacteria, wherein the 3 or more strains comprise Ruminococcus torques, Eubacterium ventriosum and Blautia ovatus, wherein each strain of anti-inflammatory bacteria selected for inclusion in the composition is capable of increasing secretion of Interleukin 10 (IL-10) by a population of peripheral blood mononuclear cells (PBMCs) in vitro, and wherein the isolated population of anti-inflammatory bacterial cells increases IL-10secretion by PBMCs by at least 80% or more relative to each strain individually; and a pharmaceutically acceptable carrier.

30. The pharmaceutical composition of claim 29, wherein the 3or more strains of anti-inflammatory bacteria comprise Ruminococcus torques, Eubacterium ventriosum, and Bacteroides ovatus.