Patent Grant
9610307
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.
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.
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.
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).
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.
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 Vivinal™ (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.
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.
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.
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
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
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:
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
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
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
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.
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
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
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 (
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:
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):
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 (
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
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.
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 (
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).
Eubacterium saburreum
Eubacterium sp. oral clone
Alicyclobacillus
acidocaldarius
Clostridium baratii
Clostridium colicanis
Clostridium paraputrificum
Clostridium sardiniense
Eubacterium budayi
Eubacterium moniliforme
Eubacterium multiforme
Eubacterium nitritogenes
Anoxybacillus flavithermus
Bacillus aerophilus
Bacillus aestuarii
Bacillus amyloliquefaciens
Bacillus anthracis
Bacillus atrophaeus
Bacillus badius
Bacillus cereus
Bacillus circulans
Bacillus firmus
Bacillus flexus
Bacillus fordii
Bacillus halmapalus
Bacillus herbersteinensis
Bacillus idriensis
Bacillus lentus
Bacillus licheniformis
Bacillus megaterium
Bacillus nealsonii
Bacillus niabensis
Bacillus niacini
Bacillus pocheonensis
Bacillus pumilus
Bacillus safensis
Bacillus simplex
Bacillus sonorensis
Bacillus sp. 10403023
Bacillus sp. 2_A_57_CT2
Bacillus sp. 2008724126
Bacillus sp. 2008724139
Bacillus sp. 7_16AIA
Bacillus sp. AP8
Bacillus sp. B27(2008)
Bacillus sp. BT1B_CT2
Bacillus sp. GB1.1
Bacillus sp. GB9
Bacillus sp. HU19.1
Bacillus sp. HU29
Bacillus sp. HU33.1
Bacillus sp. JC6
Bacillus sp. oral taxon F79
Bacillus sp. SRC_DSF1
Bacillus sp. SRC_DSF10
Bacillus sp. SRC_DSF2
Bacillus sp. SRC_DSF6
Bacillus sp. tc09
Bacillus sp. zh168
Bacillus sphaericus
Bacillus sporothermodurans
Bacillus subtilis
Bacillus thermoamylovorans
Bacillus thuringiensis
Bacillus weihenstephanensis
Geobacillus kaustophilus
Geobacillus
stearothermophilus
Geobacillus
thermodenitrificans
Geobacillus
thermoglucosidasius
Lysinibacillus sphaericus
Clostridiales sp. SS3_4
Clostridium beijerinckii
Clostridium botulinum
Clostridium butyricum
Clostridium chauvoei
Clostridium favososporum
Clostridium histolyticum
Clostridium isatidis
Clostridium limosum
Clostridium sartagoforme
Clostridium septicum
Clostridium sp. 7_2_43FAA
Clostridium sporogenes
Clostridium tertium
Clostridium carnis
Clostridium celatum
Clostridium disporicum
Clostridium gasigenes
Clostridium quinii
Clostridium hylemonae
Clostridium scindens
Clostridium
glycyrrhizinilyticum
Clostridium nexile
Coprococcus comes
Ruminococcus lactaris
Ruminococcus torques
Paenibacillus lautus
Paenibacillus polymyxa
Paenibacillus sp. HGF5
Paenibacillus sp. HGF7
Eubacterium sp. oral clone
Alicyclobacillus
contaminans
Alicyclobacillus herbarius
Alicyclobacillus pomorum
Blautia coccoides
Blautia glucerasea
Blautia glucerasei
Blautia hansenii
Blautia luti
Blautia producta
Blautia schinkii
Blautia sp. M25
Blautia stercoris
Blautia wexlerae
Bryantella formatexigens
Clostridium coccoides
Eubacterium cellulosolvens
Ruminococcus hansenii
Ruminococcus obeum
Ruminococcus sp.
Ruminococcus sp. K_1
Syntrophococcus
sucromutans
Bacillus alcalophilus
Bacillus clausii
Bacillus gelatini
Bacillus halodurans
Bacillus sp. oral taxon F26
Clostridium innocuum
Clostridium sp. HGF2
Clostridium perfringens
Sarcina ventriculi
Clostridium bartlettii
Clostridium bifermentans
Clostridium ghonii
Clostridium glycolicum
Clostridium mayombei
Clostridium sordellii
Clostridium sp. MT4 E
Eubacterium tenue
Clostridium argentinense
Clostridium sp. JC122
Clostridium sp. NMBHI_1
Clostridium subterminale
Clostridium sulfidigenes
Dorea formicigenerans
Dorea longicatena
Ruminococcus gnavus
Ruminococcus sp. ID8
Blautia hydrogenotrophica
Lactonifactor longoviformis
Robinsoniella peoriensis
Eubacterium infirmum
Eubacterium sp. WAL
Eubacterium biforme
Eubacterium cylindroides
Eubacterium dolichum
Eubacterium sp. 3_1_31
Eubacterium tortuosum
Bulleidia extructa
Solobacterium moorei
Coprococcus catus
Clostridium cochlearium
Clostridium malenominatum
Clostridium tetani
Acetivibrio ethanolgignens
Anaerosporobacter mobilis
Bacteroides pectinophilus
Clostridium aminovalericum
Clostridium
phytofermentans
Eubacterium hallii
Eubacterium xylanophilum
Ruminococcus callidus
Ruminococcus
champanellensis
Ruminococcus sp. 18P13
Ruminococcus sp. 9SE51
Anaerostipes caccae
Anaerostipes sp.
Clostridium aerotolerans
Clostridium aldenense
Clostridium
algidixylanolyticum
Clostridium amygdalinum
Clostridium asparagiforme
Clostridium bolteae
Clostridium celerecrescens
Clostridium citroniae
Clostridium clostridiiformes
Clostridium clostridioforme
Clostridium hathewayi
Clostridium indolis
Clostridium lavalense
Clostridium
saccharolyticum
Clostridium sp. M62_1
Clostridium sp. SS2_1
Clostridium sphenoides
Clostridium symbiosum
Clostridium xylanolyticum
Eubacterium hadrum
Clostridium difficile
Eubacterium sp. AS15b
Eubacterium sp. OBRC9
Eubacterium sp. oral clone
Eubacterium yurii
Clostridium acetobutylicum
Clostridium algidicarnis
Clostridium cadaveris
Clostridium carboxidivorans
Clostridium estertheticum
Clostridium fallax
Clostridium felsineum
Clostridium frigidicarnis
Clostridium kluyveri
Clostridium magnum
Clostridium putrefaciens
Clostridium sp. HPB_46
Clostridium tyrobutyricum
Sutterella parvirubra
Acetanaerobacterium
elongatum
Clostridium cellulosi
Ethanoligenens harbinense
Eubacterium rectale
Eubacterium sp. oral clone
Lachnobacterium bovis
Roseburia cecicola
Roseburia faecalis
Roseburia faecis
Roseburia hominis
Roseburia intestinalis
Roseburia inulinivorans
Brevibacillus brevis
Brevibacillus laterosporus
Bacillus coagulans
Sporolactobacillus inulinus
Kocuria palustris
Nocardia farcinica
Bacillus sp. oral taxon F28
Catenibacterium mitsuokai
Clostridium sp. TM_40
Coprobacillus cateniformis
Coprobacillus sp. 29_1
Clostridium rectum
Eubacterium nodatum
Eubacterium saphenum
Eubacterium sp. oral clone
Eubacterium sp. oral clone
Faecalibacterium prausnitzii
Gemmiger formicilis
Subdoligranulum variabile
Clostridium sp. MLG055
Clostridium cocleatum
Clostridium ramosum
Clostridium saccharogumia
Clostridium spiroforme
Coprobacillus sp. D7
Clostridium sp. SY8519
Eubacterium ramulus
Erysipelothrix inopinata
Erysipelothrix rhusiopathiae
Erysipelothrix tonsillarum
Holdemania filiformis
Coxiella burnetii
Clostridium hiranonis
Clostridium irregulare
Clostridium orbiscindens
Clostridium sp. NML
Flavonifractor plautii
Pseudoflavonifractor
capillosus
Acetivibrio cellulolyticus
Clostridium aldrichii
Clostridium clariflavum
Clostridium stercorarium
Clostridium straminisolvens
Clostridium thermocellum
Fusobacterium nucleatum
Eubacterium barkeri
Eubacterium callanderi
Eubacterium limosum
Anaerotruncus colihominis
Clostridium
methylpentosum
Clostridium sp. YIT 12070
Hydrogenoanaerobacterium
saccharovorans
Ruminococcus albus
Ruminococcus flavefaciens
Clostridium haemolyticum
Clostridium novyi
Clostridium sp. LMG 16094
Eubacterium ventriosum
Bacteroides galacturonicus
Eubacterium eligens
Lachnospira multipara
Lachnospira pectinoschiza
Lactobacillus rogosae
Bacillus horti
Bacillus sp. 9_3AIA
Eubacterium brachy
Filifactor alocis
Filifactor villosus
Clostridium leptum
Clostridium sp. YIT 12069
Clostridium
sporosphaeroides
Eubacterium
coprostanoligenes
Ruminococcus bromii
Eubacterium siraeum
Clostridium viride
Oscillibacter sp. G2
Oscillibacter valericigenes
Oscillospira guilliermondii
Butyrivibrio crossotus
Clostridium sp. L2_50
Coprococcus eutactus
Coprococcus sp. ART55_1
Eubacterium ruminantium
Collinsella aerofaciens
Alkaliphilus
metalliredigenes
Alkaliphilus oremlandii
Clostridium sticklandii
Turicibacter sanguinis
Fulvimonas sp. NML
Desulfitobacterium frappieri
Desulfitobacterium
hafniense
Desulfotomaculum
nigrificans
Lutispora thermophila
Brachyspira pilosicoli
Eggerthella lenta
Streptomyces albus
Chlamydiales bacterium
Anaerofustis
stercorihominis
Butyricicoccus
pullicaecorum
Eubacterium desmolans
Papillibacter cinnamivorans
Sporobacter termitidis
Deferribacteres sp. oral
Clostridium colinum
Clostridium
lactatifermentans
Clostridium piliforme
Saccharomonospora viridis
Thermobifida fusca
Leptospira licerasiae
Moorella thermoacetica
Thermoanaerobacter
pseudethanolicus
Flexistipes sinusarabici
Gloeobacter violaceus
Eubacterium sp. oral clone
Clostridium oroticum
Clostridium sp. D5
Eubacterium contortum
Eubacterium fissicatena
Corynebacterium coyleae
Corynebacterium
mucifaciens
Corynebacterium
ureicelerivorans
Corynebacterium appendicis
Corynebacterium genitalium
Corynebacterium glaucum
Corynebacterium imitans
Corynebacterium riegelii
Corynebacterium sp.
Corynebacterium sp. NML
Corynebacterium
sundsvallense
Corynebacterium tuscaniae
Prevotella maculosa
Prevotella oris
Prevotella salivae
Prevotella sp. ICM55
Prevotella sp. oral clone
Prevotella sp. oral clone
Prevotella sp. oral taxon
Prevotella corporis
Bacteroides sp. 4_1_36
Bacteroides sp. AR20
Bacteroides sp. D20
Bacteroides sp. F_4
Bacteroides uniformis
Prevotella nanceiensis
Prevotella sp. oral taxon 299
Prevotella bergensis
Prevotella buccalis
Prevotella timonensis
Prevotella oralis
Prevotella sp. SEQ072
Leuconostoc carnosum
Leuconostoc gasicomitatum
Leuconostoc inhae
Leuconostoc kimchii
Edwardsiella tarda
Photorhabdus asymbiotica
Psychrobacter arcticus
Psychrobacter cibarius
Psychrobacter
cryohalolentis
Psychrobacter faecalis
Psychrobacter nivimaris
Psychrobacter pulmonis
Pseudomonas aeruginosa
Pseudomonas sp. 2_1_26
Corynebacterium confusum
Corynebacterium
propinquum
Corynebacterium
pseudodiphtheriticum
Bartonella bacilliformis
Bartonella grahamii
Bartonella henselae
Bartonella quintana
Bartonella tamiae
Bartonella washoensis
Brucella abortus
Brucella canis
Brucella ceti
Brucella melitensis
Brucella microti
Brucella ovis
Brucella sp. 83_13
Brucella sp. BO1
Brucella suis
Ochrobactrum anthropi
Ochrobactrum intermedium
Ochrobactrum
pseudintermedium
Prevotella genomosp. C2
Prevotella
multisaccharivorax
Prevotella sp. oral clone
Prevotella sp. oral taxon 292
Prevotella sp. oral taxon 300
Prevotella marshii
Prevotella sp. oral clone
Prevotella sp. oral taxon 781
Prevotella stercorea
Prevotella brevis
Prevotella ruminicola
Prevotella sp. sp24
Prevotella sp. sp34
Prevotella albensis
Prevotella copri
Prevotella oulorum
Prevotella sp. BI_42
Prevotella sp. oral clone
Prevotella sp. oral taxon
Prevotella amnii
Bacteroides caccae
Bacteroides finegoldii
Bacteroides intestinalis
Bacteroides sp. XB44A
Bifidobacterium
adolescentis
Bifidobacterium angulatum
Bifidobacterium animalis
Bifidobacterium breve
Bifidobacterium
catenulatum
Bifidobacterium dentium
Bifidobacterium gallicum
Bifidobacterium infantis
Bifidobacterium
kashiwanohense
Bifidobacterium longum
Bifidobacterium
pseudocatenulatum
Bifidobacterium
pseudolongum
Bifidobacterium scardovii
Bifidobacterium sp. HM2
Bifidobacterium sp.
Bifidobacterium sp. M45
Bifidobacterium sp. MSX5B
Bifidobacterium sp. TM_7
Bifidobacterium
thermophilum
Leuconostoc citreum
Leuconostoc lactis
Alicyclobacillus
acidoterrestris
Alicyclobacillus
cycloheptanicus
Acinetobacter baumannii
Acinetobacter calcoaceticus
Acinetobacter genomosp.
Acinetobacter haemolyticus
Acinetobacter johnsonii
Acinetobacter junii
Acinetobacter lwoffii
Acinetobacter parvus
Acinetobacter schindleri
Acinetobacter sp. 56A1
Acinetobacter sp. CIP
Acinetobacter sp. CIP
Acinetobacter sp. M16_22
Acinetobacter sp. RUH2624
Acinetobacter sp. SH024
Lactobacillus jensenii
Alcaligenes faecalis
Alcaligenes sp. CO14
Alcaligenes sp. S3
Oligella ureolytica
Oligella urethralis
Eikenella corrodens
Kingella denitrificans
Kingella genomosp. P1 oral
Kingella kingae
Kingella oralis
Kingella sp. oral clone
Neisseria elongata
Neisseria genomosp. P2 oral
Neisseria sp. oral clone
Neisseria sp. SMC_A9199
Simonsiella muelleri
Corynebacterium
glucuronolyticum
Corynebacterium
pyruviciproducens
Rothia aeria
Rothia dentocariosa
Rothia sp. oral taxon 188
Corynebacterium accolens
Corynebacterium
macginleyi
Corynebacterium
pseudogenitalium
Corynebacterium
tuberculostearicum
Lactobacillus casei
Lactobacillus paracasei
Lactobacillus zeae
Prevotella dentalis
Prevotella sp. oral clone
Prevotella sp. oral clone
Prevotella sp. oral clone
Prevotella sp. oral clone
Prevotella genomosp. P9
Prevotella sp. oral clone
Prevotella sp. oral clone
Prevotella sp. oral clone
Actinomyces genomosp. C1
Actinomyces genomosp. C2
Actinomyces genomosp. P1
Actinomyces georgiae
Actinomyces israelii
Actinomyces massiliensis
Actinomyces meyeri
Actinomyces odontolyticus
Actinomyces orihominis
Actinomyces sp. CCUG
Actinomyces sp. ICM34
Actinomyces sp. ICM41
Actinomyces sp. ICM47
Actinomyces sp. ICM54
Actinomyces sp. oral clone
Actinomyces sp. oral taxon
Actinomyces sp. oral taxon
Actinomyces sp. TeJ5
Haematobacter sp. BC14248
Paracoccus denitrificans
Paracoccus marcusii
Grimontia hollisae
Shewanella putrefaciens
Afipia genomosp. 4
Rhodopseudomonas
palustris
Methylobacterium
extorquens
Methylobacterium podarium
Methylobacterium
radiotolerans
Methylobacterium sp. 1sub
Methylobacterium sp. MM4
Achromobacter denitrificans
Achromobacter piechaudii
Achromobacter
xylosoxidans
Bordetella bronchiseptica
Bordetella holmesii
Bordetella parapertussis
Bordetella pertussis
Microbacterium chocolatum
Microbacterium flavescens
Microbacterium lacticum
Microbacterium oleivorans
Microbacterium oxydans
Microbacterium
paraoxydans
Microbacterium
phyllosphaerae
Microbacterium schleiferi
Microbacterium sp. 768
Microbacterium sp. oral
Microbacterium testaceum
Corynebacterium atypicum
Corynebacterium mastitidis
Corynebacterium sp. NML
Mycobacterium elephantis
Mycobacterium paraterrae
Mycobacterium phlei
Mycobacterium sp. 1776
Mycobacterium sp. 1781
Mycobacterium sp.
Mycobacterium sp.
Mycobacterium sp.
Mycobacterium sp.
Mycobacterium sp.
Mycobacterium sp.
Anoxybacillus contaminans
Bacillus aeolius
Brevibacterium
frigoritolerans
Geobacillus sp. E263
Geobacillus sp. WCH70
Geobacillus
thermocatenulatus
Geobacillus
thermoleovorans
Lysinibacillus fusiformis
Planomicrobium koreense
Sporosarcina newyorkensis
Sporosarcina sp. 2681
Ureibacillus composti
Ureibacillus suwonensis
Ureibacillus terrenus
Ureibacillus thermophilus
Ureibacillus
thermosphaericus
Prevotella micans
Prevotella sp. oral clone
Prevotella sp. SEQ053
Treponema socranskii
Treponema sp. 6:H:D15A_4
Treponema sp. oral taxon
Treponema sp. oral taxon
Porphyromonas
endodontalis
Porphyromonas sp. oral
Porphyromonas sp. oral
Porphyromonas sp. oral
Porphyromonas sp. oral
Acidovorax sp. 98_63833
Comamonas sp. NSP5
Delftia acidovorans
Xenophilus aerolatus
Oribacterium sp. oral taxon
Oribacterium sp. oral taxon
Weissella cibaria
Weissella confusa
Weissella hellenica
Weissella kandleri
Weissella koreensis
Weissella
paramesenteroides
Weissella sp. KLDS 7.0701
Mobiluncus curtisii
Enhydrobacter aerosaccus
Moraxella osloensis
Moraxella sp. GM2
Brevibacterium casei
Brevibacterium epidermidis
Brevibacterium sanguinis
Brevibacterium sp. H15
Acinetobacter radioresistens
Lactobacillus alimentarius
Lactobacillus farciminis
Lactobacillus kimchii
Lactobacillus nodensis
Lactobacillus tucceti
Pseudomonas mendocina
Pseudomonas
pseudoalcaligenes
Pseudomonas sp. NP522b
Pseudomonas stutzeri
Paenibacillus barcinonensis
Paenibacillus barengoltzii
Paenibacillus chibensis
Paenibacillus cookii
Paenibacillus durus
Paenibacillus glucanolyticus
Paenibacillus lactis
Paenibacillus pabuli
Paenibacillus popilliae
Paenibacillus sp. CIP
Paenibacillus sp. JC66
Paenibacillus sp. R_27413
Paenibacillus sp. R_27422
Paenibacillus timonensis
Rothia mucilaginosa
Rothia nasimurium
Prevotella sp. oral taxon 302
Prevotella sp. oral taxon F68
Prevotella tannerae
Porphyromonas
asaccharolytica
Porphyromonas gingivalis
Porphyromonas macacae
Porphyromonas sp. UQD
Porphyromonas uenonis
Leptotrichia buccalis
Leptotrichia hofstadii
Leptotrichia sp. oral clone
Leptotrichia sp. oral taxon
Bacteroides fluxus
Bacteroides helcogenes
Parabacteroides johnsonii
Parabacteroides merdae
Treponema denticola
Treponema genomosp. P5
Treponema putidum
Treponema sp. oral clone
Treponema sp. oral taxon
Treponema sp. oral taxon
Treponema sp. oral taxon
Anaerococcus hydrogenalis
Anaerococcus sp. 8404299
Anaerococcus sp. gpac215
Anaerococcus vaginalis
Propionibacterium
acidipropionici
Propionibacterium avidum
Propionibacterium
granulosum
Propionibacterium jensenii
Propionibacterium
propionicum
Propionibacterium sp. H456
Propionibacterium thoenii
Bifidobacterium bifidum
Leuconostoc mesenteroides
Leuconostoc
pseudomesenteroides
Johnsonella ignava
Propionibacterium acnes
Propionibacterium sp.
Propionibacterium sp. LG
Propionibacterium sp.
Alicyclobacillus sp. CCUG
Actinomyces cardiffensis
Actinomyces funkei
Actinomyces sp. HKU31
Actinomyces sp. oral taxon
Kerstersia gyiorum
Pigmentiphaga daeguensis
Aeromonas
allosaccharophila
Aeromonas enteropelogenes
Aeromonas hydrophila
Aeromonas jandaei
Aeromonas salmonicida
Aeromonas trota
Aeromonas veronii
Marvinbryantia
formatexigens
Rhodobacter sp. oral taxon
Rhodobacter sphaeroides
Lactobacillus antri
Lactobacillus coleohominis
Lactobacillus fermentum
Lactobacillus gastricus
Lactobacillus mucosae
Lactobacillus oris
Lactobacillus pontis
Lactobacillus reuteri
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Lactobacillus sp. oral taxon
Lactobacillus vaginalis
Brevibacterium aurantiacum
Brevibacterium linens
Lactobacillus pentosus
Lactobacillus plantarum
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Agrobacterium radiobacter
Agrobacterium tumefaciens
Corynebacterium
argentoratense
Corynebacterium
diphtheriae
Corynebacterium
pseudotuberculosis
Corynebacterium renale
Corynebacterium ulcerans
Aurantimonas coralicida
Aureimonas altamirensis
Lactobacillus acidipiscis
Lactobacillus salivarius
Lactobacillus sp. KLDS
Lactobacillus buchneri
Lactobacillus genomosp. C1
Lactobacillus genomosp. C2
Lactobacillus hilgardii
Lactobacillus kefiri
Lactobacillus parabuchneri
Lactobacillus parakefiri
Lactobacillus curvatus
Lactobacillus sakei
Aneurinibacillus
aneurinilyticus
Aneurinibacillus danicus
Aneurinibacillus migulanus
Aneurinibacillus
terranovensis
Staphylococcus aureus
Staphylococcus auricularis
Staphylococcus capitis
Staphylococcus caprae
Staphylococcus carnosus
Staphylococcus cohnii
Staphylococcus condimenti
Staphylococcus epidermidis
Staphylococcus equorum
Staphylococcus
haemolyticus
Staphylococcus hominis
Staphylococcus lugdunensis
Staphylococcus pasteuri
Staphylococcus
pseudintermedius
Staphylococcus
saccharolyticus
Staphylococcus
saprophyticus
Staphylococcus sp. clone
Staphylococcus sp. H292
Staphylococcus sp. H780
Staphylococcus succinus
Staphylococcus warneri
Staphylococcus xylosus
Cardiobacterium hominis
Cardiobacterium valvarum
Pseudomonas fluorescens
Pseudomonas gessardii
Pseudomonas monteilii
Pseudomonas poae
Pseudomonas putida
Pseudomonas sp. G1229
Pseudomonas tolaasii
Pseudomonas viridiflava
Listeria grayi
Listeria innocua
Listeria ivanovii
Listeria monocytogenes
Listeria welshimeri
Capnocytophaga sp. oral
Capnocytophaga sputigena
Leptotrichia genomosp. C1
Leptotrichia shahii
Leptotrichia sp.
Leptotrichia sp. oral clone
Leptotrichia sp. oral clone
Bacteroides sp. 20_3
Bacteroides sp. 3_1_19
Bacteroides sp. 3_2_5
Parabacteroides distasonis
Parabacteroides goldsteinii
Parabacteroides gordonii
Parabacteroides sp. D13
Capnocytophaga genomosp.
Capnocytophaga ochracea
Capnocytophaga sp. GEJ8
Capnocytophaga sp. oral
Capnocytophaga sp. S1b
Paraprevotella clara
Bacteroides heparinolyticus
Prevotella heparinolytica
Treponema genomosp. P4
Treponema genomosp. P6
Treponema sp. oral taxon
Treponema sp. oral taxon
Treponema sp. oral taxon
Chlamydia muridarum
Chlamydia trachomatis
Chlamydia psittaci
Chlamydophila pneumoniae
Chlamydophila psittaci
Anaerococcus octavius
Anaerococcus sp. 8405254
Anaerococcus sp. 9401487
Anaerococcus sp. 9403502
Gardnerella vaginalis
Campylobacter lari
Anaerobiospirillum
succiniciproducens
Anaerobiospirillum thomasii
Ruminobacter amylophilus
Succinatimonas hippei
Actinomyces europaeus
Actinomyces sp. oral clone
Moraxella catarrhalis
Moraxella lincolnii
Moraxella sp. 16285
Psychrobacter sp. 13983
Actinobaculum massiliae
Actinobaculum schaalii
Actinobaculum sp.
Actinobaculum sp. P2P_19
Actinomyces sp. oral clone
Actinomyces sp. oral taxon
Actinomyces neuii
Mobiluncus mulieris
Blastomonas natatoria
Novosphingobium
aromaticivorans
Sphingomonas sp. oral
Sphingopyxis alaskensis
Oxalobacter formigenes
Veillonella atypica
Veillonella dispar
Veillonella genomosp. P1
Veillonella parvula
Veillonella sp. 3_1_44
Veillonella sp. 6_1_27
Veillonella sp. ACP1
Veillonella sp. AS16
Veillonella sp. BS32b
Veillonella sp. ICM51a
Veillonella sp. MSA12
Veillonella sp. NVG 100cf
Veillonella sp. OK11
Veillonella sp. oral clone
Veillonella sp. oral clone
Veillonella sp. oral clone
Veillonella sp. oral taxon
Kocuria marina
Kocuria rhizophila
Kocuria rosea
Kocuria varians
Micrococcus antarcticus
Micrococcus luteus
Micrococcus lylae
Micrococcus sp. 185
Lactobacillus brevis
Lactobacillus parabrevis
Pediococcus acidilactici
Pediococcus pentosaceus
Lactobacillus dextrinicus
Lactobacillus perolens
Lactobacillus rhamnosus
Lactobacillus saniviri
Lactobacillus sp. BT6
Mycobacterium mageritense
Mycobacterium neoaurum
Mycobacterium smegmatis
Mycobacterium sp. HE5
Dysgonomonas gadei
Dysgonomonas mossii
Porphyromonas levii
Porphyromonas somerae
Bacteroides barnesiae
Bacteroides coprocola
Bacteroides coprophilus
Bacteroides dorei
Bacteroides massiliensis
Bacteroides plebeius
Bacteroides sp. 3_1_33FAA
Bacteroides sp. 3_1_40A
Bacteroides sp. 4_3_47FAA
Bacteroides sp. 9_1_42FAA
Bacteroides sp. NB_8
Bacteroides vulgatus
Bacteroides ovatus
Bacteroides sp. 1_1_30
Bacteroides sp. 2_1_22
Bacteroides sp. 2_2_4
Bacteroides sp. 3_1_23
Bacteroides sp. D1
Bacteroides sp. D2
Bacteroides sp. D22
Bacteroides xylanisolvens
Treponema lecithinolyticum
Treponema parvum
Treponema sp. oral clone
Treponema sp. oral taxon
Parascardovia denticolens
Scardovia inopinata
Scardovia wiggsiae
Mogibacterium diversum
Mogibacterium neglectum
Mogibacterium pumilum
Mogibacterium timidum
Borrelia burgdorferi
Borrelia garinii
Borrelia sp. NE49
Caldimonas manganoxidans
Lautropia mirabilis
Lautropia sp. oral clone
Peptoniphilus
asaccharolyticus
Peptoniphilus duerdenii
Peptoniphilus harei
Peptoniphilus indolicus
Peptoniphilus lacrimalis
Peptoniphilus sp. gpac077
Peptoniphilus sp. JC140
Peptoniphilus sp. oral taxon
Peptoniphilus sp. oral taxon
Dialister pneumosintes
Dialister sp. oral taxon 502
Cupriavidus metallidurans
Herbaspirillum seropedicae
Herbaspirillum sp. JC206
Janthinobacterium sp. SY12
Massilia sp. CCUG 43427A
Ralstonia pickettii
Ralstonia sp. 5_7_47FAA
Francisella novicida
Francisella philomiragia
Francisella tularensis
Ignatzschineria indica
Ignatzschineria sp. NML
Streptococcus mutans
Lactobacillus gasseri
Lactobacillus hominis
Lactobacillus iners
Lactobacillus johnsonii
Lactobacillus senioris
Lactobacillus sp. oral clone
Weissella beninensis
Sphingomonas echinoides
Sphingomonas sp. oral
Sphingomonas sp. oral
Zymomonas mobilis
Arcanobacterium
haemolyticum
Arcanobacterium pyogenes
Trueperella pyogenes
Lactococcus garvieae
Lactococcus lactis
Brevibacterium mcbrellneri
Brevibacterium paucivorans
Brevibacterium sp. JC43
Selenomonas artemidis
Selenomonas sp. FOBRC9
Selenomonas sp. oral taxon
Desmospora activa
Desmospora sp. 8437
Paenibacillus sp. oral taxon
Corynebacterium
ammoniagenes
Corynebacterium
aurimucosum
Corynebacterium bovis
Corynebacterium canis
Corynebacterium casei
Corynebacterium durum
Corynebacterium efficiens
Corynebacterium falsenii
Corynebacterium flavescens
Corynebacterium
glutamicum
Corynebacterium jeikeium
Corynebacterium
kroppenstedtii
Corynebacterium
lipophiloflavum
Corynebacterium
matruchotii
Corynebacterium
minutissimum
Corynebacterium resistens
Corynebacterium simulans
Corynebacterium singulare
Corynebacterium sp. 1 ex
Corynebacterium sp. NML
Corynebacterium striatum
Corynebacterium
urealyticum
Corynebacterium variabile
Aerococcus sanguinicola
Aerococcus urinae
Aerococcus urinaeequi
Aerococcus viridans
Fusobacterium naviforme
Moryella indoligenes
Selenomonas genomosp. P5
Selenomonas sp. oral clone
Selenomonas sputigena
Hyphomicrobium
sulfonivorans
Methylocella silvestris
Legionella pneumophila
Lactobacillus coryniformis
Arthrobacter agilis
Arthrobacter arilaitensis
Arthrobacter bergerei
Arthrobacter globiformis
Arthrobacter nicotianae
Mycobacterium abscessus
Mycobacterium chelonae
Bacteroides salanitronis
Paraprevotella xylaniphila
Barnesiella intestinihominis
Barnesiella viscericola
Parabacteroides sp. NS31_3
Tannerella forsythia
Tannerella sp.
6_1_58FAA_CT1
Mycoplasma amphoriforme
Mycoplasma genitalium
Mycoplasma pneumoniae
Mycoplasma penetrans
Ureaplasma parvum
Ureaplasma urealyticum
Treponema genomosp. P1
Treponema sp. oral taxon
Treponema sp. oral taxon
Treponema sp. oral taxon
Treponema sp. oral taxon
Treponema sp. oral taxon
Treponema sp. ovine footrot
Treponema vincentii
Parasutterella
excrementihominis
Parasutterella secunda
Sutterella morbirenis
Sutterella sanguinus
Sutterella sp. YIT 12072
Sutterella stercoricanis
Sutterella wadsworthensis
Propionibacterium
freudenreichii
Propionibacterium sp. oral
Tessaracoccus sp. oral taxon
Peptoniphilus ivorii
Peptoniphilus sp. gpac007
Peptoniphilus sp. gpac018A
Peptoniphilus sp. gpac148
Flexispira rappini
Helicobacter bilis
Helicobacter cinaedi
Helicobacter sp. None
Brevundimonas
subvibrioides
Hyphomonas neptunium
Phenylobacterium zucineum
Streptococcus downei
Streptococcus sp. SHV515
Acinetobacter sp. CIP 53.82
Halomonas elongata
Halomonas johnsoniae
Butyrivibrio fibrisolvens
Roseburia sp. 11SE37
Roseburia sp. 11SE38
Shuttleworthia satelles
Shuttleworthia sp. MSX8B
Shuttleworthia sp. oral
Bdellovibrio sp. MPA
Desulfobulbus sp. oral clone
Desulfovibrio desulfuricans
Desulfovibrio fairfieldensis
Desulfovibrio piger
Desulfovibrio sp. 3_1_syn3
Geobacter bemidjiensis
Brachybacterium
alimentarium
Brachybacterium
conglomeratum
Brachybacterium
tyrofermentans
Dermabacter hominis
Aneurinibacillus
thermoaerophilus
Brevibacillus agri
Brevibacillus centrosporus
Brevibacillus choshinensis
Brevibacillus invocatus
Brevibacillus parabrevis
Brevibacillus reuszeri
Brevibacillus sp. phR
Brevibacillus thermoruber
Lactobacillus murinus
Lactobacillus oeni
Lactobacillus ruminis
Lactobacillus vini
Gemella haemolysans
Gemella morbillorum
Gemella morbillorum
Gemella sanguinis
Gemella sp. oral clone
Gemella sp. oral clone
Gemella sp. oral clone
Gemella sp. WAL 1945J
Sporolactobacillus
nakayamae
Gluconacetobacter entanii
Gluconacetobacter
europaeus
Gluconacetobacter hansenii
Gluconacetobacter
oboediens
Gluconacetobacter xylinus
Auritibacter ignavus
Dermacoccus sp. Ellin185
Janibacter limosus
Janibacter melonis
Acetobacter aceti
Acetobacter fabarum
Acetobacter lovaniensis
Acetobacter malorum
Acetobacter orientalis
Acetobacter pasteurianus
Acetobacter pomorum
Acetobacter syzygii
Acetobacter tropicalis
Gluconacetobacter
azotocaptans
Gluconacetobacter
diazotrophicus
Gluconacetobacter johannae
Nocardia brasiliensis
Nocardia cyriacigeorgica
Nocardia puris
Nocardia sp. 01_Je_025
Rhodococcus equi
Oceanobacillus caeni
Oceanobacillus sp. Ndiop
Ornithinibacillus bavariensis
Ornithinibacillus sp.
Virgibacillus proomii
Corynebacterium
amycolatum
Corynebacterium hansenii
Corynebacterium xerosis
Staphylococcus fleurettii
Staphylococcus sciuri
Staphylococcus vitulinus
Stenotrophomonas
maltophilia
Stenotrophomonas sp. FG_6
Mycobacterium africanum
Mycobacterium alsiensis
Mycobacterium avium
Mycobacterium
colombiense
Mycobacterium gordonae
Mycobacterium
intracellulare
Mycobacterium kansasii
Mycobacterium lacus
Mycobacterium leprae
Mycobacterium
lepromatosis
Mycobacterium mantenii
Mycobacterium marinum
Mycobacterium microti
Mycobacterium
parascrofulaceum
Mycobacterium seoulense
Mycobacterium sp. 1761
Mycobacterium sp. 1791
Mycobacterium sp. 1797
Mycobacterium sp.
Mycobacterium sp.
Mycobacterium sp. W
Mycobacterium tuberculosis
Mycobacterium ulcerans
Mycobacterium vulneris
Xanthomonas campestris
Xanthomonas sp. kmd_489
Dietzia natronolimnaea
Dietzia sp. BBDP51
Dietzia sp. CA149
Dietzia timorensis
Gordonia bronchialis
Gordonia
polyisoprenivorans
Gordonia sp. KTR9
Gordonia sputi
Gordonia terrae
Leptotrichia goodfellowii
Leptotrichia sp. oral clone
Leptotrichia sp. oral clone
Butyricimonas virosa
Odoribacter laneus
Odoribacter splanchnicus
Capnocytophaga gingivalis
Capnocytophaga granulosa
Capnocytophaga sp. oral
Capnocytophaga sp. oral
Capnocytophaga sp. oral
Capnocytophaga canimorsus
Capnocytophaga sp. oral
Lactobacillus catenaformis
Lactobacillus vitulinus
Cetobacterium somerae
Fusobacterium
gonidiaformans
Fusobacterium mortiferum
Fusobacterium necrogenes
Fusobacterium necrophorum
Fusobacterium sp. 12_1B
Fusobacterium sp. 3_1_5R
Fusobacterium sp. D12
Fusobacterium ulcerans
Fusobacterium varium
Mycoplasma arthritidis
Mycoplasma faucium
Mycoplasma hominis
Mycoplasma orale
Mycoplasma salivarium
Mitsuokella jalaludinii
Mitsuokella multacida
Mitsuokella sp. oral taxon
Mitsuokella sp. oral taxon
Selenomonas genomosp. C1
Selenomonas genomosp. P8
Selenomonas ruminantium
Alloscardovia omnicolens
Alloscardovia sp. OB7196
Bifidobacterium urinalis
Prevotella loescheii
Prevotella sp. oral clone
Prevotella sp. oral clone
Prevotella sp. oral taxon 472
Selenomonas dianae
Selenomonas flueggei
Selenomonas genomosp. C2
Selenomonas genomosp. P6
Selenomonas genomosp. P7
Selenomonas infelix
Selenomonas noxia
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral taxon
Agrococcus jenensis
Microbacterium
gubbeenense
Pseudoclavibacter sp.
Timone
Tropheryma whipplei
Zimmermannella bifida
Legionella hackeliae
Legionella longbeachae
Legionella sp. D3923
Legionella sp. D4088
Legionella sp. H63
Legionella sp. NML 93L054
Legionella steelei
Tatlockia micdadei
Helicobacter pullorum
Roseomonas cervicalis
Roseomonas mucosa
Roseomonas sp.
Roseomonas sp.
Roseomonas sp.
Roseomonas sp.
Rickettsia akari
Rickettsia conorii
Rickettsia prowazekii
Rickettsia rickettsii
Rickettsia slovaca
Rickettsia typhi
Anaeroglobus geminatus
Megasphaera genomosp. C1
Megasphaera
micronuciformis
Tsukamurella
paurometabola
Tsukamurella
tyrosinosolvens
Abiotrophia para_adiacens
Carnobacterium divergens
Carnobacterium
maltaromaticum
Enterococcus avium
Enterococcus caccae
Enterococcus casseliflavus
Enterococcus durans
Enterococcus faecalis
Enterococcus faecium
Enterococcus gallinarum
Enterococcus gilvus
Enterococcus hawaiiensis
Enterococcus hirae
Enterococcus italicus
Enterococcus mundtii
Enterococcus raffinosus
Enterococcus sp.
Enterococcus sp.
Enterococcus sp. F95
Enterococcus sp. RfL6
Enterococcus thailandicus
Fusobacterium canifelinum
Fusobacterium genomosp.
Fusobacterium genomosp.
Fusobacterium
periodonticum
Fusobacterium sp.
Fusobacterium sp. 11_3_2
Fusobacterium sp. 2_1_31
Fusobacterium sp. 3_1_27
Fusobacterium sp. 3_1_33
Fusobacterium sp.
Fusobacterium sp. AC18
Fusobacterium sp. ACB2
Fusobacterium sp. AS2
Fusobacterium sp. CM1
Fusobacterium sp. CM21
Fusobacterium sp. CM22
Fusobacterium sp. oral
Fusobacterium sp. oral
Granulicatella adiacens
Granulicatella elegans
Granulicatella paradiacens
Granulicatella sp. oral clone
Granulicatella sp. oral clone
Granulicatella sp. oral clone
Granulicatella sp. oral clone
Tetragenococcus halophilus
Tetragenococcus koreensis
Vagococcus fluvialis
Chryseobacterium anthropi
Chryseobacterium gleum
Chryseobacterium hominis
Treponema refringens
Treponema sp. oral clone
Treponema sp. oral taxon
Treponema sp. oral taxon
Alistipes finegoldii
Alistipes onderdonkii
Alistipes putredinis
Alistipes shahii
Alistipes sp. HGB5
Alistipes sp. JC50
Alistipes sp. RMA 9912
Mycoplasma agalactiae
Mycoplasma bovoculi
Mycoplasma fermentans
Mycoplasma flocculare
Mycoplasma
ovipneumoniae
Arcobacter butzleri
Arcobacter cryaerophilus
Campylobacter curvus
Campylobacter rectus
Campylobacter showae
Campylobacter sp.
Campylobacter sp.
Campylobacter sp. oral
Campylobacter sputorum
Bacteroides ureolyticus
Campylobacter gracilis
Campylobacter hominis
Dialister invisus
Dialister micraerophilus
Dialister microaerophilus
Dialister propionicifaciens
Dialister succinatiphilus
Megasphaera elsdenii
Megasphaera genomosp.
Megasphaera sp.
Megasphaera sp. UPII
Chromobacterium
violaceum
Laribacter hongkongensis
Methylophilus sp. ECd5
Finegoldia magna
Parvimonas micra
Parvimonas sp. oral taxon
Peptostreptococcus micros
Peptostreptococcus sp. oral
Peptostreptococcus sp.
Helicobacter pylori
Anaplasma marginale
Anaplasma
phagocytophilum
Ehrlichia chaffeensis
Neorickettsia risticii
Neorickettsia sennetsu
Pseudoramibacter
alactolyticus
Veillonella montpellierensis
Veillonella sp. oral clone
Veillonella sp. oral clone
Inquilinus limosus
Sphingomonas sp. oral
Anaerococcus lactolyticus
Anaerococcus prevotii
Anaerococcus sp. gpac104
Anaerococcus sp. gpac126
Anaerococcus sp. gpac155
Anaerococcus sp. gpac199
Anaerococcus tetradius
Bacteroides coagulans
Peptostreptococcus sp.
Peptostreptococcus sp. oral
Tissierella praeacuta
Helicobacter canadensis
Peptostreptococcus
anaerobius
Peptostreptococcus stomatis
Bilophila wadsworthia
Desulfovibrio vulgaris
Actinomyces nasicola
Cellulosimicrobium funkei
Lactococcus raffinolactis
Flavobacterium sp. NF2_1
Myroides odoratimimus
Myroides sp. MY15
Chlamydophila pecorum
Parachlamydia sp. UWE25
Fusobacterium russii
Streptobacillus moniliformis
Abiotrophia defectiva
Abiotrophia sp. oral clone
Catonella genomosp. P1
Catonella morbi
Catonella sp. oral clone
Eremococcus coleocola
Facklamia hominis
Granulicatella sp.
Campylobacter coli
Campylobacter concisus
Campylobacter fetus
Campylobacter jejuni
Campylobacter upsaliensis
Atopobium minutum
Atopobium parvulum
Atopobium rimae
Atopobium sp. BS2
Atopobium sp. F0209
Atopobium sp. ICM42b10
Atopobium sp. ICM57
Atopobium vaginae
Actinomyces naeslundii
Actinomyces oricola
Actinomyces oris
Actinomyces sp. 7400942
Actinomyces sp. ChDC
Actinomyces sp. GEJ15
Actinomyces sp.
Actinomyces sp. oral clone
Actinomyces sp. oral clone
Actinomyces sp. oral clone
Actinomyces sp. oral clone
Actinomyces sp. oral taxon
Actinomyces sp. oral taxon
Actinomyces urogenitalis
Actinomyces viscosus
Orientia tsutsugamushi
Megamonas funiformis
Megamonas hypermegale
Aeromicrobium marinum
Aeromicrobium sp. JC14
Luteococcus sanguinis
Rhodococcus
corynebacterioides
Rhodococcus erythropolis
Rhodococcus fascians
Segniliparus rotundus
Segniliparus rugosus
Exiguobacterium acetylicum
Macrococcus caseolyticus
Streptomyces sp. 1
Streptomyces sp. SD 524
Streptomyces sp. SD 528
Streptomyces
thermoviolaceus
Borrelia afzelii
Borrelia crocidurae
Borrelia duttonii
Borrelia hermsii
Borrelia hispanica
Borrelia persica
Borrelia recurrentis
Borrelia spielmanii
Borrelia turicatae
Borrelia valaisiana
Providencia alcalifaciens
Providencia rettgeri
Providencia rustigianii
Providencia stuartii
Treponema pallidum
Treponema phagedenis
Treponema sp. clone
Acholeplasma laidlawii
Mycoplasma putrefaciens
Spiroplasma insolitum
Collinsella intestinalis
Collinsella stercoris
Collinsella tanakaei
Caminicella sporogenes
Acidaminococcus
fermentans
Acidaminococcus intestini
Acidaminococcus sp. D21
Phascolarctobacterium
faecium
Phascolarctobacterium sp.
Phascolarctobacterium
succinatutens
Acidithiobacillus ferrivorans
Catabacter hongkongensis
Christensenella minuta
Heliobacterium
modesticaldum
Alistipes indistinctus
Candidatus Sulcia muelleri
Cytophaga xylanolytica
Gramella forsetii
Sphingobacterium faecium
Sphingobacterium mizutaii
Sphingobacterium
multivorum
Sphingobacterium
spiritivorum
Jonquetella anthropi
Pyramidobacter piscolens
Synergistes genomosp. C1
Synergistes sp. RMA 14551
Synergistetes bacterium
Candidatus Arthromitus sp.
Gracilibacter thermotolerans
Brachyspira aalborgi
Brachyspira sp. HIS3
Brachyspira sp. HIS4
Brachyspira sp. HIS5
Adlercreutzia equolifaciens
Cryptobacterium curtum
Eggerthella sinensis
Eggerthella sp. 1_3_56FAA
Eggerthella sp. HGA1
Eggerthella sp. YY7918
Gordonibacter pamelaeae
Gordonibacter pamelaeae
Slackia equolifaciens
Slackia exigua
Slackia faecicanis
Slackia heliotrinireducens
Slackia isoflavoniconvertens
Slackia piriformis
Slackia sp. NATTS
Victivallis vadensis
Streptomyces griseus
Streptomyces sp. SD 511
Streptomyces sp. SD 534
Cloacibacillus evryensis
Deferribacteres sp. oral
Deferribacteres sp. oral
Peptococcus sp. oral clone
Helicobacter winghamensis
Wolinella succinogenes
Olsenella genomosp. C1
Olsenella profusa
Olsenella sp. F0004
Olsenella sp. oral taxon 809
Olsenella uli
Nocardiopsis dassonvillei
Peptococcus niger
Peptococcus sp. oral taxon
Akkermansia muciniphila
Opitutus terrae
Leptospira borgpetersenii
Leptospira broomii
Leptospira interrogans
Methanobrevibacter
gottschalkii
Methanobrevibacter
millerae
Methanobrevibacter oralis
Methanobrevibacter thaueri
Methanobrevibacter smithii
Deinococcus radiodurans
Deinococcus sp. R_43890
Thermus aquaticus
Actinomyces sp. c109
Anaerobaculum
hydrogeniformans
Microcystis aeruginosa
Prochlorococcus marinus
Methanobrevibacter
acididurans
Methanobrevibacter
arboriphilus
Methanobrevibacter
curvatus
Methanobrevibacter
cuticularis
Methanobrevibacter
filiformis
Methanobrevibacter woesei
Roseiflexus castenholzii
Methanobrevibacter olleyae
Methanobrevibacter
ruminantium
Methanobrevibacter wolinii
Methanosphaera stadtmanae
Halorubrum lipolyticum
Methanobacterium
formicicum
Acidilobus saccharovorans
Hyperthermus butylicus
Ignicoccus islandicus
Metallosphaera sedula
Thermofilum pendens
Prevotella melaninogenica
Prevotella sp. ICM1
Prevotella sp. oral clone
Prevotella sp. oral clone
Prevotella sp. SEQ116
Streptococcus anginosus
Streptococcus milleri
Streptococcus sp. 16362
Streptococcus sp. 69130
Streptococcus sp. AC15
Streptococcus sp. CM7
Streptococcus sp. OBRC6
Burkholderia ambifaria
Burkholderia cenocepacia
Burkholderia cepacia
Burkholderia mallei
Burkholderia multivorans
Burkholderia oklahomensis
Burkholderia pseudomallei
Burkholderia rhizoxinica
Burkholderia sp. 383
Burkholderia xenovorans
Prevotella buccae
Prevotella genomosp. P8
Prevotella sp. oral clone
Prevotella bivia
Prevotella disiens
Bacteroides faecis
Bacteroides fragilis
Bacteroides nordii
Bacteroides salyersiae
Bacteroides sp. 1_1_14
Bacteroides sp. 1_1_6
Bacteroides sp. 2_1_56FAA
Bacteroides sp. AR29
Bacteroides sp. B2
Bacteroides
thetaiotaomicron
Actinobacillus minor
Haemophilus parasuis
Vibrio cholerae
Vibrio fluvialis
Vibrio furnissii
Vibrio mimicus
Vibrio parahaemolyticus
Vibrio sp. RC341
Vibrio vulnificus
Lactobacillus acidophilus
Lactobacillus amylolyticus
Lactobacillus amylovorus
Lactobacillus crispatus
Lactobacillus delbrueckii
Lactobacillus helveticus
Lactobacillus kalixensis
Lactobacillus
kefiranofaciens
Lactobacillus leichmannii
Lactobacillus sp. 66c
Lactobacillus sp. KLDS
Lactobacillus sp. KLDS
Lactobacillus sp. oral clone
Lactobacillus ultunensis
Prevotella intermedia
Prevotella nigrescens
Prevotella pallens
Prevotella sp. oral taxon 310
Prevotella genomosp. C1
Prevotella sp. CM38
Prevotella sp. oral taxon 317
Prevotella sp. SG12
Prevotella denticola
Prevotella genomosp. P7
Prevotella histicola
Prevotella multiformis
Prevotella sp. JCM 6330
Prevotella sp. oral clone
Prevotella sp. oral taxon 782
Prevotella sp. oral taxon
Prevotella sp. SEQ065
Prevotella veroralis
Bacteroides acidifaciens
Bacteroides cellulosilyticus
Bacteroides clarus
Bacteroides eggerthii
Bacteroides oleiciplenus
Bacteroides pyogenes
Bacteroides sp. 315_5
Bacteroides sp. 31SF15
Bacteroides sp. 31SF18
Bacteroides sp. 35AE31
Bacteroides sp. 35AE37
Bacteroides sp. 35BE34
Bacteroides sp. 35BE35
Bacteroides sp. WH2
Bacteroides sp. XB12B
Bacteroides stercoris
Actinobacillus
pleuropneumoniae
Actinobacillus ureae
Haemophilus aegyptius
Haemophilus ducreyi
Haemophilus haemolyticus
Haemophilus influenzae
Haemophilus
parahaemolyticus
Haemophilus parainfluenzae
Haemophilus
paraphrophaemolyticus
Haemophilus somnus
Haemophilus sp. 70334
Haemophilus sp. HK445
Haemophilus sp. oral clone
Haemophilus sp. oral clone
Haemophilus sp. oral clone
Haemophilus sp. oral clone
Haemophilus sp. oral taxon
Haemophilus sputorum
Histophilus somni
Mannheimia haemolytica
Pasteurella bettyae
Moellerella wisconsensis
Morganella morganii
Morganella sp. JB_T16
Proteus mirabilis
Proteus penneri
Proteus sp. HS7514
Proteus vulgaris
Oribacterium sinus
Oribacterium sp. ACB1
Oribacterium sp. ACB7
Oribacterium sp. CM12
Oribacterium sp. ICM51
Oribacterium sp. OBRC12
Oribacterium sp. oral taxon
Actinobacillus
actinomycetemcomitans
Actinobacillus succinogenes
Aggregatibacter
actinomycetemcomitans
Aggregatibacter aphrophilus
Aggregatibacter segnis
Averyella dalhousiensis
Bisgaard Taxon
Bisgaard Taxon
Bisgaard Taxon
Bisgaard Taxon
Buchnera aphidicola
Cedecea davisae
Citrobacter amalonaticus
Citrobacter braakii
Citrobacter farmeri
Citrobacter freundii
Citrobacter gillenii
Citrobacter koseri
Citrobacter murliniae
Citrobacter rodentium
Citrobacter sedlakii
Citrobacter sp. 30_2
Citrobacter sp. KMSI_3
Citrobacter werkmanii
Citrobacter youngae
Cronobacter malonaticus
Cronobacter sakazakii
Cronobacter turicensis
Enterobacter aerogenes
Enterobacter asburiae
Enterobacter cancerogenus
Enterobacter cloacae
Enterobacter cowanii
Enterobacter hormaechei
Enterobacter sp. 247BMC
Enterobacter sp. 638
Enterobacter sp. JC163
Enterobacter sp. SCSS
Enterobacter sp. TSE38
Escherichia albertii
Escherichia coli
Escherichia fergusonii
Escherichia hermannii
Escherichia sp. 1_1_43
Escherichia sp. 4_1_40B
Escherichia sp. B4
Escherichia vulneris
Ewingella americana
Haemophilus genomosp. P2
Haemophilus genomosp. P3
Haemophilus sp. oral clone
Hafnia alvei
Klebsiella oxytoca
Klebsiella pneumoniae
Klebsiella sp. AS10
Klebsiella sp. Co9935
Klebsiella sp. enrichment
Klebsiella sp. OBRC7
Klebsiella sp. SP_BA
Klebsiella sp. SRC_DSD1
Klebsiella sp. SRC_DSD11
Klebsiella sp. SRC_DSD12
Klebsiella sp. SRC_DSD15
Klebsiella sp. SRC_DSD2
Klebsiella sp. SRC_DSD6
Klebsiella variicola
Kluyvera ascorbata
Kluyvera cryocrescens
Leminorella grimontii
Leminorella richardii
Pantoea agglomerans
Pantoea ananatis
Pantoea brenneri
Pantoea citrea
Pantoea conspicua
Pantoea septica
Pasteurella dagmatis
Pasteurella multocida
Plesiomonas shigelloides
Raoultella ornithinolytica
Raoultella planticola
Raoultella terrigena
Salmonella bongori
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella typhimurium
Salmonella typhimurium
Serratia fonticola
Serratia liquefaciens
Serratia marcescens
Serratia odorifera
Serratia proteamaculans
Shigella boydii
Shigella dysenteriae
Shigella flexneri
Shigella sonnei
Tatumella ptyseos
Trabulsiella guamensis
Yersinia aldovae
Yersinia aleksiciae
Yersinia bercovieri
Yersinia enterocolitica
Yersinia frederiksenii
Yersinia intermedia
Yersinia kristensenii
Yersinia mollaretii
Yersinia pestis
Yersinia pseudotuberculosis
Yersinia rohdei
Yokenella regensburgei
Conchiformibius kuhniae
Morococcus cerebrosus
Neisseria bacilliformis
Neisseria cinerea
Neisseria flavescens
Neisseria gonorrhoeae
Neisseria lactamica
Neisseria macacae
Neisseria meningitidis
Neisseria mucosa
Neisseria pharyngis
Neisseria polysaccharea
Neisseria sicca
Neisseria sp. KEM232
Neisseria sp. oral clone
Neisseria sp. oral strain
Neisseria sp. oral taxon 014
Neisseria sp. TM10_1
Neisseria subflava
Okadaella gastrococcus
Streptococcus agalactiae
Streptococcus alactolyticus
Streptococcus australis
Streptococcus bovis
Streptococcus canis
Streptococcus constellatus
Streptococcus cristatus
Streptococcus dysgalactiae
Streptococcus equi
Streptococcus equinus
Streptococcus gallolyticus
Streptococcus genomosp.
Streptococcus genomosp.
Streptococcus genomosp.
Streptococcus genomosp.
Streptococcus genomosp.
Streptococcus genomosp.
Streptococcus genomosp.
Streptococcus genomosp.
Streptococcus gordonii
Streptococcus infantarius
Streptococcus infantis
Streptococcus intermedius
Streptococcus lutetiensis
Streptococcus massiliensis
Streptococcus mitis
Streptococcus
oligofermentans
Streptococcus oralis
Streptococcus parasanguinis
Streptococcus pasteurianus
Streptococcus peroris
Streptococcus pneumoniae
Streptococcus porcinus
Streptococcus
pseudopneumoniae
Streptococcus
pseudoporcinus
Streptococcus pyogenes
Streptococcus ratti
Streptococcus sanguinis
Streptococcus sinensis
Streptococcus sp.
Streptococcus sp. 2285_97
Streptococcus sp. ACS2
Streptococcus sp. AS20
Streptococcus sp. BS35a
Streptococcus sp. C150
Streptococcus sp. CM6
Streptococcus sp. ICM10
Streptococcus sp. ICM12
Streptococcus sp. ICM2
Streptococcus sp. ICM4
Streptococcus sp. ICM45
Streptococcus sp. M143
Streptococcus sp. M334
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral taxon
Streptococcus sp. oral taxon
Streptococcus sp. oral taxon
Streptococcus sp. oral taxon
Streptococcus suis
Streptococcus thermophilus
Streptococcus salivarius
Streptococcus uberis
Streptococcus urinalis
Streptococcus vestibularis
Streptococcus viridans
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
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
Blautia uncultured bacterium clone SJTU_B_14_30
Blautia uncultured bacterium clone SJTU_C_14_16
Blautia uncultured bacterium clone S1-5 (GenBank: GQ898099.1)
Blautia uncultured PAC000178_s
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
Anaerotruncus colihominis strain 13
Blautia producta strain 6
Clostridium bolteae strain 7
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
Oscillibacter valericigenes
Ruminococcus sp. ID8 strain 14
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
Escherichia coli
Escherichia coli S88
Eubacterium eligens
Eubacterium rectale
Faecalibacterium prausnitzii
Odoribacter splanchnicus
Parabacteroides merdae
Roseburia intestinalis
Ruminococcus torques
Streptococcus thermophilus
Akkermansia muciniphila
Enterococcus faecalis
Klebsiella oxytoca
Lactobacillus rhamnosus
Staphylococcus epidermidis
Streptococcus viridans
Veillonella dispar
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
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
Escherichia coli
Escherichia coli S88
Eubacterium eligens
Eubacterium fissicatena
Eubacterium rectale
Faecalibacterium prausnitzii
Fusobacterium mortiferum
Fusobacterium nucleatum
Hydrogenoanaerobacterium saccharovorans
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
Blautia producta
Clostridium bartlettii
Clostridium bolteae
Clostridium botulinum
Clostridium butyricum
Clostridium celatum
Clostridium clostridioforme
Clostridium disporicum
Clostridium glycolicum
Clostridium mayombei
Clostridium paraputrificum
Clostridium sordellii
Clostridium sp. 7_2_43FAA
Clostridium symbiosum
Clostridium tertium
Clostridium paraputrificum
Clostridium disporicum
Clostridium glycolicum
Clostridium bartlettii
Clostridium butyricum
Ruminococcus bromii
Eubacterium hadrum
Turicibacter sanguinis
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
Eubacterium rectale
Clostridium viride
Ruminococcus sp. K_1
Clostridium symbiosum
Ruminococcus torques
Clostridium algidicarnis
Clostridium paraputrificum
Clostridium bartlettii
Clostridium disporicum
Ruminococcus bromii
Eubacterium hadrum
Clostridium butyricum
Roseburia sp. 11SE37
Clostridium perfringens
Clostridium glycolicum
Clostridium hylemonae
Clostridium orbiscindens
Ruminococcus lactaris
Clostridium symbiosum
Blautia hansenii
Turicibacter sanguinis
Clostridium straminisolvens
Clostridium botulinum
Roseburia cecicola
Ruminococcus sp. K_1
Clostridium bifermentans
Eubacterium rectale
Clostridium quinii
Clostridium viride
Clostridium kluyveri
Clostridium tyrobutyricum
Oscillibacter sp. G2
Clostridium sp. JC122
Clostridium aldenense
Ruminococcus torques
Clostridium sp. 7_2_43FAA
Clostridium celatum
Eubacterium sp. WAL_14571
Eubacterium tenue
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
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)
B. coccoides (EPV22)
B. hydrogenotrophica (EPV23)
Eubacterium rectale (EPV35)
B. hansenii (EPV24)
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.
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.
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| 20160143962 A1 | May 2016 | US |
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| 62084536 | Nov 2014 | US | |
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| 62084540 | Nov 2014 | US |
