# Patent

## Title
Compositions and methods

## Abstract

Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 14/884,655 filed Oct. 15, 2015, which is a continuation of U.S. application Ser. No. 14/313,828 filed Jun. 24, 2014, now U.S. Pat. No. 9,180,147, issued Nov. 10, 2015, which is a divisional of U.S. application Ser. No. 14/197,044, filed Mar. 4, 2014, now U.S. Pat. No. 9,011,834, issued Apr. 21, 2015, which is a continuation of International Application No. PCT/US2014/014745, filed Feb. 4, 2014, which claims priority to U.S. Provisional Application No. 61/760,584, filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,585, filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,574, filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,606, filed Feb. 4, 2013, and U.S. Provisional Application No. 61/926,918, filed Jan. 13, 2014. These applications are all incorporated by reference in their entirety for all purposes.


REFERENCE TO A SEQUENCE LISTING
This application includes a Sequence Listing with 2043 sequences submitted electronically as a text file named 30735_US_CRF_Sequence_Listing.txt, created on Jan. 28, 2016, with a size of 4,325,376 bytes. The sequence listing is incorporated by reference.
BACKGROUND
Mammals are colonized by microbes in the gastrointestinal (GI) tract, on the skin, and in other epithelial and tissue niches such as the oral cavity, eye surface and vagina. The gastrointestinal tract harbors an abundant and diverse microbial community. It is a complex system, providing an environment or niche for a community of many different species or organisms, including diverse strains of bacteria. Hundreds of different species may form a commensal community in the GI tract in a healthy person, and this complement of organisms evolves from the time of birth to ultimately form a functionally mature microbial population by about 3 years of age. Interactions between microbial strains in these populations and between microbes and the host, e.g. the host immune system, shape the community structure, with availability of and competition for resources affecting the distribution of microbes. Such resources may be food, location and the availability of space to grow or a physical structure to which the microbe may attach. For example, host diet is involved in shaping the GI tract flora.
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 of the gut. For instance, subjects become more susceptible to pathogenic infections when the normal intestinal microbiota has been disturbed due to use of broad-spectrum antibiotics. Many of these diseases and disorders are chronic conditions that significantly decrease a subject's quality of life and can be ultimately fatal.
Manufacturers of probiotics have asserted that their preparations of bacteria promote mammalian health by preserving the natural microflora in the GI tract and reinforcing the normal controls on aberrant immune responses. See, e.g., U.S. Pat. No. 8,034,601. Probiotics, however, have been limited to a very narrow group of genera and a correspondingly limited number of species; as such, they do not adequately replace the missing natural microflora of the GI tract in many situations.
Thus practitioners have a need for a method of populating a subject's gastrointestinal tract with a diverse and useful selection of microbiota in order to alter a dysbiosis.
Therefore, in response to the need for durable, efficient, and effective compositions and methods for treatment of GI diseases by way of restoring or enhancing microbiota functions, we address these and other shortcomings of the prior art by providing compositions and methods for treating subjects.
SUMMARY OF THE INVENTION
Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A provides a schematic of 16S rRNA gene and denotes the coordinates of hypervariable regions 1-9 (V1-V9). Coordinates of V1-V9 are 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294, and 1435-1465 respectively, based on numbering using E. coli system of nomenclature defined by Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene (16S rRNA) from Escherichia coli, PNAS 75(10):4801-4805 (1978). FIG. 1B highlights in bold the nucleotide sequences for each hypervariable region in the exemplary reference E. coli 16S sequence described by Brosius et al. FIG. 1B discloses SEQ ID NO: 2043.
FIG. 2 shows a photograph of a CsCl gradient demonstrating the spore separation from other residual habitat material.
FIG. 3 shows three phase contrast image demonstrating the progressive enrichment of spores from a fecal suspension; ethanol treated, CsCl purified spore preparation; and an ethanol treated, CsCl purified, sucrose purified spore preparation.
FIG. 4 shows a set of survival curves demonstrating efficacy of the spore population in a mouse prophylaxis model of C. difficile. 
FIG. 5 provides a set of survival curves demonstrating efficacy of the spore population in a hamster relapse prevention model of C. difficile. 
FIG. 6 demonstrates the cell viability under a variety of ethanol and heat treatments for varying lengths of time.
FIG. 7 demonstrates cell survivability from four donor fecal samples after heat treatment at 60 C for 5 minutes.
FIG. 8 demonstrates that ethanol reduces both anaerobic and aerobic bacterial species by several orders of magnitude in seconds.
FIG. 9 demonstrates the spore concentration of fecal donations from multiple donors over time.
FIG. 10 shows the strong correlation and linear correspondence between the measurement of DPA concentration by a coupled fluorescence assay and the viable spore colony forming units
FIG. 11 demonstrates the effect on various germination treatments on the ability to cultivate vegetative bacteria from a spore population.
FIG. 12 demonstrates the increase in bacterial diversity from using a germinant treatment to grow vegetative bacteria from spore populations.
FIG. 13 demonstrates the role of heat activation at various temperatures on spores from three different donor fecal samples.
FIG. 14 demonstrates a lysozyme treatment with heat activation improves germination at most temperatures.
FIG. 15 demonstrates spore concentrations present in a fecal sample grown on various medias.
FIG. 16 demonstrates similar spore production from incubating plates for 2 and 7 days after a spore population was germinated on plates with various medias.
FIG. 17 demonstrates the protective efficacy of the spore population in mice challenged with C. difficile as measured by the change in weight of mice over the course of the experiment. Each plot tracks the change in the individual mouse's weight relative to day −1 over the course of the experiment. The number of deaths over the course of the experiment is indicated at the top of the chart and demonstrated by a line termination prior to day 6. The top panels (from left to right) are the vehicle control arm, the fecal suspension arm, and the untreated naive control arm, while the bottom panels are the ethanol treated, gradient purified spore preparation; the ethanol treated, gradient purified, “germinable” spore preparation, and ethanol treated, gradient purified, “sporulatable” preparation.
FIG. 18 demonstrates the microbial diversity measured in the ethanol treated spore treatment sample and patient pre- and post-treatment samples. Total microbial diversity is defined using the Chao1 Alpha-Diversity Index and is measured at the same genomic sampling depths to confirm adequate sequence coverage to assay the microbiome in the target samples. The patient pretreatment (purple) harbored a microbiome that was significantly reduced in total diversity as compared to the ethanol treated spore treatment (red) and patient post treatment at days 5 (blue), 14 (orange), and 25 (green).
FIG. 19 demonstrates how the patient microbial ecology is shifted by treatment with an ethanol treated spore treatment from a dysbiotic state to a state of health. Principle coordinates analysis based on the total diversity and structure of the microbiome (Bray Curtis Beta Diversity) of the patient pre- and post-treatment delineates that the combination of engraftment of the OTUs from the spore treatment and the augmentation of the patient microbial ecology leads to a microbial ecology that is distinct from both the pretreatment microbiome and the ecology of the ethanol treated spore treatment.
FIG. 20 demonstrates the augmentation of bacteroides species in patients treated with the spore population. Comparing the number of Bacteroides colonies from fecal suspensions pre-treatment and in week 4 post treatment reveals an increase of 4 logs or greater. Colonies were enumerated by serial dilution and plating on Bacteroides Bile Esculin agar which is highly selective for the B. fragilis group. Species were determined by 16S full-length sequence identification.
FIG. 21 demonstrates the increase in number of species engrafting and species augmenting in patient's microbiomes after treatment with an ethanol-treated spore population. Relative abundance of species that engrafted or augmented as described were determined based on the number of 16S sequence reads. Each plot is from a different patient treated with the ethanol-treated spore population for recurrent C. difficile. 
The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.



DESCRIPTION OF THE TABLES
Table 1. 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.
Table 2 contains bacterial OTUs identified from the 16s analysis of the ethanol treated spore population before and after a CsCl gradient purification.
Table 3 contains the mortality and weight change of mice treated with a donor fecal suspension and an ethanol and/or heat-treated spore preparation at various dilutions,
Table 4 contains OTUs identified from spore forming species generated by picking colonies from a spore preparation involving various heat treatments
Table 5 contains OTUs not identified in untreated fecal slurries, but identified in ethanol treated or heat treated spore populations.
Table 6 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor A.
Table 7 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor B.
Table 8 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor C.
Table 9 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor D.
Table 10 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor E.
Table 11 contains OTUs identified from an ethanol treated spore population isolated from a microbiome sample from donor F.
Table 12 contains OTUs identified from growing ethanol treated spore populations on various media types.
Table 13. Species identified as “germinable” and “sporulatable” by colony picking approach
Table YYY. Species identified as “germinable” using 16S-V4 NGS approach.
Table ZZZ. Species identified as “sporulatable” using 16s-V4 NGS approach.
Table AC shows spore content data from 3 different ethanol treated spore preparations used to successfully treat 3 patients suffering from recurrent C. difficile infection.
Table AD. DPA doses in Table AC when normalized to 4×105 SCFU per dose
Table GB. OTUs detected by a minimum of ten 16S-V4 sequence reads in at least a one ethanol treated spore preparation (pan-microbiome). OTUs that engraft in a treated patients and the percentage of patients in which they engraft are denoted, as are the clades, spore forming status, and Keystone OTU status. Starred OTUs occur in ≧80% of the ethanol preps and engraft in ≧50% of the treated patients.
Table GC ranks the top 20 OTUs by CES with the further requirement that an OTU must be shown to engraft to be a considered an element of a core ecology.
Table GD: Subsets of the Core Ecology tested in the C. difficile mouse model
Table GE: Results of bacterial compositions tested in a C. difficile mouse model.
Table GF. OTUs and their clade assignments tested in ternary combinations with results in the in vitro inhibition assay
Table ZA. Microbial compositions administered via oral gavage on Day −1
Table TAB. Population of OTUs on Days 2, 3 and 4 following dosing with Microbial Compositions
Table TAC. Population of clades on Days 2, 3 and 4 following dosing with Microbial Compositions
Table TAD. Mortality by experimental group in mice challenged with 104.5 C. difficile spores on Day 0
DETAILED DESCRIPTION
Overview
Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health. While spore-based compositions are known, these are generally prepared according to various techniques such as lyophilization or spray-drying of liquid bacterial cultures, resulting in poor efficacy, instability, substantial variability and lack of adequate safety and efficacy.
It has now been found that populations of bacterial spores can be obtained from biological materials obtained from mammalian subjects, including humans. These populations are formulated into compositions as provided herein, and administered to mammalian subjects using the methods as provided herein.
Definitions
“Microbiota” refers to the community of microorganisms that occur (sustainably or transiently) in and on an animal subject, typically a mammal such as a human, including eukaryotes, archaea, bacteria, and viruses (including bacterial viruses i.e., 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 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 humans. 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 was 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” or simply “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 and microbiological techniques) from the administered therapeutic microbial composition, (ii) absent, undetectable, or present at low frequencies in the host niche (as example: gastrointestinal tract, skin, anterior-nares, or vagina) before the delivery of the microbial composition, and (iii) are found 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 were 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 microbial composition induces an environmental shift in the target niche that promotes favorable conditions for the growth of these commensal microbes. In the absence of treatment with a therapeutic microbial composition, 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 that 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 health state.
“Ecological Niche” or simply “Niche” refers to the ecological space in which a an organism or group of organisms 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 when predators, parasites and pathogens are scarce) and how it in turn alters those same factors (e.g., limiting access to resources by other organisms, acting as a food source for predators and a consumer of prey).
“Dysbiosis” refers to a state of the microbiota of the gut or other body area in a subject, including mucosal or skin surfaces in which the normal diversity and/or function of the ecological network is disrupted. This unhealthy state can be due to a decrease in diversity, the overgrowth of one or more pathogens or pathobionts, symbiotic organisms able to cause disease only when certain genetic and/or environmental conditions are present in a subject, or the shift to an ecological microbial network that no longer provides an essential function to the host subject, and therefore no longer promotes health.
“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.
“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 M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. 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 M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.). 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.
“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, or 95% 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 (including bacterial viruses (i.e., phage)), fungal, 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. Thus, 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, 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.
“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 that is a distinct monophyletic evolutionary unit and that share some extent of sequence similarity.
In microbiology, “16S sequencing” or “16S-rRNA” or “16S” refers to sequence derived by characterizing the nucleotides that comprise the 16S ribosomal RNA gene(s). The bacterial 16S rDNA is approximately 1500 nucleotides in length and 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. Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978). 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.
The term “subject” refers to any animal subject including humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), and household pets (e.g., dogs, cats, and rodents). 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.
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.
The term “Network Ecology” refers to a consortium of 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. OTUs) and edges (connections between specific OTUs) relate to one another to define the structural ecology of a consortium of OTUs. Any given Network Ecology will possess inherent phylogenetic diversity and functional properties. A Network Ecology can also be defined in terms of function 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 pathways (www.genome.jp/kegg/).
The terms “Network Class”, “Core Network” and “Core Network 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 Core Network 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.
The term “Keystone OTU” refers to one or more OTUs that are common to many network ecologies and are members of networks ecologies that occur in many subjects (i.e. are pervasive) (FIG. 1). Due to the ubiquitous nature of Keystone OTUs, 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 may exist in low, moderate, or high abundance in subjects.
The term “non-Keystone OTU” refers to an OTU that is observed in a Network Ecology and is not a keystone OTU.
The term “Phylogenetic Diversity” refers to the biodiversity present in a given Network Ecology or Core Network Ecology based on the OTUs that comprise the network. Phylogenetic diversity is a relative term, meaning that a Network Ecology or Core Network 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.
“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.
In one embodiment, the spore preparation comprises spore forming species wherein residual non-spore forming species have been inactivated by chemical or physical treatments including ethanol, detergent, heat, sonication, and the like; or wherein the non-spore forming species have been removed from the spore preparation by various separations steps including density gradients, centrifugation, filtration and/or chromatography; or wherein inactivation and separation methods are combined to make the spore preparation. In yet another embodiment, the spore preparation comprises spore forming species that are enriched over viable non-spore formers or vegetative forms of spore formers. In this embodiment, spores are enriched by 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold or greater than 10,000-fold compared to all vegetative forms of bacteria. In yet another embodiment, the spores in the spore preparation undergo partial germination during processing and formulation such that the final composition comprises spores and vegetative bacteria derived from spore forming species.
A “germinant” is a material or composition or physical-chemical process capable of inducing vegetative growth of a bacterium that is in a dormant spore form, or group of bacteria in the spore form, either directly or indirectly in a host organism and/or in vitro.
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 spores” 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).
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 any other microbiotal niche) by a pathogenic bacterium includes a reduction in the residence time of the pathogen in the gastrointestinal tract as well as a reduction in the number (or concentration) of the pathogen in the gastrointestinal tract or adhered to the luminal surface of the gastrointestinal tract. Measuring reductions of adherent pathogens may be demonstrated, e.g., by 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.
A “cytotoxic” activity or bacterium includes the ability to kill a bacterial cell, such as a pathogenic bacterial cell. A “cytostatic” activity or bacterium includes the ability to inhibit, partially or fully, growth, metabolism, and/or proliferation of a bacterial cell, such as a pathogenic bacterial cell.
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.
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, analogs.
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.
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.
Compositions of the Invention
Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health. While spore-based compositions are known, these are generally prepared according to various techniques such as lyophilization or spray-drying of liquid bacterial cultures, resulting in poor efficacy, instability, substantial variability and lack of adequate safety and efficacy.
It has now been found that populations of bacterial spores can be obtained from biological materials obtained from mammalian subjects, including humans. These populations are formulated into compositions as provided herein, and administered to mammalian subjects using the methods as provided herein.
Provided herein are therapeutic compositions containing a purified population of bacterial spores. 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 spores, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired bacterial spore, 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 spores at or above an acceptable amount and/or concentration. In other embodiments, the ratio of desired-to-undesired activity (e.g. spores compared to vegetative bacteria), has changed by 2-, 5-, 10-, 30-, 100-, 300-, 1×104, 1×105, 1×106, 1×107, 1×108, or greater than 1×108. In other embodiments, the purified population of bacterial spores 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%, 90%, 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 spores 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%, 90%, 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 spores have reduced sensory components as compared to fecal material, such as reduced odor, taste, appearance, and umami.
Provided are purified populations of bacterial spores that are substantially free of residual habitat products. In certain embodiments, this means that the bacterial spore 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 spores may be 100% free, 99% free, 98% free, 97% free, 96% free, or 95% 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 spore 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%, 1×10−4%, 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%, 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 spore populations are substantially free of an acellular material, e.g., DNA, viral coat material, or non-viable bacterial material. Alternatively, the purified spore population 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 spore populations can be demonstrated by genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, microscopic analysis, microbial analysis including germination and culturing, and methods using instrumentation such as flow cytometry with reagents that distinguish desired bacterial spores from non-desired, contaminating materials.
Exemplary biological materials include fecal materials such as feces or materials isolated from the various segments of the small and large intestines. Fecal materials are obtained from a mammalian donor subject, or can be obtained from more than one donor subject, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or from greater than 1000 donors, where such materials are then pooled prior to purification of the desired bacterial spores. In another embodiment, fecal materials can be obtained from a single donor subject over multiple times and pooled from multiple samples e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 32, 35, 40, 45, 48, 50, 100 samples from a single donor.
In alternative embodiments, the desired bacterial spores are purified from a single fecal material sample obtained from a single donor, and after such purification are combined with purified spore populations from other purifications, either from the same donor at a different time, or from one or more different donors, or both.
Mammalian donor subjects are generally of good health and have microbiota consistent with such good health. Often, the donor subjects have not been administered antibiotic compounds within a certain period prior to the collection of the fecal material. In certain embodiments, the donor subjects are not obese or overweight, and may have body mass index (BMI) scores of below 25, such as between 18.5 and 24.9. In other embodiments, the donor subjects are not mentally ill or have no history or familial history of mental illness, such as anxiety disorder, depression, bipolar disorder, autism spectrum disorders, schizophrenia, panic disorders, attention deficit (hyperactivity) disorders, eating disorders or mood disorders. In other embodiments, the donor subjects do not have irritable bowel disease (e.g., crohn's disease, ulcerative colitis), irritable bowel syndrome, celiac disease, colorectal cancer or a family history of these diseases. In other embodiments, donors have been screened for blood borne pathogens and fecal transmissible pathogens using standard techniques known to one in the art (e.g. nucleic acid testing, serological testing, antigen testing, culturing techniques, enzymatic assays, assays of cell free fecal filtrates looking for toxins on susceptible cell culture substrates).
In some embodiments, donors are also selected for the presence of certain genera and/or species that provide increased efficacy of therapeutic compositions containing these genera or species. In other embodiments, donors are preferred that produce relatively higher concentrations of spores in fecal material than other donors. In further embodiments, donors are preferred that provide fecal material from which spores having increased efficacy are purified; this increased efficacy is measured using in vitro or in animal studies as described below. In some embodiments, the donor may be subjected to one or more pre-donation treatments in order to reduce undesired material in the fecal material, and/or increase desired spore populations.
It is advantageous to screen the health of the donor subject prior to and optionally, one or more times after, the collection of the fecal material. Such screening identifies donors carrying pathogenic materials such as viruses (HIV, hepatitis, polio) and pathogenic bacteria. Post-collection, donors are screened about one week, two weeks, three weeks, one month, two months, three months, six months, one year or more than one year, and the frequency of such screening may be daily, weekly, bi-weekly, monthly, bi-monthly, semi-yearly or yearly. Donors that are screened and do not test positive, either before or after donation or both, are considered “validated” donors.
Solvent Treatments. To purify the bacterial spores, the fecal material is subjected to one or more solvent treatments. A solvent treatment is a miscible solvent treatment (either partially miscible or fully miscible) or an immiscible solvent treatment. Miscibility is the ability of two liquids to mix with each to form a homogeneous solution. Water and ethanol, for example, are fully miscible such that a mixture containing water and ethanol in any ratio will show only one phase. Miscibility is provided as a wt/wt %, or weight of one solvent in 100 g of final solution. If two solvents are fully miscible in all proportions, their miscibility is 100%. Provided as fully miscible solutions with water are alcohols, e.g., methanol, ethanol, isopropanol, butanol, propanediol, butanediol, etc. The alcohols can be provided already combined with water; e.g., a solution containing 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 85%, 90%, 95% or greater than 95%. Other solvents are only partially miscible, meaning that only some portion will dissolve in water. Diethyl ether, for example, is partially miscible with water. Up to 7 grams of diethyl ether will dissolve in 93 g of water to give a 7% (wt/wt %) solution. If more diethyl ether is added, a two-phase solution will result with a distinct diethyl ether layer above the water. Other partially miscible materials include ethers, propanoate, butanoate, chloroform, dimethoxyethane, or tetrahydrofuran. In contrast, an oil such as an alkane and water are immiscible and form two phases. Further, immiscible treatments are optionally combined with a detergent, either an ionic detergent or a non-ionic detergent. Exemplary detergents include Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, or a polyol. The solvent treatment steps reduces the viability of non-spore forming bacterial species by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, and it may optionally reduce the viability of contaminating protists, parasites and/or viruses.
Chromatography Treatments. To purify spore populations, the fecal materials are subjected to one or more chromatographic treatments, either sequentially or in parallel. In a chromatographic treatment, a solution containing the fecal material is contacted with a solid medium containing a hydrophobic interaction chromatographic (HIC) medium or an affinity chromatographic medium. In an alternative embodiment, a solid medium capable of absorbing a residual habitat product present in the fecal material is contacted with a solid medium that adsorbs a residual habitat product. In certain embodiments, the HIC medium contains sepharose or a derivatized sepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, or butyl-s sepharose. In other embodiments, the affinity chromatographic medium contains material derivatized with mucin type I, II, III, IV, V, or VI, or oligosaccharides derived from or similar to those of mucins type I, II, III, IV, V, or VI. Alternatively, the affinity chromatographic medium contains material derivatized with antibodies that recognize spore-forming bacteria.
Mechanical Treatments. Provided herein is the physical disruption of the fecal material, particularly by one or more mechanical treatment such as blending, mixing, shaking, vortexing, impact pulverization, and sonication. As provided herein, the mechanical disrupting treatment substantially disrupts a non-spore material present in the fecal material and does not substantially disrupt a spore present in the fecal material, or it may disrupt the spore material less than the non-spore material, e.g. 2-fold less, 5-, 10-, 30-, 100-, 300-, 1000- or greater than 1000-fold less. Furthermore, mechanical treatment homogenizes the material for subsequent sampling, testing, and processing. Mechanical treatments optionally include filtration treatments, where the desired spore populations are retained on a filter while the undesirable (non-spore) fecal components to pass through, and the spore fraction is then recovered from the filter medium. Alternatively, undesirable particulates and eukaryotic cells may be retained on a filter while bacterial cells including spores pass through. In some embodiments the spore fraction retained on the filter medium is subjected to a diafiltration step, wherein the retained spores are contacted with a wash liquid, typically a sterile saline-containing solution or other diluent such as a water compatible polymer including a low-molecular polyethylene glycol (PEG) solution, in order to further reduce or remove the undesirable fecal components.
Thermal Treatments. Provided herein is the thermal disruption of the fecal material. Generally, the fecal material is mixed in a saline-containing solution such as phosphate-buffered saline (PBS) and subjected to a heated environment, such as a warm room, incubator, water-bath, or the like, such that efficient heat transfer occurs between the heated environment and the fecal material. Preferably the fecal material solution is mixed during the incubation to enhance thermal conductivity and disrupt particulate aggregates. Thermal treatments can be modulated by the temperature of the environment and/or the duration of the thermal treatment. For example, the fecal material or a liquid comprising the fecal material is subjected to a heated environment, e.g., a hot water bath of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or greater than 100 degrees Celsius, for at least about 1, 5, 10, 15, 20, 30, 45 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 hours. In certain embodiments the thermal treatment occurs at two different temperatures, such as 30 seconds in a 100 degree Celsius environment followed by 10 minutes in a 50 degree Celsius environment. In preferred embodiments the temperature and duration of the thermal treatment are sufficient to kill or remove pathogenic materials while not substantially damaging or reducing the germination-competency of the spores. In other preferred embodiments, the temperature and duration of the thermal treatment is short enough to reduce the germination of the spore population.
Irradiation Treatments. Provided are methods of treating the fecal material or separated contents of the fecal material with ionizing radiation, typically gamma irradiation, ultraviolet irradiation or electron beam irradiation provided at an energy level sufficient to kill pathogenic materials while not substantially damaging the desired spore populations. For example, ultraviolet radiation at 254 nm provided at an energy level below about 22,000 microwatt seconds per cm2 will not generally destroy desired spores.
Centrifugation and Density Separation Treatments. Provided are methods of separating desired spore populations from the other components of the fecal material by centrifugation. A solution containing the fecal material is subjected to one or more centrifugation treatments, e.g., at about 200×g, 1000×g, 2000×g, 3000×g, 4000×g, 5000×g, 6000×g, 7000×g, 8000×g or greater than 8000×g. Differential centrifugation separates desired spores from undesired non-spore material; at low forces the spores are retained in solution, while at higher forces the spores are pelleted while smaller impurities (e.g., virus particles, phage, microscopic fibers, biological macromolecules such as free protein, nucleic acids and lipids) are retained in solution. For example, a first low force centrifugation pellets fibrous materials; a second, higher force centrifugation pellets undesired eukaryotic cells, and a third, still higher force centrifugation pellets the desired spores while smaller contaminants remain in suspension. In some embodiments density or mobility gradients or cushions (e.g., step cushions), such as CsCl, Percoll, Ficoll, Nycodenz, Histodenz or sucrose gradients, are used to separate desired spore populations from other materials in the fecal material.
Also provided herein are methods of producing spore populations that combine two or more of the treatments described herein in order to synergistically purify the desired spores while killing or removing undesired materials and/or activities from the spore population. It is generally desirable to retain the spore populations under non-germinating and non-growth promoting conditions and media, in order to minimize the growth of pathogenic bacteria present in the spore populations and to minimize the germination of spores into vegetative bacterial cells.
Purified Spore Populations. As described herein, purified spore populations contain combinations of commensal bacteria of the human gut microbiota with the capacity to meaningfully provide functions of a healthy microbiota when administered to a mammalian subject. Without being limited to a specific mechanism, it is thought that such compositions inhibit the growth of a pathogen such as C. difficile, Salmonella spp., enteropathogenic E. coli, Fusobacterium spp., Klebsiella spp. and vancomycin-resistant Enterococcus spp., so that a healthy, diverse and protective microbiota can be maintained or, in the case of pathogenic bacterial infections such as C. difficile infection, repopulate the intestinal lumen to reestablish ecological control over potential pathogens. 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 and 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 then present initially.
Preferred bacterial genera include 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, Turicibacter. 
Preferred bacterial species are provided at Table 1 and demarcated as spore formers. 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 some embodiments, spore-forming bacteria are identified by the presence of nucleic acid sequences that modulate sporulation. In particular, signature sporulation genes are highly conserved across members of distantly related genera including Clostridium and Bacillus. Traditional approaches of forward genetics have identified many, if not all, genes that are essential for sporulation (spo). The developmental program of sporulation is governed in part by the successive action of four compartment-specific sigma factors (appearing in the order σF, σE, σG and σK), whose activities are confined to the forespore (σF and σG) or the mother cell (σE and σK). In other embodiments, spore-forming bacteria are identified by the biochemical activity of DPA producing enzymes or by analyzing DPA content of cultures. As part of the bacterial sporulation, large amounts of DPA are produced, and comprise 5-15% of the mass of a spore. Because not all viable spores germinate and grow under known media conditions, it is difficult to assess a total spore count in a population of bacteria. As such, a measurement of DPA content highly correlates with spore content and is an appropriate measure for characterizing total spore content in a bacterial population.
[Provided are spore populations containing more than one type of bacterium. 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 embodiments all or essentially all of the bacterial spores present in a purified population are obtained from a fecal material treated as described herein or otherwise known in the art. In alternative embodiments, one or more than one bacterial spores or types of bacterial spores are generated in culture and combined to form a purified spore population. In other alternative embodiments, one or more of these culture-generated spore populations are combined with a fecal material-derived spore population to generate a hybrid spore population. Bacterial compositions may contain at least two types of these preferred bacteria, including strains of the same species. For instance, a bacterial composition may comprise at least 2, at least 3, at least 4, 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 or more than 20 types of bacteria, as defined by species or operational taxonomic unit (OTU) encompassing such species.
Thus, provided herein are methods for production of a composition containing a population of bacterial spores suitable for therapeutic administration to a mammalian subject in need thereof. And the composition is 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 spores is produced from the fecal material. The composition is formulated such that a single oral dose contains at least about 1×104 colony forming units of the bacterial spores, and a single oral dose will typically contain about 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 CFUs of the bacterial spores. The presence and/or concentration of a given type of bacterial spore may be known or unknown in a given purified spore population. If known, for example the concentration of spores of a given strain, or the aggregate of all strains, is e.g., 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 viable bacterial spores per gram of composition or per administered dose.
In some formulations, the 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 bacterial spore compositions 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 bacterial spore compositions may be formulated with a germinant to enhance engraftment, or efficacy. In yet other embodiments, the bacterial spore compositions may be co-formulated or co-administered with prebiotic substances, to enhance engraftment or efficacy.
The bacterial spore compositions may be formulated to be effective in a given mammalian subject in a single administration or over multiple administrations. For example, a single administration is substantially effective to reduce Cl. difficile and/or Cl. difficile toxin content in a mammalian subject to whom the composition is administered. Substantially effective means that Cl. difficile and/or Cl. difficile toxin content in the subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99% following administration of the composition. Alternatively, efficacy may be measured by the absence of diarrheal symptoms or the absence of carriage of C. difficile or C. difficile toxin after 2 day, 4 days, 1 week, 2 weeks, 4 weeks, 8 weeks or longer than 8 weeks.
Bacterial Compositions
Provided are bacteria and combinations of bacteria of the human gut microbiota with the capacity to meaningfully provide functions of a healthy microbiota when administered to mammalian hosts. Without being limited to a specific mechanism, it is thought that such compositions inhibit the growth, proliferation, and/or colonization of one or a plurality of pathogenic bacteria in the dysbiotic microbiotal niche, so that a healthy, diverse and protective microbiota colonizes and populates the intestinal lumen to establish or reestablish ecological control over pathogens or potential pathogens (e.g., some bacteria are pathogenic bacteria only when present in a dysbiotic environment). Inhibition of pathogens includes those pathogens such as C. difficile, Salmonella spp., enteropathogenic E coli, multi-drug resistant bacteria such as Klebsiella, and E. coli, Carbapenem-resistent Enterobacteriaceae (CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), and vancomycin-resistant Enterococci (VRE).
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.
Bacterial compositions may comprise two types of bacteria (termed “binary combinations” or “binary pairs”) or greater than two types of bacteria. For instance, a bacterial composition may comprise at least 2, at least 3, at least 4, 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, at least 20, or at least 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or at least 40, at least 50 or greater than 50 types of bacteria, as defined by species or operational taxonomic unit (OTU), or otherwise as provided herein.
In another embodiment, the number of types of bacteria present in a bacterial composition is at or below a known value. For example, in such embodiments the bacterial composition comprises 50 or fewer types of bacteria, such as 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer, or 9 or fewer types of bacteria, 8 or fewer types of bacteria, 7 or fewer types of bacteria, 6 or fewer types of bacteria, 5 or fewer types of bacteria, 4 or fewer types of bacteria, or 3 or fewer types of bacteria. In another embodiment, a bacterial 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, or from 2 to no more than 5 types of bacteria.
Bacterial Compositions Described by Species
Bacterial compositions may be prepared comprising at least two types of isolated bacteria, chosen from the species in Table 1.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum, Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus). In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum, Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus). In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In another embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Acidaminococcus intestinalis, Bacteroides ovatus, two strains of Bifidobacterium adolescentis, two strains of Bifidobacterium longum, Blautia producta, Clostridium cocleatum, Collinsella aerofaciens, two strains of Dorea longicatena, Escherichia coli, Eubacterium desmolans, Eubacterium eligens, Eubacterium limosum, four strains of Eubacterium rectale, Eubacterium ventriosumi, Faecalibacterium prausnitzii, Lachnospira pectinoshiza, Lactobacillus casei, Lactobacillus casei/paracasei, Paracateroides distasonis, Raoultella sp., one strain of Roseburia (chosen from Roseburia faecalis or Roseburia faecis), Roseburia intestinalis, two strains of Ruminococcus torques, two strains of Ruminococcus obeum, and Streptococcus mitis. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In yet another embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Barnesiella intestinihominis; Lactobacillus reuteri; a species characterized as one of Enterococcus hirae, Enterococus faecium, or Enterococcus durans; a species characterized as one of Anaerostipes caccae or Clostridium indolis; a species characterized as one of Staphylococcus warneri or Staphylococcus pasteuri; and Adlercreutzia equolifaciens. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In other embodiments, the bacterial composition comprises at least one and preferably more than one of the following: Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bartlettii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium camis, Clostridium celatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridium cochlearium, Clostridium difficile, Clostridium fallax, Clostridium felsineum, Clostridium ghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, Clostridium limosum, Clostridium malenominatum, Clostridium novyi, Clostridium oroticum, Clostridium paraputrificum, Clostridium perfringens, Clostridium piliforme, Clostridium putrefaciens, Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridium tertium, Clostridium tetani, Clostridium welchii, and Clostridium villosum. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Clostridium innocuum, Clostridum bifermentans, Clostridium butyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides uniformis, three strains of Escherichia coli, and Lactobacillus sp. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Clostridium bifermentans, Clostridium innocuum, Clostridium butyricum, three strains of Escherichia coli, three strains of Bacteroides, and Blautia producta. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides sp., Escherichia coli, and non pathogenic Clostridia, including Clostridium innocuum, Clostridium bifermentans and Clostridium ramosum. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides species, Escherichia coli and non-pathogenic Clostridia, such as Clostridium butyricum, Clostridium bifermentans and Clostridium innocuum. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroides gracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae, Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris, Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, and Bacteroides vulgatus. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides, Eubacteria, Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas, and Peptostreptococcus. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens, Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previously known as Peptostreptococcus productus II), Bacteroides fragilis ss. Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacterium aerofaciens III, Blautia producta (previously known as Peptostreptococcus productus I), Ruminococcus bronii, Bifidobacterium adolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacterium siraeum, Ruminococcus torques, Eubacterium rectale III-H, Eubacterium rectale IV, Eubacterium eligens, Bacteroides eggerthii, Clostridium leptum, Bacteroides fragilis ss. A, Eubacterium biforme, Bifidobacterium infantis, Eubacterium rectale III-F, Coprococcus comes, Bacteroides capillosus, Ruminococcus albus, Eubacterium formicigenerans, Eubacterium hallii, Eubacterium ventriosum I, Fusobacterium russii, Ruminococcus obeum, Eubacterium rectale II, Clostridium ramosum I, Lactobacillus leichmanii, Ruminococcus cailidus, Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilis ss. fragilis, Bacteroides AR, Coprococcus catus, Eubacterium hadrum, Eubacterium cylindroides, Eubacterium ruminantium, Eubacterium CH-1, Staphylococcus epidermidis, Peptostreptococcus BL, Eubacterium limosum, Bacteroides praeacutus, Bacteroides L, Fusobacterium mortiferum I, Fusobacterium naviforme, Clostridium innocuum, Clostridium ramosum, Propionibacterium acnes, Ruminococcus flavefaciens, Ruminococcus AT, Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, -AN; Bacteroides fragilis ss. ovatus, -ss. d, -ss. f; Bacteroides L-1, L-5; Fusobacterium nucleatum, Fusobacterium mortiferum, Escherichia coli, Streptococcus morbiliorum, Peptococcus magnus, Peptococcus G, AU-2; Streptococcus intermedius, Ruminococcus lactaris, Ruminococcus CO Gemmiger X, Coprococcus BH, -CC; Eubacterium tenue, Eubacterium ramulus, Eubacterium AE, -AG-H, -AG-M, -AJ, -BN-1; Bacteroides clostridiiformis ss. clostridliformis, Bacteroides coagulans, Bacteroides orails, Bacteroides ruminicola ss. brevis, -ss. ruminicola, Bacteroides splanchnicus, Desuifomonas pigra, Bacteroides L-4, -N-i; Fusobacterium H, Lactobacillus G, and Succinivibrio A. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
Bacterial Compositions Described by Operational Taxonomic Unit (OTUs)
Bacterial compositions may be prepared comprising at least two types of isolated bacteria, chosen from the species in Table 1.
In one embodiment, the OTUs can be characterized by one or more of the variable regions of the 16S sequence (V1-V9). 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., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978)). 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.
Bacterial Compositions Exclusive of Certain Bacterial Species Or Strains
In one embodiment, the bacterial composition does not comprise at least one of Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum, Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus).
In another embodiment, the bacterial composition does not comprise at least one of Acidaminococcus intestinalis, Bacteroides ovatus, two species of Bifidobacterium adolescentis, two species of Bifidobacterium longum, Collinsella aerofaciens, two species of Dorea longicatena, Escherichia coli, Eubacterium eligens, Eubacterium limosum, four species of Eubacterium rectale, Eubacterium ventriosumi, Faecalibacterium prausnitzii, Lactobacillus casei, Lactobacillus paracasei, Paracateroides distasonis, Raoultella sp., one species of Roseburia (chosen from Roseburia faecalis or Roseburia faecis), Roseburia intestinalis, two species of Ruminococcus torques, and Streptococcus mitis. 
In yet another embodiment, the bacterial composition does not comprise at least one of Bamesiella intestinihominis; Lactobacillus reuteri; a species characterized as one of Enterococcus hirae, Enterococus faecium, or Enterococcus durans; a species characterized as one of Anaerostipes caccae or Clostridium indolis; a species characterized as one of Staphylococcus warneri or Staphylococcus pasteuri; and Adlercreutzia equolifaciens. 
In other embodiments, the bacterial composition does not comprise at least one of Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium camis, Clostridium celatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridium cochlearium, Clostridium difficile, Clostridium fallax, Clostridium felsineum, Clostridium ghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulars, Clostridium limosum, Clostridium malenominatum, Clostridium novyi, Clostridium oroticum, Clostridium paraputrificum, Clostridium perfringens, Clostridium piliforme, Clostridium putrefaciens, Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridium tertium, Clostridium tetani, Clostridium welchii, and Clostridium villosum. 
In another embodiment, the bacterial composition does not comprise at least one of Clostridium innocuum, Clostridum bifermentans, Clostridium butyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides uniformis, three strains of Escherichia coli, and Lactobacillus sp.
In another embodiment, the bacterial composition does not comprise at least one of Clostridium bifermentans, Clostridium innocuum, Clostridium butyricum, three strains of Escherichia coli, three strains of Bacteroides, and Blautia producta (previously known as Peptostreptococcus productus).
In another embodiment, the bacterial composition does not comprise at least one of Bacteroides sp., Escherichia coli, and non pathogenic Clostridia, including Clostridium innocuum, Clostridium bifermentans and Clostridium ramosum. 
In another embodiment, the bacterial composition does not comprise at least one of more than one Bacteroides species, Escherichia coli and non-pathogenic Clostridia, such as Clostridium butyricum, Clostridium bifermentans and Clostridium innocuum. 
In another embodiment, the bacterial composition does not comprise at least one of Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroides gracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae, Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris, Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, and Bacteroides vulgatus. 
In another embodiment, the bacterial composition does not comprise at least one of Bacteroides, Eubacteria, Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas, and Peptostreptococcus. 
In another embodiment, the bacterial composition does not comprise at least one of Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens, Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previously known as Peptostreptococcus productus II), Bacteroides fragilis ss. Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacterium aerofaciens III, Blautia producta (previously known as Peptostreptococcus productus I), Ruminococcus bromii, Bifidobacterium adolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacterium siraeum, Ruminococcus torques, Eubacterium rectale III-H, Eubacterium rectale IV, Eubacterium eligens, Bacteroides eggerthii, Clostridium leptum, Bacteroides fragilis ss. A, Eubacterium biforme, Bifidobacterium infantis, Eubacterium rectale III-F, Coprococcus comes, Bacteroides capillosus, Ruminococcus albus, Eubacterium formicigenerans, Eubacterium hallii, Eubacterium ventriosum I, Fusobacterium russii, Ruminococcus obeum, Eubacterium rectale II, Clostridium ramosum I, Lactobacillus leichmanii, Ruminococcus cailidus, Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilis ss. fragilis, Bacteroides AR, Coprococcus catus, Eubacterium hadrum, Eubacterium cylindroides, Eubacterium ruminantium, Eubacterium CH-1, Staphylococcus epidermidis, Peptostreptococcus BL, Eubacterium limosum, Bacteroides praeacutus, Bacteroides L, Fusobacterium mortiferum I, Fusobacterium naviforme, Clostridium innocuum, Clostridium ramosum, Propionibacterium acnes, Ruminococcus flavefaciens, Ruminococcus AT, Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, -AN; Bacteroides fragilis ss. ovatus, -ss. d, -ss. f; Bacteroides L-1, L-5; Fusobacterium nucleatum, Fusobacterium mortiferum, Escherichia coli, Streptococcus morbiliorum, Peptococcus magnus, Peptococcus G, AU-2; Streptococcus intermedius, Ruminococcus lactaris, Ruminococcus CO Gemmiger X, Coprococcus BH, -CC, Eubacterium tenue, Eubacterium ramulus, Eubacterium AE, -AG-H, -AG-M, -AJ, -BN-1, Bacteroides clostridiiformis ss. clostridliformis, Bacteroides coagulans, Bacteroides orails, Bacteroides ruminicola ss. brevis, -ss. ruminicola, Bacteroides splanchnicus, Desuifomonas pigra, Bacteroides L-4, -N-i; Fusobacterium H, Lactobacillus G, and Succinivibrio A.
Inhibition of Bacterial Pathogens
In some embodiments, the bacterial composition provides a protective or therapeutic effect against infection by one or more GI pathogens of interest.
A list of exemplary bacterial pathogens is provided in Table 1 as indicated by pathogen status.
In some embodiments, the pathogenic bacterium is selected from the group consisting of Yersinia, Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, Bacillus, multi-drug resistant bacteria, extended spectrum beta-lactam resistant Enterococci (ESBL), Carbapenem-resistent Enterobacteriaceae (CRE), and vancomycin-resistant Enterococci (VRE).
In some embodiments, these pathogens include, but are not limited to, Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia coli (such as, but not limited to, LT and/or ST), Escherichia coli 0157:H7, Helicobacter pylori, Klebsiellia pneumonia, Lysteria monocytogenes, Plesiomonas shigelloides, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Shigella spp., Staphylococcus spp., Staphylococcus aureus, vancomycin-resistant enterococcus spp., Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica. 
In one embodiment, the pathogen of interest is at least one pathogen chosen from Clostridium difficile, Salmonella spp., pathogenic Escherichia coli, vancomycin-resistant Enterococcus spp., and extended spectrum beta-lactam resistant Enterococci (ESBL).
Purified Spore Populations
In some embodiments, the bacterial compositions comprise purified spore populations. Purified spore populations contain combinations of commensal bacteria of the human gut microbiota with the capacity to meaningfully provide functions of a healthy microbiota when administered to a mammalian subject. Without being limited to a specific mechanism, it is thought that such compositions inhibit the growth of a pathogen such as C. difficile, Salmonella spp., enteropathogenic E. coli, and vancomycin-resistant Enterococcus spp., so that a healthy, diverse and protective microbiota can be maintained or, in the case of pathogenic bacterial infections such as C. difficile infection, repopulate the intestinal lumen to reestablish ecological control over potential pathogens. In some embodiments, yeast spores and other fungal spores are also purified and selected for therapeutic use.
Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health. These compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health. While spore-based compositions are known, these are generally prepared according to various techniques such as lyophilization or spray-drying of liquid bacterial cultures, resulting in poor efficacy, instability, substantial variability and lack of adequate safety.
It has now been found that populations of bacterial spores can be obtained from biological materials obtained from mammalian subjects, including humans. These populations are formulated into compositions as provided herein, and administered to mammalian subjects using the methods as provided herein.
Provided herein are therapeutic compositions containing a purified population of bacterial spores. 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 spores, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired bacterial spore, 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 spores at or above an acceptable amount and/or concentration. In other embodiments, the purified population of bacterial spores 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%, 90%, 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 certain embodiments, the purified populations of bacterial spores have reduced or undetectable levels of one or more pathogenic activities, such as toxicity, an infection of the mammalian recipient subject, an 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%, 90%, 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 spores have reduced sensory components as compared to fecal material, such as reduced odor, taste, appearance, and umami.
Provided are purified populations of bacterial spores that are substantially free of residual habitat products. In certain embodiments, this means that the bacterial spore 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 spores may be 100% free, 99% free, 98% free, 97% free, 96% free, or 95% 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 spore 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%, 1×10−4%, 1×10−5%, 1×10−5%, 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%, 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 spore populations are substantially free of an acellular material, e.g., DNA, viral coat material, or non-viable bacterial material.
As described herein, purified spore populations can be demonstrated by genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired bacterial spores from non-desired, contaminating materials.
Exemplary biological materials include fecal materials such as feces or materials isolated from the various segments of the small and large intestines. Fecal materials are obtained from a mammalian donor subject, or can be obtained from more than one donor subject, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or from greater than 1000 donors, where such materials are then pooled prior to purification of the desired bacterial spores.
In alternative embodiments, the desired bacterial spores are purified from a single fecal material sample obtained from a single donor, and after such purification are combined with purified spore populations from other purifications, either from the same donor at a different time, or from one or more different donors, or both.
Preferred bacterial genera include Acetonema, Alkaliphilus, Alicyclobacillus, Amphibacillus, Ammonifex, Anaerobacter, Anaerofustis, Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Blautia, Brevibacillus, Bryantella, Caldicellulosiruptor, Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Clostridium, Cohnella, Coprococcus, Dendrosporobacter Desulfitobacterium, Desulfosporosinus, Desulfotomaculum, Dorea, Eubacterium, Faecalibacterium, Filifactor, Geobacillus, Halobacteroides, Heliobacillus, Heliobacterium, Heliophilum, Heliorestis, Lachnoanaerobaculum, Lysinibacillus, Moorella, Oceanobacillus, Orenia (S.), Oxalophagus, Oxobacter, Paenibacillus, Pelospora, Pelotomaculum, Propionispora, Roseburia, Ruminococcus, Sarcina, Sporobacterium, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporosarcina, Sporotomaculum, Subdoligranulum, Symbiobacterium, Syntrophobotulus, Syntrophospora, Terribacillus, Thermoanaerobacter, and Thermosinus. 
Preferred bacterial species are provided at Table X4. 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 some embodiments, spore-forming bacteria are identified by the presence of nucleic acid sequences that modulate sporulation. In particular, signature sporulation genes are highly conserved across members of distantly related genera including Clostridium and Bacillus. Traditional approaches of forward genetics have identified many, if not all, genes that are essential for sporulation (spo). The developmental program of sporulation is governed in part by the successive action of four compartment-specific sigma factors (appearing in the order σF, σE, σG and σK), whose activities are confined to the forespore (σF and σG) or the mother cell (σE and σK).
Provided are spore populations containing more than one type of bacterium. 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 embodiments, all or essentially all of the bacterial spores present in a purified population are obtained from a fecal material treated as described herein or otherwise known in the art. In alternative embodiments, one or more than one bacterial spores or types of bacterial spores are generated in culture and combined to form a purified spore population. In other alternative embodiments, one or more of these culture-generated spore populations are combined with a fecal material-derived spore population to generate a hybrid spore population. Bacterial compositions may contain at least two types of these preferred bacteria, including strains of the same species. For instance, a bacterial composition may comprise at least 2, at least 3, at least 4, 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 or more than 20 types of bacteria, as defined by species or operational taxonomic unit (OTU) encompassing such species.
Thus, provided herein are methods for production of a composition containing a population of bacterial spores suitable for therapeutic administration to a mammalian subject in need thereof. And the composition is 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 spores is produced from the fecal material. The composition is formulated such that a single oral dose contains at least about 1×104 colony forming units of the bacterial spores, and a single oral dose will typically contain about 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 CFUs of the bacterial spores. The presence and/or concentration of a given type of bacteria spore may be known or unknown in a given purified spore population. If known, for example the concentration of spores of a given strain, or the aggregate of all strains, is e.g., 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 viable bacterial spores per gram of composition or per administered dose.
In some formulations, the composition contains at least about 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 bacterial spore compositions 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.
The bacterial spore compositions may be formulated to be effective in a given mammalian subject in a single administration or over multiple administrations. For example, a single administration is substantially effective to reduce Cl. difficile and/or Cl. difficile toxin content in a mammalian subject to whom the composition is administered. Substantially effective means that Cl. difficile and/or Cl. difficile toxin content in the subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99% following administration of the composition.
Methods of the Invention
Methods for Determining 16S Sequences
OTUs can be defined either by full 16S sequencing of the rRNA gene, by sequencing of a specific hypervariable region of this gene (i.e. V1, V2, V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination of hypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial 16S rDNA is approximately 1500 nucleotides in length and 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 microbes.
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 can 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.
Methods for Preparing a Bacterial Composition for Administration to a Subject
Methods for producing bacterial compositions can include three main processing steps, combined with one or more mixing steps. The steps include organism banking, organism production, and preservation.
For banking, the strains included in the bacterial composition may be (1) isolated directly from a specimen or taken from a banked stock, (2) optionally cultured on a nutrient agar or broth that supports growth to generate viable biomass, and (3) the biomass optionally preserved in multiple aliquots in long-term storage.
In embodiments that use a culturing step, the agar or broth can contain nutrients that provide essential elements and specific factors that enable growth. An example would be a medium composed of 20 g/L glucose, 10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/L sodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/L magnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1 mg/L menadione. A variety of microbiological media and variations are well known in the art (e.g. R. M. Atlas, Handbook of Microbiological Media (2010) CRC Press). Medium can be added to the culture at the start, may be added during the culture, or may be intermittently/continuously flowed through the culture. The strains in the bacterial composition may be cultivated alone, as a subset of the bacterial composition, or as an entire collection comprising the bacterial composition. As an example, a first strain may be cultivated together with a second strain in a mixed continuous culture, at a dilution rate lower than the maximum growth rate of either cell to prevent the culture from washing out of the cultivation.
The inoculated culture is incubated under favorable conditions for a time sufficient to build biomass. For bacterial compositions for human use, this is often at 37° C. temperature, pH, and other parameter with values similar to the normal human niche. The environment can be actively controlled, passively controlled (e.g., via buffers), or allowed to drift. For example, for anaerobic bacterial compositions (e.g., gut microbiota), an anoxic/reducing environment can be employed. This can be accomplished by addition of reducing agents such as cysteine to the broth, and/or stripping it of oxygen. As an example, a culture of a bacterial composition can be grown at 37° C., pH 7, in the medium above, pre-reduced with 1 g/L cysteine HCl.
When the culture has generated sufficient biomass, it can be preserved for banking. The organisms can be placed into a chemical milieu that protects from freezing (adding ‘cryoprotectants’), drying (‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensing into multiple (optionally identical) containers to create a uniform bank, and then treating the culture for preservation. Containers are generally impermeable and have closures that assure isolation from the environment. Cryopreservation treatment is accomplished by freezing a liquid at ultra-low temperatures (e.g., at or below −80° C.). Dried preservation removes water from the culture by evaporation (in the case of spray drying or ‘cool drying’) or by sublimation (e.g., for freeze drying, spray freeze drying). Removal of water improves long-term bacterial composition storage stability at temperatures elevated above cryogenic. If the bacterial composition comprises spore forming species and results in the production of spores, the final composition can be purified by additional means, such as density gradient centrifugation preserved using the techniques described above. Bacterial composition banking can be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank. As an example of cryopreservation, a bacterial composition culture can be harvested by centrifugation to pellet the cells from the culture medium, the supernate decanted and replaced with fresh culture broth containing 15% glycerol. The culture can then be aliquoted into 1 mL cryotubes, sealed, and placed at −80° C. for long-term viability retention. This procedure achieves acceptable viability upon recovery from frozen storage.
Organism production can be conducted using similar culture steps to banking, including medium composition and culture conditions. It can be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there can be several subcultivations of the bacterial composition prior to the final cultivation. At the end of cultivation, the culture is harvested to enable further formulation into a dosage form for administration. This can involve concentration, removal of undesirable medium components, and/or introduction into a chemical milieu that preserves the bacterial composition and renders it acceptable for administration via the chosen route. For example, a bacterial composition can be cultivated to a concentration of 1010 CFU/mL, then concentrated 20-fold by tangential flow microfiltration; the spent medium can be exchanged by diafiltering with a preservative medium consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphate buffer. The suspension can then be freeze-dried to a powder and titrated.
After drying, the powder can be blended to an appropriate potency, and mixed with other cultures and/or a filler such as microcrystalline cellulose for consistency and ease of handling, and the bacterial composition formulated as provided herein.
Administration of Bacterial Compositions.
The bacterial compositions of the invention are suitable for administration to mammals and non-mammalian animals in need thereof. In certain embodiments, the mammalian subject is a human subject who has one or more symptoms of a dysbiosis, including but not limited to overgrowth of an undesired pathobiont or pathogen, reduced representation of key bacterial taxa such as the Bacteroidetes or Firmicutes or genera or species thereof, or reduced diversity of microbial species compared to a healthy individual, or reduced overall abundance of anaerobic bacteria.
When the mammalian subject is suffering from a disease, disorder or condition characterized by an aberrant microbiota, the bacterial compositions described herein are suitable for treatment thereof. In some embodiments, the mammalian subject has not received antibiotics in advance of treatment with the bacterial compositions. For example, the mammalian subject has not been administered at least two doses of vancomycin, metronidazole and/or or similar antibiotic compound within one week prior to administration of the therapeutic composition. In other embodiments, the mammalian subject has not previously received an antibiotic compound in the one month prior to administration of the therapeutic composition. In other embodiments, the mammalian subject has received one or more treatments with one or more different antibiotic compounds and such treatment(s) resulted in no improvement or a worsening of symptoms. In some embodiments, the spore composition is administered following a successful course of antibiotics to prevent dysbiosis and enhance recovery of a diverse, healthy microbiota.
In some embodiments, the gastrointestinal disease, disorder or condition is diarrhea caused by C. difficile including recurrent C. difficile infection, ulcerative colitis, colitis, Crohn's disease, or irritable bowel disease. Beneficially, the therapeutic composition is administered only once prior to improvement of the disease, disorder or condition. 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. Or the preparation may be 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 another embodiment, the preparation may be administered on a long-term basis to individuals who are at risk for infection with or who may be carriers of these pathogens, including individuals who will have an invasive medical procedure (such as surgery), who will be hospitalized, who live in a long-term care or rehabilitation facility, who are exposed to pathogens by virtue of their profession (livestock and animal processing workers), or who could be carriers of pathogens (including hospital workers such as physicians, nurses, and other healthcare professionals).
Also provided are methods of treating or preventing a mammalian subject suffering from or at risk of developing a metabolic disease, and disorder or condition selected from the group consisting of diabetes, metabolic syndrome, obesity, heart disease, autoimmune disease, liver disease, and autism using the therapeutic compositions provided herein.
In embodiments, the bacterial spore 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 bacterial 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, it is administered to all regions of the gastrointestinal tract. The bacterial compositions may be administered orally in the form of medicaments such as powders, capsules, tablets, gels or liquids. The bacterial 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.
If the composition is administered colonoscopically and, optionally, if the bacterial composition 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 the bacterial 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.
To evaluate the subject, symptoms of dysbiosis are evaluated post treatment ranging from 1 day to 6 months after administration of the purified spore population. Fecal material is collected during this period and the microbes present in the gastrointestinal tract can be assessed by 16S rDNA or metagenomic sequencing analysis or other analyses commonly used by the skilled artisan. Repopulation by species provided by the spore population as well as Augmentation by commensal microbes not present in the spore population will occur in this time as the spore population catalyzes a reshaping of the gut ecology to a state of healthy biosis. The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments and should not be construed to limit the scope. The skilled artisan readily recognizes that many other embodiments are encompassed. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art.
Methods of Treating a Subject
In some embodiments, the compositions disclosed herein are administered to a patient or a user (sometimes collectively referred to as a “subject”). As used herein “administer” and “administration” encompasses embodiments in which one person directs another to consume a bacterial composition in a certain manner and/or for a certain purpose, and also situations in which a user uses a bacteria composition in a certain manner and/or for a certain purpose independently of or in variance to any instructions received from a second person. Non-limiting examples of embodiments in which one person directs another to consume a bacterial composition in a certain manner and/or for a certain purpose include when a physician prescribes a course of conduct and/or treatment to a patient, when a parent commands a minor user (such as a child) to consume a bacterial composition, when a trainer advises a user (such as an athlete) to follow a particular course of conduct and/or treatment, and when a manufacturer, distributer, or marketer recommends conditions of use to an end user, for example through advertisements or labeling on packaging or on other materials provided in association with the sale or marketing of a product.
The bacterial compositions offer a protective and/or therapeutic effect against infection by one or more GI pathogens of interest and can be administered after an acute case of infection has been resolved in order to prevent relapse, during an acute case of infection as a complement to antibiotic therapy if the bacterial composition is not sensitive to the same antibiotics as the GI pathogen, or to prevent infection or reduce transmission from disease carriers.
The present bacterial compositions can be useful in a variety of clinical situations. For example, the bacterial compositions can be administered as a complementary treatment to antibiotics when a patient is suffering from an acute infection, to reduce the risk of recurrence after an acute infection has subsided, or when a patient will be in close proximity to others with or at risk of serious gastrointestinal infections (physicians, nurses, hospital workers, family members of those who are ill or hospitalized).
The present bacterial compositions can be administered to animals, including humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), and household pets (e.g., dogs, cats, rodents).
In the present method, the bacterial composition can be administered enterically, in other words, by a route of access to the gastrointestinal tract. This includes oral administration, rectal administration (including enema, suppository, or colonoscopy), by an oral or nasal tube (nasogastric, nasojejunal, oral gastric, or oral jejunal), as detailed more fully herein.
Pretreatment Protocols
Prior to administration of the bacterial composition, the patient can optionally have a pretreatment protocol to prepare the gastrointestinal tract to receive the bacterial composition. In certain embodiments, the pretreatment protocol is advisable, such as when a patient has an acute infection with a highly resilient pathogen. In other embodiments, the pretreatment protocol is entirely optional, such as when the pathogen causing the infection is not resilient, or the patient has had an acute infection that has been successfully treated but where the physician is concerned that the infection may recur. In these instances, the pretreatment protocol can enhance the ability of the bacterial 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 before the bacterial composition is administered. In one embodiment, the antibiotic can be discontinued 1, 2, or 3 days before the administration of the bacterial composition. In another embodiment, the antibiotic can be discontinued 3, 4, 5, 6, or 7 antibiotic half-lives before administration of the bacterial composition. In another embodiment, the antibiotic can be chosen so the constituents in the bacterial composition have an MIC50 that is higher than the concentration of the antibiotic in the gut.
MIC50 of a bacterial composition or the elements in the composition can be determined by methods well known in the art. Reller et al., Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices, Clinical Infectious Diseases 49(11):1749-1755 (2009). In such an embodiment, the additional time between antibiotic administration and administration of the bacterial 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 constituents in the bacterial composition are not sensitive to the antibiotic.
Routes of Administration
The bacterial compositions of the invention are suitable for administration to mammals and non-mammalian animals in need thereof. In certain embodiments, the mammalian subject is a human subject who has one or more symptoms of a dysbiosis.
When a mammalian subject is suffering from a disease, disorder or condition characterized by an aberrant microbiota, the bacterial compositions described herein are suitable for treatment thereof. In some embodiments, the mammalian subject has not received antibiotics in advance of treatment with the bacterial compositions. For example, the mammalian subject has not been administered at least two doses of vancomycin, metronidazole and/or or similar antibiotic compound within one week prior to administration of the therapeutic composition. In other embodiments, the mammalian subject has not previously received an antibiotic compound in the one month prior to administration of the therapeutic composition. In other embodiments, the mammalian subject has received one or more treatments with one or more different antibiotic compounds and such treatment(s) resulted in no improvement or a worsening of symptoms.
In some embodiments, the gastrointestinal disease, disorder or condition is diarrhea caused by C. difficile including recurrent C. difficile infection, ulcerative colitis, colitis, Crohn's disease, or irritable bowel disease. Beneficially, the therapeutic composition is administered only once prior to improvement of the disease, disorder or condition. 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 can be 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 another embodiment, the preparation may be administered on a long-term basis to subjects who are at risk for infection with or who may be carriers of these pathogens, including subjects who will have an invasive medical procedure (such as surgery), who will be hospitalized, who live in a long-term care or rehabilitation facility, who are exposed to pathogens by virtue of their profession (livestock and animal processing workers), or who could be carriers of pathogens (including hospital workers such as physicians, nurses, and other health care professionals).
In certain embodiments, the bacterial 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 bacterial 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 bacterial 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.
If the composition is administered colonoscopically and, optionally, if the bacterial 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 composition relative to the other organisms previously residing in the gastrointestinal tract of the subject. Any ordinarily acceptable colon-cleansing preparation may be used such as those typically provided when a subject undergoes a colonoscopy.
Dosages and Schedule for Administration
In some embodiments, the bacteria and bacterial compositions are provided in a dosage form. In certain embodiments, the dosage form is designed for administration of at least one OTU or combination thereof disclosed herein, wherein the total amount of bacterial 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 bacterial 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, 105 and 1012 microorganisms 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 bacterial composition having from 107 to 1011 bacteria per ml or per gram, or a capsule, tablet or suppository having from 1 mg to 1000 mg lyophilized powder having from 107 to 1011 bacteria. 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 preparations 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. In one embodiment, the preparation can be administered on a long-term basis to individuals who are at risk for infection with or who may be carriers of these pathogens, including individuals who will have an invasive medical procedure (such as surgery), who will be hospitalized, who live in a long-term care or rehabilitation facility, who are exposed to pathogens by virtue of their profession (livestock and animal processing workers), or who could be carriers of pathogens (including hospital workers such as physicians, nurses, and other health care professionals).
Patient Selection
Particular bacterial compositions can be selected for individual patients or for patients with particular profiles. For example, 16S sequencing can be performed for a given patient to identify the bacteria present in his or her microbiota. The sequencing can either profile the patient's entire microbiome using 16S sequencing (to the family, genera, or species level), a portion of the patient's microbiome using 16S sequencing, or it can be used to detect the presence or absence of specific candidate bacteria that are biomarkers for health or a particular disease state, such as markers of multi-drug resistant organisms or specific genera of concern such as Escherichia. Based on the biomarker data, a particular composition can be selected for administration to a patient to supplement or complement a patient's microbiota in order to restore health or treat or prevent disease. In another embodiment, patients can be screened to determine the composition of their microbiota to determine the likelihood of successful treatment.
Combination Therapy
The bacterial compositions can be administered with other agents in a combination therapy mode, including anti-microbial agents and prebiotics. Administration can be sequential, over a period of hours or days, or simultaneous.
In one embodiment, the bacterial compositions 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.
Examples of antifungal compounds 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 bacterial compositions are included in combination therapy 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.
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 essential nutritional components 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.
Methods for Testing Bacterial Compositions for Populating Effect
In Vivo Assay for Determining Whether a Bacterial Composition Populates a Subject's Gastrointestinal Tract
In order to determine that the bacterial composition populates the gastrointestinal tract of a subject, an animal model, such as a mouse model, can be used. The model can begin by evaluating the microbiota of the mice. Qualitative assessments can be accomplished using 16S profiling of the microbial community in the feces of normal mice. It can also be accomplished by full genome sequencing, whole genome shotgun sequencing (WGS), or traditional microbiological techniques. Quantitative assessments can be conducted using quantitative PCR (qPCR), described below, or by using traditional microbiological techniques and counting colony formation.
Optionally, the mice can receive an antibiotic treatment to mimic the condition of dysbiosis. Antibiotic treatment can decrease the taxonomic richness, diversity, and evenness of the community, including a reduction of abundance of a significant number of bacterial taxa. Dethlefsen et al., The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing, PLoS Biology 6(11):3280 (2008). At least one antibiotic can be used, and antibiotics are well known. Antibiotics can include aminoglycoside antibiotic (amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, and apramycin), amoxicillin, ampicillin, Augmentin (an amoxicillin/clavulanate potassium combination), cephalosporin (cefaclor, defadroxil, cefazolin, cefixime, fefoxitin, cefprozil, ceftazimdime, cefuroxime, cephalexin), clavulanate potassium, clindamycin, colistin, gentamycin, kanamycin, metronidazole, or vancomycin. As an individual, nonlimiting specific example, the mice can be provided with drinking water containing a mixture of the antibiotics kanamycin, colistin, gentamycin, metronidazole and vancomycin at 40 mg/kg, 4.2 mg/kg, 3.5 mg/kg, 21.5 mg/kg, and 4.5 mg/kg (mg per average mouse body weight), respectively, for 7 days. Alternatively, mice can be administered ciprofloxacin at a dose of 15-20 mg/kg (mg per average mouse body weight), for 7 days. If the mice are provided with an antibiotic, a wash out period of from one day to three days may be provided with no antibiotic treatment and no bacterial composition treatment.
Subsequently, the test bacterial composition is administered to the mice by oral gavage. The test bacterial composition may be administered in a volume of 0.2 ml containing 104 CFUs of each type of bacteria in the bacterial composition. Dose-response may be assessed by using a range of doses, including, but not limited to 102, 103, 104, 105, 106, 107, 108, 109, and/or 1010.
The mice can be evaluated using 16S sequencing, full genome sequencing, whole genome shotgun sequencing (WGS), or traditional microbiological techniques to determine whether the test bacterial composition has populated the gastrointestinal tract of the mice. For example only, one day, three days, one week, two weeks, and one month after administration of the bacterial composition to the mice, 16S profiling is conducted to determine whether the test bacterial composition has populated the gastrointestinal tract of the mice. Quantitative assessments, including qPCR and traditional microbiological techniques such as colony counting, can additionally or alternatively be performed, at the same time intervals.
Furthermore, the number of sequence counts that correspond exactly to those in the bacterial composition over time can be assessed to determine specifically which components of the bacterial composition reside in the gastrointestinal tract over a particular period of time. In one embodiment, the strains of the bacterial composition persist for a desired period of time. In another embodiment, the components of the bacterial composition persist for a desired period of time, while also increasing the ability of other microbes (such as those present in the environment, food, etc.) to populate the gastrointestinal tract, further increasing overall diversity, as discussed below.
Ability of Bacterial Compositions to Populate Different Regions of the Gastrointestinal Tract
The present bacterial compositions can also be assessed for their ability to populate different regions on the gastrointestinal tract. In one embodiment, a bacterial composition can be chosen for its ability to populate one or more than one region of the gastrointestinal tract, including, but not limited to the stomach, the small intestine (duodenum, jejunum, and ileum), the large intestine (the cecum, the colon (the ascending, transverse, descending, and sigmoid colon), and the rectum).
An in vivo study can be conducted to determine which regions of the gastrointestinal tract a given bacterial composition will populate. A mouse model similar to the one described above can be conducted, except instead of assessing the feces produced by the mice, particular regions of the gastrointestinal tract can be removed and studied individually. For example, at least one particular region of the gastrointestinal tract can be removed and a qualitative or quantitative determination can be performed on the contents of that region of the gastrointestinal tract. In another embodiment, the contents can optionally be removed and the qualitative or quantitative determination may be conducted on the tissue removed from the mouse.
qPCR
As one quantitative method for determining whether a bacterial composition populates the gastrointestinal tract, quantitative PCR (qPCR) can be performed. Standard techniques can be followed to generate a standard curve for the bacterial composition of interest, either for all of the components of the bacterial composition collectively, individually, or in subsets (if applicable). Genomic DNA can be extracted from samples using commercially-available kits, such as the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), the Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.
In some embodiments, qPCR can be conducted using HotMasterMix (5PRIME, Gaithersburg, Md.) and primers specific for the bacterial composition of interest, and may be conducted on a MicroAmp Fast® Optical 96-well Reaction Plate with Barcode (0.1 mL) (Life Technologies, Grand Island, N.Y.) and performed on a BioRad C1000TM Thermal Cycler equipped with a CFX96TM Real-Time System (BioRad, Hercules, Calif.), with fluorescent readings of the FAM and ROX channels. The Cq value for each well on the FAM channel is determined by the CFX ManagerTM software version 2.1. The log10 (cfu/ml) of each experimental sample is calculated by inputting a given sample's Cq value into linear regression model generated from the standard curve comparing the Cq values of the standard curve wells to the known log10 (cfu/ml) of those samples. The skilled artisan may employ alternative qPCR modes.
Methods for Characterization of Bacterial Compositions
In certain embodiments, provided are methods for testing certain characteristics of bacterial compositions. For example, the sensitivity of bacterial compositions to certain environmental variables is determined, e.g., in order to select for particular desirable characteristics in a given composition, formulation and/or use. For example, the constituents in the bacterial composition can be tested for pH resistance, bile acid resistance, and/or antibiotic sensitivity, either individually on a constituent-by-constituent basis or collectively as a bacterial composition comprised of multiple bacterial constituents (collectively referred to in this section as bacterial composition).
pH Sensitivity Testing
If a bacterial composition will be administered other than to the colon or rectum (i.e., for example, an oral route), optionally testing for pH resistance enhances the selection of bacterial compositions that will survive at the highest yield possible through the varying pH environments of the distinct regions of the GI tract. Understanding how the bacterial compositions react to the pH of the GI tract also assists in formulation, so that the number of bacteria in a dosage form can be increased if beneficial and/or so that the composition may be administered in an enteric-coated capsule or tablet or with a buffering or protective composition. As the pH of the stomach can drop to a pH of 1 to 2 after a high-protein meal for a short time before physiological mechanisms adjust it to a pH of 3 to 4 and often resides at a resting pH of 4 to 5, and as the pH of the small intestine can range from a pH of 6 to 7.4, bacterial compositions can be prepared that survive these varying pH ranges (specifically wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or as much as 100% of the bacteria can survive gut transit times through various pH ranges). This can be tested by exposing the bacterial composition to varying pH ranges for the expected gut transit times through those pH ranges. Therefore, as a nonlimiting example only, 18-hour cultures of bacterial compositions can be grown in standard media, such as gut microbiota medium (“GMM”, see Goodman et al., Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice, PNAS 108(15):6252-6257 (2011)) or another animal-products-free medium, with the addition of pH adjusting agents for a pH of 1 to 2 for 30 minutes, a pH of 3 to 4 for 1 hour, a pH of 4 to 5 for 1 to 2 hours, and a pH of 6 to 7.4 for 2.5 to 3 hours. An alternative method for testing stability to acid is described in U.S. Pat. No. 4,839,281. Survival of bacteria may be determined by culturing the bacteria and counting colonies on appropriate selective or non-selective media.
Bile Acid Sensitivity Testing
Additionally, in some embodiments, testing for bile-acid resistance enhances the selection of bacterial compositions that will survive exposures to bile acid during transit through the GI tract. Bile acids are secreted into the small intestine and can, like pH, affect the survival of bacterial compositions. This can be tested by exposing the bacterial compositions to bile acids for the expected gut exposure time to bile acids. For example, bile acid solutions can be prepared at desired concentrations using 0.05 mM Tris at pH 9 as the solvent. After the bile acid is dissolved, the pH of the solution may be adjusted to 7.2 with 10% HCl. Bacterial compositions can be cultured in 2.2 ml of a bile acid composition mimicking the concentration and type of bile acids in the patient, 1.0 ml of 10% sterile-filtered feces media and 0.1 ml of an 18-hour culture of the given strain of bacteria. Incubations may be conducted for from 2.5 to 3 hours or longer. An alternative method for testing stability to bile acid is described in U.S. Pat. No. 4,839,281. Survival of bacteria may be determined by culturing the bacteria and counting colonies on appropriate selective or non-selective media.
Antibiotic Sensitivity Testing
As a further optional sensitivity test, bacterial compositions can be tested for sensitivity to antibiotics. In one embodiment, bacterial compositions can be chosen so that the bacterial constituents are sensitive to antibiotics such that if necessary they can be eliminated or substantially reduced from the patient's gastrointestinal tract by at least one antibiotic targeting the bacterial composition.
Adherence to Gastrointestinal Cells
The bacterial compositions may optionally be tested for the ability to adhere to gastrointestinal cells. A method for testing adherence to gastrointestinal cells is described in U.S. Pat. No. 4,839,281.
Methods for Purifying Spores
Solvent Treatments
To purify the bacterial spores, the fecal material is subjected to one or more solvent treatments. A solvent treatment is a miscible solvent treatment (either partially miscible or fully miscible) or an immiscible solvent treatment. Miscibility is the ability of two liquids to mix with each to form a homogeneous solution. Water and ethanol, for example, are fully miscible such that a mixture containing water and ethanol in any ratio will show only one phase. Miscibility is provided as a wt/wt %, or weight of one solvent in 100 g of final solution. If two solvents are fully miscible in all proportions, their miscibility is 100%. Provided as fully miscible solutions with water are alcohols, e.g., methanol, ethanol, isopropanol, butanol, etc. The alcohols can be provided already combined with water; e.g., a solution containing 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 85%, 90%, 95% or greater than 95% Other solvents are only partially miscible, meaning that only some portion will dissolve in water. Diethyl ether, for example, is partially miscible with water. Up to 7 grams of diethyl ether will dissolve in 93 g of water to give a 7% (wt/wt %) solution. If more diethyl ether is added, a two phase solution will result with a distinct diethyl ether layer above the water. Other miscible materials include ethers, dimethoxyethane, or tetrahydrofuran In contrast, an oil such as an alkane and water are immiscible and form two phases. Further, immiscible treatments are optionally combined with a detergent, either an ionic detergent or a non-ionic detergent. Exemplary detergents include Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, or a polyol.
Chromatography Treatments
To purify spore populations, the fecal materials are subjected to one or more chromatographic treatments, either sequentially or in parallel. In a chromatographic treatment, a solution containing the fecal material is contacted with a solid medium containing a hydrophobic interaction chromatographic (HIC) medium or an affinity chromatographic medium. In an alternative embodiment, a solid medium capable of absorbing a residual habitat product present in the fecal material is contacted with a solid medium that adsorbs a residual habitat product. In certain embodiments, the HIC medium contains sepharose or a derivatized sepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, or butyl-s sepharose. In other embodiments, the affinity chromatographic medium contains material derivatized with mucin type I, II, III, IV, V, or VI, or oligosaccharides derived from or similar to those of mucins type I, II, III, IV, V, or VI. Alternatively, the affinity chromatographic medium contains material derivatized with antibodies that recognize spore-forming bacteria.
Mechanical Treatments
Provided herein is the physical disruption of the fecal material, particularly by one or more mechanical treatment such as blending, mixing, shaking, vortexing, impact pulverization, and sonication. As provided herein, the mechanical disrupting treatment substantially disrupts a non-spore material present in the fecal material and does not substantially disrupt a spore present in the fecal material. Mechanical treatments optionally include filtration treatments, where the desired spore populations are retained on a filter while the undesirable (non-spore) fecal components to pass through, and the spore fraction is then recovered from the filter medium. Alternatively, undesirable particulates and eukaryotic cells may be retained on a filter while bacterial cells including spores pass through. In some embodiments the spore fraction retained on the filter medium is subjected to a diafiltration step, wherein the retained spores are contacted with a wash liquid, typically a sterile saline-containing solution or other diluent, in order to further reduce or remove the undesirable fecal components.
Thermal Treatments
Provided herein is the thermal disruption of the fecal material. Generally, the fecal material is mixed in a saline-containing solution such as phosphate-buffered saline (PBS) and subjected to a heated environment, such as a warm room, incubator, water-bath, or the like, such that efficient heat transfer occurs between the heated environment and the fecal material. Preferably the fecal material solution is mixed during the incubation to enhance thermal conductivity and disrupt particulate aggregates. Thermal treatments can be modulated by the temperature of the environment and/or the duration of the thermal treatment. For example, the fecal material or a liquid comprising the fecal material is subjected to a heated environment, e.g., a hot water bath of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or greater than 100 degrees Celsius, for at least about 1, 5, 10, 15, 20, 30, 45 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 hours. In certain embodiments the thermal treatment occurs at two different temperatures, such as 30 seconds in a 100 degree Celsius environment followed by 10 minutes in a 50 degree Celsius environment. In preferred embodiments the temperature and duration of the thermal treatment are sufficient to kill or remove pathogenic materials while not substantially damaging or reducing the germination-competency of the spores.
Irradiation Treatments
Provided are methods of treating the fecal material or separated contents of the fecal material with ionizing radiation, typically gamma irradiation, ultraviolet irradiation or electron beam irradiation provided at an energy level sufficient to kill pathogenic materials while not substantially damaging the desired spore populations. For example, ultraviolet radiation at 254 nm provided at an energy level below about 22,000 microwatt seconds per cm2 will not generally destroy desired spores.
Centrifugation and Density Separation Treatments
Provided are methods of separating desired spore populations from the other components of the fecal material by centrifugation. A solution containing the fecal material is subjected to one or more centrifugation treatments, e.g., at about 1000×g, 2000×g, 3000×g, 4000×g, 5000×g, 6000×g, 7000×g, 8000×g or greater than 8000×g. Differential centrifugation separates desired spores from undesired non-spore material; at low forces the spores are retained in solution, while at higher forces the spores are pelleted while smaller impurities (e.g., virus particles, phage) are retained in solution. For example, a first low force centrifugation pellets fibrous materials; a second, higher force centrifugation pellets undesired eukaryotic cells, and a third, still higher force centrifugation pellets the desired spores while small contaminants remain in suspension. In some embodiments density or mobility gradients or cushions (e.g., step cushions), such as Percoll, Ficoll, Nycodenz, Histodenz or sucrose gradients, are used to separate desired spore populations from other materials in the fecal material.
Also provided herein are methods of producing spore populations that combine two or more of the treatments described herein in order to synergistically purify the desired spores while killing or removing undesired materials and/or activities from the spore population. It is generally desirable to retain the spore populations under non-germinating and non-growth promoting conditions and media, in order to minimize the growth of pathogenic bacteria present in the spore populations and to minimize the germination of spores into vegetative bacterial cells.
Pharmaceutical Compositions and Formulations of the Invention
Formulations
Provided are formulations for administration to humans and other subjects in need thereof. Generally the bacterial compositions are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.
In some embodiments, the composition 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 CnH2nOn. 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.
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 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 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 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 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 bacterial 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 bacterial 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 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 (up to 100 mg) of lyophilized powder (108 to 1011 bacteria), 160 mg microcrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesium stearate. In an alternative embodiment, from 105 to 1012 bacteria may be used, 105 to 107, 106 to 107, or 108 to 1010, 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.
EXAMPLES
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
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).
Example 1
Provision of Fecal Material
Fresh fecal samples were obtained from healthy human donors who have been screened for general good health and for the absence of infectious diseases, and meet inclusion and exclusion criteria, inclusion criteria include being in good general health, without significant medical history, physical examination findings, or clinical laboratory abnormalities, regular bowel movements with stool appearance typically Type 2, 3, 4, 5 or 6 on the Bristol Stool Scale, and having a BMI ≧18 kg/m2 and ≦25 kg/m2. Exclusion criteria generally included significant chronic or acute medical conditions including renal, hepatic, pulmonary, gastrointestinal, cardiovascular, genitourinary, endocrine, immunologic, metabolic, neurologic or hematological disease, a family history of, inflammatory bowel disease including Crohn's disease and ulcerative colitis, Irritable bowel syndrome, colon, stomach or other gastrointestinal malignancies, or gastrointestinal polyposis syndromes, or recent use of yogurt or commercial probiotic materials in which an organism(s) is a primary component. Samples were collected directly using a commode specimen collection system, which contains a plastic support placed on the toilet seat and a collection container that rests on the support. Feces were deposited into the container, and the lid was then placed on the container and sealed tightly. The sample was then delivered on ice within 1-4 hours for processing. Samples were mixed with a sterile disposable tool, and 2-4 g aliquots were weighed and placed into tubes and flash frozen in a dry ice/ethanol bath. Aliquots are frozen at −80 degrees Celsius until use.
Optionally, the fecal material was suspended in a solution, and/or fibrous and/or particulate materials were removed. A frozen aliquot containing a known weight of feces was removed from storage at −80 degrees Celsius and allowed to thaw at room temperature. Sterile 1×PBS was added to create a 10% w/v suspension, and vigorous vortexing was performed to suspend the fecal material until the material appeared homogeneous. The material was then left to sit for 10 minutes at room temperature to sediment fibrous and particulate matter. The suspension above the sediment was then carefully removed into a new tube and contains a purified spore population. Optionally, the suspension was then centrifuged at a low speed, e.g., 1000×g, for 5 minutes to pellet particulate matter including fibers. The pellet was discarded and the supernatant, which contained vegetative organisms and spores, was removed into a new tube. The supernatant was then centrifuged at 6000×g for 10 minutes to pellet the vegetative organisms and spores. The pellet was then resuspended in 1×PBS with vigorous vortexing until the material appears homogenous.
Example 2
Spore Purification from Alcohol Treatment of Fecal Material
A 10% w/v suspension of human fecal material in PBS was mixed with absolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. The suspension was incubated at 37 degrees Celsius for 1 hour. After incubation the suspension was centrifuged at 13,000 rpm for 5 minutes to pellet spores. The supernatant was discarded and the pellet was resuspended in an equal volume of PBS. Glycerol was added to a final concentration of 15% and then the purified spore fraction is stored at −80 degrees Celsius.
Example 2A
Generation of a Spore Preparation from Alcohol Treatment of Fecal Material
A 10% w/v suspension of human fecal material in PBS was mixed with absolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. The suspension was incubated at 37 degrees Celsius for 1 hour. After incubation the suspension is centrifuged at 13,000 rpm for 5 minutes to concentrate spores into a pellet containing a purified spore-containing preparation. The supernatant was discarded and the pellet resuspended in an equal volume of PBS. Glycerol was added to a final concentration of 15% and then the purified spore preparation was stored at −80 degrees Celsius.
Example 3
Spore Purification from Thermal Treatment of Fecal Material
A 10% w/v suspension of human fecal material in PBS was incubated in a water bath at 80 degrees Celsius for 30 minutes. Glycerol was added to a final concentration of 15% and then the enriched spore containing material was stored at −80 degrees Celsius.
Example 4
Spore Purification from Alcohol Treatment and Thermal Treatment of Fecal Material
A 10% w/v suspension of human feces in PBS was mixed with absolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. The suspension was incubated in a water bath under aerobic conditions at 37 degrees Celsius for 1 hour. After incubation the suspension was centrifuged at 13,000 rpm for 5 minutes to pellet spores. The supernatant was discarded and the pellet was resuspended in equal volume PBS. The ethanol treated spore population was then incubated in a water bath at 80 degrees Celsius for 30 minutes. Glycerol was added to a final concentration of 15% and the purified spore fraction was stored at −80 C.
Example 5
Spore Purification from Detergent Treatment of Fecal Material
A 10% w/v suspension of human feces in PBS is prepared to contain a final concentration of 0.5 to 2% Triton X-100. After shaking incubation for 30 minutes at 25 to 37 degrees Celsius, the sample is centrifuged at 1000 g for 5-10 minutes to pellet particulate matter and large cells. The bacterial spores are recovered in the supernatant fraction, where the purified spore population is optionally further treated, such as in Example 4. Without being bound by theory, detergent addition to the fecal mixture produces better spore populations, at least in part by enhancing separation of the spores from particulates thereby resulting in higher yields of spores.
Example 6
Spore Purification by Chromatographic Separation of Fecal Material
A spore-enriched population such as obtained from Examples 1-5 above, is mixed with NaCl to a final concentration of 4M total salt and contacted with octyl Sepharose 4 Fast Flow to bind the hydrophobic spore fraction. The resin is washed with 4M NaCl to remove less hydrophobic components, and the spores are eluted with distilled water, and the desired enriched spore fraction is collected via UV absorbance.
Example 7
Spore Purification by Filtration of Fecal Material
A spore-enriched population such as obtained from Examples 1-6 above is diluted 1:10 with PBS, and placed in the reservoir vessel of a tangential flow microfiltration system. A 0.2 um pore size mixed cellulose ester hydrophilic tangential flow filter is connected to the reservoir such as by a tubing loop. The diluted spore preparation is recirculated through the loop by pumping, and the pressure gradient across the walls of the microfilter forces the supernatant liquid through the filter pores. By appropriate selection of the filter pore size the desired bacterial spores are retained, while smaller contaminants such as cellular debris, and other contaminants in feces such as bacteriophage pass through the filter. Fresh PBS buffer is added to the reservoir periodically to enhance the washout of the contaminants. At the end of the diafiltration, the spores are concentrated approximately ten-fold to the original concentration. The purified spores are collected from the reservoir and stored as provided herein.
Example 8
Characterization of Purified Spore Populations
Counts of viable spores are determined by performing 10 fold serial dilutions in PBS and plating to Brucella Blood Agar Petri plates or applicable solid media. Plates are incubated at 37 degrees Celsius for 2 days. Colonies are counted from a dilution plate with 50-400 colonies and used to back-calculate the number of viable spores in the population. The ability to germinate into vegetative bacteria is also demonstrated. Visual counts are determined by phase contrast microscopy. A spore preparation is either diluted in PBS or concentrated by centrifugation, and a 5 microliter aliquot is placed into a Petroff Hauser counting chamber for visualization at 400× magnification. Spores are counted within ten 0.05 mm×0.05 mm grids and an average spore count per grid is determined and used to calculate a spore count per ml of preparation. Lipopolysaccharide (LPS) reduction in purified spore populations is measured using a Limulus amebocyte lysate (LAL) assay such as the commercially available ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, N.J.) or other standard methods known to those skilled in the art.
Example 9
Determination of Bacterial Pathogens in Purified Spore Populations
Bacterial pathogens present in a purified spore population are determined by qPCR using specific oligonucleotide primers as follows.
Standard Curve Preparation. The standard curve is generated from wells containing the pathogen of interest at a known concentration or simultaneously quantified by selective spot plating. Serial dilutions of duplicate cultures are performed in sterile phosphate-buffered saline. Genomic DNA is then extracted from the standard curve samples along with the other samples.
Genomic DNA Extraction.
Genomic DNA may be extracted from 100 μl of fecal samples, fecal-derived samples, or purified spore preparations using the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) according to the manufacturer's instructions with two exceptions: the beadbeating is performed for 2×4:40 minutes using a BioSpec Mini-Beadbeater-96 (BioSpec Products, Bartlesville, Okla.) and the DNA is eluted in 50 μl of Solution C6. Alternatively the genomic DNA could be isolated using the Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), the Sigma-Aldrich Extract-N-AmpTM Plant PCR Kit, the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.
[qPCR Composition and Conditions.
The qPCR reaction to detect C. difficile contains 1× HotMasterMix (5PRIME, Gaithersburg, Md.), 900 nM of Wr-tcdB-F (AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG, (SEQ ID NO: 2040) IDT, Coralville, Iowa), 900 nM of Wr-tcdB-R (CATGCTTTTTTAGTTTCTGGATTGAA, (SEQ ID NO: 2041) IDT, Coralville, Iowa), 250 nM of We-tcdB-P (6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQ ID NO: 2042), Life Technologies, Grand Island, N.Y.), and PCR Water (Mo Bio Laboratories, Carlsbad, Calif.) to 18 μl (Primers adaped from: Wroblewski, D. et al. Rapid Molecular Characterization of Clostridium difficile and Assessment of Populations of C. difficile in Stool Specimens. Journal of Clinical Microbiology 47:2142-2148 (2009)). This reaction mixture is aliquoted to wells of a MicroAmp® Fast Optical 96-well Reaction Plate with Barcode (0.1 mL) (Life Technologies, Grand Island, N.Y.). To this reaction mixture, 2 μl of extracted genomic DNA is added. The qPCR is performed on a BioRad C1000TM Thermal Cycler equipped with a CFX96TM Real-Time System (BioRad, Hercules, Calif.). The thermocycling conditions are 95° C. for 2 minutes followed by 45 cycles of 95° C. for 3 seconds, 60° C. for 30 seconds, and fluorescent readings of the FAM and ROX channels. Other bacterial pathogens can be detected by using primers and a probe specific for the pathogen of interest.
Data Analysis.
The Cq value for each well on the FAM channel is determined by the CFX ManagerTM Software Version 2.1. The log 10 (cfu/ml) of each experimental sample is calculated by inputting a given sample's Cq value into linear regression model generated from the standard curve comparing the Cq values of the standard curve wells to the known log 10 (cfu/ml) of those samples.
[Viral pathogens present in a purified spore population are determined by qPCR as described herein and otherwise known in the art.
Example 10
Species Identification
The identity of the spore-forming species which grew up from a complex fraction can be determined in multiple ways. First, individual colonies can be picked into liquid media in a 96 well format, grown up and saved as 15% glycerol stocks at −80 C. Aliquots of the cultures can be placed into cell lysis buffer and colony PCR methods can be used to amplify and sequence the 16S rDNA gene (Example 2). Alternatively, colonies may be streaked to purity in several passages on solid media. Well separated colonies are streaked onto the fresh plates of the same kind and incubated for 48-72 hours at 37 C. The process is repeated multiple times in order to ensure purity. Pure cultures can be analyzed by phenotypic- or sequence-based methods, including 16S rDNA amplification and sequencing as described in Examples 11 & 12. Sequence characterization of pure isolates or mixed communities e.g. plate scrapes and spore fractions can also include whole genome shotgun sequencing. The latter is valuable to determine the presence of genes associated with sporulation, antibiotic resistance, pathogenicity, and virulence. Colonies can also be scraped from plates en masse and sequenced using a massively parallel sequencing method as described in Examples 11 & 12 such that individual 16S signatures can be identified in a complex mixture. Optionally, the sample can be sequenced prior to germination (if appropriate DNA isolation procedures are used to lsye and release the DNA from spores) in order to compare the diversity of germinable species with the total number of species in a spore sample. As an alternative or complementary approach to 16S analysis, MALDI-TOF-mass spec can also be used for species identification (as reviewed in Anaerobe 22:123).
Example 11
16s Sequencing to Determine Operational Taxonomic Unit (OTU)
Method for Determining 16S Sequence
OTUs may be defined either by full 16S sequencing of the rRNA gene, by sequencing of a specific hypervariable region of this gene (i.e. V1, V2, V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination of hypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial 16S rDNA is approximately 1500 nucleotides in length and 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 microbes.
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.
In addition to the 16S rRNA gene, one may define an OTU by sequencing a selected set of genes that are known to be marker genes for a given species or taxonomic group of OTUs. These genes may alternatively be assayed using a PCR-based screening strategy. As example, various strains of pathogenic Escherichia coli can be distinguished using DNAs from the genes that encode heat-labile (LTI, LTIIa, and LTIIb) and heat-stable (STI and STII) toxins, verotoxin types 1, 2, and 2e (VT1, VT2, and VT2e, respectively), cytotoxic necrotizing factors (CNF1 and CNF2), attaching and effacing mechanisms (eaeA), enteroaggregative mechanisms (Eagg), and enteroinvasive mechanisms (Einv). The optimal genes to utilize for taxonomic assignment of OTUs by use of marker genes will be familiar to one with ordinary skill of the art of sequence based taxonomic identification.
Genomic DNA Extraction
Genomic DNA is extracted from pure microbial cultures using a hot alkaline lysis method. 1 μl of microbial culture is added to 9 μl of Lysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the mixture is incubated at 95° C. for 30 minutes. Subsequently, the samples are cooled to 4° C. and neutralized by the addition of 10 μl of Neutralization Buffer (40 mM Tris-HCl) and then diluted 10-fold in Elution Buffer (10 mM Tris-HCl). Alternatively, genomic DNA is extracted from pure microbial cultures using commercially available kits such as the Mo Bio Ultraclean® Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) or by standard methods known to those skilled in the art.
Amplification of 16S Sequences for Downstream Sanger Sequencing
To amplify bacterial 16S rDNA (FIG. 1A), 2 μl of extracted gDNA is added to a 20 μl final volume PCR reaction. For full-length 16 sequencing the PCR reaction also contains 1× HotMasterMix (5FRIME, Gaithersburg, Md.), 250 nM of 27f (AGRGTTTGATCMTGGCTCAG (SEQ ID NO: 2033), IDT, Coralville, Iowa), and 250 nM of 1492r (TACGGYTACCTTGTTAYGACTT (SEQ ID NO: 2034), IDT, Coralville, Iowa), with PCR Water (Mo Bio Laboratories, Carlsbad, Calif.) for the balance of the volume. Alternatively, other universal bacterial primers or thermostable polymerases known to those skilled in the art are used. For example primers are available to those skilled in the art for the sequencing of the “V1-V9 regions” of the 16S rRNA (FIG. 1A). These regions refer 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. Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978). 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 (in FIG. 1A) by comparing the candidate sequence in question to the reference sequence (FIG. 1B) and identifying the hypervariable regions based on similarity to the reference hypervariable regions.
The PCR is performed on commercially available thermocyclers such as a BioRad MyCyclerTM Thermal Cycler (BioRad, Hercules, Calif.). The reactions are run at 94° C. for 2 minutes followed by 30 cycles of 94° C. for 30 seconds, 51° C. for 30 seconds, and 68° C. for 1 minute 30 seconds, followed by a 7 minute extension at 72° C. and an indefinite hold at 4° C. Following PCR, gel electrophoresis of a portion of the reaction products is used to confirm successful amplification of a −1.5 kb product.
To remove nucleotides and oligonucleotides from the PCR products, 2 μl of HT ExoSap-IT (Affymetrix, Santa Clara, Calif.) is added to 5 μl of PCR product followed by a 15 minute incubation at 37° C. and then a 15 minute inactivation at 80° C.
Amplification of 16S Sequences for Downstream Characterization by Massively Parallel Sequencing Technologies
Amplification performed for downstream sequencing by short read technologies such as Illumina require amplification using primers known to those skilled in the art that additionally include a sequence-based barcoded tag. As example, to amplify the 16s hypervariable region V4 region of bacterial 16S rDNA, 2 μl of extracted gDNA is added to a 20 μl final volume PCR reaction. The PCR reaction also contains 1× HotMasterMix (5FRIME, Gaithersburg, Md.), 200 nM of V4_515f_adapt (AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 2035), IDT, Coralville, Iowa), and 200 nM of barcoded 806rbc (CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 2036)_12 bpGolayBarcode_AGTCAGTCAGCCGGACTACHVGGGTVVTCTAAT (SEQ ID NO: 2037), IDT, Coralville, Iowa), with PCR Water (Mo Bio Laboratories, Carlsbad, Calif.) for the balance of the volume. These primers incorporate barcoded adapters for Illumina sequencing by synthesis. Optionally, identical replicate, triplicate, or quadruplicate reactions may be performed. Alternatively other universal bacterial primers or thermostable polymerases known to those skilled in the art are used to obtain different amplification and sequencing error rates as well as results on alternative sequencing technologies.
The PCR amplification is performed on commercially available thermocyclers such as a BioRad MyCyclerTM Thermal Cycler (BioRad, Hercules, Calif.). The reactions are run at 94° C. for 3 minutes followed by 25 cycles of 94° C. for 45 seconds, 50° C. for 1 minute, and 72° C. for 1 minute 30 seconds, followed by a 10 minute extension at 72° C. and a indefinite hold at 4° C. Following PCR, gel electrophoresis of a portion of the reaction products is used to confirm successful amplification of a  ̃1.5 kb product. PCR cleanup is performed as specified in the previous example.
Sanger Sequencing of Target Amplicons from Pure Homogeneous Samples
To detect nucleic acids for each sample, two sequencing reactions are performed to generate a forward and reverse sequencing read. For full-length 16s sequencing primers 27f and 1492r are used. 40 ng of ExoSap-IT-cleaned PCR products are mixed with 25 pmol of sequencing primer and Mo Bio Molecular Biology Grade Water (Mo Bio Laboratories, Carlsbad, Calif.) to 15 μl total volume. This reaction is submitted to a commercial sequencing organization such as Genewiz (South Plainfield, N.J.) for Sanger sequencing.
Massively Parallel Sequencing of Target Amplicons from Heterogeneous Samples
DNA Quantification & Library Construction. The cleaned PCR amplification products are quantified using the Quant-iTTM PicoGreene® dsDNA Assay Kit (Life Technologies, Grand Island, N.Y.) according to the manufacturer's instructions. Following quantification, the barcoded cleaned PCR products are combined such that each distinct PCR product is at an equimolar ratio to create a prepared Illumina library.
Nucleic Acid Detection. The prepared library is sequenced on Illumina HiSeq or MiSeq sequencers (Illumina, San Diego, Calif.) with cluster generation, template hybridization, iso-thermal amplification, linearization, blocking and denaturization and hybridization of the sequencing primers performed according to the manufacturer's instructions. 16SV4SeqFw (TATGGTAATTGTGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 2038)), 16SV4SeqRev (AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO: 2037)), and 16SV4Index (ATTAGAWACCCBDGTAGTCCGGCTGACTGACT (SEQ ID NO: 2039)) (IDT, Coralville, Iowa) are used for sequencing. Other sequencing technologies can be used such as but not limited to 454, Pacific Biosciences, Helicos, Ion Torrent, and Nanopore using protocols that are standard to someone skilled in the art of genomic sequencing.
Example 12
Sequence Read Annotation
Primary Read Annotation
Nucleic acid sequences are analyzed and annotations are to define taxonomic assignments using sequence similarity and phylogenetic placement methods or a combination of the two strategies. A similar approach can be used to annotate protein names, transcription factor names, and any other classification schema for nucleic acid sequences. Sequence similarity based methods include those familiar to individuals skilled in the art including, but not limited to BLAST, BLASTx, tBLASTn, tBLASTx, RDP-classifier, DNAclust, and various implementations of these algorithms such as Qiime or Mothur. These methods rely on mapping a sequence read to a reference database and selecting the match with the best score and e-value. Common databases include, but are not limited to the Human Microbiome Project, NCBI non-redundant database, Greengenes, RDP, and Silva. Phylogenetic methods can be used in combination with sequence similarity methods to improve the calling accuracy of an annotation or taxonomic assignment. Here tree topologies and nodal structure are used to refine the resolution of the analysis. In this approach we analyze nucleic acid sequences using one of numerous sequence similarity approaches and leverage phylogenetic methods that are well known to those skilled in the art, including but not limited to maximum likelihood phylogenetic reconstruction (see e.g. Liu K, Linder C R, and Warnow T. 2011. RAxML and FastTree: Comparing Two Methods for Large-Scale Maximum Likelihood Phylogeny Estimation. PLoS ONE 6: e27731. McGuire G, Denham M C, and Balding D J. 2001. Models of sequence evolution for DNA sequences containing gaps. Mol. Biol. Evol 18: 481-490. Wróbel B. 2008. Statistical measures of uncertainty for branches in phylogenetic trees inferred from molecular sequences by using model-based methods. J. Appl. Genet. 49: 49-67.) Sequence reads are placed into a reference phylogeny comprised of appropriate reference sequences. Annotations are made based on the placement of the read in the phylogenetic tree. The certainty or significance of the OTU annotation is defined based on the OTU's sequence similarity to a reference nucleic acid sequence and the proximity of the OTU sequence relative to one or more reference sequences in the phylogeny. As an example, the specificity of a taxonomic assignment is defined with confidence at the the level of Family, Genus, Species, or Strain with the confidence determined based on the position of bootstrap supported branches in the reference phylogenetic tree relative to the placement of the OTU sequence being interrogated.
Clade Assignments
The ability of 16S-V4 OTU identification to assign an OTU as a specific species depends in part on the resolving power of the 16S-V4 region of the 16S gene for a particular species or group of species. Both the density of available reference 16S sequences for different regions of the tree as well as the inherent variability in the 16S gene between different species will determine the definitiveness of a taxonomic annotation. Given the topological nature of a phylogenetic tree and the fact that tree represents hierarchical relationships of OTUs to one another based on their sequence similarity and an underlying evolutionary model, taxonomic annotations of a read can be rolled up to a higher level using a clade-based assignment procedure (Table 1). Using this approach, clades are defined based on the topology of a phylogenetic tree that is constructed from full-length 16S sequences using maximum likelihood or other phylogenetic models 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 (generally, 1-5 bootstraps), and (ii) within a 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. 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. The power of clade based analysis is that members of the same clade, due to their evolutionary relatedness, are likely to 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.
Notably, 16S sequences of isolates of a given OTU are phylogenetically placed within their respective clades, sometimes in conflict with the microbiological-based assignment of species and genus that may have preceded 16S-based assignment. Discrepancies between taxonomic assignment based on microbiological characteristics versus genetic sequencing are known to exist from the literature.
Example 13
Germinating Spores
Germinating a spore fraction increases the number of viable spores that will grow on various media types. To germinate a population of spores, the sample is moved to the anaerobic chamber, resuspended in prereduced PBS, mixed and incubated for 1 hour at 37 C to allow for germination. Germinants can include amino-acids (e.g., alanine, glycine), sugars (e.g., fructose), nucleosides (e.g., inosine), bile salts (e.g., cholate and taurocholate), metal cations (e.g., Mg2+, Ca2+), fatty acids, and long-chain alkyl amines (e.g., dodecylamine, Germination of bacterial spores with alkyl primary amines” J. Bacteriology, 1961.). Mixtures of these or more complex natural mixtures, such as rumen fluid or Oxgall, can be used to induce germination. Oxgall is dehydrated bovine bile composed of fatty acids, bile acids, inorganic salts, sulfates, bile pigments, cholesterol, mucin, lecithin, glycuronic acids, porphyrins, and urea. The germination can also be performed in a growth medium like prereduced BHIS/oxgall germination medium, in which BHIS (Brain heart infusion powder (37 g/L), yeast extract (5 g/L), L-cysteine HCl (1 g/L)) provides peptides, amino acids, inorganic ions and sugars in the complex BHI and yeast extract mixtures and Oxgall provides additional bile acid germinants.
In addition, pressure may be used to germinate spores. The selection of germinants can vary with the microbe being sought. Different species require different germinants and different isolates of the same species can require different germinants for optimal germination. Finally, it is important to dilute the mixture prior to plating because some germinants are inhibitory to growth of the vegetative-state microorganisms. For instance, it has been shown that alkyl amines must be neutralized with anionic lipophiles in order to promote optimal growth. Bile acids can also inhibit growth of some organisms despite promoting their germination, and must be diluted away prior to plating for viable cells.
For example, BHIS/oxgall solution is used as a germinant and contains 0.5×BHIS medium with 0.25% oxgall (dehydrated bovine bile) where 1×BHIS medium contains the following per L of solution: 6 g Brain Heart Infusion from solids, 7 g peptic digest of animal tissue, 14.5 g of pancreatic digest of casein, 5 g of yeast extract, 5 g sodium chloride, 2 g glucose, 2.5 g disodium phosphate, and 1 g cysteine. Additionally, Ca-DPA is a germinant and contains 40 mM CaCl2, and 40 mM dipicolinic acid (DPA). Rumen fluid (Bar Diamond, Inc.) is also a germinant. Simulated gastric fluid (Ricca Chemical) is a germinant and is 0.2% (w/v) Sodium Chloride in 0.7% (v/v) Hydrochloric Acid. Mucin medium is a germinant and prepared by adding the following items to 1 L of distilled sterile water: 0.4 g KH2PO4, 0.53 g Na2HPO4, 0.3 g NH4Cl, 0.3 g NaCl, 0.1 g MgCl2×6H2O, 0.11 g CaCl2, 1 ml alkaline trace element solution, 1 ml acid trace element solution, 1 ml vitamin solution, 0.5 mg resazurin, 4 g NaHCO3, 0.25 g Na2S×9H2O. The trace element and vitamin solutions prepared as described previously (Stams et al., 1993). All compounds were autoclaved, except the vitamins, which were filter-sterilized. The basal medium was supplemented with 0.7% (v/v) clarified, sterile rumen fluid and 0.25% (v/v) commercial hog gastric mucin (Type III; Sigma), purified by ethanol precipitation as described previously (Miller & Hoskins, 1981). This medium is referred herein as mucin medium.
Fetal Bovine Serum (Gibco) can be used as a germinant and contains 5% FBS heat inactivated, in Phosphate Buffered Saline (PBS, Fisher Scientific) containing 0.137M Sodium Chloride, 0.0027M Potassium Chloride, 0.0119M Phosphate Buffer. Thioglycollate is a germinant as described previously (Kamiya et al Journal of Medical Microbiology 1989) and contains 0.25M (pH10) sodium thioglycollate. Dodecylamine solution containing 1 mM dodecylamine in PBS is a germinant. A sugar solution can be used as a germinant and contains 0.2% fructose, 0.2% glucose, and 0.2% mannitol. Amino acid solution can also be used as a germinant and contains 5 mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mM proline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine. A germinant mixture referred to herein as Germix 3 can be a germinant and contains 5 mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mM proline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine, 0.2% taurocholate, 0.2% fructose, 0.2% mannitol, 0.2% glucose, 1 mM inosine, 2.5 mM Ca-DPA, and 5 mM KCl. BHIS medium+DPA is a germinant mixture and contains BHIS medium and 2 mM Ca-DPA. Escherichia coli spent medium supernatant referred to herein as EcSN is a germinant and is prepared by growing E. coli MG1655 in SweetB/Fos inulin medium anaerobically for 48 hr, spinning down cells at 20,000 rcf for 20 minutes, collecting the supernatant and heating to 60 C for 40 min. Finally, the solution is filter sterilized and used as a germinant solution.
Example 14
Selection of Media for Growth
It is important to select appropriate media to support growth, including preferred carbon sources. For example, some organisms prefer complex sugars such as cellobiose over simple sugars. Examples of media used in the isolation of sporulating organisms include EYA, BHI, BHIS, and GAM (see below for complete names and references). Multiple dilutions are plated out to ensure that some plates will have well isolated colonies on them for analysis, or alternatively plates with dense colonies may scraped and suspended in PBS to generate a mixed diverse community.
Plates are incubated anaerobically or aerobically at 37 C for 48-72 or more hours, targeting anaerobic or aerobic spore formers, respectively.
Solid plate media include:

    
    
        Gifu Anaerobic Medium (GAM, Nissui) without dextrose supplemented with fructooligosaccharides/inulin (0.4%), mannitol (0.4%), inulin (0.4%), or fructose (0.4%), or a combination thereof.
        Sweet GAM [Gifu Anaerobic Medium (GAM, Nissui)] modified, supplemented with glucose, cellobiose, maltose, L-arabinose, fructose, fructooligosaccharides/inulin, mannitol and sodium lactate)
        Brucella Blood Agar (BBA, Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010)
        PEA sheep blood (Anaerobe Systems; 5% Sheep Blood Agar with Phenylethyl Alcohol)
        Egg Yolk Agar (EYA) (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010)
        Sulfite polymyxin milk agar (Mevissen-Verhage et al., J. Clin. Microbiol. 25:285-289 (1987))
        Mucin agar (Derrien et al., IJSEM 54: 1469-1476 (2004))
        Polygalacturonate agar (Jensen & Canale-Parola, Appl. Environ. Microbiol. 52:880-997 (1986))
        M2GSC (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010)
        M2 agar (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010) supplemented with starch (1%), mannitol (0.4%), lactate (1.5 g/L) or lactose (0.4%)
        Sweet B—Brain Heart Infusion agar (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010) supplemented with yeast extract (0.5%), hemin, cysteine (0.1%), maltose (0.1%), cellobiose (0.1%), soluble starch (sigma, 1%), MOPS (50 mM, pH 7).
        PY-salicin (peptone-yeast extract agar supplemented with salicin) (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010).
        Modified Brain Heart Infusion (M-BHI) [[sweet and sour]] contains the following per L: 37.5 g Brain Heart Infusion powder (Remel), 5 g yeast extract, 2.2 g meat extract, 1.2 g liver extract, 1 g cystein HCl, 0.3 g sodium thioglycolate, 10 mg hemin, 2 g soluble starch, 2 g FOS/Inulin, 1 g cellobiose, 1 g L-arabinose, 1 g mannitol, 1 Na-lactate, 1 mL Tween 80, 0.6 g MgSO4×7H2O, 0.6 g CaCl2, 6 g (NH4)2SO4, 3 g KH2PO4, 0.5 g K2HPO4, 33 mM Acetic acid, 9 mM propionic acid, 1 mM Isobutyric acid, 1 mM isovaleric acid, 15 g agar, and after autoclaving add 50 mL of 8% NaHCO3 solution and 50 mL 1M MOPS-KOH (pH 7).
        Noack-Blaut Eubacterium agar (See Noack et al. J. Nutr. (1998) 128:1385-1391)
        BHIS az1/ge2-BHIS az/ge agar (Reeves et. al. Infect. Immun. 80:3786-3794 (2012)) [Brain Heart Infusion agar (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010) supplemented with yeast extract 0.5%, cysteine 0.1%, 0.1% cellobiose, 0.1% inulin, 0.1% maltose, aztreonam 1 mg/L, gentamycin 2 mg/L]
        BHIS CInM az1/ge2-BHIS CInM [Brain Heart Infusion agar (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010) supplemented with yeast extract 0.5%, cysteine 0.1%, 0.1% cellobiose, 0.1% inulin, 0.1% maltose, aztreonam 1 mg/L, gentamycin 2 mg/L]
    
    


Example 15
The Purification and Isolation of a Spore Forming Fraction from Feces
To purify and selectively isolate efficacious spores from fecal material a donation is first blended with saline using a homogenization device (e.g., laboratory blender) to produce a 20% slurry (w/v). 100% ethanol is added for an inactivation treatment that lasts 10 seconds to 1 hour. The final alcohol concentration can range from 30-90%, preferably 50-70%. High speed centrifugation (3200 rcf for 10 min) is performed to remove solvent and the pellet is retained and washed. Subsequently, once the washed pellet is resuspended, a low speed centrifugation step (200 rcf for 4 min) is performed to remove large particulate vegetative matter and the supernatant containing the spores is retained. High speed centrifugation (3200 rcf for 10 min) is performed on the supernatant to concentrate the spore material. The pellet is then washed and resuspended to generate a 20% slurry. This is the ethanol treated spore preparation. The concentrated slurry is then separated with a density based gradient e.g. a CsCl gradient, sucrose gradient or combination of the two generating a ethanol treated, gradient-purified spore preparation. For example, a CsCl gradient is performed by loading a 20% volume of spore suspension on top a 80% volume of a stepwise CsCl gradient (w/v) containing the steps of 64%, 50%, 40% CsCl (w/v) and centrifuging for 20 min at 3200 rcf. The spore fraction is then run on a sucrose step gradient with steps of 67%, 50%, 40%, and 30% (w/v). When centrifuged in a swinging bucket rotor for 10 min at 3200 rcf. The spores run roughly in the 30% and 40% sucrose fractions. The lower spore fraction (FIG. 2) is then removed and washed to produce a concentrated ethanol treated, gradient-purified spore preparation. Taking advantage of the refractive properties of spores observed by phase contrast microscopy (spores are bright and refractive while germinated spores and vegetative cells are dark) one can see an enrichment of the spore fraction from a fecal bacterial cell suspension (FIG. 3, left) compared to an ethanol treated, CsCl gradient purified, spore preparation (FIG. 3, center), and to an ethanol treated, CsCl gradient purified, sucrose gradient purified, spore preparation (FIG. 3, right).
Furthermore, growth of spores after treatment with a germinant can also be used to quantify a viable spore population. Briefly, samples were incubated with a germinant (Oxgall, 0.25% for up to 1 hour), diluted and plated anaerobically on BBA (Brucella Blood Agar) or similar media (e.g. see Examples 4 and 5). Individual colonies were picked and DNA isolated for full-length 16S sequencing to identify the species composition (e.g. see examples 2 and 3). Analysis revealed that 22 species were observed in total (Table 2) with a vast majority present in both the material purified with the gradient and without the gradient, indicating no or inconsequential shift in the ecology as a result of gradient purification. Spore yield calculations demonstrate an efficient recovery of 38% of the spores from the initial fecal material as measured by germination and plating of spores on BBA or measuring DPA count in the sample.
Example 16
Bacterial Compositions Prevent C. difficile Infection in a Mouse Model
To test the therapeutic potential of the bacterial compositions a prophylactic mouse model of C. difficile infection (model based on Chen, et al., A mouse model of Clostridium difficile associated disease, Gastroenterology 135(6):1984-1992) was used. Two cages of five mice each were tested for each arm of the experiment. All mice received an antibiotic cocktail consisting of 10% glucose, kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) and Vancomycin (0.056 mg/ml) in their drinking water on days −14 through −5 and a dose of 10 mg/kg Clindamycin by oral gavage on day −3. On day −1, they received either the test article or vehicle control via oral gavage. On day 0 they were challenged by administration of approximately 4.5 log 10 cfu of C. difficile (ATCC 43255) via oral gavage. Optionally a positive control group received vancomycin from day −1 through day 3 in addition to the antibiotic protocol and C. difficile challenge specified above. Feces were collected from the cages for analysis of bacterial carriage, mortality was assessed every day from day 0 to day 6 and the weight and subsequent weight change of the animal was assessed with weight loss being associated with C. difficile infection. Mortality and reduced weight loss of the test article compared to the vehicle were used to assess the success of the test article. Additionally, a C. difficile symptom scoring was performed each day from day −1 through day 6. Clinical Score was based on a 0-4 scale by combining scores for Appearance (0-2 pts based on normal, hunched, piloerection, or lethargic), and Clinical Signs (0-2 points based on normal, wet tail, cold-to-the-touch, or isolation from other animals).
In a naive control arm, animals were challenged with C. difficile. In the vancomycin positive control arm animals were dosed with C. difficile and treated with vancomycin from day −1 through day 3. The negative control was gavaged with PBS alone and no bacteria. The test arms of the experiment tested 1×, 0.1×, 0.01× dilutions derived from a single donor preparation of ethanol treated spores (e.g. see example 6) or the heat treated feces prepared by treating a 20% slurry for 30 min at 80 C. Dosing for CFU counts was determined from the final ethanol treated spores and dilutions of total spores were administered at 1×, 0.1×, 0.01× of the spore mixture for the ethanol treated fraction and a 1× dose for the heat treated fraction.
Weight loss and mortality were assessed on day 3. The negative control, treated with C. difficile only, exhibits 20% mortality and weight loss on Day 3, while the positive control of 10% human fecal suspension displays no mortality or weight loss on Day 3 (Table 3). EtOH-treated feces prevents mortality and weight loss at three dilutions, while the heat-treated fraction was protective at the only dose tested. These data indicate that the spore fraction is efficacious in preventing C. difficile infection in the mouse.
Example 17
The Prophylactic and Relapse Prevention Hamster Models
Previous studies with hamsters using toxigenic and nontoxigenic strains of C. difficile demonstrated the utility of the hamster model in examining relapse post antibiotic treatment and the effects of prophylaxis treatments with cecal flora in C. difficile infection (Wilson et al. 1981, Wilson et al. 1983, Borriello et al. 1985) and more broadly gastrointestinal infectious disease. To demonstrate prophylactic use of a test article to ameliorate C. difficile infection, the following hamster model is used. In a prophylactic model, Clindamycin (10 mg/kg s.c.) is given on day −5, the test article or control is administered on day −3, and C. difficile challenge occurs on day 0. In the positive control arm, vancomycin is then administered on day 1-5 (and vehicle control is delivered on day −3). Feces are collected on day −5, −4, −1, 1, 3, 5, 7, 9 and fecal samples are assessed for pathogen carriage and reduction by microbiological methods, 16S sequencing approaches or other methods utilized by one skilled in the art. Mortality is assessed throughout the experiment through 21 days post C. difficile challenge. The percentage survival curves show that ethanol treated spores and ethanol treated, gradient-purified spores better protect the hamsters compared to the Vancomycin control, and vehicle control.
See FIG. 4: Prophylaxis model with the ethanol treated spore preparation and the ethanol treated, gradient-purified spore preparation.
In the relapse prevention model, hamsters are challenged with toxigenic C. difficile strains on day 0, and treated with clindamycin by oral gavage on day 1, and vancomycin dosing day 2-6. Test or control treatment was then administered on day 7, 8, and 9. The groups of hamsters for each arm consist of 8 hamsters per group. Fecal material is collected on day −1, 1, 3, 5, 7, 10 and 13 and hamster mortality is assessed throughout. Survival curves are used to assess the success of the test article e.g. ethanol treated or ethanol treated, gradient purified spores versus the control treatment in preventing hamster death. The survival curves demonstrate maximum efficacy for the ethanol treated, gradient-purified spores followed by the ethanol treated spores. Both treatments improved survival percentage over vancomycin treatment alone.
See FIG. 5: Relapse prevention model with ethanol treated spores and ethanol treated, gradient purified spores
Example 18
Clinical Treatment of Recurrent C. difficile in Patients
To assess the efficacy of a test article (e.g., ethanol treated spore preparations, see Example 15) to treat recurrent C. difficile in human patients, the following procedure was performed to take feces from a healthy donor, inactivate via the ethanol treated spore preparation protocol described below, and treat recurrent C. difficile in patients presenting with this indication. Non-related donors were screened for general health history for absence of chronic medical conditions (including inflammatory bowel disease; irritable bowel syndrome; Celiac disease; or any history of gastrointestinal malignancy or polyposis), absence of risk factors for transmissible infections, antibiotic non-use in the previous 6 months, and negative results in laboratory assays for blood-borne pathogens (HIV, HTLV, HCV, HBV, CMV, HAV and Treponema pallidum) and fecal bacterial pathogens (Salmonella, Shigella, Yersinia, Campylobacter, E. coli 0157), ova and parasites, and other infectious agents (Giardia, Cryptosporidium Cyclospora, Isospora) prior to stool donation.
Donor stool was frozen shortly after donation and sampled for testing. At the time of use, approximately 75 g of donor stool was thawed and resuspended in 500 mL of non-bacteriostatic normal saline and mixed in a single use glass or plastic blender. The resulting slurry was sequentially passed through sterile, disposable mesh screens that remove particles of size 600, 300 and 200 microns. The slurry was then centrifuged briefly (200 rcf for 4 min) to separate fibrous and particulate materials, and the supernatant (containing bacterial cells and spores) was transferred to a fresh container. Ethanol was added to a final concentration of 50% and the resulting  ̃1500 ml slurry was incubated at room temperature for 1 hr with continuous mixing to inactivate vegetative bacterial cells. Midway through inactivation the slurry was transferred to a new bottle to ensure complete contact with the ethanol. The solid matter was pelleted in a centrifuge and washed 3 times with normal saline to remove residual ethanol. The final pellet was resuspended in 100% sterile, USP glycerol at a minimum volume, and filled into approximately 30 size 0 delayed release capsules (hypromellose DRcaps, Capsugel, Inc.) at 0.65 mL suspension each. The capsules were immediately capped and placed onto an aluminum freezing block held at −80° C. via dry ice to freeze. The frozen capsules were in turn over-capsulated with size 00 DRcaps to enhance capsule stability, labeled, and placed into <−65° C. storage immediately. The final product was stored at <−65° C. until the day and time of use. Encapsulated product may be stored for indefinitely at <−65° C. On the day of dosing capsules were warmed on wet ice for 1 to 2 hours to improve tolerability, and were then dosed with water ad libitium.
Patient 1 is a 45-year old woman with a history of C. difficile infection and diarrhea for at least 1 year prior to treatment. She has been previously treated with multiple courses of antibiotics followed each time by recurrence of C. difficile-associated diarrhea.
Patient 2 is an 81-year old female who has experienced recurrent C. difficile infection for 6 months prior to treatment despite adequate antibiotic therapy following each recurrence.
24 hours prior to starting oral treatment, CDAD antibiotic therapy was discontinued. Each patient received a colon preparation procedure intended to reduce the competing microbial burden in the gastrointestinal tract and to facilitate repopulation by the spore forming organisms in the investigational product.
On the morning of the first treatment day, the patients received a dose of delayed release capsules containing the investigational product with water ad libitum. Patients were requested to avoid food for 1 hour thereafter. The next day, the patient returned to the clinic to receive an additional dose. Patients were asked to avoid food for 4 hours prior to receiving their second dose and for 1 hour following dosing.
Both patients were followed closely for evidence of relapse or adverse symptoms following treatment. Patients were contacted by phone on Day 2, Day 4, and Weeks 1, 2 and 4 and each was queried about her general status and the condition of her CDAD and related symptoms. Stool samples were collected at baseline and Weeks 1, 2, 4 and 8 post-treatment to assess changes in the gut microbiota via 16S sequencing and spore count with methods explained previously (e.g. see Examples 11 and 12). Through 4 weeks post treatment, each patient has gradually improved with no evidence of C. difficile recurrence.
Six other patients with recurrent C. difficile-associated diarrhea were treated in a similar fashion, with no CDI recurrence and no requirement for resumption of antibiotics (total of 8 patients). Additionally, there were no treatment-related serious adverse events.
Example 19
Treatment of Fecal Suspensions with Ethanol or Heat Drastically Reduces Vegetative Cell Numbers and Results in an Enrichment of Spore Forming Species
Treatment of a sample, preferably a human fecal sample, in a manner to inactivate or kill substantially all of the vegetative forms of bacteria present in the sample results in selection and enrichment of the spore fraction. Methods for inactivation can include heating, sonication, detergent lysis, enzymatic digestion (such as lysozyme and/or proteinase K), ethanol or acid treatment, exposure to solvents (Tetrahydrofuran, 1-butanol, 2-butanol, 1,2 propanediol, 1,3 propanediol, butanoate, propanoate, chloroform, dimethyl ether and a detergent like triton X-100, diethyl ether), or a combination of these methods. To demonstrate the efficacy of ethanol induced inactivation of vegetative cells, a 10% fecal suspension was mixed with absolute ethanol in a 1:1 ratio and vortexed to mix for 1 min. The suspension was incubated at room temperature for 30 min, 1 h, 4 h or 24 h. After incubation the suspension was centrifuged at 13,000 rpm for 5 min to pellet spores. The supernatant is discarded and the pellet is resuspended in equal volume of PBS. Viable cells were measured as described below.
To demonstrate the efficacy of heat treatment on vegetative cell inactivation a 10-20% fecal suspension was incubated at 70 C, 80 C, 90 C or 100 C for 10 min or 1 h.
After ethanol or heat treatment, remaining viable cells were measured after 24 h incubation on plates by determining the bacterial titer on Brucella blood agar (BBA) as a function of treatment and time (See FIG. 6). Ethanol treatment for 1 h and 25 h have similar effects, reducing the number of viable cells by approximately 4 logs, while increasing temperature and time at high temperature leads to higher losses in viable cell number, with no colonies detectable at 100° C. at either 10 min or 1 h. In this experiment no germinants were used. After several days of additional growth on plates, a number of colonies were picked from these treated samples and identified by 16S rDNA analysis (e.g. see Examples 11 and 12). These included known spore forming Clostridium spp. as well as species not previously reported to be spore formers including Ruminococcus bromii, and Anaerotruncus colihominis (Lawson, et al 2004), and a Eubacterium sp. (Table 4). See FIG. 6: Heat and ethanol treatments reduce cell viability
To demonstrate that vegetative cells are greatly reduced by ethanol treatment, known non-spore forming bacteria are ethanol treated as described previously (e.g. see Example 15) and viability was determined by plating on BBA in anaerobic conditions (e.g. see Example 14). Fecal material from four independent donors was exposed to 60 C for 5 min and subsequently plated on three types of selective media under either aerobic (+O2) or anaerobic conditions (−O2) (BBA+aerobic, MacConkey lactose+aerobic, Bacteroides Bile esculin+anaerobic) to identify known nonsporeforming Enterobacteria (survivors on MacConkey agar) and Bacteroides fragilis group species (survivors on Bacteroides Bile Esculin plates). The detectable limit for these assays was roughly 20 cfu/mL. Germinants were not used in this experiment (FIG. 7). Both ethanol and heat inactivation greatly reduces the cell viability from fecal material to the limit of detection under using MacConkey lactose agar and BBE agar. The remaining cells identified on BBA media grown in anaerobic conditions comprise the non-germinant dependent spore forming species. See FIG. 7: Reduction in non-spore forming vegetative cells by treatment at 60° C. for 5 min
Additionally, the ethanol treatment was shown to rapidly kill both aerobic and non-spore forming anaerobic colony forming units in 10% fecal suspensions as determined by plating on rich (BBA) media. The reduction of plated CFUs decreases four orders of magnitude in seconds as shown in FIG. 8.
See FIG. 8: Time course demonstrates ethanol reduces both anaerobic and aerobic bacterial CFUs
Example 20
Species Identified and Isolated as Spore Formers by Ethanol Treatment
To demonstrate that spore-forming species are enriched by heat or ethanol treatment methods, a comparison of >7000 colony isolates was performed to identify species in a repeatable fashion (e.g., identified independently in multiple preparations, see examples 1, 2, and 3) only isolated from fecal suspensions treated with 50% ethanol or heat treatment and not from untreated fecal suspensions (Table 5). These data demonstrate the ability to select for spore forming species from fecal material, and identify organisms as spore formers not previously described as such in the literature. In each case, organisms were picked as an isolated colony, grown anaerobically, and then subjected to full-length 16S sequencing in order to assign species identity.
To further identify spore formers, ethanol treated fecal samples from donors A, B, C, D, E and F were plated to a variety of solid media types, single colonies were picked and grown up in broth in a 96 well format (Table 6-11). The 16S rRNA gene was then amplified by PCR and direct cycle sequencing was performed (See examples 11 and 12). The ID is based on the forward read from direct cycle sequencing of the 16S rRNA gene.
There is surprising heterogeneity in the microbiome from one individual to another (Clemente et al., 2012) and this has consequences for determining the potential efficacy of various donors to generate useful spore compositions. The method described below is useful for screening donors when, for instance, a particular quantity or diversity of spore forming organisms is useful or desired for repopulating the microbiome following antibiotic treatment or treating a particular disease or condition. Further, such screening is useful when there is a need to screen donors for the purpose of isolating microorganisms capable of spore formation, or when a purified preparation of spore forming organisms is desired from a particular donor.
Total spore count is also a measure of potency of a particular donation or purified spore preparation and is vital to determine the quantity of material required to achieve a desired dose level. To understand the variability in total spore counts, donor samples were collected and processed as described in prior examples. Donor spore counts in CFU/g were then determined by growth on media plates at various titrations to determine the spore content of the donation. Furthermore, DPA assays were used to assess spore content (expressed as spore equivalents) as described in Example 21. As seen in FIG. 9, there is as much as two logs difference in an individual donor over time and can be up to three logs difference between donors. One possible reason for the difference in spore content measures is that nonviable spores and non-germinable spores will not be observed by plating but will have measurable DPA content. Another possibility is the variability between species of DPA content in spores making some complex mixtures containing high DPA spores while other mixtures contain low DPA content spores. Selecting donors with high spore counts is important in determining productivity of isolating spores from fecal donations by identifying preferred donors.
See FIG. 9: Donation Spore concentrations from clinical donors
A fresh fecal sample from donor F was treated as described in Example 15 to generate an ethanol treated spore fraction, germinated with BHIS/Oxgall for 1 h as a described (e.g. see Example 13), then plated to a variety of media (e.g. See example 14). Colonies were picked with a focus on picking several of each type of morphologically distinct colony on each plate to capture as much diversity as possible. Colonies are counted on a plate of each media type with well isolated colonies such that the number of colony forming units per ml can be calculated (Table 12). Colonies were picked into one of several liquid media and the 16S rDNA sequences (e.g. see Examples 11 and 12) were determined and analyzed as described above. The number of unique OTUs for each media type is shown below with the media with the most unique OTUs at the top (Table 12). Combinations of 3 to 5 of the top 5 media types capture diversity, and some other can be chosen to target specific species of interest. Colony forming units can be calculated for a given species using the 16S data, and could be used to determine whether a sufficient level of a given organism is present. The spore complement from Donor F as determined in this experiment includes these 52 species as determined by 16S sequencing (Table 12).
To screen human donors for the presence of a diversity of spore forming bacteria and/or for specific spore-forming bacteria, fecal samples were prepared using germinants and selective plating conditions and colonies were picked (e.g. see Examples 13 and 14) and analyzed for 16S diversity as described previously (see Examples 11 and 12). An assessment of donor diversity could include the cfu/ml of ethanol resistant cells on a given media type, or cfu/ml of a given species using the 16S analysis of colonies picked from that media to determine the level of spores of a given species of interest. This type of culture-based analysis could be complemented by culture-independent methods such as qPCR with probes specific to species or genera of interest or metagenomic sequencing of spore preparations, or 16S profiling of spore preparations using Illumina 16S variable region sequencing approaches (e.g. see Examples 11 and 12).
Example 21
Quantification of Spore Concentrations Using DPA Assay
Methods to assess spore concentration in complex mixtures typically require the separation and selection of spores and subsequent growth of individual species to determine the colony forming units. The art does not teach how to quantitatively germinate all the spores in a complex mixture as there are many species for which appropriate germinants have not been identified. Furthermore, sporulation is thought to be a stochastic process as a result of evolutionary selection, meaning that not all spores from a single species germinate with same response to germinant concentration, time and other environmental conditions. Alternatively, a key metabolite of bacterial spores, dipicolinic acid (DPA) has been developed to quantify spores particles in a sample and avoid interference from fecal contaminants. The assay utilizes the fact that DPA chelates Terbium 3+ to form a luminescent complex (Fichte) et al, FEMS Microbiology Ecology, 2007; Kort et al, Applied and Environmental Microbiology, 2005; Shafaat and Ponce, Applied and Environmental Microbiology, 2006; Yang and Ponce, International Journal of Food Microbiology, 2009; Hindle and Hall, Analyst, 1999). A time-resolved fluorescence assay detects terbium luminescence in the presence of DPA giving a quantitative measurement of DPA concentration in a solution.
To perform the assay 1 mL of the spore standard to be measured was transferred to a 2 mL microcentrifuge tube. The samples were centrifuged at 13000 RCF for 10 min and the sample is washed in 1 mL sterile deionized H2O. Wash an additional time by repeating the centrifugation. Transfer the 1 mL solution to hungate tubes and autoclave samples on a steam cycle for 30 min at 250 C. Add 100 uL of 30 uM TbCl3 solution (400 mM sodium acetate, pH 5.0, 30 μM TbCl3) to the sample. Make serial dilutions of the autoclaved material and measure the fluorescence of each sample by exciting with 275 nm light and measuring the emission wavelength of 543 nm for an integration time of 1.25 ms and a 0.1 ms delay.
Purified spores are produced as described previously (e.g. see http://www.epa.gov/pesticides/methods/MB-28-00.pdf). Serial dilutions of purified spores from C. bifermentans, C. sporogenes, and C. butyricum cultures were prepared and measured by plating on BBA media and incubating overnight at 37 C to determine CFU/ml. FIG. 10 shows the linear correspondence across different spore producing bacteria across several logs demonstrating the DPA assay as means to assess spore content.
See FIG. 10: Linear range of DPA assay compared to CFU counts/ml
The discrepancy for complex spore populations between spore counts measured by germinable spore CFU and by DPA has important implications for determining the potency of an ethanol treated spore preparation for clinical use. Table AC shows spore content data from 3 different ethanol treated spore preparations used to successfully treat 3 patients suffering from recurrent C. difficile infection. The spore content of each spore preparation is characterized using the two described methods.







TABLE AC







Spore quantitation for ethanol treated spore preparations


using spore CFU (SCFU) assay and DPA assay













SCFU/
DPA SEq/
Ratio



Preparation
30 capsules
30 capsules
SCFU/DPA







Preparation 1
4.0 × 10{circumflex over ( )}5
6.8 × 10{circumflex over ( )}7
5.9 × 10−3



Preparation 2
2.1 × 10{circumflex over ( )}7
9.2 × 10{circumflex over ( )}8
0.023



Preparation 3
6.9 × 10{circumflex over ( )}9
9.6 × 10{circumflex over ( )}9
0.72










What is immediately apparent is that spore content varies greatly per 30 capsules. As measured by germinable SCFU, spore content varies by greater than 10,000-fold. As measured by DPA, spore content varies by greater than 100-fold. In the absence of the DPA assay, it would be difficult to set a minimum dose for administration to a patient. For instance, without data from the DPA assay, one would conclude that a minimum effective dose of spores is 4×105 or less using the SCFU assay (e.g. Preparation 1, Table AC). If that SCFU dose was used to normalize dosing in a clinical setting, however, then the actual spore doses given to patients would be much lower for other ethanol treated spore preparations as measured as by the DPA assay (Table AD).







TABLE AD







DPA doses in Table AC when normalized


to 4 × 105 SCFU per dose











SCFU/
DPA SEq/
Fraction of Prep-


Preparation
30 capsules
30 capsules
aration 1 Dose













Preparation 1
4.0 × 10{circumflex over ( )}5
6.8 × 10{circumflex over ( )}7
1.0


Preparation 2
4.0 × 10{circumflex over ( )}5
1.8 × 10{circumflex over ( )}7
0.26


Preparation 3
4.0 × 10{circumflex over ( )}5
5.6 × 10{circumflex over ( )}5
0.0082









It becomes clear from the variability of SCFU and DPA counts across various donations that using SCFU as the measure of potency would lead to significant underdosing in certain cases. For instance, setting a dose specification of 4×105 SCFU (the apparent effective dose from donor Preparation 1) for product Preparation 3 would lead to a potential underdosing of more than 100-fold. This can be rectified only by setting potency specifications based on the DPA assay, which better reflects total spore counts in an ethanol treated spore preparation. The unexpected finding of this work is that the DPA assay is uniquely suited to set potency and determine dosing for an ethanol treated spore preparation.
Example 22
Demonstration of Enhanced Growth with a Germinant
To enhance the ethanol treated spores germination capability and demonstrate spore viability, spores from three different donors were germinated by various treatments and plated on various media. Germination with BHIS/oxgall (BHIS ox), Ca-DPA, rumen fluid (RF), simulated gastric fluid (SGF), mucin medium (Muc), fetal bovine serum (FBS), or thioglycollate (Thi) for 1 hour at 37 C in anaerobic conditions was performed as described previously (e.g. see Examples 13 and 14) with samples derived from two independent donors (FIG. 11). The spore-germinant mixture was serially diluted and plated on different plate media including BBA, Sweet B, Sweet B+lysozyme (2 ug/ml), M2GSC and M2GSC+lysozyme (2 ug/ml) as previously described (e.g. see Examples 13 and 14) to determine spore germination. Colony forming units were tallied and titers were determined using standard techniques by one skilled in the art. As FIG. 11 shows, maximum colony forming units are derived from BHI-oxgall treatment. This germination treatment also greatly increases the diversity as measured by the number of OTUs identified when samples were submitted for 16S sequencing (e.g. see Examples 11 and 12) compared to plating without a germination step (FIG. 12). As shown in FIG. 11: Different germinant treatments have variable effects on CFU counts from donor A (upper left) and donor B (lower right). The Y-Axes are spore CFU per ml. As shown in FIG. 12: Germinates greatly increase the diversity of cultured spore forming OTUs.
To test the effect of heat activation to promote germination, ethanol treated fecal samples were treated for 15 min at room temperature, 55 C, 65 C, 75 C or 85 C from three different donors and germinated subsequently with BHIS+ Oxgall for 1 hr at 37 C then plated on BBA media (FIG. 13) as previously described (e.g. see Examples 13 and 14). Pretreatment at room temperature produced equal if not more spores than the elevated temperatures in all three donors. The temperature of germinating was also examined by incubating samples at room temperature or 37 C for 1 hr in anaerobic conditions before plating on BBA. No difference in the number of CFUs was observed between the two conditions. Lysozyme addition to the plates (2 ug/ml) was also tested on a single donor sample by the testing of various activation temperature followed by an incubation in the presence or absence of lysozyme. The addition of lysozyme had a small effect when plated on Sweet B or M2GSC media but less so than treatment with BHIS oxgall without lysozyme for 1 hr (FIG. 14).
As shown in FIG. 13: Heat Activation as a germination treatment with BHIS+oxgall. As shown in FIG. 14: Effect of lysozyme slightly enhances germination.
Germination time was also tested by treating a 10% suspension of a single donor ethanol treated feces (e.g. see Example 15) incubated in either BHIS, taurocholate, oxgall, or germix for 0, 15, 30, or 60 minutes and subsequently plated on BHIS, EYA, or BBA media (e.g. see Examples 13 and 14). 60 minutes resulted in the most CFU units across all various combinations germinates and plate media tested.
Example 23
Demonstrating Efficacy of Germinable and Sporulatable Fractions of Ethanol Treated Spores
To define methods for characterization and purification, and to improve (e.g., such as by modulating the diversity of the compositions) the active spore forming ecology derived from fecal donations, the ethanol treated spore population (as described in Example 15) was further fractionated. A “germinable fraction” was derived by treating the ethanol-treated spore preparation with oxgall, plating to various solid media, and then, after 2 days or 7 days of growth, scraping all the bacterial growth from the plates into 5 mL of PBS per plate to generate a bacterial suspension. A “sporulatable fraction” was derived as above except that the cells were allowed to grow on solid media for 2 days or 7 days (the time was extended to allow sporulation, as is typical in sporulation protocols), and the resulting bacterial suspension was treated with 50% ethanol to derive a population of “sporulatable” spores, or species that were capable of forming spores. In preparing these fractions, fecal material from donor A was used to generate an ethanol treated spore preparation as previously described in Example 15; then spore content was determined by DPA assay and CFU/ml grown on various media (FIG. 15) as previously described (see Example 21). See FIG. 15: Spores initially present in ethanol treated spore preparation as measured by DPA and CFU/ml grown on specified media.
To characterize the fraction that is sporulatable, the 2 day and 7 day “germinable” fractions were assessed for CFU and DPA content before and after ethanol treatment to generate a spore fraction. Bacterial suspensions were treated with ethanol, germinated with Oxgall, and plated on the same types of media that the “germinable” fraction was grown on. DPA data showed that growth on plates for 2 and 7 days produced the same amount of total spores. Colonies on the several types of media were picked for 16S sequence analysis to identify the spore forming bacteria present (Table 13).
A 2 day “germinable” fraction and a 7 day “sporulatable” fraction were used as a treatment in the mouse prophylaxis assay as described (e.g. see Example 16). As a control, a 10% fecal suspension prepared from a donor (Donor B) was also administered to mice to model fecal microbiota transplant (FMT). Weight loss and mortality of the various test and control arms of the study are plotted in FIG. 17 and summarized in Table 15 which also contains the dosing information. The data clearly shows both the “germinable” and “sporulatable” fractions are efficacious in providing protection against C. difficile challenge in a prophylaxis mouse model (e.g. see Example 16). The efficacy of these fractions further demonstrates that the species present are responsible for the efficacy of the spore fraction, as the fractionation further dilutes any potential contaminant from the original spore preparation.
See FIG. 16: Titer of “germinable” fraction after 2 days (left) and Sporulatable fraction (right) by DPA and CFU/ml. The “sporulatable” fraction made following 7 days of growth was measured as previously described using germination and growth assays or DPA content as previously described (see Example 21).
The species present in the “germinable” and “sporulatable” fractions were determined by full length 16S sequencing of colony picks and by 16S NGS sequencing of the fractions themselves. The colony pick data indicate Clostridium species are very abundant in both fractions, while the NGS data reveal other spore forming organisms that are typically found in ethanol treated spore preparations are present.
Results are shown in the following: See Table 13. Species identified as “germinable” and “sporulatable” by colony picking approach. See Table YYY. Species identified as “germinable” using 16S-V4 NGS approach. See Table ZZZ. Species identified as “sporulatable” using 16s-V4 NGS approach. See FIG. 17: Mouse prophylaxis model demonstrates “germinable” and “sporulatable” preparations are protective against C. difficile challenge. Each plot tracks the change in the individual mouse's weight relative to day −1 over the course of the experiment. The number of deaths over the course of the experiment is indicated at the top of the chart and demonstrated by a line termination prior to day 6. The top panels (from left to right) are the vehicle control arm, the fecal suspension arm, and the untreated naive control arm, while the bottom panels are the ethanol treated, gradient purified spore preparation; the ethanol treated, gradient purified, “germinable” preparation, and ethanol treated, gradient purified, “sporulatable” preparation. See Table 15: Results of the prophylaxis mouse model and dosing information
Example 24
Donor Pooling Efficacy in Prophylaxis Mouse
To test the efficacy and dosing of pooled donor samples the C. difficile prophylaxis mouse model (e.g., see Example 16) is used with donations mixed from two or more donor samples as previously described. Weight loss and mortality with the mixed spore product versus the spore product derived from a single donor at the various dosing is determine whether the two treatment schemes are equivalent or one is significantly better than the other.
Dosing of the spore product derived from a single or multiple donors is between 1E4 to 1E10 CFU/ml. The spore product is mixed from product derived from any number of donors ranging from 1-10 at either equal concentrations or different known concentrations.
Additionally, this method can be used to expand the spore fraction for production purposes. For production purposes, an enriched spore fraction (e.g.—a purified and EtOH treated fraction of a fecal sample) is preserved in multiple aliquots to form a bank of viable spore-forming organisms. An aliquot of this bank is then recovered by germinant treatment followed by cultivation in a medium, and under conditions, that are broadly permissive for spore-forming organisms and encourage sporulation. After a suitable amplification time, the amplified bacteria including spores are harvested, and this preparation is solvent or heat-treated to isolate the spore fraction. This fraction may be further purified away from non-spore forms and culture constituents. The process of amplification, spore isolation and optional purification may be repeated at increasing scales to generate large quantities for further use. When enough germinable/sporulatable material has been accumulated by amplification, it may be further purified, concentrated or diluted, and/or preserved to a state suitable for further use, e.g.—clinical dosing.
Features may be incorporated into the above process to make it suitable for further utility, especially for product applications such as clinical use. The production of the initial spore fraction may be conducted under controlled conditions (cGMP's) and validated to remove non-spore organisms to a high degree. The germination may be conducted using reagents that are more standardizable than natural products such as oxgall, e.g.—synthetic mixtures of bile salts. Amplification may be done using media with components that are preferred for clinical safety, e.g.—sourced from qualified animals, or non-animal sourced. Conditions may be arranged so as to ensure consistent compositions of sporulated organisms, are less prone to contamination, and are more amenable to scale-up, e.g.—closed stirred fermenters with feedback control loops. Sporulated organisms from the process may be isolated using procedures that alone or combined stringently eliminate non-spores and other process residuals, e.g.—solvent treatment, aqueous two-phase extraction, and/or 60° C. long-time heat treatment. Preservation may involve addition of excipients and/or adjustment of conditions to enable conversion to a preferred dosage form amenable to long-term shelf storage, e.g.—addition of trehalose, followed by lyophilization or spray drying, further blending of the powder with microcrystalline cellulose, and encapsulation in a gelatin capsule to form an orally dosable product.
Example 25
Engraftment, Augmentation and Reduction of Pathogen Carriage in Patients Treated with Spore Compositions
Complementary genomic and microbiological methods were used to characterize the composition of the microbiota from Patient 1, 2, 3, 4, and 5, 6, 7, 8, 9, and 10 at pretreatment (pretreatment) and on up to 4 weeks post-treatment.
To determine the OTUs that engraft from treatment with an ethanol treated spore preparation in the patients and how their microbiome changed in response, the microbiome was characterized by 16S-V4 sequencing prior to treatment (pretreatment) with an ethanol treated spore preparation and up to 25 days after receiving treatment. As example, the treatment of patient 1 with an ethanol treated spore preparation led to the engraftment of OTUs from the spore treatment and augmentation in the microbiome of the patient (FIG. 18 and FIG. 19). By day 25 following treatment, the total microbial carriage was dominated by species of the following taxonomic groups: Bacteroides, Sutterella, Ruminococcus, Blautia, Eubacterium, Gemmiger/Faecalibacterium, and the non-sporeforming Lactobacillus (see Table 16 and Table 2 for specific OTUs). The first two genera represent OTUs that do not form spores while the latter taxonomic groups represent OTUs that are believed to form spores.
Patient treatment with the ethanol treated spore preparation leads to the establishment of a microbial ecology that has greater diversity than prior to treatment (FIG. 18). Genomic-based microbiome characterization confirmed engraftment of a range of OTUs that were absent in the patient pretreatment (Table 16). These OTUs comprised both bacterial species that were capable and not capable of forming spores, and OTUs that represent multiple phylogenetic clades. Organisms absent in Patient 1 pre-treatment either engraft directly from the ethanol treated spore fraction or are augmented by the creation of a gut environment favoring a healthy, diverse microbiota. Furthermore, Bacteroides fragilis group species were increased by 4 and 6 logs in patients 1 and 2 (FIG. 20).
OTUs that comprise an augmented ecology are not present in the patient prior to treatment and/or exist at extremely low frequencies such that they do not comprise a significant fraction of the total microbial carriage and are not detectable by genomic and/or microbiological assay methods. OTUs that are members of the engrafting and augmented ecologies were identified by characterizing the OTUs that increase in their relative abundance post treatment and that respectively are: (i) present in the ethanol treated spore preparation and absent in the patient pretreatment, or (ii) absent in the ethanol treated spore preparation, but increase in their relative abundance through time post treatment with the preparation due to the formation of favorable growth conditions by the treatment. Notably, the latter OTUs can grow from low frequency reservoirs in the patient, or be introduced from exogenous sources such as diet. OTUs that comprise a “core” augmented or engrafted ecology can be defined by the percentage of total patients in which they are observed to engraft and/or augment; the greater this percentage the more likely they are to be part of a core ecology responsible for catalyzing a shift away from a dysbiotic ecology. The dominant OTUs in an ecology can be identified using several methods including but not limited to defining the OTUs that have the greatest relative abundance in either the augmented or engrafted ecologies and defining a total relative abundance threshold. As example, the dominant OTUs in the augmented ecology of Patient-1 were identified by defining the OTUs with the greatest relative abundance, which together comprise 60% of the microbial carriage in this patient's augmented ecology.
See FIG. 18: Microbial diversity measured in the ethanol treated spore treatment sample and patient pre- and post-treatment samples. Total microbial diversity is defined using the Chao1 Alpha-Diversity Index and is measured at different genomic sampling depths to confirm adequate sequence coverage to assay the microbiome in the target samples. The patient pretreatment (purple) harbored a microbiome that was significantly reduced in total diversity as compared to the ethanol treated spore treatment (red) and patient post treatment at days 5 (blue), 14 (orange), and 25 (green).
See FIG. 19: Patient microbial ecology is shifted by treatment with an ethanol treated spore treatment from a dysbiotic state to a state of health. Principle Coordinates Analysis based on the total diversity and structure of the microbiome (Bray-Curtis Beta-Diversity) of the patient pre- and post-treatment delineates that the engraftment of OTUs from the spore treatment and the augmentation of the patient microbial ecology leads to a microbial ecology that is distinct from both the pretreatment microbiome and the ecology of the ethanol treated spore treatment (Table 16).
See FIG. 20: Augmentation of Bacteroides species in patients. Comparing the number of Bacteroides fragilis groups species per cfu/g of feces pre-treatment and in week 4 post treatment reveals an increase of 4 logs or greater. The ability of 16S-V4 OTU identification to assign an OTU as a specific species depends in part on the resolution of the 16S-V4 region of the 16S gene for a particular species or group of species. Both the density of available reference 16S sequences for different regions of the tree as well as the inherent variability in the 16S gene between different species will determine the definitiveness of a taxonomic annotation to a given sequence read. Given the topological nature of a phylogenetic tree and that the tree represents hierarchical relationships of OTUs to one another based on their sequence similarity and an underlying evolutionary model, taxonomic annotations of a read can be rolled up to a higher level using a clade-based assignment procedure (Table 1). Using this approach, clades are defined based on the topology of a phylogenetic tree that is constructed from full-length 16S sequences using maximum likelihood or other phylogenetic models 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 (generally, 1-5 bootstraps), and (ii) within a 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. 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. The power of clade based analysis is that 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.
Stool samples were aliquoted and resuspended 10× vol/wt in either 100% ethanol (for genomic characterization) or PBS containing 15% glycerol (for isolation of microbes) and then stored at −80° C. until needed for use. For genomic 16S sequence analysis colonies picked from plate isolates had their full-length 16S sequence characterized as described in Examples 11 and 12, and primary stool samples were prepared targeting the 16S-V4 region using the method for heterogeneous samples in Example 10.
Notably, 16S sequences of isolates of a given OTU are phylogenetically placed within their respective clades despite that the actual taxonomic assignment of species and genus may suggest they are taxonomically distinct from other members of the clades in which they fall. Discrepancies between taxonomic names given to an OTU is based on microbiological characteristics versus genetic sequencing are known to exist from the literature. The OTUs footnoted in this table are known to be discrepant between the different methods for assigning a taxonomic name.
Engraftment of OTUs from the ethanol treated spore preparation treatment into the patient as well as the resulting augmentation of the resident microbiome led to a significant decrease in and elimination of the carriage of pathogenic species other than C. difficile in the patient. 16S-V4 sequencing of primary stool samples demonstrated that at pretreatment, 20% of reads were from the genus Klebsiella and an additional 19% were assigned to the genus Fusobacterium. These striking data are evidence of a profoundly dysbiotic microbiota associated with recurrent C. difficile infection and chronic antibiotic use. In healthy individuals, Klebsiella is a resident of the human microbiome in only about 2% of subjects based on an analysis of HMP database (www.hmpdacc.org), and the mean relative abundance of Klebsiella is only about 0.09% in the stool of these people. It's surprising presence at 20% relative abundance in Patient 1 before treatment is an indicator of a proinflammatory gut environment enabling a “pathobiont” to overgrow and outcompete the commensal organisms normally found in the gut. Similarly, the dramatic overgrowth of Fusobacterium indicates a profoundly dysbiotic gut microbiota. One species of Fusobacterium, F. nucleatum (an OTU phylogenetically indistinguishable from Fusobacterium sp. 3_1_33 based on 16S-V4), has been termed “an emerging gut pathogen” based on its association with IBD, Crohn's disease, and colorectal cancer in humans and its demonstrated causative role in the development of colorectal cancer in animal models [Allen-Vercoe, Gut Microbes (2011) 2:294-8]. Importantly, neither Klebsiella nor Fusobacterium was detected in the 16S-V4 reads by Day 25 (Table 18).
To further characterize the colonization of the gut by Klebsiella and other Enterobacteriaceae and to speciate these organisms, pretreatment and Day 25 fecal samples stored at −80 C as PBS-glycerol suspensions were plated on a variety of selective media including MacConkey lactose media (selective for gram negative enterobacteria) and Simmons Citrate Inositol media (selective for Klebsiella spp) [Van Cregten et al, J. Clin. Microbiol. (1984) 20: 936-41]. Enterobacteria identified in the patient samples included K. pneumoniae, Klebsiella sp. Co_9935 and E. coli. Strikingly, each Klebsiella species was reduced by 2-4 logs whereas E. coli, a normal commensal organism present in a healthy microbiota, was reduced by less than 1 log (Table 19). This decrease in Klebsiella spp. carriage is consistent across multiple patients (Table 19). Four separate patients were evaluated for the presence of Klebsiella spp. pre treatment and 4 weeks post treatment. Klebsiella spp. were detected by growth on selective Simmons Citrate Inositol media as previously described. Serial dilution and plating, followed by determining cfu/mL titers of morphologically distinct species and 16S full length sequence identification of representatives of those distinct morphological classes, allowed calculation of titers of specific species.
The genus Bacteroides is an important member of the gastrointestinal microbiota; 100% of stool samples from the Human Microbiome Project contain at least one species of Bacteroides with total relative abundance in these samples ranging from 0.96% to 93.92% with a median relative abundance of 52.67% (www.hmpdacc.org reference data set HMSMCP). Bacteroides in the gut has been associated with amino acid fermentation and degradation of complex polysaccharides. Its presence in the gut is enhanced by diets rich in animal-derived products as found in the typical western diet [David, L. A. et al, Nature (2013) doi:10.1038/nature12820]. Strikingly, prior to treatment, fewer than 0.008% of the 16S-V4 reads from Patient 1 mapped to the genus Bacteroides strongly suggesting that Bacteroides species were absent or that viable Bacteroides were reduced to an extremely minor component of the patient's gut microbiome. Post treatment, ≧42% of the 16S-V4 reads could be assigned to the genus Bacteroides within 5 days of treatment and by Day 25 post treatment 59.48% of the patients gut microbiome was comprised of Bacteroides. These results were confirmed microbiologically by the absence of detectable Bacteroides in the pretreatment sample plated on two different Bacteroides selective media: Bacteroides Bile Esculin (BBE) agar which is selective for Bacteroides fragilis group species [Livingston, S. J. et al J. Clin. Microbiol (1978). 7: 448-453] and Polyamine Free Arabinose (PFA) agar [Noack et al. J. Nutr. (1998) 128: 1385-1391; modified by replacing glucose with arabinose]. The highly selective BBE agar had a limit of detection of <2×103 cfu/g, while the limit of detection for Bacteroides on PFA agar was approximately 2×107 cfu/g due to the growth of multiple non-Bacteroides species in the pretreatment sample on that medium. Colony counts of Bacteroides species on Day 25 were up to 2×1010 cfu/g, consistent with the 16S-V4 sequencing, demonstrating a profound reconstitution of the gut microbiota in Patient 1 (Table 20).
The significant abundance of Bacteroides in Patient 1 on Day 25 (and as early as Day 5 as shown by 16S-V4 sequencing) is remarkable. Viable Bacteroides fragilis group species were not present in the ethanol treated spore population based on microbiological plating (limit of detection of 10 cfu/ml). Thus, administration of the ethanol treated spore population to Patient 1 resulted not only in the engraftment of spore-forming species, but also the restoration of high levels of non-spore forming species commonly found in healthy individuals through the creation of a niche that allowed for the repopulation of Bacteroides species. These organisms were most likely either present at extremely low abundance in the GI tract of Patient 1, or present in a reservoir in the GI tract from which they could rebound to high titer. Those species may also be reinoculated via oral uptake from food following treatment. We term this healthy repopulation of the gut with OTUs that are not present in the ethanol treated spore population “Augmentation.” Augmentation is an important phenomenon in that it shows the ability to use an ethanol treated spore ecology to restore a healthy microbiota by seeding a diverse array or commensal organisms beyond the actual component organisms in the ethanol treated spore population itself; specifically the spore treatment itself and the engraftment of OTUs from the spore composition create a niche that enables the outgrowth of OTUs required to shift a dysbiotic microbiome to a microbial ecology that is associated with health. The diversity of Bacteroides species and their approximate relative abundance in the gut of Patient 1 is shown in Table 21, comprising at least 8 different species.
See FIG. 21: Species Engrafting versus Species Augmenting in patients microbiomes after treatment with an ethanol-treated spore population. Relative abundance of species that engrafted or augmented as described were determined based on the number of 16S sequence reads. Each plot is from a different patient treated with the ethanol-treated spore population for recurrent C. difficile. 
The impact of ethanol treated spore population treatment on carriage of imipenem resistant Enterobacteriaceae was assessed by plating pretreatment and Day 28 clinical samples from Patients 2, 4 and 5 on MacConkey lactose plus 1 ug/mL of imipenem. Resistant organisms were scored by morphology, enumerated and DNA was submitted for full length 16S rDNA sequencing as described above. Isolates were identified as Morganella morganii, Providencia rettgeri and Proteus pennerii. Each of these are gut commensal organisms; overgrowth can lead to bacteremia and/or urinary tract infections requiring aggressive antibiotic treatment and, in some cases, hospitalization [Kim, B-N, et al Scan J. Inf Dis (2003) 35: 98-103; Lee, I-K and Liu, J-W J. Microbiol Immunol Infect (2006) 39: 328-334; O'Hara et al, Clin Microbiol Rev (2000) 13: 534]. The titer of organisms at pretreatment and Day 28 by patient is shown in Table 22. Importantly, administration of the ethanol treated spore preparation resulted in greater than 100-fold reduction in 4 of 5 cases of Enterobacteriaceae carriage with multiple imipenem resistant organisms (Table 22).
In addition to speciation and enumeration, multiple isolates of each organism from Patient 4 were grown overnight in 96-well trays containing a 2-fold dilution series of imipenem in order to quantitatively determine the minimum inhibitory concentration (MIC) of antibiotic. Growth of organisms was detected by light scattering at 600 nm on a SpectraMax M5e plate reader. In the clinical setting, these species are considered resistant to imipenem if they have an MIC of 1 ug/mL or greater. M. morganii isolates from pretreatment samples from Patient D had MICs of 2-4 ug/mL and P. pennerii isolates had MICs of 4-8 ug/mL. Thus the ethanol treated spore population administered to Patient 4 caused the clearance of 2 imipenem resistant organisms (Table 16).
Example 26
Enrichment and Purification of Bacteria
To purify individual bacterial strains, dilution plates were selected in which the density enables distinct separation of single colonies. Colonies were picked with a sterile implement (either a sterile loop or toothpick) and re-streaked to BBA or other solid media. Plates were incubated at 37° C. for 3-7 days. One or more well-isolated single colonies of the major morphology type were re-streaked. This process was repeated at least three times until a single, stable colony morphology is observed. The isolated microbe was then cultured anaerobically in liquid media for 24 hours or longer to obtain a pure culture of 106-1010 cfu/ml. Liquid growth medium might include Brain Heart Infusion-based medium (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010) supplemented with yeast extract, hemin, cysteine, and carbohydrates (for example, maltose, cellobiose, soluble starch) or other media described previously (e.g. see example 14). The culture was centrifuged at 10,000×g for 5 min to pellet the bacteria, the spent culture media was removed, and the bacteria were resuspended in sterile PBS. Sterile 75% glycerol was added to a final concentration of 20%. An aliquot of glycerol stock was titered by serial dilution and plating. The remainder of the stock was frozen on dry ice for 10-15 min and then placed at −80 C for long term storage.
Example 27
Cell Bank Preparation
Cell banks (RCBs) of bacterial strains were prepared as follows. Bacterial strains were struck from −80° C. frozen glycerol stocks to Brucella blood agar with Hemin or Vitamin K (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010), M2GSC (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press, 2010) or other solid growth media and incubated for 24 to 48 h at 37° C. in an anaerobic chamber with a gas mixture of H2: CO2: N2 of 10:10:80. Single colonies were then picked and used to inoculate 250 ml to 1 L of Wilkins-Chalgren broth, Brain-Heart Infusion broth, M2GSC broth or other growth media, and grown to mid to late exponential phase or into the stationary phase of growth. Alternatively, the single colonies may be used to inoculate a pilot culture of 10 ml, which were then used to inoculate a large volume culture. The growth media and the growth phase at harvest were selected to enhance cell titer, sporulation (if desired) and phenotypes that might be associated desired in vitro or in vivo. Optionally, Cultures were grown static or shaking, depending which yielded maximal cell titer. The cultures were then concentrated 10 fold or more by centrifugation at 5000 rpm for 20 min, and resuspended in sterile phosphate buffered saline (PBS) plus 15% glycerol. 1 ml aliquots were transferred into 1.8 ml cryovials which were then frozen on dry ice and stored at −80 C. The identity of a given cell bank was confirmed by PCR amplification of the 16S rDNA gene, followed by Sanger direct cycle sequencing, and comparison to a curated rDNA database to determine a taxonomic ID. Each bank was confirmed to yield colonies of a single morphology upon streaking to Brucella blood agar or M2GSC agar. When more than one morphology was observed, colonies were confirmed to be the expected species by PCR and sequencing analysis of the 16S rDNA gene. Variant colony morphologies can be observed within pure cultures, and in a variety of bacteria the mechanisms of varying colony morphologies have been well described (van der Woude, Clinical Microbiology Reviews, 17:518, 2004), including in Clostridium species (Wadsworth-KTL Anaerobic Bacteriology Manual, 6th Ed, Jousimie-Somer, et al 2002). For obligate anaerobes, RCBs were confirmed to lack aerobic colony forming units at a limit of detection of 10 cfu/ml.
Example 28
Titer Determination
The number of viable cells per ml was determined on the freshly harvested, washed and concentrated culture by plating serial dilutions of the RCB to Brucella blood agar or other solid media, and varied from 106 to 1010 cfu/ml. The impact of freezing on viability was determined by titering the banks after one or two freeze-thaw cycles on dry ice or at −80° C., followed by thawing in an anaerobic chamber at room temperature. Some strains displayed a 1-3 log drop in viable cfu/ml after the 1st and/or 2nd freeze thaw, while the viability of others were unaffected.
Example 29
Preparation of Bacterial Compositions
Individual strains were typically thawed on ice and combined in an anaerobic chamber to create mixtures, followed by a second freeze at −80° C. to preserve the mixed samples. When making combinations of strains for in vitro or in vivo assays, the cfu in the final mixture was estimated based on the second freeze-thaw titer of the individual strains. For experiments in rodents, strains may be combined at equal counts in order to deliver between 1e4 and 1e10 per strain. Additionally, some bacteria may not grow to sufficient titer to yield cell banks that allowed the production of compositions where all bacteria were present at 1e10.
Example 30
Identification of Keystone OTUs and Functions
The human body is an ecosystem in which the microbiota, and the microbiome, play a significant role in the basic healthy function of human systems (e.g. metabolic, immunological, and neurological). The microbiota and resulting microbiome comprise an ecology of microorganisms that co-exist within single subjects interacting with one another and their host (i.e., the mammalian subject) to form a dynamic unit with inherent biodiversity and functional characteristics. Within these networks of interacting microbes (i.e. ecologies), particular members can contribute more significantly than others; as such these members are also found in many different ecologies, and the loss of these microbes from the ecology can have a significant impact on the functional capabilities of the specific ecology. Robert Paine coined the concept “Keystone Species” in 1969 (see Paine R T. 1969. A note on trophic complexity and community stability. The American Naturalist 103: 91-93.) to describe the existence of such lynchpin species that are integral to a given ecosystem regardless of their abundance in the ecological community. Paine originally describe the role of the starfish Pisaster ochraceus in marine systems and since the concept has been experimentally validated in numerous ecosystems.
Keystone OTUs and/or Functions are computationally-derived by analysis of network ecologies elucidated from a defined set of samples that share a specific phenotype. Keystone OTUs and/or Functions are defined as all Nodes within a defined set of networks that meet two or more of the following criteria. Using Criterion 1, the node is frequently observed in networks, and the networks in which the node is observed are found in a large number of individual subjects; the frequency of occurrence of these Nodes in networks and the pervasiveness of the networks in individuals indicates these Nodes perform an important biological function in many individuals. Using Criterion 2, the node is frequently observed in networks, and each the networks in which the node is observed contain a large number of Nodes—these Nodes are thus “super-connectors”, meaning that they form a nucleus of a majority of networks and as such have high biological significance with respect to their functional contributions to a given ecology. Using Criterion 3, the node is found in networks containing a large number of Nodes (i.e. they are large networks), and the networks in which the node is found occur in a large number of subjects; these networks are potentially of high interest as it is unlikely that large networks occurring in many individuals would occur by chance alone strongly suggesting biological relevance. Optionally, the required thresholds for the frequency at which a node is observed in network ecologies, the frequency at which a given network is observed across subject samples, and the size of a given network to be considered a Keystone node are defined by the 50th, 70th, 80th, or 90th percentiles of the distribution of these variables. Optionally, the required thresholds are defined by the value for a given variable that is significantly different from the mean or median value for a given variable using standard parametric or non-parametric measures of statistical significance. In another embodiment a Keystone node is defined as one that occurs in a sample phenotype of interest such as but not limited to “health” and simultaneously does not occur in a sample phenotype that is not of interest such as but not limited to “disease.” Optionally, a Keystone Node is defined as one that is shown to be significantly different from what is observed using permuted test datasets to measure significance.
Example 31
Identifying the Core Ecology from the Ethanol Treated Spore Preparation
Ten different ethanol treated spore preparations were made from 6 different donors (as described in Example 15). The spore preparations were used to treat 10 patients, each suffering from recurrent C. difficile infection. Patients were identified using the inclusion/exclusion criteria described in Example 18, and donors were identified using the criteria described in Example 1. None of the patients experienced a relapse of C. difficile in the 4 weeks of follow up after treatment, whereas the literature would predict that 70-80% of subjects would experience a relapse following cessation of antibiotic [Van Nood, et al, NEJM (2013)]. Thus, the ethanol treated spore preparations derived from multiple different donors and donations showed remarkable clinical efficacy.
To define the Core Ecology underlying the remarkable clinical efficacy of the ethanol treated spore preparation, the following analysis was carried out. The OTU composition of the spore preparation was determined by 16S-V4 rDNA sequencing and computational assignment of OTUs per Example 12. A requirement to detect at least ten sequence reads in the ethanol treated spore preparation was set as a conservative threshold to define only OTUs that were highly unlikely to arise from errors during amplification or sequencing. Methods routinely employed by those familiar to the art of genomic-based microbiome characterization use a read relative abundance threshold of 0.005% (see e.g. Bokulich, A. et al. 2013. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nature Methods 10: 57-59), which would equate to ≧2 reads given the sequencing depth obtained for the samples analyzed in this example, as cut-off which is substantially lower than the ≧10 reads used in this analysis. All taxonomic and clade assignments were made for each OTU as described in Examples 12. The resulting list of OTUs, clade assignments, and frequency of detection in the spore preparations are shown in Table GB. OTUs that engraft in a treated patients and the percentage of patients in which they engraft are denoted, as are the clades, spore forming status, and Keystone OTU status. Starred OTUs occur in ≧80% of the ethanol preps and engraft in ≧50% of the treated patients.







TABLE GB







OTUs detected by a minimum of ten 16S-V4 sequence reads in at


least a one ethanol treated spore preparation (pan-microbiome).














% of Spore
% of Pa-






Preps
tients OTU
Spore
Keystone


OTU
Clade
with OTU
Engrafts
Former
OTU





Prevotella_maculosa
clade_104
10%
 0%
N
N


Prevotella_copri
clade_168
20%
 0%
N
N


Bacteroides_caccae
clade_170
30%
 0%
N
Y


Bifidobacterium_sp_TM_7*
clade_172
90%
60%
N
N


Bifidobacterium_gallicum
clade_172
70%
20%
N
N


Bifidobacterium_dentium
clade_172
50%
 0%
N
N


Lactobacillus_casei
clade_198
20%
10%
N
N


Actinomyces_odontolyticus
clade_212
20%
30%
N
N


Clostridium_colicanis
clade_223
10%
10%
Y
N


Clostridiales_sp_SS3_4*
clade_246
100% 
70%
Y
N


Clostridium_sporogenes
clade_252
40%
40%
Y
N


Clostridium_butyricum
clade_252
20%
20%
Y
N


Clostridium_disporicum
clade_253
40%
30%
Y
N


Clostridium_hylemonae*
clade_260
100% 
50%
Y
N


Clostridium_scindens
clade_260
10%
60%
Y
N


Coprococcus_comes*
clade_262
90%
80%
Y
Y


Lachnospiraceae_bacterium_1_4_56FAA*
clade_262
90%
80%
Y
Y


Ruminococcus_torques
clade_262
30%
70%
Y
Y


Parabacteroides_merdae
clade_286
30%
20%
N
Y


Bifidobacterium_bifidum
clade_293
10%
 0%
N
N


Johnsonella_ignava
clade_298
10%
10%
N
N


Blautia_glucerasea*
clade_309
100% 
80%
Y
N


Blautia_sp_M25*
clade_309
100% 
70%
Y
Y


Lachnospiraceae_bacterium_6_1_63FAA*
clade_309
100% 
60%
Y
N


Eubacterium_cellulosolvens
clade_309
10%
30%
Y
Y


Lactobacillus_fermentum
clade_313
10%
 0%
N
N


Sarcina_ventriculi
clade_353
10%
10%
Y
N


Clostridium_bartlettii*
clade_354
90%
70%
Y
N


Clostridium_bifermentans
clade_354
70%
70%
Y
N


Clostridium_mayombei
clade_354
50%
50%
Y
N


Dorea_longicatena*
clade_360
100% 
60%
Y
Y


Lachnospiraceae_bacterium_9_1_43BFAA
clade_360
100% 
30%
Y
N


Lachnospiraceae_bacterium_2_1_58FAA*
clade_360
80%
80%
Y
N


Lachnospiraceae_bacterium_2_1_46FAA
clade_360
50%
50%
Y
N


Lactobacillus_perolens
clade_373
10%
 0%
N
N


Bacteroides_dorei
clade_378
60%
50%
N
Y


Eubacterium_biforme
clade_385
10%
 0%
Y
N


Peptoniphilus_sp_gpac077
clade_389
10%
20%
N
N


Coprococcus_catus*
clade_393
100% 
70%
Y
Y


Eubacterium_hallii*
clade_396
90%
60%
Y
Y


Anaerosporobacter_mobilis
clade_396
40%
60%
Y
N


Bacteroides_pectinophilus
clade_396
10%
60%
Y
N


Lactobacillus_hominis
clade_398
10%
 0%
N
N


Lactococcus_lactis
clade_401
40%
40%
N
N


Ruminococcus_champanellensis*
clade_406
80%
50%
Y
N


Ruminococcus_callidus
clade_406
10%
10%
Y
N


Clostridium_clostridioforme*
clade_408
100% 
60%
Y
Y


Eubacterium_hadrum*
clade_408
100% 
90%
Y
Y


Clostridium_symbiosum
clade_408
30%
50%
Y
Y


Anaerostipes_caccae
clade_408
10%
50%
Y
N


Parasutterella_excrementihominis
clade_432
10%
 0%
N
N


Sutterella_stercoricanis
clade_432
10%
 0%
N
N


Eubacterium_rectale*
clade_444
100% 
80%
Y
Y


Lachnobacterium_bovis*
clade_444
100% 
80%
Y
N


Desulfovibrio_desulfuricans
clade_445
10%
 0%
N
Y


Eubacterium_sp_oral_clone_JS001*
clade_476
80%
70%
Y
N


Faecalibacterium_prausnitzii*
clade_478
100% 
60%
Y
Y


Subdoligranulum_variabile*
clade_478
100% 
80%
Y
Y


Coprobacillus_sp_D7*
clade_481
90%
60%
Y
N


Clostridium_cocleatum
clade_481
60%
20%
Y
N


Clostridium_spiroforme
clade_481
40%
50%
Y
N


Eubacterium_ramulus*
clade_482
80%
60%
Y
N


Flavonifractor_plautii
clade_494
70%
60%
Y
Y


Pseudoflavonifractor_capillosus
clade_494
60%
60%
Y
Y


Ruminococcaceae_bacterium_D16
clade_494
30%
50%
Y
Y


Acetivibrio_cellulolyticus*
clade_495
70%
80%
Y
N


Clostridium_stercorarium
clade_495
40%
50%
Y
N


Enterococcus_durans
clade_497
10%
10%
N
N


Enterococcus_faecium
clade_497
10%
10%
N
N


Dialister_invisus
clade_506
50%
10%
N
N


Eubacterium_limosum
clade_512
20%
 0%
Y
N


Ruminococcus_flavefaciens
clade_516
60%
60%
Y
N


Eubacterium_ventriosum
clade_519
30%
60%
Y
Y


Bilophila_wadsworthia
clade_521
90%
 0%
N
Y


Lachnospira_pectinoschiza
clade_522
40%
60%
Y
N


Eubacterium_eligens
clade_522
30%
50%
Y
Y


Catonella_morbi
clade_534
20%
 0%
N
N


Clostridium_sporosphaeroides*
clade_537
100% 
80%
Y
N


Ruminococcus_bromii
clade_537
60%
30%
Y
Y


Clostridium_leptum
clade_537
40%
70%
Y
Y


Clostridium_sp_YIT_12069
clade_537
40%
60%
Y
N


Clostridium_viride
clade_540
10%
10%
Y
N


Megamonas_funiformis
clade_542
50%
 0%
N
N


Eubacterium_ruminantium*
clade_543
80%
90%
Y
N


Coprococcus_eutactus
clade_543
20%
20%
Y
N


Collinsella_aerofaciens
clade_553
50%
10%
Y
Y


Alkaliphilus_metalliredigenes
clade_554
40%
10%
Y
N


Turicibacter_sanguinis
clade_555
80%
40%
Y
N


Phascolarctobacterium_faecium
clade_556
20%
 0%
N
N


Clostridiales_bacterium_oral_clone_P4PA*
clade_558
80%
50%
N
N


Lutispora_thermophila
clade_564
100% 
 0%
Y
N


Coriobacteriaceae_bacterium_JC110
clade_566
70%
 0%
N
N


Eggerthella_sp_1_3_56FAA
clade_566
70%
30%
N
N


Adlercreutzia_equolifaciens
clade_566
40%
 0%
N
N


Gordonibacter_pamelaeae
clade_566
30%
 0%
N
Y


Slackia_isoflavoniconvertens
clade_566
10%
 0%
N
N


Eubacterium_desmolans*
clade_572
90%
70%
Y
N


Papillibacter_cinnamivorans*
clade_572
90%
80%
Y
N


Clostridium_colinum
clade_576
30%
30%
Y
N


Akkermansia_muciniphila
clade_583
60%
10%
N
Y


Clostridiales_bacterium_oral_taxon_F32
clade_584
60%
30%
N
N


Prochlorococcus_marinus
clade_592
30%
 0%
N
N


Methanobrevibacter_wolinii
clade_595
30%
 0%
N
N


Bacteroides_fragilis
clade_65
20%
30%
N
Y


Lactobacillus_delbrueckii
clade_72
10%
 0%
N
N


Escherichia_coli
clade_92
50%
 0%
N
Y


Clostridium_sp_D5
clade_96
80%
60%
Y
N


Streptococcus_thermophilus
clade_98
90%
20%
N
Y


Streptococcus_sp_CM6
clade_98
20%
10%
N
N


Streptococcus_sp_oral_clone_ASCE05
clade_98
10%
 0%
N
N









Next, it was reasoned that for an OTU to be considered a member of the Core Ecology of the spore preparation, that OTU must be shown to engraft in a patient. Engraftment is important for two reasons. First, engraftment is a sine qua non of the mechanism to reshape the microbiome and eliminate C. difficile colonization. OTUs that engraft with higher frequency are highly likely to be a component of the Core Ecology of the spore preparation. Second, OTUs detected by sequencing the spore preparation (as in Table GB) may include non-viable spores or other contaminant DNA molecules not associated with spores. The requirement that an OTU must be shown to engraft in the patient eliminates OTUs that represent non-viable spores or contaminating sequences. Table GB also identifies all OTUs detected in the spore preparation that also were shown to engraft in at least one patient post-treatment. OTUs that are present in a large percentage of the ethanol spore preparations analyzed and that engraft in a large number of patients represent a subset of the Core Ecology that are highly likely to catalyze the shift from a dysbiotic disease ecology to a healthy microbiome.
A third lens was applied to further refine insights into the Core Ecology of the spore preparation. Computational-based, network analysis has enabled the description of microbial ecologies that are present in the microbiota of a broad population of healthy individuals. These network ecologies are comprised of multiple OTUs, some of which are defined as Keystone OTUs. Keystone OTUs are computationally defined as described in Example 30. Keystone OTUs form a foundation to the microbially ecologies in that they are found and as such are central to the function of network ecologies in healthy subjects. Keystone OTUs associated with microbial ecologies associated with healthy subjects are often are missing or exist at reduced levels in subjects with disease. Keystone OTUs may exist in low, moderate, or high abundance in subjects. Table GB further notes which of the OTUs in the spore preparation are Keystone OTUs exclusively associated with individuals that are healthy and do not harbor disease.
There are several important findings from this data. A relatively small number of species, 16 in total, are detected in all of the spore preparations from 6 donors and 10 donations. This is surprising because the HMP database (www.hmpdacc.org) describes the enormous variability of commensal species across healthy individuals. The presence of a small number of consistent OTUs lends support to the concept of a Core Ecology. The engraftment data further supports this conclusion. A regression analysis shows a significant correlation between frequency of detection in a spore preparation and frequency of engraftment in a donor: R=0.43 (p<0.001). There is no a priori requirement that an OTU detected frequently in the spore preparation will or should engraft. For instance, Lutispora thermophila, a spore former found in all ten spore preparations, did not engraft in any of the patients. Bilophila wadsworthia, a gram negative anaerobe, is present in 9 of 10 donations, yet it does not engraft in any patient, indicating that it is likely a non-viable contaminant in the ethanol treated spore preparation. Finally, it is worth noting the high preponderance of previously defined Keystone OTUs among the most frequent OTUs in the spore preparations.
These three factors—prevalence in the spore preparation, frequency of engraftment, and designation as a Keystone OTUs—enabled the creation of a “Core Ecology Score” (CES) to rank individual OTUs. CES was defined as follows:

    
    
        40% weighting for presence of OTU in spore preparation
        multiplier of 1 for presence in 1-3 spore preparations
        multiplier of 2.5 for presence in 4-8 spore preparations
        multiplier of 5 for presences in 9 spore preparations
        40% weighting for engraftment in a patient
        multiplier of 1 for engraftment in 1-4 patients
        multiplier of 2.5 for engraftment in 5-6 patients
        multiplier of 5 for engraftment in 7 patients
        20% weighting to Keystone OTUs
        multiplier of 1 for a Keystone OTU
        multiplier of 0 for a non-Keystone OTU
    
    


Using this guide, the CES has a maximum possible score of 5 and a minimum possible score of 0.8. As an example, an OTU found in 8 of the 10 spore preparations that engrafted in 3 patients and was a Keystone OTU would be assigned the follow CES:

CES=(0.4×2.5)+(0.4×1)+(0.2×1)=1.6

Table GC ranks the top 20 OTUs by CES with the further requirement that an OTU must be shown to engraft to be a considered an element of a core ecology.







TABLE GC







Top 20 OTUs ranked by CES














Spore
Keystone


OTU
Clade
CES
Former
OTU





Eubacterium_hadrum
clade_408
4.2
Y
Y


Eubacterium_rectale
clade_444
4.2
Y
Y


Subdoligranulum_variabile
clade_478
4.2
Y
Y


Blautia_sp_M25
clade_309
4.2
Y
Y


Coprococcus_catus
clade_393
4.2
Y
Y


Lachnospiraceae_bacte-
clade_262
4.2
Y
Y


rium_1_4_56FAA


Coprococcus_comes
clade_262
4.2
Y
Y


Blautia_glucerasea
clade_309
4.0
Y
N


Lachnobacterium_bovis
clade_444
4.0
Y
N


Clostridium_sporosphaeroides
clade_537
4.0
Y
N


Clostridiales_sp_SS3_4
clade_246
4.0
Y
N


Papillibacter_cinnamivorans
clade_572
4.0
Y
N


Clostridium_bartlettii
clade_354
4.0
Y
N


Eubacterium_desmolans
clade_572
4.0
Y
N


Clostridium_clostridioforme
clade_408
3.2
Y
Y


Dorea_longicatena
clade_360
3.2
Y
Y


Faecalibacterium_prausnitzii
clade_478
3.2
Y
Y


Eubacterium_hallii
clade_396
3.2
Y
Y


Clostridium_leptum
clade_537
3.2
Y
Y


Lachnospiraceae_bacte-
clade_309
3.0
Y
N


rium_6_1_63FAA









Example 32
Defining Efficacious Subsets of the Core Ecology
The number of organisms in the human gastrointestinal tract, as well as the diversity between healthy individuals, is indicative of the functional redundancy of a healthy gut microbiome ecology (see The Human Microbiome Consortia. 2012. Structure, function and diversity of the healthy human microbiome. Nature 486: 207-214). This redundancy makes it highly likely that subsets of the Core Ecology describe therapeutically beneficial components of the ethanol treated spore preparation and that such subsets may themselves be useful compositions for the treatment of C. difficile infection given the ecologies functional characteristics. Using the CES, individual OTUs can be prioritized for evaluation as an efficacious subset of the Core Ecology.
Another aspect of functional redundancy is that evolutionarily related organisms (i.e. those close to one another on the phylogenetic tree, e.g. those grouped into a single clade) will also be effective substitutes in the Core Ecology or a subset thereof for treating C. difficile. 
To one skilled in the art, the selection of appropriate OTU subsets for testing in vitro (e.g. see Example 33 below) or in vivo (e.g. see Examples 16 or 17) is straightforward. Subsets may be selected by picking any 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 OTUs from Table GB, with a particular emphasis on those with higher CES, such as the OTUs described in Table GC. In addition, using the clade relationships defined in Example 12 and Table 1 above, related OTUs can be selected as substitutes for OTUs with acceptable CES values. These organisms can be cultured anaerobically in vitro using the appropriate media (selected from those described in Example 14 above), and then combined in a desired ratio. A typical experiment in the mouse C. difficile model utilizes at least 104 and preferably at least 105, 106, 107, 108, 109 or more than 109 colony forming units of a each microbe in the composition. Variations in the culture yields may sometimes mean that organisms are combined in unequal ratios, e.g. 1:10, 1:100, 1:1,000, 1:10,000, 1:100,000, or greater than 1:100,000. What is important in these compositions is that each strain be provided in a minimum amount so that the strain's contribution to the efficacy of the Core Ecology subset can be measured. Using the principles and instructions described here, it is straightforward for one of skill in the art to make clade-based substitutions to test the efficacy of subsets of the Core Ecology. Table GB describes the clades for each OTU detected in a spore preparation and Table 1 describes the OTUs that can be used for substitutions based on clade relationships.
Example 33
Testing Subsets of the Core Ecology in the Mouse Model
Several subsets of the Core Ecology were tested in the C. difficile mouse model. The negative control was phosphate buffered saline and the positive control was a 10% human fecal suspension. The subsets are described in Table GD.







TABLE GD







Subsets of the Core Ecology tested in the C. difficile mouse model











Substitute For OTU


Subset
OTU
in Table 1 (Clade)





Subset 1
Collinsella aerofaciens
none (Clade_553)



Clostridium tertium
C. sporogenes




(Clade_252)



Clostridium disporicum
none (Clade_253)



Clostridium innocuum
Clostridium_sp_HGF2




(Clade_351)



Clostridium mayombei
none (Clade_354)



Clostridium butyricum
none (Clade_252)



Coprococcus comes
none (Clade_262)



Clostridium hylemonae
none (Clade_260)



Clostridium bolteae
E. hadrum




(Clade_408)



Clostridium symbiosum
C. clostridioforme




(Clade_408)



Clostridium orbiscindens
R._bacterium_D16




(Clade_494)



Lachnospiraceae bacte-
C. scindens



rium_5_1_57FAA
(Clade_260)



Blautia producta
Blautia_sp_M25




(Clade_309)



Ruminococcus gnavus
D. longicatena




(Clade_360)



Ruminococcus bromii
none (Clade_537)


Subset 2
Collinsella aerofaciens
none (Clade_553)



Clostridium butyricum
none (Clade_252)



Clostridium hylemonae
none (Clade_260)



Blautia producta
Blautia_sp_M25




(Clade_309)


Subset 3
Collinsella aerofaciens
none (Clade_553)



Clostridium innocuum
Clostridium_sp_HGF2




(Clade_351)



Coprococcus comes
none (Clade_262)



Ruminococcus bromii
none (Clade_537)


Subset 4
Clostridium butyricum
none (Clade_252)



Clostridium hylemonae
none (Clade_260)



Blautia producta
Blautia_sp_M25




(Clade_309)


Subset 5
Clostridium butyricum
none (Clade_252)



Clostridium hylemonae
none (Clade_260)


Subset 6
Blautia producta
Blautia_sp_M25




(Clade_309)



Clostridium butyricum
none (Clade_252)


Subset 7
Clostridium orbiscindens
R._bacterium_D16




(Clade_494)



Lachnospiraceae bacte-
C. scindens



rium_5_1_57FAA
(Clade_260)



Eubacterium rectale
none (Clade_444)









Two cages of five mice each were tested for each arm of the experiment. All mice received an antibiotic cocktail consisting of 10% glucose, kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) and Vancomycin (0.056 mg/ml) in their drinking water on days −14 through −5 and a dose of 10 mg/kg Clindamycin by oral gavage on day −3. On day −1, they received either the test articles or control articles via oral gavage. On day 0 they were challenged by administration of approximately 4.5 log 10 cfu of C. difficile (ATCC 43255) via oral gavage. Mortality was assessed every day from day 0 to day 6 and the weight and subsequent weight change of the animal was assessed with weight loss being associated with C. difficile infection. Mortality and reduced weight loss of the test article compared to the empty vehicle was used to assess the success of the test article. Additionally, a C. difficile symptom scoring was performed each day from day −1 through day 6. Symptom scoring was based on Appearance (0-2 pts based on normal, hunched, piloerection, or lethargic), Respiration (0-2 pts based on normal, rapid or shallow, with abdominal breathing), Clinical Signs (0-2 points based on normal, wet tail, cold-to-the-touch, or isolation from other animals).
In addition to compiling the cumulative mortality for each arm, the average minimum relative weight is calculated as the mean of each mouse's minimum weight relative to Day −1 and the average maximum clinical score is calculated as the mean of each mouse's maximum combined clinical score with a score of 4 assigned in the case of death. The results are reported in Table GE.







TABLE GE







Results of bacterial compositions tested


in a C. difficile mouse model.













Cumu-
Avg.
Avg. Maxi-




lative
Minimum
mum Clini-




Mortality
Relative
cal Score


Group
Dose
(%)
Weight
(Death = 4)














Vehicle
—
40
0.87
2.8


Control












Feces
5.8e8
cfu total
0
0.99
0


Control


Subset 1
1e8
cfu/OTU
0
0.98
0


Subset 2
1e8
cfu/OTU
10
0.84
2.1


Subset 3
1e8
cfu/OTU
10
0.84
2.2


Subset 4
1e8
cfu/OTU
0
0.87
2


Subset 5
1e8
cfu/OTU
20
0.91
1.7


Subset 6
1e8
cfu/OTU
40
0.82
2.8


Subset 7
1e8
cfu/OTU
0
0.90
1









Example 34
Defining Subsets of the Core Ecology in the In Vitro C. Difficile Inhibition Assay
Vials of −80° C. glycerol stock banks were thawed and diluted to 1e8 CFU/mL. Selected strains and their clade assignment are given in Table GF. Each strain was then diluted 10× (to a final concentration of 1e7 CFU/mL of each strain) into 200 uL of PBS+15% glycerol in the wells of a 96-well plate. Plates were then frozen at −80° C. When needed for the assay, plates were removed from −80° C. and thawed at room temperature under anaerobic conditions when testing in a in vitro C. difficile inhibition assay (CivSim).
An overnight culture of Clostridium difficile is grown under anaerobic conditions in SweetB-Fosln or other suitable media for the growth of C. difficile. SweetB-Fosln is a complex media composed of brain heart infusion, yeast extract, cysteine, cellobiose, maltose, soluble starch, and fructooligosaccharides/inulin, and hemin, and is buffered with MOPs. After 24 hr of growth the culture is diluted 100,000 fold into a complex media such as SweetB-Fosln which is suitable for the growth of a wide variety of anaerobic bacterial species. The diluted C. difficile mixture is then aliquoted to wells of a 96-well plate (180 uL to each well). 20 uL of a subset Core Ecology is then added to each well at a final concentration of 1e6 CFU/mL of each species. Alternatively the assay can be tested each species at different initial concentrations (1e9 CFU/mL, 1e8 CFU/mL, 1e7 CFU/mL, 1e5 CFU/mL, 1e4 CFU/mL, 1e3 CFU/mL, 1e2 CFU/mL). Control wells only inoculated with C. difficile are included for a comparison to the growth of C. difficile without inhibition. Additional wells are used for controls that either inhibit or do not inhibit the growth of C. difficile. One example of a positive control that inhibits growth is a combination of Blautia producta, Clostridium bifermentans and Escherichia coli. One example of a control that shows reduced inhibition of C. difficile growth is a combination of Bacteroides thetaiotaomicron, Bacteroides ovatus and Bacteroides vulgatus. Plates are wrapped with parafilm and incubated for 24 hr at 37° C. under anaerobic conditions. After 24 hr the wells containing C. difficile alone are serially diluted and plated to determine titer. The 96-well plate is then frozen at −80 C before quantifying C. difficile by qPCR assay.
A standard curve is generated from a well on each assay plate containing only pathogenic C. difficile grown in SweetB+Fosln media and quantified by selective spot plating. Serial dilutions of the culture are performed in sterile phosphate-buffered saline. Genomic DNA is extracted from the standard curve samples along with the other wells.
Genomic DNA is extracted from 5 μl of each sample using a dilution, freeze/thaw, and heat lysis protocol. 5 μL of thawed samples is added to 45 μL of UltraPure water (Life Technologies, Carlsbad, Calif.) and mixed by pipetting. The plates with diluted samples are frozen at −20° C. until use for qPCR which includes a heated lysis step prior to amplification. Alternatively the genomic DNA is isolated using the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.
The qPCR reaction mixture contains 1× SsoAdvanced Universal Probes Supermix, 900 nM of Wr-tcdB-F primer (AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG (SEQ ID NO: 2040), IDT, Coralville, Iowa), 900 nM of Wr-tcdB-R primer (CATGCTTTTTTAGTTTCTGGATTGAA (SEQ ID NO: 2041), IDT, Coralville, Iowa), 250 nM of Wr-tcdB-P probe (6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQ ID NO: 2042), Life Technologies, Grand Island, N.Y.), and Molecular Biology Grade Water (Mo Bio Laboratories, Carlsbad, Calif.) to 18 μl (Primers adapted from: Wroblewski, D. et al., Rapid Molecular Characterization of Clostridium difficile and Assessment of Populations of C. difficile in Stool Specimens, Journal of Clinical Microbiology 47:2142-2148 (2009)). This reaction mixture is aliquoted to wells of a Hard-shell Low-Profile Thin Wall 96-well Skirted PCR Plate (BioRad, Hercules, Calif.). To this reaction mixture, 2 μl of diluted, frozen, and thawed samples are added and the plate sealed with a Microseal ‘B’ Adhesive Seal (BioRad, Hercules, Calif.). The qPCR is performed on a BioRad C1000TM Thermal Cycler equipped with a CFX96TM Real-Time System (BioRad, Hercules, Calif.). The thermocycling conditions are 95° C. for 15 minutes followed by 45 cycles of 95° C. for 5 seconds, 60° C. for 30 seconds, and fluorescent readings of the FAM channel. Alternatively, the qPCR is performed with other standard methods known to those skilled in the art.
The Cq value for each well on the FAM channel is determined by the CFX ManagerTM 3.0 software. The log10 (cfu/mL) of C. difficile each experimental sample is calculated by inputting a given sample's Cq value into a linear regression model generated from the standard curve comparing the Cq values of the standard curve wells to the known log10 (cfu/mL) of those samples. The log inhibition is calculated for each sample by subtracting the log10 (cfu/mL) of C. difficile in the sample from the log10 (cfu/mL) of C. difficile in the sample on each assay plate used for the generation of the standard curve that has no additional bacteria added. The mean log inhibition is calculated for all replicates for each composition.
A histogram of the range and standard deviation of each composition is plotted. Ranges or standard deviations of the log inhibitions that are distinct from the overall distribution are examined as possible outliers. If the removal of a single log inhibition datum from one of the binary pairs that is identified in the histograms would bring the range or standard deviation in line with those from the majority of the samples, that datum is removed as an outlier, and the mean log inhibition is recalculated.
The pooled variance of all samples evaluated in the assay is estimated as the average of the sample variances weighted by the sample's degrees of freedom. The pooled standard error is then calculated as the square root of the pooled variance divided by the square root of the number of samples. Confidence intervals for the null hypothesis are determined by multiplying the pooled standard error to the z score corresponding to a given percentage threshold. Mean log inhibitions outside the confidence interval are considered to be inhibitory if positive or stimulatory if negative with the percent confidence corresponding to the interval used. Ternary combinations with mean log inhibition greater than 0.312 are reported as ++++ (≧99% confidence interval (C.I.) of the null hypothesis), those with mean log inhibition between 0.221 and 0.312 as +++ (95%<C.I <99%), those with mean log inhibition between 0.171 and 0.221 as ++ (90%<C.I <95%), those with mean log inhibition between 0.113 and 0.171 as + (80%<C.I <90%), those with mean log inhibition between −0.113 and −0.171 as − (80%<C.I <90%), those with mean log inhibition between −0.171 and −0.221 as −− (90%<C.I <95%), those with mean log inhibition between −0.221 and −0.312 as −−− (95%<C.I <99%), and those with mean log inhibition less than −0.312 as −−−− (99%<C.I.).







TABLE GF







OTUs and their clade assignments tested in ternary combinations with results in the in vitro inhibition assay













OTU1
Clade1
OTU2
Clade2
OTU3
Clade3
Results





Clostridium_bolteae
clade_408
Blautia_producta
clade_309
Eubacterium_rectale
clade_444
++++


Clostridium_bolteae
clade_408
Clostridium_symbiosum
clade_408
Blautia_producta
clade_309
++++


Clostridium_bolteae
clade_408
Clostridium_symbiosum
clade_408
Eubacterium_rectale
clade_444
−


Clostridium_bolteae
clade_408
Clostridium_symbiosum
clade_408
Faecalibacte-
clade_478
−






rium_prausnitzii




Clostridium_bolteae
clade_408
Clostridium_symbiosum
clade_408
Lachnospiraceae_bacte-
clade_260







rium_5_1_57FAA




Clostridium_bolteae
clade_408
Faecalibacte-
clade_478
Blautia_producta
clade_309
++++




rium_prausnitzii






Clostridium_bolteae
clade_408
Faecalibacte-
clade_478
Eubacterium_rectale
clade_444





rium_prausnitzii






Clostridium_bolteae
clade_408
Faecalibacte-
clade_478
Lachnospiraceae_bacte-
clade_260
++++




rium_prausnitzii

rium_5_1_57FAA




Clostridium_bolteae
clade_408
Lachnospiraceae_bacte-
clade_260
Blautia_producta
clade_309
++++




rium_5_1_57FAA






Clostridium_bolteae
clade_408
Lachnospiraceae_bacte-
clade_260
Eubacterium_rectale
clade_444
+




rium_5_1_57FAA






Clostridium_symbiosum
clade_408
Blautia_producta
clade_309
Eubacterium_rectale
clade_444
++++


Clostridium_symbiosum
clade_408
Faecalibacte-
clade_478
Blautia_producta
clade_309
++++




rium_prausnitzii






Clostridium_symbiosum
clade_408
Faecalibacte-
clade_478
Eubacterium_rectale
clade_444





rium_prausnitzii






Clostridium_symbiosum
clade_408
Faecalibacte-
clade_478
Lachnospiraceae_bacte-
clade_260
+




rium_prausnitzii

rium_5_1_57FAA




Clostridium_symbiosum
clade_408
Lachnospiraceae_bacte-
clade_260
Blautia_producta
clade_309
++++




rium_5_1_57FAA






Clostridium_symbiosum
clade_408
Lachnospiraceae_bacte-
clade_260
Eubacterium_rectale
clade_444





rium_5_1_57FAA






Collinsella_aerofaciens
clade_553
Blautia_producta
clade_309
Eubacterium_rectale
clade_444
++++


Collinsella_aerofaciens
clade_553
Clostridium_bolteae
clade_408
Blautia_producta
clade_309
++++


Collinsella_aerofaciens
clade_553
Clostridium_bolteae
clade_408
Clostridium_symbiosum
clade_408
++++


Collinsella_aerofaciens
clade_553
Clostridium_bolteae
clade_408
Eubacterium_rectale
clade_444
++++


Collinsella_aerofaciens
clade_553
Clostridium_bolteae
clade_408
Faecalibacte-
clade_478
++++






rium_prausnitzii




Collinsella_aerofaciens
clade_553
Clostridium_bolteae
clade_408
Lachnospiraceae_bacte-
clade_260
++++






rium_5_1_57FAA




Collinsella_aerofaciens
clade_553
Clostridium_symbiosum
clade_408
Blautia_producta
clade_309
++++


Collinsella_aerofaciens
clade_553
Clostridium_symbiosum
clade_408
Eubacterium_rectale
clade_444



Collinsella_aerofaciens
clade_553
Clostridium_symbiosum
clade_408
Faecalibacte-
clade_478







rium_prausnitzii




Collinsella_aerofaciens
clade_553
Clostridium_symbiosum
clade_408
Lachnospiraceae_bacte-
clade_260
+






rium_5_1_57FAA




Collinsella_aerofaciens
clade_553
Coprococcus_comes
clade_262
Blautia_producta
clade_309
++++


Collinsella_aerofaciens
clade_553
Coprococcus_comes
clade_262
Clostridium_bolteae
clade_408
++++


Collinsella_aerofaciens
clade_553
Coprococcus_comes
clade_262
Clostridium_symbiosum
clade_408
+++


Collinsella_aerofaciens
clade_553
Coprococcus_comes
clade_262
Eubacterium_rectale
clade_444
+++


Collinsella_aerofaciens
clade_553
Coprococcus_comes
clade_262
Faecalibacte-
clade_478
++++






rium_prausnitzii




Collinsella_aerofaciens
clade_553
Coprococcus_comes
clade_262
Lachnospiraceae_bacte-
clade_260
+++






rium_5_1_57FAA




Collinsella_aerofaciens
clade_553
Faecalibacte-
clade_478
Blautia_producta
clade_309
++++




rium_prausnitzii






Collinsella_aerofaciens
clade_553
Faecalibacte-
clade_478
Eubacterium_rectale
clade_444
+++




rium_prausnitzii






Collinsella_aerofaciens
clade_553
Faecalibacte-
clade_478
Lachnospiraceae_bacte-
clade_260
+++




rium_prausnitzii

rium_5_1_57FAA




Collinsella_aerofaciens
clade_553
Lachnospiraceae_bacte-
clade_260
Blautia_producta
clade_309
++++




rium_5_1_57FAA






Collinsella_aerofaciens
clade_553
Lachnospiraceae_bacte-
clade_260
Eubacterium_rectale
clade_444
++++




rium_5_1_57FAA






Coprococcus_comes
clade_262
Blautia_producta
clade_309
Eubacterium_rectale
clade_444
++++


Coprococcus_comes
clade_262
Clostridium_bolteae
clade_408
Blautia_producta
clade_309
++++


Coprococcus_comes
clade_262
Clostridium_bolteae
clade_408
Clostridium_symbiosum
clade_408



Coprococcus_comes
clade_262
Clostridium_bolteae
clade_408
Eubacterium_rectale
clade_444
−−


Coprococcus_comes
clade_262
Clostridium_bolteae
clade_408
Faecalibacte-
clade_478
+++






rium_prausnitzii




Coprococcus_comes
clade_262
Clostridium_bolteae
clade_408
Lachnospiraceae_bacte-
clade_260
+++






rium_5_1_57FAA




Coprococcus_comes
clade_262
Clostridium_symbiosum
clade_408
Blautia_producta
clade_309
++++


Coprococcus_comes
clade_262
Clostridium_symbiosum
clade_408
Eubacterium_rectale
clade_444
−−−


Coprococcus_comes
clade_262
Clostridium_symbiosum
clade_408
Faecalibacte-
clade_478







rium_prausnitzii




Coprococcus_comes
clade_262
Clostridium_symbiosum
clade_408
Lachnospiraceae_bacte-
clade_260







rium_5_1_57FAA




Coprococcus_comes
clade_262
Faecalibacte-
clade_478
Blautia_producta
clade_309
++++




rium_prausnitzii






Coprococcus_comes
clade_262
Faecalibacte-
clade_478
Eubacterium_rectale
clade_444
−




rium_prausnitzii






Coprococcus_comes
clade_262
Faecalibacte-
clade_478
Lachnospiraceae_bacte-
clade_260





rium_prausnitzii

rium_5_1_57FAA




Coprococcus_comes
clade_262
Lachnospiraceae_bacte-
clade_260
Blautia_producta
clade_309
++++




rium_5_1_57FAA






Coprococcus_comes
clade_262
Lachnospiraceae_bacte-
clade_260
Eubacterium_rectale
clade_444





rium_5_1_57FAA






Faecalibacte-
clade_478
Blautia_producta
clade_309
Eubacterium_rectale
clade_444
++++


rium_prausnitzii








Faecalibacte-
clade_478
Lachnospiraceae_bacte-
clade_260
Blautia_producta
clade_309
++++


rium_prausnitzii

rium_5_1_57FAA






Faecalibacte-
clade_478
Lachnospiraceae_bacte-
clade_260
Eubacterium_rectale
clade_444



rium_prausnitzii

rium_5_1_57FAA






Lachnospiraceae_bacte-
clade_260
Blautia_producta
clade_309
Eubacterium_rectale
clade_444
++++


rium_5_1_57FAA









The CivSim shows that many ternary combinations inhibit C. difficile. 39 of 56 combinations show inhibition with a confidence interval >80%; 36 of 56 with a C.I. >90%; 36 of 56 with a C.I. >95%; 29 of 56 with a C.I. of >99%. Non-limiting but exemplary ternary combinations include those with mean log reduction greater than 0.171, e.g. any combination shown in Table 6 with a score of ++++, such as Colinsella aerofaciens, Coprococcus comes, and Blautia producta. Equally important, the CivSim assay describes ternary combinations that do not effectively inhibit C. difficile. 5 of 56 combinations promote growth with >80% confidence; 2 of 56 promote growth with >90% confidence; 1 of 56, Coprococcus comes, Clostridium symbiosum and Eubacterium rectale, promote growth with >95% confidence. 12 of 56 combinations are neutral in the assay, meaning they neither promote nor inhibit C. difficile growth to the limit of measurement.
It is straightforward for one of skill in the art to use the CivSim method to determine efficacious subsets of the Core Ecology derived from the ethanol treated spore fraction shown to be efficacious in treating C. difficile in humans.
Example AAZA
Bacterial Compositions Populating the Gut in a Mouse Model
Two bacterial compositions were evaluated in a mouse model to demonstrate the ability to populate the gastrointestinal tract. Bacteria were grown as described in ***Example 14. Compositions were pre-made under anaerobic conditions and suspended in PBS+15% glycerol and stored at −70° C. prior to
Groups of mice (10 females/group; 5 per cage) were pre-treated on Days −14 to −5 with an antibiotic cocktail consisting of 10% glucose, kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) and vancomycin (0.056 mg/ml) in their drinking water. On Day −3 they received 10 mg/kg Clindamycin by oral gavage. On Day −1, they were dosed with a microbial compositions by oral gavage in a volume of 0.2 mL (Table ZA). Microbial compositions comprised approximately equal numbers of each OTU and were dosed at approximately 1×109, 1×108 and 1×107 per OTU for each composition (e.g. microbial composition 1, comprising 15 strains, was dosed at approximately 1.5×1010, 1.5×109, and 1.5×108 total CFU). Fecal samples were collected from each cage on Day −1 (approximately 1 hour before dosing) and on Days 2, 3 and 4 post-dosing. Feces were stored frozen prior to processing and sequencing. Weight gain of mice treated with either microbial compositions was similar to that of naive, control mice.
In parallel, groups of animals treated with the same microbial compositions on Day −1 were challenged on Day 0 with approximately 104.5 spores of Clostridium difficile (ATCC 43255) via oral gavage. Mortality for C. difficile challenged animals was assessed every day from Day 0 to Day 6 and the weight and subsequent weight change of the animal was assessed with weight loss being associated with C. difficile infection. Mortality and reduced weight loss of the test article compared to the empty vehicle was used to assess the success of the test article.







TABLE ZA







Microbial compositions administered via oral gavage on Day −1










OTU
Clade













Microbial
Clostridium_butyricum
clade_252


Composition 1
Clostridium_disporicum
clade_253



Clostridium_hylemonae
clade_260



Clostridium_orbiscindens
clade_494



Clostridium_symbiosum
clade_408



Collinsella_aerofaciens
clade_553



Coprococcus_comes
clade_262



Lachnospiraceae_bacterium_5_1_57FAA
clade_260



Ruminococcus_bromii
clade_537



Blautia_producta
clade_309



Clostridium_bolteae
clade_408



Clostridium_innocuum
clade_351



Clostridium_mayombei
clade_354



Clostridium_tertium
clade_252



Ruminococcus_gnavus
clade_360


Microbial
Clostridium_disporicum
clade_253


Composition 2
Clostridium_orbiscindens
clade_494



Clostridium_symbiosum
clade_408



Collinsella_aerofaciens
clade_553



Eubacterium_rectale
clade_444



Lachnospiraceae_bacterium_5_1_57FAA
clade_260



Blautia_producta
clade_309



Clostridium_innocuum
clade_351



Clostridium_mayombei
clade_354









Fecal samples were processed by isolating and sequencing DNA according to ***Example 11 and 12. The OTU assignment of fecal samples from Days −1, 2, 3 and 4 was determined by analyzing 16S-V4 sequence reads and assigning OTUs as described in ***Example 11. clades were assigned as described in ***Example 11. Total read counts were determined for each OTU or each clade by summing the results from cages of the same experimental group. Samples with 10 or fewer sequence reads for a given OTU or clade were considered to be below background and were not included in the summation process. Results are shown by OTU (Table TAB) and by clade (Table TAC).







TABLE TAB







Population of OTUs on Days 2, 3 and 4 following dosing with Microbial Compositions











1 × 109 per OTU
1 × 108 per OTU
1 × 107 per OTU




















−1
2
3
4
−1
2
3
4
−1
2
3
4






















Microbial comp 1














Cl_butyricum
0
106
51
32
0
10
0
34
195
0
0
0


Cl_disporicum
10
1746
1190
887
0
1746
769
1011
201
11175
1531
1152


Cl_hylemonae
0
258
258
84
0
203
164
77
0
265
214
90


Cl_orbiscindens
0
188
192
471
0
188
138
276
0
221
174
341


Cl_symbiosum
0
485
482
486
0
444
379
447
0
562
427
775


Co_aerofaciens
0
0
0
0
0
0
0
0
0
0
0
0


C_comes
0
0
0
0
0
0
0
0
0
0
0
0


L_bacterium_5_1_57FAA
0
341
336
354
0
351
182
356
0
256
240
300


R_bromii
0
0
0
0
0
0
0
0
0
0
0
0


B_producta
0
0
0
0
0
0
0
0
0
0
0
0


Cl_bolteae
0
0
0
0
0
0
0
0
0
0
0
0


Cl_innocuum
0
0
0
0
0
0
0
0
0
0
0
0


Cl_mayombei
0
0
0
0
0
0
0
0
0
0
0
0


Cl_tertium
0
0
0
0
0
0
0
0
0
0
0
0


R_gnavus
0
0
0
0
0
0
0
0
0
0
0
0


Microbial comp 2














Cl_disporicum
29
11810
10948
14672
0
11349
13978
3942
0
11995
7005
6268


Cl_orbiscindens
0
510
408
764
0
332
545
544
0
310
319
432


Cl_symbiosum
0
559
508
375
0
665
494
450
0
396
639
650


Co_aerofaciens
0
0
0
0
0
0
1172
0
0
0
247
0


E_rectale
0
0
0
0
0
0
0
12
0
0
0
261


L_bacterium_5_1_57FAA
0
972
801
596
0
860
962
844
0
636
1910
1269


B_producta
0
0
0
0
0
0
0
0
0
0
0
0


Cl_innocuum
0
0
0
0
0
0
0
0
0
0
0
0


Cl_mayombei
0
0
0
0
0
0
0
0
0
0
0
0
















TABLE TAC







Population of Clades on Days 2, 3 and 4 following dosing with Microbial Compositions











1 × 109 per OTU
1 × 108 per OTU
1 × 107 per OTU




















−1
2
3
4
−1
2
3
4
−1
2
3
4






















Microbial comp 1














clade_252
0
444
252
87
0
198
122
125
209
394
231
88


clade_253
10
1746
1190
887
0
1746
769
1011
201
11175
1531
1152


clade_260
0
599
594
438
0
554
346
433
0
521
454
390


clade_262
0
14
151
51
0
0
0
0
0
12
21
57


clade_309
0
11093
9750
4023
0
9991
5208
5145
19
9311
6369
4951


clade_351
0
9064
10647
7751
0
6528
7259
8213
0
8903
10049
8701


clade_354
0
0
0
0
0
0
0
31
173
0
0
0


clade_360
0
14300
10220
11036
0
12553
12989
6889
0
9308
13483
9292


clade_408
13
8892
12985
12101
23
3952
7260
10652
43
4079
8581
14929


clade_494
0
226
227
565
0
188
184
411
0
221
200
351


clade_537
0
0
68
225
0
0
0
0
0
0
0
55


clade_553
0
0
0
0
0
0
0
0
0
0
0
0


Microbial comp 2














clade_253
29
11810
10948
14672
0
11349
13978
3942
0
11995
7005
6268


clade_260
0
1125
1312
854
0
1049
1295
1250
0
792
2121
1637


clade_309
54
12513
13731
7849
0
11610
12004
12672
0
7407
14111
10858


clade_351
0
7651
9939
5936
0
8495
9724
9207
0
6005
9833
7655


clade_354
149
0
127
429
0
0
0
39
12
0
0
0


clade_408
18
2242
4989
10480
12
1688
5580
3789
0
1068
1561
6281


clade_444
41
0
49
202
0
18
0
12
0
14
82
1578


clade_494
0
510
465
1054
0
332
565
596
0
310
319
476


clade_553
0
0
0
0
0
0
1172
0
0
0
247
0









Upon examining the OTU data in Table TAB, several patterns emerge. First, there are a group of OTUs with no sequence reads on Day −1 that show subsequent and large numbers of sequence reads on Days 2, 3, or 4; this group includes Cl. butyricum, Cl. hylemonae, Cl. orbiscindens, Cl. symbiosum, and L. bacterium_5_1_57 FAA. Cl. disporicum is comparable to this group as it has sequence reads on Day −1 that are very close to background (10 and 29 in compositions 1 and 2, respectively), which subsequently increase by as much as 1000-fold on Days 2, 3 or 4. Second, there are OTUs such as Co. aerofaciens, C. comes, R. bromii, B. producta, Cl. bolteae, Cl. mayombei, Cl. innocuum, Cl. tertium and R. gnavus which are not detectable at the OTU level in either the Day −1 sample or in subsequent samples. In composition 2, Co. aerofaciens is detected transiently on Day 2 in the 1×108 and 1×107 dose groups; E. rectale in the same experimental groups is detected on Day 3, suggesting a possible relationship between transient population by Co. aerofaciens followed by E. rectale in these groups of mice. A striking observation is that the observed number of OTU sequence reads is not highly dose dependent. Overall, the data is consistent with a model whereby OTUs populate rapidly following oral administration.
The clade-based analysis in Table TAC was performed to more thoroughly evaluate the population of the GI tract. Clade-based analysis obscures some of the details afforded by an OTU analysis. For instance, Cl. tertium and Cl. butyricum are members of the same clade and thus a clade-based analysis cannot distinguish the dynamics of these individual OTUs. However, clade-based analysis has the compensatory benefit that it is sensitive to measuring population changes that can be missed by an OTU-based analysis. The ability of 16S-V4 OTU identification to assign an OTU as a specific species depends in part on the resolving power of the 16S-V4 region for a particular species or group of species. Both the density of available reference 16S sequences for different regions of the tree as well as the inherent variability in the 16S gene between different species will determine the definitiveness of a taxonomic annotation. So in some cases, the population of a species can be followed using clade-based assignments when OTU based-detection is insensitive in a complex population. For instance, the clade-based analysis in Table 2B supports the case that R. bromii, B. producta, Cl. innocuum, and R. gnavus were able to populate since each OTU is a sole member of a clade in the microbial compositions and sequence reads went from undetectable on Day −1 to well above background on Days 2, 3 or 4. 16S V4 sequencing and clade-based analysis could not determine whether Cl. tertium or Cl. bolteae populated due to the fact that other members of their clades (Cl. butyricum and Cl. symbiosum, respectively) were present and shown to populate at the OTU level in the mice.
In the mice challenged in parallel with C. difficile, animals were significantly protected as shown in Table TAD. Mice gavaged with vehicle (phosphate buffered saline) experienced 100% mortality while microbial compositions 1 and 2 protected at all dose levels with between 0 and 10% mortality by Day 6, the last day of the experiment. In addition, weight loss in animals treated with microbial compositions 1 and 2 was minimal compared to animals receiving the vehicle gavage. These data confirm that population of the gastrointestinal tract with microbial compositions confers a clinical benefit by restoring a state of dysbiosis so that animals can resist infection by a pathogen.







TABLE TAD







Mortality by experimental group in mice challenged


with 104.5 C. difficile spores on Day 0












Dose
Deaths



Group
(CFU per OTU)
(% mortality)
















Vehicle control
N/A
10
(100%)



Microbial
109
1
(10%)



composition 1
108
1
(10%)




107
0
(0%)



Microbial
109
0
(0%)



composition 2
108
1
(10%)




107
1
(10%)










Example 36
Prophylactic Use and Treatment in a Mouse Model of Vancomycin Resistant Enterococcus (VRE) Colonization
The emergence and spread of highly antibiotic-resistant bacteria represent a major clinical challenge (Snitkin et al Science Translational Medicine, 2012). In recent years, the numbers of infections caused by organisms such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, vancomycin-resistant Enterococcus (VRE), and Clostridium difficile have increased markedly, and many of these strains are acquiring resistance to the few remaining active antibiotics. Most infections produced by highly antibiotic-resistant bacteria are acquired during hospitalizations, and preventing patient-to-patient transmission of these pathogens is one of the major challenges confronting hospitals and clinics. Most highly antibiotic-resistant bacterial strains belong to genera that colonize mucosal surfaces, usually at low densities. The highly complex microbiota that normally colonizes mucosal surfaces inhibits expansion of and domination by bacteria such as Enterobacteriaceae and Enterococcaceae. Destruction of the normal flora by antibiotic administration, however, disinhibition antibiotic-resistant members of these bacterial families, leading to their expansion to very high densities (Ubeda et al Journal of Clinical Investigation 2010). High-density colonization by these organisms can be calamitous for the susceptible patient, resulting in bacteremia and sepsis (Taur et al, Clinical Infectious Disease, 2012).
To test prophylactic use and treatment of a bacterial composition test article e.g. spore population, a VRE infection mouse model is used as previously described (Ubeda et al, Infectious Immunity 2013, Ubeda et al, Journal of clinical investigation, 2010). Briefly, experiments are done with 7-week-old C57BL/6J female mice purchased from Jackson Laboratory, housed with irradiated food, and provided with acidified water. Mice are individually housed to avoid contamination between mice due to coprophagia. For experimental infections with VRE, mice are treated with ampicillin (0.5 g/liter) in their drinking water, which is changed every 3 days.
In the treatment model, on day 1, mice are infected by means of oral gavage with 108 CFU of the vancomycin-resistant Enterococcus faecium strain purchased from ATCC (ATCC 700221). One day after infection (day 1), antibiotic treatment is stopped and VRE levels are determined at different time points by plating serial dilutions of fecal pellets on Enterococcosel agar plates (Difco) with vancomycin (8 ug/ml, Sigma). VRE colonies are identified by appearance and confirmed by Gram staining or other methods previously described (e.g. see example 1, 2 and 3). In addition, as previously described (Ubeda et al Journal of Clinical Investigation 2010), PCR of the vanA gene, which confers resistance to vancomycin, confirms the presence of VRE in infected mice. The test article e.g. bacterial composition, or ethanol treated, gradient purified spore preparation (as described herein), fecal suspension, or antibiotic treatment is delivered in PBS on days 1-3 while the negative control contains only PBS and is also delivered on days 1-3 by oral gavage. Fresh fecal stool pellets are obtained daily for the duration of the experiment from days −7 to day 10. The samples are immediately frozen and stored at −80° C. DNA was extracted using standard techniques and analyzed with 16S or comparable methods (e.g. see example 2 and 3).
In the colonization model, ampicillin is administered as described above for day −7 to day 1, treatment with the test article or vehicle control is administered on day 0-2 and the VRE resistant bacteria at 108 CFU are administered on day 14. Fecal samples are taken throughout the experiment daily from −7 to day 21 and submitted for 16S sequencing as previously described (e.g. see examples 2 and 3).
In both models titers of VRE in feces are used to evaluate the success of the test article versus the negative control. Furthermore, microbiota composition is assessed for the ability of the test article to induce a healthy microbiome.
Example 37
Prophylactic Use and Treatment of a Mouse Model of Carbapenem Resistant Klebsiella (CRKB) Colonization
The emergence of Klebsiella pneumoniae strains with decreased susceptibility to carbapenems is a significant threat to hospitalized patients. Resistance to carbapenems in these organisms is most frequently mediated by K. pneumoniae carbapenemase (KPC), a class A beta-lactamase that also confers resistance to broad-spectrum cephalosporins and commercially available beta-lactam/beta-lactamase inhibitor combinations (Queenan et al, Clinical Microbiology Review, 2007). KPC-producing K. pneumoniae (KPC-Kp) strains often harbor resistance determinants against several other classes of antimicrobials, including aminoglycosides and fluoroquinolones, resulting in truly multidrug-resistant (MDR) organisms (Hirsch et al, Journal of Antimicrobial Chemotherapy, 2009). Considering the limited antimicrobial options, infections caused by KPC-Kp pose a tremendous therapeutic challenge and are associated with poor clinical outcomes
A treatment protocol in a mouse model as previously described (e.g. Perez et al, Antimicrobial Agents Chemotherapy, 2011) is used to evaluate the test article e.g. bacterial composition for treating carbapenem resistant Klebsiella and reducing carriage in the GI tract. Female CF1 mice (Harlan Sprague-Dawley, Indianapolis, Ind.) are used and are individually housed and weighed between 25 and 30 g.
The thoroughly characterized strain of K. pneumoniae, VA-367 (8, 9, 25) is used in this study. This clinical isolate is genetically related to the KPC-Kp strain circulating in the Eastern United States. Characterization of the resistance mechanisms in K. pneumoniae VA-367 with PCR and DNA sequence analysis revealed the presence of blaKPC-3, blaTEM-1, blaSLV-11, and blaSHV-12 as well as qnrB19 and aac(6′)-lb. Additionally, PCR and DNA sequencing revealed disruptions in the coding sequences of the following outer membrane protein genes: ompK35, ompK36, and ompK37. Antibiotic susceptibility testing (AST) was performed with the agar dilution method and interpreted according to current recommendations from the Clinical and Laboratory Standards Institute (CLSI). A modified Hodge test was performed, according to a method described previously (e.g. see Anderson et al, Journal of Clinical Microbiology, 2007) with ertapenem, meropenem, and imipenem. Tigecycline and polymyxin E were evaluated by Etest susceptibility assays (AB bioM ́erieux, Solna, Sweden). Results for tigecycline were interpreted as suggested by the U.S. Food and Drug Administration (FDA) and according to CLSI recommendations (criteria for Pseudomonas) for polymyxin E.
Mice (10 per group) are assigned to either a test article e.g. bacterial composition, ethanol treated, spore preparation (e.g. see example 6), antibiotic clindamycin, piperacillin-tazobactam, tigecycline, ertapenem, cefepime, ciprofloxacin, or combination thereof or control group receiving only the vehicle. They are administered the test article daily from day −10 to day 0, on day 0, 103 CFU of KPC-Kp VA-367 diluted in 0.5 ml phosphate-buffered saline (PBS) was administered by oral gavage using a stainless-steel feeding tube (Perfektum, Popper & Sons, New Hyde Park, N.Y.). Stool samples were collected 1, 4, 6, and 11 days after the administration of KPC-Kp in order to measure the concentration of carbapenem-resistant K. pneumoniae. Stool samples (100 mg diluted in 800 ml of PBS) are plated onto MacConkey agar with and without 0.5 ug/ml of imipenem, and the number of CFU per gram of stool was determined. Alternatively other methods may be used to measure the levels of carbapenem-resistant K. pneumoniae e.g. pcr, antigen testing, as one who's skilled in the art could perform.
Stool samples were collected after 5 days of treatment to assess the effects of the antibiotics on the stool microflora and to measure antibiotic levels in stool. To assess the effects on the microflora, fresh stool samples as previously described (e.g. see examples 2 and 3). Additional experiments are performed to examine whether the administration the test article e.g. bacterial composition resulted in the elimination or persistence of colonization with KPC-Kp VA-367.
Mice are treated with subcutaneous clindamycin to reduce the normal intestinal flora 1 day before receiving 104 CFU of KPC-Kp VA-367 by oral gavage, and the mice continued to receive subcutaneous clindamycin every other day for 7 days. Concurrently, for 7 days after oral gavage with KPC-Kp, mice received oral gavage of normal saline (control group), or the bacterial composition as specified. An additional dose of subcutaneous clindamycin was administered 20 days after the administration of KPC-Kp VA-367 to assess whether low levels of carbapenem-resistant K. pneumoniae were present that could be augmented by the elimination of the anaerobic microflora. Stool samples were collected at baseline and at 3, 6, 8, 11, 16, and 21 days after KPC-Kp VA-367 was given by gavage. The bacterial composition will be examined by the reduction of CRKB in feces.
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.
Additional Tables







TABLE 1







List of Operational Taxonomic Units (OTU) with taxonomic assignments


made to Genus, Species, and Phylogenetic Clade













SEQ ID
Public DB

Spore
Pathogen


OTU
Number
Accession
Clade
Former
Status















Eubacterium saburreum
858
AB525414
clade_178
Y
N


Eubacterium sp. oral clone IR009
866
AY349376
clade_178
Y
N


Lachnospiraceae bacterium ICM62
1061
HQ616401
clade_178
Y
N


Lachnospiraceae bacterium MSX33
1062
HQ616384
clade_178
Y
N


Lachnospiraceae bacterium oral taxon 107
1063
ADDS01000069
clade_178
Y
N


Alicyclobacillus acidocaldarius
122
NR_074721
clade_179
Y
N


Clostridium baratii
555
NR_029229
clade_223
Y
N


Clostridium colicanis
576
FJ957863
clade_223
Y
N


Clostridium paraputrificum
611
AB536771
clade_223
Y
N


Clostridium sardiniense
621
NR_041006
clade_223
Y
N


Eubacterium budayi
837
NR_024682
clade_223
Y
N


Eubacterium moniliforme
851
HF558373
clade_223
Y
N


Eubacterium multiforme
852
NR_024683
clade_223
Y
N


Eubacterium nitritogenes
853
NR_024684
clade_223
Y
N


Anoxybacillus flavithermus
173
NR_074667
clade_238
Y
N


Bacillus aerophilus
196
NR_042339
clade_238
Y
N


Bacillus aestuarii
197
GQ980243
clade_238
Y
N


Bacillus amyloliquefaciens
199
NR_075005
clade_238
Y
N


Bacillus anthracis
200
AAEN01000020
clade_238
Y
Category-A


Bacillus atrophaeus
201
NR_075016
clade_238
Y
OP


Bacillus badius
202
NR_036893
clade_238
Y
OP


Bacillus cereus
203
ABDJ01000015
clade_238
Y
OP


Bacillus circulans
204
AB271747
clade_238
Y
OP


Bacillus firmus
207
NR_025842
clade_238
Y
OP


Bacillus flexus
208
NR_024691
clade_238
Y
OP


Bacillus fordii
209
NR_025786
clade_238
Y
OP


Bacillus halmapalus
211
NR_026144
clade_238
Y
OP


Bacillus herbersteinensis
213
NR_042286
clade_238
Y
OP


Bacillus idriensis
215
NR_043268
clade_238
Y
OP


Bacillus lentus
216
NR_040792
clade_238
Y
OP


Bacillus licheniformis
217
NC_006270
clade_238
Y
OP


Bacillus megaterium
218
GU252124
clade_238
Y
OP


Bacillus nealsonii
219
NR_044546
clade_238
Y
OP


Bacillus niabensis
220
NR_043334
clade_238
Y
OP


Bacillus niacini
221
NR_024695
clade_238
Y
OP


Bacillus pocheonensis
222
NR_041377
clade_238
Y
OP


Bacillus pumilus
223
NR_074977
clade_238
Y
OP


Bacillus safensis
224
JQ624766
clade_238
Y
OP


Bacillus simplex
225
NR_042136
clade_238
Y
OP


Bacillus sonorensis
226
NR_025130
clade_238
Y
OP


Bacillus sp. 10403023 MM10403188
227
CAET01000089
clade_238
Y
OP


Bacillus sp. 2_A_57_CT2
230
ACWD01000095
clade_238
Y
OP


Bacillus sp. 2008724126
228
GU252108
clade_238
Y
OP


Bacillus sp. 2008724139
229
GU252111
clade_238
Y
OP


Bacillus sp. 7_16AIA
231
FN397518
clade_238
Y
OP


Bacillus sp. AP8
233
JX101689
clade_238
Y
OP


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


Bacillus sp. BT1B_CT2
235
ACWC01000034
clade_238
Y
OP


Bacillus sp. GB1.1
236
FJ897765
clade_238
Y
OP


Bacillus sp. GB9
237
FJ897766
clade_238
Y
OP


Bacillus sp. HU19.1
238
FJ897769
clade_238
Y
OP


Bacillus sp. HU29
239
FJ897771
clade_238
Y
OP


Bacillus sp. HU33.1
240
FJ897772
clade_238
Y
OP


Bacillus sp. JC6
241
JF824800
clade_238
Y
OP


Bacillus sp. oral taxon F79
248
HM099654
clade_238
Y
OP


Bacillus sp. SRC_DSF1
243
GU797283
clade_238
Y
OP


Bacillus sp. SRC_DSF10
242
GU797292
clade_238
Y
OP


Bacillus sp. SRC_DSF2
244
GU797284
clade_238
Y
OP


Bacillus sp. SRC_DSF6
245
GU797288
clade_238
Y
OP


Bacillus sp. tc09
249
HQ844242
clade_238
Y
OP


Bacillus sp. zh168
250
FJ851424
clade_238
Y
OP


Bacillus sphaericus
251
DQ286318
clade_238
Y
OP


Bacillus sporothermodurans
252
NR_026010
clade_238
Y
OP


Bacillus subtilis
253
EU627588
clade_238
Y
OP


Bacillus thermoamylovorans
254
NR_029151
clade_238
Y
OP


Bacillus thuringiensis
255
NC_008600
clade_238
Y
OP


Bacillus weihenstephanensis
256
NR_074926
clade_238
Y
OP


Geobacillus kaustophilus
933
NR_074989
clade_238
Y
N


Geobacillus stearothermophilus
936
NR_040794
clade_238
Y
N


Geobacillus thermodenitrificans
938
NR_074976
clade_238
Y
N


Geobacillus thermoglucosidasius
939
NR_043022
clade_238
Y
N


Lysinibacillus sphaericus
1193
NR_074883
clade_238
Y
N


Clostridiales sp. SS3_4
543
AY305316
clade_246
Y
N


Clostridium beijerinckii
557
NR_074434
clade_252
Y
N


Clostridium botulinum
560
NC_010723
clade_252
Y
Category-A


Clostridium butyricum
561
ABDT01000017
clade_252
Y
N


Clostridium chauvoei
568
EU106372
clade_252
Y
N


Clostridium favososporum
582
X76749
clade_252
Y
N


Clostridium histolyticum
592
HF558362
clade_252
Y
N


Clostridium isatidis
597
NR_026347
clade_252
Y
N


Clostridium limosum
602
FR870444
clade_252
Y
N


Clostridium sartagoforme
622
NR_026490
clade_252
Y
N


Clostridium septicum
624
NR_026020
clade_252
Y
N


Clostridium sp. 7_2_43FAA
626
ACDK01000101
clade_252
Y
N


Clostridium sporogenes
645
ABKW02000003
clade_252
Y
N


Clostridium tertium
653
Y18174
clade_252
Y
N


Clostridium carnis
564
NR_044716
clade_253
Y
N


Clostridium celatum
565
X77844
clade_253
Y
N


Clostridium disporicum
579
NR_026491
clade_253
Y
N


Clostridium gasigenes
585
NR_024945
clade_253
Y
N


Clostridium quinii
616
NR_026149
clade_253
Y
N


Clostridium hylemonae
593
AB023973
clade_260
Y
N


Clostridium scindens
623
AF262238
clade_260
Y
N


Lachnospiraceae bacterium 5_1_57FAA
1054
ACTR01000020
clade_260
Y
N


Clostridium glycyrrhizinilyticum
588
AB233029
clade_262
Y
N


Clostridium nexile
607
X73443
clade_262
Y
N


Coprococcus comes
674
ABVR01000038
clade_262
Y
N


Lachnospiraceae bacterium 1_1_57FAA
1048
ACTM01000065
clade_262
Y
N


Lachnospiraceae bacterium 1_4_56FAA
1049
ACTN01000028
clade_262
Y
N


Lachnospiraceae bacterium 8_1_57FAA
1057
ACWQ01000079
clade_262
Y
N


Ruminococcus lactaris
1663
ABOU02000049
clade_262
Y
N


Ruminococcus torques
1670
AAVP02000002
clade_262
Y
N


Paenibacillus lautus
1397
NR_040882
clade_270
Y
N


Paenibacillus polymyxa
1399
NR_037006
clade_270
Y
N


Paenibacillus sp. HGF5
1402
AEXS01000095
clade_270
Y
N


Paenibacillus sp. HGF7
1403
AFDH01000147
clade_270
Y
N


Eubacterium sp. oral clone JI012
868
AY349379
clade_298
Y
N


Alicyclobacillus contaminans
124
NR_041475
clade_301
Y
N


Alicyclobacillus herbarius
126
NR_024753
clade_301
Y
N


Alicyclobacillus pomorum
127
NR_024801
clade_301
Y
N


Blautia coccoides
373
AB571656
clade_309
Y
N


Blautia glucerasea
374
AB588023
clade_309
Y
N


Blautia glucerasei
375
AB439724
clade_309
Y
N


Blautia hansenii
376
ABYU02000037
clade_309
Y
N


Blautia luti
378
AB691576
clade_309
Y
N


Blautia producta
379
AB600998
clade_309
Y
N


Blautia schinkii
380
NR_026312
clade_309
Y
N


Blautia sp. M25
381
HM626178
clade_309
Y
N


Blautia stercoris
382
HM626177
clade_309
Y
N


Blautia wexlerae
383
EF036467
clade_309
Y
N


Bryantella formatexigens
439
ACCL02000018
clade_309
Y
N


Clostridium coccoides
573
EF025906
clade_309
Y
N


Eubacterium cellulosolvens
839
AY178842
clade_309
Y
N


Lachnospiraceae bacterium 6_1_63FAA
1056
ACTV01000014
clade_309
Y
N


Ruminococcus hansenii
1662
M59114
clade_309
Y
N


Ruminococcus obeum
1664
AY169419
clade_309
Y
N


Ruminococcus sp. 5_1_39BFAA
1666
ACII01000172
clade_309
Y
N


Ruminococcus sp. K_1
1669
AB222208
clade_309
Y
N


Syntrophococcus sucromutans
1911
NR_036869
clade_309
Y
N


Bacillus alcalophilus
198
X76436
clade_327
Y
N


Bacillus clausii
205
FN397477
clade_327
Y
OP


Bacillus gelatini
210
NR_025595
clade_327
Y
OP


Bacillus halodurans
212
AY144582
clade_327
Y
OP


Bacillus sp. oral taxon F26
246
HM099642
clade_327
Y
OP


Clostridium innocuum
595
M23732
clade_351
Y
N


Clostridium sp. HGF2
628
AENW01000022
clade_351
Y
N


Clostridium perfringens
612
ABDW01000023
clade_353
Y
Category-B


Sarcina ventriculi
1687
NR_026146
clade_353
Y
N


Clostridium bartlettii
556
ABEZ02000012
clade_354
Y
N


Clostridium bifermentans
558
X73437
clade_354
Y
N


Clostridium ghonii
586
AB542933
clade_354
Y
N


Clostridium glycolicum
587
FJ384385
clade_354
Y
N


Clostridium mayombei
605
FR733682
clade_354
Y
N


Clostridium sordellii
625
AB448946
clade_354
Y
N


Clostridium sp. MT4 E
635
FJ159523
clade_354
Y
N


Eubacterium tenue
872
M59118
clade_354
Y
N


Clostridium argentinense
553
NR_029232
clade_355
Y
N


Clostridium sp. JC122
630
CAEV01000127
clade_355
Y
N


Clostridium sp. NMBHI_1
636
JN093130
clade_355
Y
N


Clostridium subterminale
650
NR_041795
clade_355
Y
N


Clostridium sulfidigenes
651
NR_044161
clade_355
Y
N


Dorea formicigenerans
773
AAXA02000006
clade_360
Y
N


Dorea longicatena
774
AJ132842
clade_360
Y
N


Lachnospiraceae bacterium 2_1_46FAA
1050
ADLB01000035
clade_360
Y
N


Lachnospiraceae bacterium 2_1_58FAA
1051
ACT001000052
clade_360
Y
N


Lachnospiraceae bacterium 4_1_37FAA
1053
ADCR01000030
clade_360
Y
N


Lachnospiraceae bacterium 9_1_43BFAA
1058
ACTX01000023
clade_360
Y
N


Ruminococcus gnavus
1661
X94967
clade_360
Y
N


Ruminococcus sp. ID8
1668
AY960564
clade_360
Y
N


Blautia hydrogenotrophica
377
ACBZ01000217
clade_368
Y
N


Lactonifactor longoviformis
1147
DQ100449
clade_368
Y
N


Robinsoniella peoriensis
1633
AF445258
clade_368
Y
N


Eubacterium infirmum
849
U13039
clade_384
Y
N


Eubacterium sp. WAL 14571
864
FJ687606
clade_384
Y
N


Erysipelotrichaceae bacterium 5_2_54FAA
823
ACZW01000054
clade_385
Y
N


Eubacterium biforme
835
ABYT01000002
clade_385
Y
N


Eubacterium cylindroides
842
FP929041
clade_385
Y
N


Eubacterium dolichum
844
L34682
clade_385
Y
N


Eubacterium sp. 3_1_31
861
ACTL01000045
clade_385
Y
N


Eubacterium tortuosum
873
NR_044648
clade_385
Y
N


Bulleidia extructa
441
ADFR01000011
clade_388
Y
N


Solobacterium moorei
1739
AECQ01000039
clade_388
Y
N


Coprococcus catus
673
EU266552
clade_393
Y
N


Lachnospiraceae bacterium oral taxon F15
1064
HM099641
clade_393
Y
N


Clostridium cochlearium
574
NR_044717
clade_395
Y
N


Clostridium malenominatum
604
FR749893
clade_395
Y
N


Clostridium tetani
654
NC_004557
clade_395
Y
N


Acetivibrio ethanolgignens
6
FR749897
clade_396
Y
N


Anaerosporobacter mobilis
161
NR_042953
clade_396
Y
N


Bacteroides pectinophilus
288
ABVQ01000036
clade_396
Y
N


Clostridium aminovalericum
551
NR_029245
clade_396
Y
N


Clostridium phytofermentans
613
NR_074652
clade_396
Y
N


Eubacterium hallii
848
L34621
clade_396
Y
N


Eubacterium xylanophilum
875
L34628
clade_396
Y
N


Ruminococcus callidus
1658
NR_029160
clade_406
Y
N


Ruminococcus champanellensis
1659
FP929052
clade_406
Y
N


Ruminococcus sp. 18P13
1665
AJ515913
clade_406
Y
N


Ruminococcus sp. 9SE51
1667
FM954974
clade_406
Y
N


Anaerostipes caccae
162
ABAX03000023
clade_408
Y
N


Anaerostipes sp. 3_2_56FAA
163
ACWB01000002
clade_408
Y
N


Clostridiales bacterium 1_7_47FAA
541
ABQR01000074
clade_408
Y
N


Clostridiales sp. SM4_1
542
FP929060
clade_408
Y
N


Clostridiales sp. SSC_2
544
FP929061
clade_408
Y
N


Clostridium aerotolerans
546
X76163
clade_408
Y
N


Clostridium aldenense
547
NR_043680
clade_408
Y
N


Clostridium algidixylanolyticum
550
NR_028726
clade_408
Y
N


Clostridium amygdalinum
552
AY353957
clade_408
Y
N


Clostridium asparagiforme
554
ACCJ01000522
clade_408
Y
N


Clostridium bolteae
559
ABCC02000039
clade_408
Y
N


Clostridium celerecrescens
566
JQ246092
clade_408
Y
N


Clostridium citroniae
569
ADLJ01000059
clade_408
Y
N


Clostridium clostridiiformes
571
M59089
clade_408
Y
N


Clostridium clostridioforme
572
NR_044715
clade_408
Y
N


Clostridium hathewayi
590
AY552788
clade_408
Y
N


Clostridium indolis
594
AF028351
clade_408
Y
N


Clostridium lavalense
600
EF564277
clade_408
Y
N


Clostridium saccharolyticum
620
CP002109
clade_408
Y
N


Clostridium sp. M62_1
633
ACFX02000046
clade_408
Y
N


Clostridium sp. SS2_1
638
ABGC03000041
clade_408
Y
N


Clostridium sphenoides
643
X73449
clade_408
Y
N


Clostridium symbiosum
652
ADLQ01000114
clade_408
Y
N


Clostridium xylanolyticum
658
NR_037068
clade_408
Y
N


Eubacterium hadrum
847
FR749933
clade_408
Y
N


Lachnospiraceae bacterium 3_1_57FAA_CT1
1052
ACTP01000124
clade_408
Y
N


Lachnospiraceae bacterium 5_1_63FAA
1055
ACTS01000081
clade_408
Y
N


Lachnospiraceae bacterium A4
1059
DQ789118
clade_408
Y
N


Lachnospiraceae bacterium DJF VP30
1060
EU728771
clade_408
Y
N


Lachnospiraceae genomosp. C1
1065
AY278618
clade_408
Y
N


Clostridium difficile
578
NC_013315
clade_409
Y
OP


Eubacterium sp. AS15b
862
HQ616364
clade_428
Y
N


Eubacterium sp. OBRC9
863
HQ616354
clade_428
Y
N


Eubacterium sp. oral clone OH3A
871
AY947497
clade_428
Y
N


Eubacterium yurii
876
AEES01000073
clade_428
Y
N


Clostridium acetobutylicum
545
NR_074511
clade_430
Y
N


Clostridium algidicarnis
549
NR_041746
clade_430
Y
N


Clostridium cadaveris
562
AB542932
clade_430
Y
N


Clostridium carboxidivorans
563
FR733710
clade_430
Y
N


Clostridium estertheticum
580
NR_042153
clade_430
Y
N


Clostridium fallax
581
NR_044714
clade_430
Y
N


Clostridium felsineum
583
AF270502
clade_430
Y
N


Clostridium frigidicarnis
584
NR_024919
clade_430
Y
N


Clostridium kluyveri
598
NR_074165
clade_430
Y
N


Clostridium magnum
603
X77835
clade_430
Y
N


Clostridium putrefaciens
615
NR_024995
clade_430
Y
N


Clostridium sp. HPB_46
629
AY862516
clade_430
Y
N


Clostridium tyrobutyricum
656
NR_044718
clade_430
Y
N


Sutterella parvirubra
1899
AB300989
clade_432
Y
N


Acetanaerobacterium elongatum
4
NR_042930
clade_439
Y
N


Clostridium cellulosi
567
NR_044624
clade_439
Y
N


Ethanoligenens harbinense
832
AY675965
clade_439
Y
N


Eubacterium rectale
856
FP929042
clade_444
Y
N


Eubacterium sp. oral clone GI038
865
AY349374
clade_444
Y
N


Lachnobacterium bovis
1045
GU324407
clade_444
Y
N


Roseburia cecicola
1634
GU233441
clade_444
Y
N


Roseburia faecalis
1635
AY804149
clade_444
Y
N


Roseburia faecis
1636
AY305310
clade_444
Y
N


Roseburia hominis
1637
AJ270482
clade_444
Y
N


Roseburia intestinalis
1638
FP929050
clade_444
Y
N


Roseburia inulinivorans
1639
AJ270473
clade_444
Y
N


Brevibacillus brevis
410
NR_041524
clade_448
Y
N


Brevibacillus laterosporus
414
NR_037005
clade_448
Y
N


Bacillus coagulans
206
DQ297928
clade_451
Y
OP


Sporolactobacillus inulinus
1752
NR_040962
clade_451
Y
N


Kocuria palustris
1041
EU333884
clade_453
Y
N


Nocardia farcinica
1353
NC_006361
clade_455
Y
N


Bacillus sp. oral taxon F28
247
HM099650
clade_456
Y
OP


Catenibacterium mitsuokai
495
AB030224
clade_469
Y
N


Clostridium sp. TM_40
640
AB249652
clade_469
Y
N


Coprobacillus cateniformis
670
AB030218
clade_469
Y
N


Coprobacillus sp. 29_1
671
ADKX01000057
clade_469
Y
N


Clostridium rectum
618
NR_029271
clade_470
Y
N


Eubacterium nodatum
854
U13041
clade_476
Y
N


Eubacterium saphenum
859
NR_026031
clade_476
Y
N


Eubacterium sp. oral clone JH012
867
AY349373
clade_476
Y
N


Eubacterium sp. oral clone JS001
870
AY349378
clade_476
Y
N


Faecalibacterium prausnitzii
880
ACOP02000011
clade_478
Y
N


Gemmiger formicilis
932
GU562446
clade_478
Y
N


Subdoligranulum variabile
1896
AJ518869
clade_478
Y
N


Clostridiaceae bacterium JC13
532
JF824807
clade_479
Y
N


Clostridium sp. MLG055
634
AF304435
clade_479
Y
N


Erysipelotrichaceae bacterium 3_1_53
822
ACTJ01000113
clade_479
Y
N


Clostridium cocleatum
575
NR_026495
clade_481
Y
N


Clostridium ramosum
617
M23731
clade_481
Y
N


Clostridium saccharogumia
619
DQ100445
clade_481
Y
N


Clostridium spiroforme
644
X73441
clade_481
Y
N


Coprobacillus sp. D7
672
ACDT01000199
clade_481
Y
N


Clostridiales bacterium SY8519
535
AB477431
clade_482
Y
N


Clostridium sp. SY8519
639
AP012212
clade_482
Y
N


Eubacterium ramulus
855
AJ011522
clade_482
Y
N


Erysipelothrix inopinata
819
NR_025594
clade_485
Y
N


Erysipelothrix rhusiopathiae
820
ACLK01000021
clade_485
Y
N


Erysipelothrix tonsillarum
821
NR_040871
clade_485
Y
N


Holdemania filiformis
1004
Y11466
clade_485
Y
N


Mollicutes bacterium pACH93
1258
AY297808
clade_485
Y
N


Coxiella burnetii
736
CP000890
clade_486
Y
Category-B


Clostridium hiranonis
591
AB023970
clade_487
Y
N


Clostridium irregulare
596
NR_029249
clade_487
Y
N


Clostridium orbiscindens
609
Y18187
clade_494
Y
N


Clostridium sp. NML 04A032
637
EU815224
clade_494
Y
N


Flavonifractor plautii
886
AY724678
clade_494
Y
N


Pseudoflavonifractor capillosus
1591
AY136666
clade_494
Y
N


Ruminococcaceae bacterium D16
1655
ADDX01000083
clade_494
Y
N


Acetivibrio cellulolyticus
5
NR_025917
clade_495
Y
N


Clostridium aldrichii
548
NR_026099
clade_495
Y
N


Clostridium clariflavum
570
NR_041235
clade_495
Y
N


Clostridium stercorarium
647
NR_025100
clade_495
Y
N


Clostridium straminisolvens
649
NR_024829
clade_495
Y
N


Clostridium thermocellum
655
NR_074629
clade_495
Y
N


Fusobacterium nucleatum
901
ADVK01000034
clade_497
Y
N


Eubacterium barkeri
834
NR_044661
clade_512
Y
N


Eubacterium callanderi
838
NR_026330
clade_512
Y
N


Eubacterium limosum
850
CP002273
clade_512
Y
N


Anaerotruncus colihominis
164
ABGD02000021
clade_516
Y
N


Clostridium methylpentosum
606
ACEC01000059
clade_516
Y
N


Clostridium sp. YIT 12070
642
AB491208
clade_516
Y
N


Hydrogenoanaerobacterium saccharovorans
1005
NR_044425
clade_516
Y
N


Ruminococcus albus
1656
AY445600
clade_516
Y
N


Ruminococcus flavefaciens
1660
NR_025931
clade_516
Y
N


Clostridium haemolyticum
589
NR_024749
clade_517
Y
N


Clostridium novyi
608
NR_074343
clade_517
Y
N


Clostridium sp. LMG 16094
632
X95274
clade_517
Y
N


Eubacterium ventriosum
874
L34421
clade_519
Y
N


Bacteroides galacturonicus
280
DQ497994
clade_522
Y
N


Eubacterium eligens
845
CP001104
clade_522
Y
N


Lachnospira multipara
1046
FR733699
clade_522
Y
N


Lachnospira pectinoschiza
1047
L14675
clade_522
Y
N


Lactobacillus rogosae
1114
GU269544
clade_522
Y
N


Bacillus horti
214
NR_036860
clade_527
Y
OP


Bacillus sp. 9_3AIA
232
FN397519
clade_527
Y
OP


Eubacterium brachy
836
U13038
clade_533
Y
N


Filifactor alocis
881
CP002390
clade_533
Y
N


Filifactor villosus
882
NR_041928
clade_533
Y
N


Clostridium leptum
601
AJ305238
clade_537
Y
N


Clostridium sp. YIT 12069
641
AB491207
clade_537
Y
N


Clostridium sporosphaeroides
646
NR_044835
clade_537
Y
N


Eubacterium coprostanoligenes
841
HM037995
clade_537
Y
N


Ruminococcus bromii
1657
EU266549
clade_537
Y
N


Eubacterium siraeum
860
ABCA03000054
clade_538
Y
N


Clostridium viride
657
NR_026204
clade_540
Y
N


Oscillibacter sp. G2
1386
HM626173
clade_540
Y
N


Oscillibacter valericigenes
1387
NR_074793
clade_540
Y
N


Oscillospira guilliermondii
1388
AB040495
clade_540
Y
N


Butyrivibrio crossotus
455
ABWN01000012
clade_543
Y
N


Clostridium sp. L2_50
631
AAYW02000018
clade_543
Y
N


Coprococcus eutactus
675
EF031543
clade_543
Y
N


Coprococcus sp. ART55_1
676
AY350746
clade_543
Y
N


Eubacterium ruminantium
857
NR_024661
clade_543
Y
N


Collinsella aerofaciens
659
AAVN02000007
clade_553
Y
N


Alkaliphilus metalliredigenes
137
AY137848
clade_554
Y
N


Alkaliphilus oremlandii
138
NR_043674
clade_554
Y
N


Clostridium sticklandii
648
L04167
clade_554
Y
N


Turicibacter sanguinis
1965
AF349724
clade_555
Y
N


Fulvimonas sp. NML 060897
892
EF589680
clade_557
Y
N


Desulfitobacterium frappieri
753
AJ276701
clade_560
Y
N


Desulfitobacterium hafniense
754
NR_074996
clade_560
Y
N


Desulfotomaculum nigrificans
756
NR_044832
clade_560
Y
N


Lutispora thermophila
1191
NR_041236
clade_564
Y
N


Brachyspira pilosicoli
405
NR_075069
clade_565
Y
N


Eggerthella lenta
778
AF292375
clade_566
Y
N


Streptomyces albus
1888
AJ697941
clade_566
Y
N


Chlamydiales bacterium NS11
505
JN606074
clade_567
Y
N


Anaerofustis stercorihominis
159
ABIL02000005
clade_570
Y
N


Butyricicoccus pullicaecorum
453
HH793440
clade_572
Y
N


Eubacterium desmolans
843
NR_044644
clade_572
Y
N


Papillibacter cinnamivorans
1415
NR_025025
clade_572
Y
N


Sporobacter termitidis
1751
NR_044972
clade_572
Y
N


Deferribacteres sp. oral clone JV006
744
AY349371
clade_575
Y
N


Clostridium colinum
577
NR_026151
clade_576
Y
N


Clostridium lactatifermentans
599
NR_025651
clade_576
Y
N


Clostridium piliforme
614
D14639
clade_576
Y
N


Saccharomonospora viridis
1671
X54286
clade_579
Y
N


Thermobifida fusca
1921
NC_007333
clade_579
Y
N


Leptospira licerasiae
1164
EF612284
clade_585
Y
OP


Moorella thermoacetica
1259
NR_075001
clade_590
Y
N


Thermoanaerobacter pseudethanolicus
1920
CP000924
clade_590
Y
N


Flexistipes sinusarabici
888
NR_074881
clade_591
Y
N


Gloeobacter violaceus
942
NR_074282
clade_596
Y
N


Eubacterium sp. oral clone JN088
869
AY349377
clade_90
Y
N


Clostridium oroticum
610
FR749922
clade_96
Y
N


Clostridium sp. D5
627
ADBG01000142
clade_96
Y
N


Eubacterium contortum
840
FR749946
clade_96
Y
N


Eubacterium fissicatena
846
FR749935
clade_96
Y
N


Corynebacterium coyleae
692
X96497
clade_100
N
N


Corynebacterium mucifaciens
711
NR_026396
clade_100
N
N


Corynebacterium ureicelerivorans
733
AM397636
clade_100
N
N


Corynebacterium appendicis
684
NR_028951
clade_102
N
N


Corynebacterium genitalium
698
ACLJ01000031
clade_102
N
N


Corynebacterium glaucum
699
NR_028971
clade_102
N
N


Corynebacterium imitans
703
AF537597
clade_102
N
N


Corynebacterium riegelii
719
EU848548
clade_102
N
N


Corynebacterium sp. L_2012475
723
HE575405
clade_102
N
N


Corynebacterium sp. NML 93_0481
724
GU238409
clade_102
N
N


Corynebacterium sundsvallense
728
Y09655
clade_102
N
N


Corynebacterium tuscaniae
730
AY677186
clade_102
N
N


Prevotella maculosa
1504
AGEK01000035
clade_104
N
N


Prevotella oris
1513
ADDV01000091
clade_104
N
N


Prevotella salivae
1517
AB108826
clade_104
N
N


Prevotella sp. ICM55
1521
HQ616399
clade_104
N
N


Prevotella sp. oral clone AA020
1528
AY005057
clade_104
N
N


Prevotella sp. oral clone GI032
1538
AY349396
clade_104
N
N


Prevotella sp. oral taxon G70
1558
GU432179
clade_104
N
N


Prevotella corporis
1491
L16465
clade_105
N
N


Bacteroides sp. 4_1_36
312
ACTC01000133
clade_110
N
N


Bacteroides sp. AR20
315
AF139524
clade_110
N
N


Bacteroides sp. D20
319
ACPT01000052
clade_110
N
N


Bacteroides sp. F_4
322
AB470322
clade_110
N
N


Bacteroides uniformis
329
AB050110
clade_110
N
N


Prevotella nanceiensis
1510
JN867228
clade_127
N
N


Prevotella sp. oral taxon 299
1548
ACWZ01000026
clade_127
N
N


Prevotella bergensis
1485
ACKS01000100
clade_128
N
N


Prevotella buccalis
1489
JN867261
clade_129
N
N


Prevotella timonensis
1564
ADEF01000012
clade_129
N
N


Prevotella oralis
1512
AEPE01000021
clade_130
N
N


Prevotella sp. SEQ072
1525
JN867238
clade_130
N
N


Leuconostoc carnosum
1177
NR_040811
clade_135
N
N


Leuconostoc gasicomitatum
1179
FN822744
clade_135
N
N


Leuconostoc inhae
1180
NR_025204
clade_135
N
N


Leuconostoc kimchii
1181
NR_075014
clade_135
N
N


Edwardsiella tarda
777
CP002154
clade_139
N
N


Photorhabdus asymbiotica
1466
Z76752
clade_139
N
N


Psychrobacter arcticus
1607
CP000082
clade_141
N
N


Psychrobacter cibarius
1608
HQ698586
clade_141
N
N


Psychrobacter cryohalolentis
1609
CP000323
clade_141
N
N


Psychrobacter faecalis
1610
HQ698566
clade_141
N
N


Psychrobacter nivimaris
1611
HQ698587
clade_141
N
N


Psychrobacter pulmonis
1612
HQ698582
clade_141
N
N


Pseudomonas aeruginosa
1592
AABQ07000001
clade_154
N
N


Pseudomonas sp. 2_1_26
1600
ACWU01000257
clade_154
N
N


Corynebacterium confusum
691
Y15886
clade_158
N
N


Corynebacterium propinquum
712
NR_037038
clade_158
N
N


Corynebacterium pseudodiphtheriticum
713
X84258
clade_158
N
N


Bartonella bacilliformis
338
NC_008783
clade_159
N
N


Bartonella grahamii
339
CP001562
clade_159
N
N


Bartonella henselae
340
NC_005956
clade_159
N
N


Bartonella quintana
341
BX897700
clade_159
N
N


Bartonella tamiae
342
EF672728
clade_159
N
N


Bartonella washoensis
343
FJ719017
clade_159
N
N


Brucella abortus
430
ACBJ01000075
clade_159
N
Category-B


Brucella canis
431
NR_044652
clade_159
N
Category-B


Brucella ceti
432
ACJD01000006
clade_159
N
Category-B


Brucella melitensis
433
AE009462
clade_159
N
Category-B


Brucella microti
434
NR_042549
clade_159
N
Category-B


Brucella ovis
435
NC_009504
clade_159
N
Category-B


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


Brucella sp. BO1
437
EU053207
clade_159
N
Category-B


Brucella suis
438
ACBK01000034
clade_159
N
Category-B


Ochrobactrum anthropi
1360
NC_009667
clade_159
N
N


Ochrobactrum intermedium
1361
ACQA01000001
clade_159
N
N


Ochrobactrum pseudintermedium
1362
DQ365921
clade_159
N
N


Prevotella genomosp. C2
1496
AY278625
clade_164
N
N


Prevotella multisaccharivorax
1509
AFJE01000016
clade_164
N
N


Prevotella sp. oral clone IDR_CEC_0055
1543
AY550997
clade_164
N
N


Prevotella sp. oral taxon 292
1547
GQ422735
clade_164
N
N


Prevotella sp. oral taxon 300
1549
GU409549
clade_164
N
N


Prevotella marshii
1505
AEEI01000070
clade_166
N
N


Prevotella sp. oral clone IK053
1544
AY349401
clade_166
N
N


Prevotella sp. oral taxon 781
1554
GQ422744
clade_166
N
N


Prevotella stercorea
1562
AB244774
clade_166
N
N


Prevotella brevis
1487
NR_041954
clade_167
N
N


Prevotella ruminicola
1516
CP002006
clade_167
N
N


Prevotella sp. sp24
1560
AB003384
clade_167
N
N


Prevotella sp. sp34
1561
AB003385
clade_167
N
N


Prevotella albensis
1483
NR_025300
clade_168
N
N


Prevotella copri
1490
ACBX02000014
clade_168
N
N


Prevotella oulorum
1514
L16472
clade_168
N
N


Prevotella sp. BI_42
1518
AJ581354
clade_168
N
N


Prevotella sp. oral clone P4PB_83 P2
1546
AY207050
clade_168
N
N


Prevotella sp. oral taxon G60
1557
GU432133
clade_168
N
N


Prevotella amnii
1484
AB547670
clade_169
N
N


Bacteroides caccae
268
EU136686
clade_170
N
N


Bacteroides finegoldii
277
AB222699
clade_170
N
N


Bacteroides intestinalis
283
ABJL02000006
clade_171
N
N


Bacteroides sp. XB44A
326
AM230649
clade_171
N
N


Bifidobacteriaceae genomosp. C1
345
AY278612
clade_172
N
N


Bifidobacterium adolescentis
346
AAXD02000018
clade_172
N
N


Bifidobacterium angulatum
347
ABYS02000004
clade_172
N
N


Bifidobacterium animalis
348
CP001606
clade_172
N
N


Bifidobacterium breve
350
CP002743
clade_172
N
N


Bifidobacterium catenulatum
351
ABXY01000019
clade_172
N
N


Bifidobacterium dentium
352
CP001750
clade_172
N
OP


Bifidobacterium gallicum
353
ABXB03000004
clade_172
N
N


Bifidobacterium infantis
354
AY151398
clade_172
N
N


Bifidobacterium kashiwanohense
355
AB491757
clade_172
N
N


Bifidobacterium longum
356
ABQQ01000041
clade_172
N
N


Bifidobacterium pseudocatenulatum
357
ABXX02000002
clade_172
N
N


Bifidobacterium pseudolongum
358
NR_043442
clade_172
N
N


Bifidobacterium scardovii
359
AJ307005
clade_172
N
N


Bifidobacterium sp. HM2
360
AB425276
clade_172
N
N


Bifidobacterium sp. HMLN12
361
JF519685
clade_172
N
N


Bifidobacterium sp. M45
362
HM626176
clade_172
N
N


Bifidobacterium sp. MSX5B
363
HQ616382
clade_172
N
N


Bifidobacterium sp. TM_7
364
AB218972
clade_172
N
N


Bifidobacterium thermophilum
365
DQ340557
clade_172
N
N


Leuconostoc citreum
1178
AM157444
clade_175
N
N


Leuconostoc lactis
1182
NR_040823
clade_175
N
N


Alicyclobacillus acidoterrestris
123
NR_040844
clade_179
N
N


Alicyclobacillus cycloheptanicus
125
NR_024754
clade_179
N
N


Acinetobacter baumannii
27
ACYQ01000014
clade_181
N
N


Acinetobacter calcoaceticus
28
AM157426
clade_181
N
N


Acinetobacter genomosp. C1
29
AY278636
clade_181
N
N


Acinetobacter haemolyticus
30
ADMT01000017
clade_181
N
N


Acinetobacter johnsonii
31
ACPL01000162
clade_181
N
N


Acinetobacter junii
32
ACPM01000135
clade_181
N
N


Acinetobacter lwoffii
33
ACPN01000204
clade_181
N
N


Acinetobacter parvus
34
AIEB01000124
clade_181
N
N


Acinetobacter schindleri
36
NR_025412
clade_181
N
N


Acinetobacter sp. 56A1
37
GQ178049
clade_181
N
N


Acinetobacter sp. CIP 101934
38
JQ638573
clade_181
N
N


Acinetobacter sp. CIP 102143
39
JQ638578
clade_181
N
N


Acinetobacter sp. M16_22
41
HM366447
clade_181
N
N


Acinetobacter sp. RUH2624
42
ACQF01000094
clade_181
N
N


Acinetobacter sp. SH024
43
ADCH01000068
clade_181
N
N


Lactobacillus jensenii
1092
ACQD01000066
clade_182
N
N


Alcaligenes faecalis
119
AB680368
clade_183
N
N


Alcaligenes sp. CO14
120
DQ643040
clade_183
N
N


Alcaligenes sp. S3
121
HQ262549
clade_183
N
N


Oligella ureolytica
1366
NR_041998
clade_183
N
N


Oligella urethralis
1367
NR_041753
clade_183
N
N


Eikenella corrodens
784
ACEA01000028
clade_185
N
N


Kingella denitrificans
1019
AEWV01000047
clade_185
N
N


Kingella genomosp. P1 oral cone MB2_C20
1020
DQ003616
clade_185
N
N


Kingella kingae
1021
AFHS01000073
clade_185
N
N


Kingella oralis
1022
ACJW02000005
clade_185
N
N


Kingella sp. oral clone ID059
1023
AY349381
clade_185
N
N


Neisseria elongata
1330
ADBF01000003
clade_185
N
N


Neisseria genomosp. P2 oral clone MB5_P15
1332
DQ003630
clade_185
N
N


Neisseria sp. oral clone JC012
1345
AY349388
clade_185
N
N


Neisseria sp. SMC_A9199
1342
FJ763637
clade_185
N
N


Simonsiella muelleri
1731
ADCY01000105
clade_185
N
N


Corynebacterium glucuronolyticum
700
ABYP01000081
clade_193
N
N


Corynebacterium pyruviciproducens
716
FJ185225
clade_193
N
N


Rothia aeria
1649
DQ673320
clade_194
N
N


Rothia dentocariosa
1650
ADDW01000024
clade_194
N
N


Rothia sp. oral taxon 188
1653
GU470892
clade_194
N
N


Corynebacterium accolens
681
ACGD01000048
clade_195
N
N


Corynebacterium macginleyi
707
AB359393
clade_195
N
N


Corynebacterium pseudogenitalium
714
ABYQ01000237
clade_195
N
N


Corynebacterium tuberculostearicum
729
ACVP01000009
clade_195
N
N


Lactobacillus casei
1074
CP000423
clade_198
N
N


Lactobacillus paracasei
1106
ABQV01000067
clade_198
N
N


Lactobacillus zeae
1143
NR_037122
clade_198
N
N


Prevotella dentalis
1492
AB547678
clade_205
N
N


Prevotella sp. oral clone ASCG10
1529
AY923148
clade_206
N
N


Prevotella sp. oral clone HF050
1541
AY349399
clade_206
N
N


Prevotella sp. oral clone ID019
1542
AY349400
clade_206
N
N


Prevotella sp. oral clone IK062
1545
AY349402
clade_206
N
N


Prevotella genomosp. P9 oral clone MB7_G16
1499
DQ003633
clade_207
N
N


Prevotella sp. oral clone AU069
1531
AY005062
clade_207
N
N


Prevotella sp. oral clone CY006
1532
AY005063
clade_207
N
N


Prevotella sp. oral clone FL019
1534
AY349392
clade_207
N
N


Actinomyces genomosp. C1
56
AY278610
clade_212
N
N


Actinomyces genomosp. C2
57
AY278611
clade_212
N
N


Actinomyces genomosp. P1 oral clone MB6_C03
58
DQ003632
clade_212
N
N


Actinomyces georgiae
59
GU561319
clade_212
N
N


Actinomyces israelii
60
AF479270
clade_212
N
N


Actinomyces massiliensis
61
AB545934
clade_212
N
N


Actinomyces meyeri
62
GU561321
clade_212
N
N


Actinomyces odontolyticus
66
ACYT01000123
clade_212
N
N


Actinomyces orihominis
68
AJ575186
clade_212
N
N


Actinomyces sp. CCUG 37290
71
AJ234058
clade_212
N
N


Actinomyces sp. ICM34
75
HQ616391
clade_212
N
N


Actinomyces sp. ICM41
76
HQ616392
clade_212
N
N


Actinomyces sp. ICM47
77
HQ616395
clade_212
N
N


Actinomyces sp. ICM54
78
HQ616398
clade_212
N
N


Actinomyces sp. oral clone IP081
87
AY349366
clade_212
N
N


Actinomyces sp. oral taxon 178
91
AEUH01000060
clade_212
N
N


Actinomyces sp. oral taxon 180
92
AEPP01000041
clade_212
N
N


Actinomyces sp. TeJ5
80
GU561315
clade_212
N
N


Haematobacter sp. BC14248
968
GU396991
clade_213
N
N


Paracoccus denitrificans
1424
CP000490
clade_213
N
N


Paracoccus marcusii
1425
NR_044922
clade_213
N
N


Grimontia hollisae
967
ADAQ01000013
clade_216
N
N


Shewanella putrefaciens
1723
CP002457
clade_216
N
N


Afipia genomosp. 4
111
EU117385
clade_217
N
N


Rhodopseudomonas palustris
1626
CP000301
clade_217
N
N


Methylobacterium extorquens
1223
NC_010172
clade_218
N
N


Methylobacterium podarium
1224
AY468363
clade_218
N
N


Methylobacterium radiotolerans
1225
GU294320
clade_218
N
N


Methylobacterium sp. 1sub
1226
AY468371
clade_218
N
N


Methylobacterium sp. MM4
1227
AY468370
clade_218
N
N


Achromobacter denitrificans
18
NR_042021
clade_224
N
N


Achromobacter piechaudii
19
ADMS01000149
clade_224
N
N


Achromobacter xylosoxidans
20
ACRC01000072
clade_224
N
N


Bordetella bronchiseptica
384
NR_025949
clade_224
N
OP


Bordetella holmesii
385
AB683187
clade_224
N
OP


Bordetella parapertussis
386
NR_025950
clade_224
N
OP


Bordetella pertussis
387
BX640418
clade_224
N
OP


Microbacterium chocolatum
1230
NR_037045
clade_225
N
N


Microbacterium flavescens
1231
EU714363
clade_225
N
N


Microbacterium lacticum
1233
EU714351
clade_225
N
N


Microbacterium oleivorans
1234
EU714381
clade_225
N
N


Microbacterium oxydans
1235
EU714348
clade_225
N
N


Microbacterium paraoxydans
1236
AJ491806
clade_225
N
N


Microbacterium phyllosphaerae
1237
EU714359
clade_225
N
N


Microbacterium schleiferi
1238
NR_044936
clade_225
N
N


Microbacterium sp. 768
1239
EU714378
clade_225
N
N


Microbacterium sp. oral strain C24KA
1240
AF287752
clade_225
N
N


Microbacterium testaceum
1241
EU714365
clade_225
N
N


Corynebacterium atypicum
686
NR_025540
clade_229
N
N


Corynebacterium mastitidis
708
AB359395
clade_229
N
N


Corynebacterium sp. NML 97_0186
725
GU238411
clade_229
N
N


Mycobacterium elephantis
1275
AF385898
clade_237
N
OP


Mycobacterium paraterrae
1288
EU919229
clade_237
N
OP


Mycobacterium phlei
1289
GU142920
clade_237
N
OP


Mycobacterium sp. 1776
1293
EU703152
clade_237
N
N


Mycobacterium sp. 1781
1294
EU703147
clade_237
N
N


Mycobacterium sp. AQ1GA4
1297
HM210417
clade_237
N
N


Mycobacterium sp. GN_10546
1299
FJ497243
clade_237
N
N


Mycobacterium sp. GN_10827
1300
FJ497247
clade_237
N
N


Mycobacterium sp. GN_11124
1301
FJ652846
clade_237
N
N


Mycobacterium sp. GN_9188
1302
FJ497240
clade_237
N
N


Mycobacterium sp. GR_2007_210
1303
FJ555538
clade_237
N
N


Anoxybacillus contaminans
172
NR_029006
clade_238
N
N


Bacillus aeolius
195
NR_025557
clade_238
N
N


Brevibacterium frigoritolerans
422
NR_042639
clade_238
N
N


Geobacillus sp. E263
934
DQ647387
clade_238
N
N


Geobacillus sp. WCH70
935
CP001638
clade_238
N
N


Geobacillus thermocatenulatus
937
NR_043020
clade_238
N
N


Geobacillus thermoleovorans
940
NR_074931
clade_238
N
N


Lysinibacillus fusiformis
1192
FN397522
clade_238
N
N


Planomicrobium koreense
1468
NR_025011
clade_238
N
N


Sporosarcina newyorkensis
1754
AFPZ01000142
clade_238
N
N


Sporosarcina sp. 2681
1755
GU994081
clade_238
N
N


Ureibacillus composti
1968
NR_043746
clade_238
N
N


Ureibacillus suwonensis
1969
NR_043232
clade_238
N
N


Ureibacillus terrenus
1970
NR_025394
clade_238
N
N


Ureibacillus thermophilus
1971
NR_043747
clade_238
N
N


Ureibacillus thermosphaericus
1972
NR_040961
clade_238
N
N


Prevotella micans
1507
AGWK01000061
clade_239
N
N


Prevotella sp. oral clone DA058
1533
AY005065
clade_239
N
N


Prevotella sp. SEQ053
1523
JN867222
clade_239
N
N


Treponema socranskii
1937
NR_024868
clade_240
N
OP


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


Treponema sp. oral taxon 265
1953
GU408850
clade_240
N
N


Treponema sp. oral taxon G85
1958
GU432215
clade_240
N
N


Porphyromonas endodontalis
1472
ACNN01000021
clade_241
N
N


Porphyromonas sp. oral clone BB134
1478
AY005068
clade_241
N
N


Porphyromonas sp. oral clone F016
1479
AY005069
clade_241
N
N


Porphyromonas sp. oral clone P2PB_52 P1
1480
AY207054
clade_241
N
N


Porphyromonas sp. oral clone P4GB_100 P2
1481
AY207057
clade_241
N
N


Acidovorax sp. 98 63833
26
AY258065
clade_245
N
N


Comamonadaceae bacterium NML000135
663
JN585335
clade_245
N
N


Comamonadaceae bacterium NML790751
664
JN585331
clade_245
N
N


Comamonadaceae bacterium NML910035
665
JN585332
clade_245
N
N


Comamonadaceae bacterium NML910036
666
JN585333
clade_245
N
N


Comamonas sp. NSP5
668
AB076850
clade_245
N
N


Delftia acidovorans
748
CP000884
clade_245
N
N


Xenophilus aerolatus
2018
JN585329
clade_245
N
N


Oribacterium sp. oral taxon 078
1380
ACIQ02000009
clade_246
N
N


Oribacterium sp. oral taxon 102
1381
GQ422713
clade_246
N
N


Weissella cibaria
2007
NR_036924
clade_247
N
N


Weissella confusa
2008
NR_040816
clade_247
N
N


Weissella hellenica
2009
AB680902
clade_247
N
N


Weissella kandleri
2010
NR_044659
clade_247
N
N


Weissella koreensis
2011
NR_075058
clade_247
N
N


Weissella paramesenteroides
2012
ACKU01000017
clade_247
N
N


Weissella sp. KLDS 7.0701
2013
EU600924
clade_247
N
N


Mobiluncus curtisii
1251
AEPZ01000013
clade_249
N
N


Enhydrobacter aerosaccus
785
ACYI01000081
clade_256
N
N


Moraxella osloensis
1262
JN175341
clade_256
N
N


Moraxella sp. GM2
1264
JF837191
clade_256
N
N


Brevibacterium casei
420
JF951998
clade_257
N
N


Brevibacterium epidermidis
421
NR_029262
clade_257
N
N


Brevibacterium sanguinis
426
NR_028016
clade_257
N
N


Brevibacterium sp. H15
427
AB177640
clade_257
N
N


Acinetobacter radioresistens
35
ACVR01000010
clade_261
N
N


Lactobacillus alimentarius
1068
NR_044701
clade_263
N
N


Lactobacillus farciminis
1082
NR_044707
clade_263
N
N


Lactobacillus kimchii
1097
NR_025045
clade_263
N
N


Lactobacillus nodensis
1101
NR_041629
clade_263
N
N


Lactobacillus tucceti
1138
NR_042194
clade_263
N
N


Pseudomonas mendocina
1595
AAUL01000021
clade_265
N
N


Pseudomonas pseudoalcaligenes
1598
NR_037000
clade_265
N
N


Pseudomonas sp. NP522b
1602
EU723211
clade_265
N
N


Pseudomonas stutzeri
1603
AM905854
clade_265
N
N


Paenibacillus barcinonensis
1390
NR_042272
clade_270
N
N


Paenibacillus barengoltzii
1391
NR_042756
clade_270
N
N


Paenibacillus chibensis
1392
NR_040885
clade_270
N
N


Paenibacillus cookii
1393
NR_025372
clade_270
N
N


Paenibacillus durus
1394
NR_037017
clade_270
N
N


Paenibacillus glucanolyticus
1395
D78470
clade_270
N
N


Paenibacillus lactis
1396
NR_025739
clade_270
N
N


Paenibacillus pabuli
1398
NR_040853
clade_270
N
N


Paenibacillus popilliae
1400
NR_040888
clade_270
N
N


Paenibacillus sp. CIP 101062
1401
HM212646
clade_270
N
N


Paenibacillus sp. JC66
1404
JF824808
clade_270
N
N


Paenibacillus sp. R_27413
1405
HE586333
clade_270
N
N


Paenibacillus sp. R_27422
1406
HE586338
clade_270
N
N


Paenibacillus timonensis
1408
NR_042844
clade_270
N
N


Rothia mucilaginosa
1651
ACVO01000020
clade_271
N
N


Rothia nasimurium
1652
NR_025310
clade_271
N
N


Prevotella sp. oral taxon 302
1550
ACZK01000043
clade_280
N
N


Prevotella sp. oral taxon F68
1556
HM099652
clade_280
N
N


Prevotella tannerae
1563
ACIJ02000018
clade_280
N
N


Prevotellaceae bacterium P4P_62 P1
1566
AY207061
clade_280
N
N


Porphyromonas asaccharolytica
1471
AENO01000048
clade_281
N
N


Porphyromonas gingivalis
1473
AE015924
clade_281
N
N


Porphyromonas macacae
1475
NR_025908
clade_281
N
N


Porphyromonas sp. UQD 301
1477
EU012301
clade_281
N
N


Porphyromonas uenonis
1482
ACLR01000152
clade_281
N
N


Leptotrichia buccalis
1165
CP001685
clade_282
N
N


Leptotrichia hofstadii
1168
ACVB02000032
clade_282
N
N


Leptotrichia sp. oral clone HE012
1173
AY349386
clade_282
N
N


Leptotrichia sp. oral taxon 223
1176
GU408547
clade_282
N
N


Bacteroides fluxus
278
AFBN01000029
clade_285
N
N


Bacteroides helcogenes
281
CP002352
clade_285
N
N


Parabacteroides johnsonii
1419
ABYH01000014
clade_286
N
N


Parabacteroides merdae
1420
EU136685
clade_286
N
N


Treponema denticola
1926
ADEC01000002
clade_288
N
OP


Treponema genomosp. P5 oral clone MB3_P23
1929
DQ003624
clade_288
N
N


Treponema putidum
1935
AJ543428
clade_288
N
OP


Treponema sp. oral clone P2PB_53 P3
1942
AY207055
clade_288
N
N


Treponema sp. oral taxon 247
1949
GU408748
clade_288
N
N


Treponema sp. oral taxon 250
1950
GU408776
clade_288
N
N


Treponema sp. oral taxon 251
1951
GU408781
clade_288
N
N


Anaerococcus hydrogenalis
144
ABXA01000039
clade_289
N
N


Anaerococcus sp. 8404299
148
HM587318
clade_289
N
N


Anaerococcus sp. gpac215
156
AM176540
clade_289
N
N


Anaerococcus vaginalis
158
ACXU01000016
clade_289
N
N


Propionibacterium acidipropionici
1569
NC_019395
clade_290
N
N


Propionibacterium avidum
1571
AJ003055
clade_290
N
N


Propionibacterium granulosum
1573
FJ785716
clade_290
N
N


Propionibacterium jensenii
1574
NR_042269
clade_290
N
N


Propionibacterium propionicum
1575
NR_025277
clade_290
N
N


Propionibacterium sp. H456
1577
AB177643
clade_290
N
N


Propionibacterium thoenii
1581
NR_042270
clade_290
N
N


Bifidobacterium bifidum
349
ABQP01000027
clade_293
N
N


Leuconostoc mesenteroides
1183
ACKV01000113
clade_295
N
N


Leuconostoc pseudomesenteroides
1184
NR_040814
clade_295
N
N


Johnsonella ignava
1016
X87152
clade_298
N
N


Propionibacterium acnes
1570
ADJM01000010
clade_299
N
N


Propionibacterium sp. 434_HC2
1576
AFIL01000035
clade_299
N
N


Propionibacterium sp. LG
1578
AY354921
clade_299
N
N


Propionibacterium sp. S555a
1579
AB264622
clade_299
N
N


Alicyclobacillus sp. CCUG 53762
128
HE613268
clade_301
N
N


Actinomyces cardiffensis
53
GU470888
clade_303
N
N


Actinomyces funkei
55
HQ906497
clade_303
N
N


Actinomyces sp. HKU31
74
HQ335393
clade_303
N
N


Actinomyces sp. oral taxon C55
94
HM099646
clade_303
N
N


Kerstersia gyiorum
1018
NR_025669
clade_307
N
N


Pigmentiphaga daeguensis
1467
JN585327
clade_307
N
N


Aeromonas allosaccharophila
104
S39232
clade_308
N
N


Aeromonas enteropelogenes
105
X71121
clade_308
N
N


Aeromonas hydrophila
106
NC_008570
clade_308
N
N


Aeromonas jandaei
107
X60413
clade_308
N
N


Aeromonas salmonicida
108
NC_009348
clade_308
N
N


Aeromonas trota
109
X60415
clade_308
N
N


Aeromonas veronii
110
NR_044845
clade_308
N
N


Marvinbryantia formatexigens
1196
AJ505973
clade_309
N
N


Rhodobacter sp. oral taxon C30
1620
HM099648
clade_310
N
N


Rhodobacter sphaeroides
1621
CP000144
clade_310
N
N


Lactobacillus antri
1071
ACLL01000037
clade_313
N
N


Lactobacillus coleohominis
1076
ACOH01000030
clade_313
N
N


Lactobacillus fermentum
1083
CP002033
clade_313
N
N


Lactobacillus gastricus
1085
AICN01000060
clade_313
N
N


Lactobacillus mucosae
1099
FR693800
clade_313
N
N


Lactobacillus oris
1103
AEKL01000077
clade_313
N
N


Lactobacillus pontis
1111
HM218420
clade_313
N
N


Lactobacillus reuteri
1112
ACGW02000012
clade_313
N
N


Lactobacillus sp. KLDS 1.0707
1127
EU600911
clade_313
N
N


Lactobacillus sp. KLDS 1.0709
1128
EU600913
clade_313
N
N


Lactobacillus sp. KLDS 1.0711
1129
EU600915
clade_313
N
N


Lactobacillus sp. KLDS 1.0713
1131
EU600917
clade_313
N
N


Lactobacillus sp. KLDS 1.0716
1132
EU600921
clade_313
N
N


Lactobacillus sp. KLDS 1.0718
1133
EU600922
clade_313
N
N


Lactobacillus sp. oral taxon 052
1137
GQ422710
clade_313
N
N


Lactobacillus vaginalis
1140
ACGV01000168
clade_313
N
N


Brevibacterium aurantiacum
419
NR_044854
clade_314
N
N


Brevibacterium linens
423
AJ315491
clade_314
N
N


Lactobacillus pentosus
1108
JN813103
clade_315
N
N


Lactobacillus plantarum
1110
ACGZ02000033
clade_315
N
N


Lactobacillus sp. KLDS 1.0702
1123
EU600906
clade_315
N
N


Lactobacillus sp. KLDS 1.0703
1124
EU600907
clade_315
N
N


Lactobacillus sp. KLDS 1.0704
1125
EU600908
clade_315
N
N


Lactobacillus sp. KLDS 1.0705
1126
EU600909
clade_315
N
N


Agrobacterium radiobacter
115
CP000628
clade_316
N
N


Agrobacterium tumefaciens
116
AJ389893
clade_316
N
N


Corynebacterium argentoratense
685
EF463055
clade_317
N
N


Corynebacterium diphtheriae
693
NC_002935
clade_317
N
OP


Corynebacterium pseudotuberculosis
715
NR_037070
clade_317
N
N


Corynebacterium renale
717
NR_037069
clade_317
N
N


Corynebacterium ulcerans
731
NR_074467
clade_317
N
N


Aurantimonas coralicida
191
AY065627
clade_318
N
N


Aureimonas altamirensis
192
FN658986
clade_318
N
N


Lactobacillus acidipiscis
1066
NR_024718
clade_320
N
N


Lactobacillus salivarius
1117
AEBA01000145
clade_320
N
N


Lactobacillus sp. KLDS 1.0719
1134
EU600923
clade_320
N
N


Lactobacillus buchneri
1073
ACGH01000101
clade_321
N
N


Lactobacillus genomosp. C1
1086
AY278619
clade_321
N
N


Lactobacillus genomosp. C2
1087
AY278620
clade_321
N
N


Lactobacillus hilgardii
1089
ACGP01000200
clade_321
N
N


Lactobacillus kefiri
1096
NR_042230
clade_321
N
N


Lactobacillus parabuchneri
1105
NR_041294
clade_321
N
N


Lactobacillus parakefiri
1107
NR_029039
clade_321
N
N


Lactobacillus curvatus
1079
NR_042437
clade_322
N
N


Lactobacillus sakei
1116
DQ989236
clade_322
N
N


Aneurinibacillus aneurinilyticus
167
AB101592
clade_323
N
N


Aneurinibacillus danicus
168
NR_028657
clade_323
N
N


Aneurinibacillus migulanus
169
NR_036799
clade_323
N
N


Aneurinibacillus terranovensis
170
NR_042271
clade_323
N
N


Staphylococcus aureus
1757
CP002643
clade_325
N
Category-B


Staphylococcus auricularis
1758
JQ624774
clade_325
N
N


Staphylococcus capitis
1759
ACFR01000029
clade_325
N
N


Staphylococcus caprae
1760
ACRH01000033
clade_325
N
N


Staphylococcus carnosus
1761
NR_075003
clade_325
N
N


Staphylococcus cohnii
1762
JN175375
clade_325
N
N


Staphylococcus condimenti
1763
NR_029345
clade_325
N
N


Staphylococcus epidermidis
1764
ACHE01000056
clade_325
N
N


Staphylococcus equorum
1765
NR_027520
clade_325
N
N


Staphylococcus haemolyticus
1767
NC_007168
clade_325
N
N


Staphylococcus hominis
1768
AM157418
clade_325
N
N


Staphylococcus lugdunensis
1769
AEQA01000024
clade_325
N
N


Staphylococcus pasteuri
1770
FJ189773
clade_325
N
N


Staphylococcus pseudintermedius
1771
CP002439
clade_325
N
N


Staphylococcus saccharolyticus
1772
NR_029158
clade_325
N
N


Staphylococcus saprophyticus
1773
NC_007350
clade_325
N
N


Staphylococcus sp. clone bottae7
1777
AF467424
clade_325
N
N


Staphylococcus sp. H292
1775
AB177642
clade_325
N
N


Staphylococcus sp. H780
1776
AB177644
clade_325
N
N


Staphylococcus succinus
1778
NR_028667
clade_325
N
N


Staphylococcus warneri
1780
ACPZ01000009
clade_325
N
N


Staphylococcus xylosus
1781
AY395016
clade_325
N
N


Cardiobacterium hominis
490
ACKY01000036
clade_326
N
N


Cardiobacterium valvarum
491
NR_028847
clade_326
N
N


Pseudomonas fluorescens
1593
AY622220
clade_326
N
N


Pseudomonas gessardii
1594
FJ943496
clade_326
N
N


Pseudomonas monteilii
1596
NR_024910
clade_326
N
N


Pseudomonas poae
1597
GU188951
clade_326
N
N


Pseudomonas putida
1599
AF094741
clade_326
N
N


Pseudomonas sp. G1229
1601
DQ910482
clade_326
N
N


Pseudomonas tolaasii
1604
AF320988
clade_326
N
N


Pseudomonas viridiflava
1605
NR_042764
clade_326
N
N


Listeria grayi
1185
ACCR02000003
clade_328
N
OP


Listeria innocua
1186
JF967625
clade_328
N
N


Listeria ivanovii
1187
X56151
clade_328
N
N


Listeria monocytogenes
1188
CP002003
clade_328
N
Category-B


Listeria welshimeri
1189
AM263198
clade_328
N
OP


Capnocytophaga sp. oral clone ASCH05
484
AY923149
clade_333
N
N


Capnocytophaga sputigena
489
ABZV01000054
clade_333
N
N


Leptotrichia genomosp. C1
1166
AY278621
clade_334
N
N


Leptotrichia shahii
1169
AY029806
clade_334
N
N


Leptotrichia sp. neutropenicPatient
1170
AF189244
clade_334
N
N


Leptotrichia sp. oral clone GT018
1171
AY349384
clade_334
N
N


Leptotrichia sp. oral clone GT020
1172
AY349385
clade_334
N
N


Bacteroides sp. 20_3
296
ACRQ01000064
clade_335
N
N


Bacteroides sp. 3_1_19
307
ADCJ01000062
clade_335
N
N


Bacteroides sp. 3_2_5
311
ACIB01000079
clade_335
N
N


Parabacteroides distasonis
1416
CP000140
clade_335
N
N


Parabacteroides goldsteinii
1417
AY974070
clade_335
N
N


Parabacteroides gordonii
1418
AB470344
clade_335
N
N


Parabacteroides sp. D13
1421
ACPW01000017
clade_335
N
N


Capnocytophaga genomosp. C1
477
AY278613
clade_336
N
N


Capnocytophaga ochracea
480
AEOH01000054
clade_336
N
N


Capnocytophaga sp. GEJ8
481
GU561335
clade_336
N
N


Capnocytophaga sp. oral strain A47ROY
486
AY005077
clade_336
N
N


Capnocytophaga sp. S1b
482
U42009
clade_336
N
N


Paraprevotella clara
1426
AFFY01000068
clade_336
N
N


Bacteroides heparinolyticus
282
JN867284
clade_338
N
N


Prevotella heparinolytica
1500
GQ422742
clade_338
N
N


Treponema genomosp. P4 oral clone MB2_G19
1928
DQ003618
clade_339
N
N


Treponema genomosp. P6 oral clone MB4_G11
1930
DQ003625
clade_339
N
N


Treponema sp. oral taxon 254
1952
GU408803
clade_339
N
N


Treponema sp. oral taxon 508
1956
GU413616
clade_339
N
N


Treponema sp. oral taxon 518
1957
GU413640
clade_339
N
N


Chlamydia muridarum
502
AE002160
clade_341
N
OP


Chlamydia trachomatis
504
U68443
clade_341
N
OP


Chlamydia psittaci
503
NR_036864
clade_342
N
Category-B


Chlamydophila pneumoniae
509
NC_002179
clade_342
N
OP


Chlamydophila psittaci
510
D85712
clade_342
N
OP


Anaerococcus octavius
146
NR_026360
clade_343
N
N


Anaerococcus sp. 8405254
149
HM587319
clade_343
N
N


Anaerococcus sp. 9401487
150
HM587322
clade_343
N
N


Anaerococcus sp. 9403502
151
HM587325
clade_343
N
N


Gardnerella vaginalis
923
CP001849
clade_344
N
N


Campylobacter lari
466
CP000932
clade_346
N
OP


Anaerobiospirillum succiniciproducens
142
NR_026075
clade_347
N
N


Anaerobiospirillum thomasii
143
AJ420985
clade_347
N
N


Ruminobacter amylophilus
1654
NR_026450
clade_347
N
N


Succinatimonas hippei
1897
AEVO01000027
clade_347
N
N


Actinomyces europaeus
54
NR_026363
clade_348
N
N


Actinomyces sp. oral clone GU009
82
AY349361
clade_348
N
N


Moraxella catarrhalis
1260
CP002005
clade_349
N
N


Moraxella lincolnii
1261
FR822735
clade_349
N
N


Moraxella sp. 16285
1263
JF682466
clade_349
N
N


Psychrobacter sp. 13983
1613
HM212668
clade_349
N
N


Actinobaculum massiliae
49
AF487679
clade_350
N
N


Actinobaculum schaalii
50
AY957507
clade_350
N
N


Actinobaculum sp. BM#101342
51
AY282578
clade_350
N
N


Actinobaculum sp. P2P_19 P1
52
AY207066
clade_350
N
N


Actinomyces sp. oral clone IO076
84
AY349363
clade_350
N
N


Actinomyces sp. oral taxon 848
93
ACUY01000072
clade_350
N
N


Actinomyces neuii
65
X71862
clade_352
N
N


Mobiluncus mulieris
1252
ACKW01000035
clade_352
N
N


Blastomonas natatoria
372
NR_040824
clade_356
N
N


Novosphingobium aromaticivorans
1357
AAAV03000008
clade_356
N
N


Sphingomonas sp. oral clone FI012
1745
AY349411
clade_356
N
N


Sphingopyxis alaskensis
1749
CP000356
clade_356
N
N


Oxalobacter formigenes
1389
ACDQ01000020
clade_357
N
N


Veillonella atypica
1974
AEDS01000059
clade_358
N
N


Veillonella dispar
1975
ACIK02000021
clade_358
N
N


Veillonella genomosp. P1 oral clone MB5_P17
1976
DQ003631
clade_358
N
N


Veillonella parvula
1978
ADFU01000009
clade_358
N
N


Veillonella sp. 3_1_44
1979
ADCV01000019
clade_358
N
N


Veillonella sp. 6_1_27
1980
ADCW01000016
clade_358
N
N


Veillonella sp. ACP1
1981
HQ616359
clade_358
N
N


Veillonella sp. AS16
1982
HQ616365
clade_358
N
N


Veillonella sp. BS32b
1983
HQ616368
clade_358
N
N


Veillonella sp. ICM51a
1984
HQ616396
clade_358
N
N


Veillonella sp. MSA12
1985
HQ616381
clade_358
N
N


Veillonella sp. NVG 100cf
1986
EF108443
clade_358
N
N


Veillonella sp. OK11
1987
JN695650
clade_358
N
N


Veillonella sp. oral clone ASCG01
1990
AY923144
clade_358
N
N


Veillonella sp. oral clone ASCG02
1991
AY953257
clade_358
N
N


Veillonella sp. oral clone OH1A
1992
AY947495
clade_358
N
N


Veillonella sp. oral taxon 158
1993
AENU01000007
clade_358
N
N


Kocuria marina
1040
GQ260086
clade_365
N
N


Kocuria rhizophila
1042
AY030315
clade_365
N
N


Kocuria rosea
1043
X87756
clade_365
N
N


Kocuria varians
1044
AF542074
clade_365
N
N


Clostridiaceae bacterium END_2
531
EF451053
clade_368
N
N


Micrococcus antarcticus
1242
NR_025285
clade_371
N
N


Micrococcus luteus
1243
NR_075062
clade_371
N
N


Micrococcus lylae
1244
NR_026200
clade_371
N
N


Micrococcus sp. 185
1245
EU714334
clade_371
N
N


Lactobacillus brevis
1072
EU194349
clade_372
N
N


Lactobacillus parabrevis
1104
NR_042456
clade_372
N
N


Pediococcus acidilactici
1436
ACXB01000026
clade_372
N
N


Pediococcus pentosaceus
1437
NR_075052
clade_372
N
N


Lactobacillus dextrinicus
1081
NR_036861
clade_373
N
N


Lactobacillus perolens
1109
NR_029360
clade_373
N
N


Lactobacillus rhamnosus
1113
ABWJ01000068
clade_373
N
N


Lactobacillus saniviri
1118
AB602569
clade_373
N
N


Lactobacillus sp. BT6
1121
HQ616370
clade_373
N
N


Mycobacterium mageritense
1282
FR798914
clade_374
N
OP


Mycobacterium neoaurum
1286
AF268445
clade_374
N
OP


Mycobacterium smegmatis
1291
CP000480
clade_374
N
OP


Mycobacterium sp. HE5
1304
AJ012738
clade_374
N
N


Dysgonomonas gadei
775
ADLV01000001
clade_377
N
N


Dysgonomonas mossii
776
ADLW01000023
clade_377
N
N


Porphyromonas levii
1474
NR_025907
clade_377
N
N


Porphyromonas somerae
1476
AB547667
clade_377
N
N


Bacteroides barnesiae
267
NR_041446
clade_378
N
N


Bacteroides coprocola
272
ABIY02000050
clade_378
N
N


Bacteroides coprophilus
273
ACBW01000012
clade_378
N
N


Bacteroides dorei
274
ABWZ01000093
clade_378
N
N


Bacteroides massiliensis
284
AB200226
clade_378
N
N


Bacteroides plebeius
289
AB200218
clade_378
N
N


Bacteroides sp. 3_1_33FAA
309
ACPS01000085
clade_378
N
N


Bacteroides sp. 3_1_40A
310
ACRT01000136
clade_378
N
N


Bacteroides sp. 4_3_47FAA
313
ACDR02000029
clade_378
N
N


Bacteroides sp. 9_1_42FAA
314
ACAA01000096
clade_378
N
N


Bacteroides sp. NB_8
323
AB117565
clade_378
N
N


Bacteroides vulgatus
331
CP000139
clade_378
N
N


Bacteroides ovatus
287
ACWH01000036
clade_38
N
N


Bacteroides sp. 1_1_30
294
ADCL01000128
clade_38
N
N


Bacteroides sp. 2_1_22
297
ACPQ01000117
clade_38
N
N


Bacteroides sp. 2_2_4
299
ABZZ01000168
clade_38
N
N


Bacteroides sp. 3_1_23
308
ACRS01000081
clade_38
N
N


Bacteroides sp. D1
318
ACAB02000030
clade_38
N
N


Bacteroides sp. D2
321
ACGA01000077
clade_38
N
N


Bacteroides sp. D22
320
ADCK01000151
clade_38
N
N


Bacteroides xylanisolvens
332
ADKP01000087
clade_38
N
N


Treponema lecithinolyticum
1931
NR_026247
clade_380
N
OP


Treponema parvum
1933
AF302937
clade_380
N
OP


Treponema sp. oral clone JU025
1940
AY349417
clade_380
N
N


Treponema sp. oral taxon 270
1954
GQ422733
clade_380
N
N


Parascardovia denticolens
1428
ADEB01000020
clade_381
N
N


Scardovia inopinata
1688
AB029087
clade_381
N
N


Scardovia wiggsiae
1689
AY278626
clade_381
N
N


Clostridiales bacterium 9400853
533
HM587320
clade_384
N
N


Mogibacterium diversum
1254
NR_027191
clade_384
N
N


Mogibacterium neglectum
1255
NR_027203
clade_384
N
N


Mogibacterium pumilum
1256
NR_028608
clade_384
N
N


Mogibacterium timidum
1257
Z36296
clade_384
N
N


Borrelia burgdorferi
389
ABGI01000001
clade_386
N
OP


Borrelia garinii
392
ABJV01000001
clade_386
N
OP


Borrelia sp. NE49
397
AJ224142
clade_386
N
OP


Caldimonas manganoxidans
457
NR_040787
clade_387
N
N


Comamonadaceae bacterium oral taxon F47
667
HM099651
clade_387
N
N


Lautropia mirabilis
1149
AEQP01000026
clade_387
N
N


Lautropia sp. oral clone AP009
1150
AY005030
clade_387
N
N


Peptoniphilus asaccharolyticus
1441
D14145
clade_389
N
N


Peptoniphilus duerdenii
1442
EU526290
clade_389
N
N


Peptoniphilus harei
1443
NR_026358
clade_389
N
N


Peptoniphilus indolicus
1444
AY153431
clade_389
N
N


Peptoniphilus lacrimalis
1446
ADDO01000050
clade_389
N
N


Peptoniphilus sp. gpac077
1450
AM176527
clade_389
N
N


Peptoniphilus sp. JC140
1447
JF824803
clade_389
N
N


Peptoniphilus sp. oral taxon 386
1452
ADCS01000031
clade_389
N
N


Peptoniphilus sp. oral taxon 836
1453
AEAA01000090
clade_389
N
N


Peptostreptococcaceae bacterium ph1
1454
JN837495
clade_389
N
N


Dialister pneumosintes
765
HM596297
clade_390
N
N


Dialister sp. oral taxon 502
767
GQ422739
clade_390
N
N


Cupriavidus metallidurans
741
GU230889
clade_391
N
N


Herbaspirillum seropedicae
1001
CP002039
clade_391
N
N


Herbaspirillum sp. JC206
1002
JN657219
clade_391
N
N


Janthinobacterium sp. SY12
1015
EF455530
clade_391
N
N


Massilia sp. CCUG 43427A
1197
FR773700
clade_391
N
N


Ralstonia pickettii
1615
NC_010682
clade_391
N
N


Ralstonia sp. 5_7_47FAA
1616
ACUF01000076
clade_391
N
N


Francisella novicida
889
ABSS01000002
clade_392
N
N


Francisella philomiragia
890
AY928394
clade_392
N
N


Francisella tularensis
891
ABAZ01000082
clade_392
N
Category-A


Ignatzschineria indica
1009
HQ823562
clade_392
N
N


Ignatzschineria sp. NML 95_0260
1010
HQ823559
clade_392
N
N


Streptococcus mutans
1814
AP010655
clade_394
N
N


Lactobacillus gasseri
1084
ACOZ01000018
clade_398
N
N


Lactobacillus hominis
1090
FR681902
clade_398
N
N


Lactobacillus iners
1091
AEKJ01000002
clade_398
N
N


Lactobacillus johnsonii
1093
AE017198
clade_398
N
N


Lactobacillus senioris
1119
AB602570
clade_398
N
N


Lactobacillus sp. oral clone HT002
1135
AY349382
clade_398
N
N


Weissella beninensis
2006
EU439435
clade_398
N
N


Sphingomonas echinoides
1744
NR_024700
clade_399
N
N


Sphingomonas sp. oral taxon A09
1747
HM099639
clade_399
N
N


Sphingomonas sp. oral taxon F71
1748
HM099645
clade_399
N
N


Zymomonas mobilis
2032
NR_074274
clade_399
N
N


Arcanobacterium haemolyticum
174
NR_025347
clade_400
N
N


Arcanobacterium pyogenes
175
GU585578
clade_400
N
N


Trueperella pyogenes
1962
NR_044858
clade_400
N
N


Lactococcus garvieae
1144
AF061005
clade_401
N
N


Lactococcus lactis
1145
CP002365
clade_401
N
N


Brevibacterium mcbrellneri
424
ADNU01000076
clade_402
N
N


Brevibacterium paucivorans
425
EU086796
clade_402
N
N


Brevibacterium sp. JC43
428
JF824806
clade_402
N
N


Selenomonas artemidis
1692
HM596274
clade_403
N
N


Selenomonas sp. FOBRC9
1704
HQ616378
clade_403
N
N


Selenomonas sp. oral taxon 137
1715
AENV01000007
clade_403
N
N


Desmospora activa
751
AM940019
clade_404
N
N


Desmospora sp. 8437
752
AFHT01000143
clade_404
N
N


Paenibacillus sp. oral taxon F45
1407
HM099647
clade_404
N
N


Corynebacterium ammoniagenes
682
ADNS01000011
clade_405
N
N


Corynebacterium aurimucosum
687
ACLH01000041
clade_405
N
N


Corynebacterium bovis
688
AF537590
clade_405
N
N


Corynebacterium canis
689
GQ871934
clade_405
N
N


Corynebacterium casei
690
NR_025101
clade_405
N
N


Corynebacterium durum
694
Z97069
clade_405
N
N


Corynebacterium efficiens
695
ACLI01000121
clade_405
N
N


Corynebacterium falsenii
696
Y13024
clade_405
N
N


Corynebacterium flavescens
697
NR_037040
clade_405
N
N


Corynebacterium glutamicum
701
BA000036
clade_405
N
N


Corynebacterium jeikeium
704
ACYW01000001
clade_405
N
OP


Corynebacterium kroppenstedtii
705
NR_026380
clade_405
N
N


Corynebacterium lipophiloflavum
706
ACHJ01000075
clade_405
N
N


Corynebacterium matruchotii
709
ACSH02000003
clade_405
N
N


Corynebacterium minutissimum
710
X82064
clade_405
N
N


Corynebacterium resistens
718
ADGN01000058
clade_405
N
N


Corynebacterium simulans
720
AF537604
clade_405
N
N


Corynebacterium singulare
721
NR_026394
clade_405
N
N


Corynebacterium sp. 1 ex sheep
722
Y13427
clade_405
N
N


Corynebacterium sp. NML 99_0018
726
GU238413
clade_405
N
N


Corynebacterium striatum
727
ACGE01000001
clade_405
N
OP


Corynebacterium urealyticum
732
X81913
clade_405
N
OP


Corynebacterium variabile
734
NR_025314
clade_405
N
N


Aerococcus sanguinicola
98
AY837833
clade_407
N
N


Aerococcus urinae
99
CP002512
clade_407
N
N


Aerococcus urinaeequi
100
NR_043443
clade_407
N
N


Aerococcus viridans
101
ADNT01000041
clade_407
N
N


Fusobacterium naviforme
898
HQ223106
clade_408
N
N


Moryella indoligenes
1268
AF527773
clade_408
N
N


Selenomonas genomosp. P5
1697
AY341820
clade_410
N
N


Selenomonas sp. oral clone IQ048
1710
AY349408
clade_410
N
N


Selenomonas sputigena
1717
ACKP02000033
clade_410
N
N


Hyphomicrobium sulfonivorans
1007
AY468372
clade_411
N
N


Methylocella silvestris
1228
NR_074237
clade_411
N
N


Legionella pneumophila
1153
NC_002942
clade_412
N
OP


Lactobacillus coryniformis
1077
NR_044705
clade_413
N
N


Arthrobacter agilis
178
NR_026198
clade_414
N
N


Arthrobacter arilaitensis
179
NR_074608
clade_414
N
N


Arthrobacter bergerei
180
NR_025612
clade_414
N
N


Arthrobacter globiformis
181
NR_026187
clade_414
N
N


Arthrobacter nicotianae
182
NR_026190
clade_414
N
N


Mycobacterium abscessus
1269
AGQU01000002
clade_418
N
OP


Mycobacterium chelonae
1273
AB548610
clade_418
N
OP


Bacteroides salanitronis
291
CP002530
clade_419
N
N


Paraprevotella xylaniphila
1427
AFBR01000011
clade_419
N
N


Barnesiella intestinihominis
336
AB370251
clade_420
N
N


Barnesiella viscericola
337
NR_041508
clade_420
N
N


Parabacteroides sp. NS31_3
1422
JN029805
clade_420
N
N


Porphyromonadaceae bacterium NML 060648
1470
EF184292
clade_420
N
N


Tannerella forsythia
1913
CP003191
clade_420
N
N


Tannerella sp. 6_1_58FAA_CT1
1914
ACWX01000068
clade_420
N
N


Mycoplasma amphoriforme
1311
AY531656
clade_421
N
N


Mycoplasma genitalium
1317
L43967
clade_421
N
N


Mycoplasma pneumoniae
1322
NC_000912
clade_421
N
N


Mycoplasma penetrans
1321
NC_004432
clade_422
N
N


Ureaplasma parvum
1966
AE002127
clade_422
N
N


Ureaplasma urealyticum
1967
AAYN01000002
clade_422
N
N


Treponema genomosp. P1
1927
AY341822
clade_425
N
N


Treponema sp. oral taxon 228
1943
GU408580
clade_425
N
N


Treponema sp. oral taxon 230
1944
GU408603
clade_425
N
N


Treponema sp. oral taxon 231
1945
GU408631
clade_425
N
N


Treponema sp. oral taxon 232
1946
GU408646
clade_425
N
N


Treponema sp. oral taxon 235
1947
GU408673
clade_425
N
N


Treponema sp. ovine footrot
1959
AJ010951
clade_425
N
N


Treponema vincentii
1960
ACYH01000036
clade_425
N
OP


Burkholderiales bacterium 1_1_47
452
ADCQ01000066
clade_432
N
OP


Parasutterella excrementihominis
1429
AFBP01000029
clade_432
N
N


Parasutterella secunda
1430
AB491209
clade_432
N
N


Sutterella morbirenis
1898
AJ832129
clade_432
N
N


Sutterella sanguinus
1900
AJ748647
clade_432
N
N


Sutterella sp. YIT 12072
1901
AB491210
clade_432
N
N


Sutterella stercoricanis
1902
NR_025600
clade_432
N
N


Sutterella wadsworthensis
1903
ADMF01000048
clade_432
N
N


Propionibacterium freudenreichii
1572
NR_036972
clade_433
N
N


Propionibacterium sp. oral taxon 192
1580
GQ422728
clade_433
N
N


Tessaracoccus sp. oral taxon F04
1917
HM099640
clade_433
N
N


Peptoniphilus ivorii
1445
Y07840
clade_434
N
N


Peptoniphilus sp. gpac007
1448
AM176517
clade_434
N
N


Peptoniphilus sp. gpac018A
1449
AM176519
clade_434
N
N


Peptoniphilus sp. gpac148
1451
AM176535
clade_434
N
N


Flexispira rappini
887
AY126479
clade_436
N
N


Helicobacter bilis
993
ACDN01000023
clade_436
N
N


Helicobacter cinaedi
995
ABQT01000054
clade_436
N
N


Helicobacter sp. None
998
U44756
clade_436
N
N


Brevundimonas subvibrioides
429
CP002102
clade_438
N
N


Hyphomonas neptunium
1008
NR_074092
clade_438
N
N


Phenylobacterium zucineum
1465
AY628697
clade_438
N
N


Streptococcus downei
1793
AEKN01000002
clade_441
N
N


Streptococcus sp. SHV515
1848
Y07601
clade_441
N
N


Acinetobacter sp. CIP 53.82
40
JQ638584
clade_443
N
N


Halomonas elongata
990
NR_074782
clade_443
N
N


Halomonas johnsoniae
991
FR775979
clade_443
N
N


Butyrivibrio fibrisolvens
456
U41172
clade_444
N
N


Roseburia sp. 11SE37
1640
FM954975
clade_444
N
N


Roseburia sp. 11SE38
1641
FM954976
clade_444
N
N


Shuttleworthia satelles
1728
ACIP02000004
clade_444
N
N


Shuttleworthia sp. MSX8B
1729
HQ616383
clade_444
N
N


Shuttleworthia sp. oral taxon G69
1730
GU432167
clade_444
N
N


Bdellovibrio sp. MPA
344
AY294215
clade_445
N
N


Desulfobulbus sp. oral clone CH031
755
AY005036
clade_445
N
N


Desulfovibrio desulfuricans
757
DQ092636
clade_445
N
N


Desulfovibrio fairfieldensis
758
U42221
clade_445
N
N


Desulfovibrio piger
759
AF192152
clade_445
N
N


Desulfovibrio sp. 3_1_syn3
760
ADDR01000239
clade_445
N
N


Geobacter bemidjiensis
941
CP001124
clade_445
N
N


Brachybacterium alimentarium
401
NR_026269
clade_446
N
N


Brachybacterium conglomeratum
402
AB537169
clade_446
N
N


Brachybacterium tyrofermentans
403
NR_026272
clade_446
N
N


Dermabacter hominis
749
FJ263375
clade_446
N
N


Aneurinibacillus thermoaerophilus
171
NR_029303
clade_448
N
N


Brevibacillus agri
409
NR_040983
clade_448
N
N


Brevibacillus centrosporus
411
NR_043414
clade_448
N
N


Brevibacillus choshinensis
412
NR_040980
clade_448
N
N


Brevibacillus invocatus
413
NR_041836
clade_448
N
N


Brevibacillus parabrevis
415
NR_040981
clade_448
N
N


Brevibacillus reuszeri
416
NR_040982
clade_448
N
N


Brevibacillus sp. phR
417
JN837488
clade_448
N
N


Brevibacillus thermoruber
418
NR_026514
clade_448
N
N


Lactobacillus murinus
1100
NR_042231
clade_449
N
N


Lactobacillus oeni
1102
NR_043095
clade_449
N
N


Lactobacillus ruminis
1115
ACGS02000043
clade_449
N
N


Lactobacillus vini
1141
NR_042196
clade_449
N
N


Gemella haemolysans
924
ACDZ02000012
clade_450
N
N


Gemella morbillorum
925
NR_025904
clade_450
N
N


Gemella morbillorum
926
ACRX01000010
clade_450
N
N


Gemella sanguinis
927
ACRY01000057
clade_450
N
N


Gemella sp. oral clone ASCE02
929
AY923133
clade_450
N
N


Gemella sp. oral clone ASCF04
930
AY923139
clade_450
N
N


Gemella sp. oral clone ASCF12
931
AY923143
clade_450
N
N


Gemella sp. WAL 1945J
928
EU427463
clade_450
N
N


Sporolactobacillus nakayamae
1753
NR_042247
clade_451
N
N


Gluconacetobacter entanii
945
NR_028909
clade_452
N
N


Gluconacetobacter europaeus
946
NR_026513
clade_452
N
N


Gluconacetobacter hansenii
947
NR_026133
clade_452
N
N


Gluconacetobacter oboediens
949
NR_041295
clade_452
N
N


Gluconacetobacter xylinus
950
NR_074338
clade_452
N
N


Auritibacter ignavus
193
FN554542
clade_453
N
N


Dermacoccus sp. Ellin185
750
AEIQ01000090
clade_453
N
N


Janibacter limosus
1013
NR_026362
clade_453
N
N


Janibacter melonis
1014
EF063716
clade_453
N
N


Acetobacter aceti
7
NR_026121
clade_454
N
N


Acetobacter fabarum
8
NR_042678
clade_454
N
N


Acetobacter lovaniensis
9
NR_040832
clade_454
N
N


Acetobacter malorum
10
NR_025513
clade_454
N
N


Acetobacter orientalis
11
NR_028625
clade_454
N
N


Acetobacter pasteurianus
12
NR_026107
clade_454
N
N


Acetobacter pomorum
13
NR_042112
clade_454
N
N


Acetobacter syzygii
14
NR_040868
clade_454
N
N


Acetobacter tropicalis
15
NR_036881
clade_454
N
N


Gluconacetobacter azotocaptans
943
NR_028767
clade_454
N
N


Gluconacetobacter diazotrophicus
944
NR_074292
clade_454
N
N


Gluconacetobacter johannae
948
NR_024959
clade_454
N
N


Nocardia brasiliensis
1351
AIHV01000038
clade_455
N
N


Nocardia cyriacigeorgica
1352
HQ009486
clade_455
N
N


Nocardia puris
1354
NR_028994
clade_455
N
N


Nocardia sp. 01_Je_025
1355
GU574059
clade_455
N
N


Rhodococcus equi
1623
ADNW01000058
clade_455
N
N


Oceanobacillus caeni
1358
NR_041533
clade_456
N
N


Oceanobacillus sp. Ndiop
1359
CAER01000083
clade_456
N
N


Ornithinibacillus bavariensis
1384
NR_044923
clade_456
N
N


Ornithinibacillus sp. 7_10AIA
1385
FN397526
clade_456
N
N


Virgibacillus proomii
2005
NR_025308
clade_456
N
N


Corynebacterium amycolatum
683
ABZU01000033
clade_457
N
OP


Corynebacterium hansenii
702
AM946639
clade_457
N
N


Corynebacterium xerosis
735
FN179330
clade_457
N
OP


Staphylococcaceae bacterium NML 92_0017
1756
AY841362
clade_458
N
N


Staphylococcus fleurettii
1766
NR_041326
clade_458
N
N


Staphylococcus sciuri
1774
NR_025520
clade_458
N
N


Staphylococcus vitulinus
1779
NR_024670
clade_458
N
N


Stenotrophomonas maltophilia
1782
AAVZ01000005
clade_459
N
N


Stenotrophomonas sp. FG_6
1783
EF017810
clade_459
N
N


Mycobacterium africanum
1270
AF480605
clade_46
N
OP


Mycobacterium alsiensis
1271
AJ938169
clade_46
N
OP


Mycobacterium avium
1272
CP000479
clade_46
N
OP


Mycobacterium colombiense
1274
AM062764
clade_46
N
OP


Mycobacterium gordonae
1276
GU142930
clade_46
N
OP


Mycobacterium intracellulare
1277
GQ153276
clade_46
N
OP


Mycobacterium kansasii
1278
AF480601
clade_46
N
OP


Mycobacterium lacus
1279
NR_025175
clade_46
N
OP


Mycobacterium leprae
1280
FM211192
clade_46
N
OP


Mycobacterium lepromatosis
1281
EU203590
clade_46
N
OP


Mycobacterium mantenii
1283
FJ042897
clade_46
N
OP


Mycobacterium marinum
1284
NC_010612
clade_46
N
OP


Mycobacterium microti
1285
NR_025234
clade_46
N
OP


Mycobacterium parascrofulaceum
1287
ADNV01000350
clade_46
N
OP


Mycobacterium seoulense
1290
DQ536403
clade_46
N
OP


Mycobacterium sp. 1761
1292
EU703150
clade_46
N
N


Mycobacterium sp. 1791
1295
EU703148
clade_46
N
N


Mycobacterium sp. 1797
1296
EU703149
clade_46
N
N


Mycobacterium sp. B10_07.09.0206
1298
HQ174245
clade_46
N
N


Mycobacterium sp. NLA001000736
1305
HM627011
clade_46
N
N


Mycobacterium sp. W
1306
DQ437715
clade_46
N
N


Mycobacterium tuberculosis
1307
CP001658
clade_46
N
Category-C


Mycobacterium ulcerans
1308
AB548725
clade_46
N
OP


Mycobacterium vulneris
1309
EU834055
clade_46
N
OP


Xanthomonas campestris
2016
EF101975
clade_461
N
N


Xanthomonas sp. kmd_489
2017
EU723184
clade_461
N
N


Dietzia natronolimnaea
769
GQ870426
clade_462
N
N


Dietzia sp. BBDP51
770
DQ337512
clade_462
N
N


Dietzia sp. CA149
771
GQ870422
clade_462
N
N


Dietzia timorensis
772
GQ870424
clade_462
N
N


Gordonia bronchialis
951
NR_027594
clade_463
N
N


Gordonia polyisoprenivorans
952
DQ385609
clade_463
N
N


Gordonia sp. KTR9
953
DQ068383
clade_463
N
N


Gordonia sputi
954
FJ536304
clade_463
N
N


Gordonia terrae
955
GQ848239
clade_463
N
N


Leptotrichia goodfellowii
1167
ADAD01000110
clade_465
N
N


Leptotrichia sp. oral clone IK040
1174
AY349387
clade_465
N
N


Leptotrichia sp. oral clone P2PB_51 P1
1175
AY207053
clade_465
N
N


Bacteroidales genomosp. P7 oral clone MB3_P19
264
DQ003623
clade_466
N
N


Butyricimonas virosa
454
AB443949
clade_466
N
N


Odoribacter laneus
1363
AB490805
clade_466
N
N


Odoribacter splanchnicus
1364
CP002544
clade_466
N
N


Capnocytophaga gingivalis
478
ACLQ01000011
clade_467
N
N


Capnocytophaga granulosa
479
X97248
clade_467
N
N


Capnocytophaga sp. oral clone AH015
483
AY005074
clade_467
N
N


Capnocytophaga sp. oral strain S3
487
AY005073
clade_467
N
N


Capnocytophaga sp. oral taxon 338
488
AEXX01000050
clade_467
N
N


Capnocytophaga canimorsus
476
CP002113
clade_468
N
N


Capnocytophaga sp. oral clone ID062
485
AY349368
clade_468
N
N


Lactobacillus catenaformis
1075
M23729
clade_469
N
N


Lactobacillus vitulinus
1142
NR_041305
clade_469
N
N


Cetobacterium somerae
501
AJ438155
clade_470
N
N


Fusobacterium gonidiaformans
896
ACET01000043
clade_470
N
N


Fusobacterium mortiferum
897
ACDB02000034
clade_470
N
N


Fusobacterium necrogenes
899
X55408
clade_470
N
N


Fusobacterium necrophorum
900
AM905356
clade_470
N
N


Fusobacterium sp. 12_1B
905
AGWJ01000070
clade_470
N
N


Fusobacterium sp. 3_1_5R
911
ACDD01000078
clade_470
N
N


Fusobacterium sp. D12
918
ACDG02000036
clade_470
N
N


Fusobacterium ulcerans
921
ACDH01000090
clade_470
N
N


Fusobacterium varium
922
ACIE01000009
clade_470
N
N


Mycoplasma arthritidis
1312
NC_011025
clade_473
N
N


Mycoplasma faucium
1314
NR_024983
clade_473
N
N


Mycoplasma hominis
1318
AF443616
clade_473
N
N


Mycoplasma orale
1319
AY796060
clade_473
N
N


Mycoplasma salivarium
1324
M24661
clade_473
N
N


Mitsuokella jalaludinii
1247
NR_028840
clade_474
N
N


Mitsuokella multacida
1248
ABWK02000005
clade_474
N
N


Mitsuokella sp. oral taxon 521
1249
GU413658
clade_474
N
N


Mitsuokella sp. oral taxon G68
1250
GU432166
clade_474
N
N


Selenomonas genomosp. C1
1695
AY278627
clade_474
N
N


Selenomonas genomosp. P8 oral clone MB5_P06
1700
DQ003628
clade_474
N
N


Selenomonas ruminantium
1703
NR_075026
clade_474
N
N


Veillonellaceae bacterium oral taxon 131
1994
GU402916
clade_474
N
N


Alloscardovia omnicolens
139
NR_042583
clade_475
N
N


Alloscardovia sp. OB7196
140
AB425070
clade_475
N
N


Bifidobacterium urinalis
366
AJ278695
clade_475
N
N


Prevotella loescheii
1503
JN867231
clade_48
N
N


Prevotella sp. oral clone ASCG12
1530
DQ272511
clade_48
N
N


Prevotella sp. oral clone GU027
1540
AY349398
clade_48
N
N


Prevotella sp. oral taxon 472
1553
ACZS01000106
clade_48
N
N


Selenomonas dianae
1693
GQ422719
clade_480
N
N


Selenomonas flueggei
1694
AF287803
clade_480
N
N


Selenomonas genomosp. C2
1696
AY278628
clade_480
N
N


Selenomonas genomosp. P6 oral clone MB3_C41
1698
DQ003636
clade_480
N
N


Selenomonas genomosp. P7 oral clone MB5_C08
1699
DQ003627
clade_480
N
N


Selenomonas infelix
1701
AF287802
clade_480
N
N


Selenomonas noxia
1702
GU470909
clade_480
N
N


Selenomonas sp. oral clone FT050
1705
AY349403
clade_480
N
N


Selenomonas sp. oral clone GI064
1706
AY349404
clade_480
N
N


Selenomonas sp. oral clone GT010
1707
AY349405
clade_480
N
N


Selenomonas sp. oral clone HU051
1708
AY349406
clade_480
N
N


Selenomonas sp. oral clone IK004
1709
AY349407
clade_480
N
N


Selenomonas sp. oral clone JI021
1711
AY349409
clade_480
N
N


Selenomonas sp. oral clone JS031
1712
AY349410
clade_480
N
N


Selenomonas sp. oral clone OH4A
1713
AY947498
clade_480
N
N


Selenomonas sp. oral clone P2PA_80 P4
1714
AY207052
clade_480
N
N


Selenomonas sp. oral taxon 149
1716
AEEJ01000007
clade_480
N
N


Veillonellaceae bacterium oral taxon 155
1995
GU470897
clade_480
N
N


Agrococcus jenensis
117
NR_026275
clade_484
N
N


Microbacterium gubbeenense
1232
NR_025098
clade_484
N
N


Pseudoclavibacter sp. Timone
1590
FJ375951
clade_484
N
N


Tropheryma whipplei
1961
BX251412
clade_484
N
N


Zimmermannella bifida
2031
AB012592
clade_484
N
N


Legionella hackeliae
1151
M36028
clade_486
N
OP


Legionella longbeachae
1152
M36029
clade_486
N
OP


Legionella sp. D3923
1154
JN380999
clade_486
N
OP


Legionella sp. D4088
1155
JN381012
clade_486
N
OP


Legionella sp. H63
1156
JF831047
clade_486
N
OP


Legionella sp. NML 93L054
1157
GU062706
clade_486
N
OP


Legionella steelei
1158
HQ398202
clade_486
N
OP


Tatlockia micdadei
1915
M36032
clade_486
N
N


Helicobacter pullorum
996
ABQU01000097
clade_489
N
N


Acetobacteraceae bacterium AT_5844
16
AGEZ01000040
clade_490
N
N


Roseomonas cervicalis
1643
ADVL01000363
clade_490
N
N


Roseomonas mucosa
1644
NR_028857
clade_490
N
N


Roseomonas sp. NML94_0193
1645
AF533357
clade_490
N
N


Roseomonas sp. NML97_0121
1646
AF533359
clade_490
N
N


Roseomonas sp. NML98_0009
1647
AF533358
clade_490
N
N


Roseomonas sp. NML98_0157
1648
AF533360
clade_490
N
N


Rickettsia akari
1627
CP000847
clade_492
N
OP


Rickettsia conorii
1628
AE008647
clade_492
N
OP


Rickettsia prowazekii
1629
M21789
clade_492
N
Category-B


Rickettsia rickettsii
1630
NC_010263
clade_492
N
OP


Rickettsia slovaca
1631
L36224
clade_492
N
OP


Rickettsia typhi
1632
AE017197
clade_492
N
OP


Anaeroglobus geminatus
160
AGCJ01000054
clade_493
N
N


Megasphaera genomosp. C1
1201
AY278622
clade_493
N
N


Megasphaera micronuciformis
1203
AECS01000020
clade_493
N
N


Clostridiales genomosp. BVAB3
540
CP001850
clade_495
N
N


Tsukamurella paurometabola
1963
X80628
clade_496
N
N


Tsukamurella tyrosinosolvens
1964
AB478958
clade_496
N
N


Abiotrophia para_adiacens
2
AB022027
clade_497
N
N


Carnobacterium divergens
492
NR_044706
clade_497
N
N


Carnobacterium maltaromaticum
493
NC_019425
clade_497
N
N


Enterococcus avium
800
AF133535
clade_497
N
N


Enterococcus caccae
801
AY943820
clade_497
N
N


Enterococcus casseliflavus
802
AEWT01000047
clade_497
N
N


Enterococcus durans
803
AJ276354
clade_497
N
N


Enterococcus faecalis
804
AE016830
clade_497
N
N


Enterococcus faecium
805
AM157434
clade_497
N
N


Enterococcus gallinarum
806
AB269767
clade_497
N
N


Enterococcus gilvus
807
AY033814
clade_497
N
N


Enterococcus hawaiiensis
808
AY321377
clade_497
N
N


Enterococcus hirae
809
AF061011
clade_497
N
N


Enterococcus italicus
810
AEPV01000109
clade_497
N
N


Enterococcus mundtii
811
NR_024906
clade_497
N
N


Enterococcus raffinosus
812
FN600541
clade_497
N
N


Enterococcus sp. BV2CASA2
813
JN809766
clade_497
N
N


Enterococcus sp. CCRI_16620
814
GU457263
clade_497
N
N


Enterococcus sp. F95
815
FJ463817
clade_497
N
N


Enterococcus sp. RfL6
816
AJ133478
clade_497
N
N


Enterococcus thailandicus
817
AY321376
clade_497
N
N


Fusobacterium canifelinum
893
AY162222
clade_497
N
N


Fusobacterium genomosp. C1
894
AY278616
clade_497
N
N


Fusobacterium genomosp. C2
895
AY278617
clade_497
N
N


Fusobacterium periodonticum
902
ACJY01000002
clade_497
N
N


Fusobacterium sp. 1_1_41FAA
906
ADGG01000053
clade_497
N
N


Fusobacterium sp. 11_3_2
904
ACUO01000052
clade_497
N
N


Fusobacterium sp. 2_1_31
907
ACDC02000018
clade_497
N
N


Fusobacterium sp. 3_1_27
908
ADGF01000045
clade_497
N
N


Fusobacterium sp. 3_1_33
909
ACQE01000178
clade_497
N
N


Fusobacterium sp. 3_1_36A2
910
ACPU01000044
clade_497
N
N


Fusobacterium sp. AC18
912
HQ616357
clade_497
N
N


Fusobacterium sp. ACB2
913
HQ616358
clade_497
N
N


Fusobacterium sp. AS2
914
HQ616361
clade_497
N
N


Fusobacterium sp. CM1
915
HQ616371
clade_497
N
N


Fusobacterium sp. CM21
916
HQ616375
clade_497
N
N


Fusobacterium sp. CM22
917
HQ616376
clade_497
N
N


Fusobacterium sp. oral clone ASCF06
919
AY923141
clade_497
N
N


Fusobacterium sp. oral clone ASCF11
920
AY953256
clade_497
N
N


Granulicatella adiacens
959
ACKZ01000002
clade_497
N
N


Granulicatella elegans
960
AB252689
clade_497
N
N


Granulicatella paradiacens
961
AY879298
clade_497
N
N


Granulicatella sp. oral clone ASC02
963
AY923126
clade_497
N
N


Granulicatella sp. oral clone ASCA05
964
DQ341469
clade_497
N
N


Granulicatella sp. oral clone ASCB09
965
AY953251
clade_497
N
N


Granulicatella sp. oral clone ASCG05
966
AY923146
clade_497
N
N


Tetragenococcus halophilus
1918
NR_075020
clade_497
N
N


Tetragenococcus koreensis
1919
NR_043113
clade_497
N
N


Vagococcus fluvialis
1973
NR_026489
clade_497
N
N


Chryseobacterium anthropi
514
AM982793
clade_498
N
N


Chryseobacterium gleum
515
ACKQ02000003
clade_498
N
N


Chryseobacterium hominis
516
NR_042517
clade_498
N
N


Treponema refringens
1936
AF426101
clade_499
N
OP


Treponema sp. oral clone JU031
1941
AY349416
clade_499
N
N


Treponema sp. oral taxon 239
1948
GU408738
clade_499
N
N


Treponema sp. oral taxon 271
1955
GU408871
clade_499
N
N


Alistipes finegoldii
129
NR_043064
clade_500
N
N


Alistipes onderdonkii
131
NR_043318
clade_500
N
N


Alistipes putredinis
132
ABFK02000017
clade_500
N
N


Alistipes shahii
133
FP929032
clade_500
N
N


Alistipes sp. HGB5
134
AENZ01000082
clade_500
N
N


Alistipes sp. JC50
135
JF824804
clade_500
N
N


Alistipes sp. RMA 9912
136
GQ140629
clade_500
N
N


Mycoplasma agalactiae
1310
AF010477
clade_501
N
N


Mycoplasma bovoculi
1313
NR_025987
clade_501
N
N


Mycoplasma fermentans
1315
CP002458
clade_501
N
N


Mycoplasma flocculare
1316
X62699
clade_501
N
N


Mycoplasma ovipneumoniae
1320
NR_025989
clade_501
N
N


Arcobacter butzleri
176
AEPT01000071
clade_502
N
N


Arcobacter cryaerophilus
177
NR_025905
clade_502
N
N


Campylobacter curvus
461
NC_009715
clade_502
N
OP


Campylobacter rectus
467
ACFU01000050
clade_502
N
OP


Campylobacter showae
468
ACVQ01000030
clade_502
N
OP


Campylobacter sp. FOBRC14
469
HQ616379
clade_502
N
OP


Campylobacter sp. FOBRC15
470
HQ616380
clade_502
N
OP


Campylobacter sp. oral clone BB120
471
AY005038
clade_502
N
OP


Campylobacter sputorum
472
NR_044839
clade_502
N
OP


Bacteroides ureolyticus
330
GQ167666
clade_504
N
N


Campylobacter gracilis
463
ACYG01000026
clade_504
N
OP


Campylobacter hominis
464
NC_009714
clade_504
N
OP


Dialister invisus
762
ACIM02000001
clade_506
N
N


Dialister micraerophilus
763
AFBB01000028
clade_506
N
N


Dialister microaerophilus
764
AENT01000008
clade_506
N
N


Dialister propionicifaciens
766
NR_043231
clade_506
N
N


Dialister succinatiphilus
768
AB370249
clade_506
N
N


Megasphaera elsdenii
1200
AY038996
clade_506
N
N


Megasphaera genomosp. type 1
1202
ADGP01000010
clade_506
N
N


Megasphaera sp. BLPYG_07
1204
HM990964
clade_506
N
N


Megasphaera sp. UPII 199_6
1205
AFIJ01000040
clade_506
N
N


Chromobacterium violaceum
513
NC_005085
clade_507
N
N


Laribacter hongkongensis
1148
CP001154
clade_507
N
N


Methylophilus sp. ECd5
1229
AY436794
clade_507
N
N


Finegoldia magna
883
ACHM02000001
clade_509
N
N


Parvimonas micra
1431
AB729072
clade_509
N
N


Parvimonas sp. oral taxon 110
1432
AFII01000002
clade_509
N
N


Peptostreptococcus micros
1456
AM176538
clade_509
N
N


Peptostreptococcus sp. oral clone FJ023
1460
AY349390
clade_509
N
N


Peptostreptococcus sp. P4P_31 P3
1458
AY207059
clade_509
N
N


Helicobacter pylori
997
CP000012
clade_510
N
OP


Anaplasma marginale
165
ABOR01000019
clade_511
N
N


Anaplasma phagocytophilum
166
NC_007797
clade_511
N
N


Ehrlichia chaffeensis
783
AAIF01000035
clade_511
N
OP


Neorickettsia risticii
1349
CP001431
clade_511
N
N


Neorickettsia sennetsu
1350
NC_007798
clade_511
N
N


Pseudoramibacter alactolyticus
1606
AB036759
clade_512
N
N


Veillonella montpellierensis
1977
AF473836
clade_513
N
N


Veillonella sp. oral clone ASCA08
1988
AY923118
clade_513
N
N


Veillonella sp. oral clone ASCB03
1989
AY923122
clade_513
N
N


Inquilinus limosus
1012
NR_029046
clade_514
N
N


Sphingomonas sp. oral clone FZ016
1746
AY349412
clade_514
N
N


Anaerococcus lactolyticus
145
ABYO01000217
clade_515
N
N


Anaerococcus prevotii
147
CP001708
clade_515
N
N


Anaerococcus sp. gpac104
152
AM176528
clade_515
N
N


Anaerococcus sp. gpac126
153
AM176530
clade_515
N
N


Anaerococcus sp. gpac155
154
AM176536
clade_515
N
N


Anaerococcus sp. gpac199
155
AM176539
clade_515
N
N


Anaerococcus tetradius
157
ACGC01000107
clade_515
N
N


Bacteroides coagulans
271
AB547639
clade_515
N
N


Clostridiales bacterium 9403326
534
HM587324
clade_515
N
N


Clostridiales bacterium ph2
539
JN837487
clade_515
N
N


Peptostreptococcus sp. 9succ1
1457
X90471
clade_515
N
N


Peptostreptococcus sp. oral clone AP24
1459
AB175072
clade_515
N
N


Tissierella praeacuta
1924
NR_044860
clade_515
N
N


Helicobacter canadensis
994
ABQS01000108
clade_518
N
N


Peptostreptococcus anaerobius
1455
AY326462
clade_520
N
N


Peptostreptococcus stomatis
1461
ADGQ01000048
clade_520
N
N


Bilophila wadsworthia
367
ADCP01000166
clade_521
N
N


Desulfovibrio vulgaris
761
NR_074897
clade_521
N
N


Actinomyces nasicola
64
AJ508455
clade_523
N
N


Cellulosimicrobium funkei
500
AY501364
clade_523
N
N


Lactococcus raffinolactis
1146
NR_044359
clade_524
N
N


Bacteroidales genomosp. P1
258
AY341819
clade_529
N
N


Bacteroidales genomosp. P2 oral clone MB1_G13
259
DQ003613
clade_529
N
N


Bacteroidales genomosp. P3 oral clone MB1_G34
260
DQ003615
clade_529
N
N


Bacteroidales genomosp. P4 oral clone MB2_G17
261
DQ003617
clade_529
N
N


Bacteroidales genomosp. P5 oral clone MB2_P04
262
DQ003619
clade_529
N
N


Bacteroidales genomosp. P6 oral clone MB3_C19
263
DQ003634
clade_529
N
N


Bacteroidales genomosp. P8 oral clone MB4_G15
265
DQ003626
clade_529
N
N


Bacteroidetes bacterium oral taxon D27
333
HM099638
clade_530
N
N


Bacteroidetes bacterium oral taxon F31
334
HM099643
clade_530
N
N


Bacteroidetes bacterium oral taxon F44
335
HM099649
clade_530
N
N


Flavobacterium sp. NF2_1
885
FJ195988
clade_530
N
N


Myroides odoratimimus
1326
NR_042354
clade_530
N
N


Myroides sp. MY15
1327
GU253339
clade_530
N
N


Chlamydiales bacterium NS16
507
JN606076
clade_531
N
N


Chlamydophila pecorum
508
D88317
clade_531
N
OP


Parachlamydia sp. UWE25
1423
BX908798
clade_531
N
N


Fusobacterium russii
903
NR_044687
clade_532
N
N


Streptobacillus moniliformis
1784
NR_027615
clade_532
N
N


Eubacteriaceae bacterium P4P_50 P4
833
AY207060
clade_533
N
N


Abiotrophia defectiva
1
ACIN02000016
clade_534
N
N


Abiotrophia sp. oral clone P4PA_155 P1
3
AY207063
clade_534
N
N


Catonella genomosp. P1 oral clone MB5_P12
496
DQ003629
clade_534
N
N


Catonella morbi
497
ACIL02000016
clade_534
N
N


Catonella sp. oral clone FL037
498
AY349369
clade_534
N
N


Eremococcus coleocola
818
AENN01000008
clade_534
N
N


Facklamia hominis
879
Y10772
clade_534
N
N


Granulicatella sp. M658_99_3
962
AJ271861
clade_534
N
N


Campylobacter coli
459
AAFL01000004
clade_535
N
OP


Campylobacter concisus
460
CP000792
clade_535
N
OP


Campylobacter fetus
462
ACLG01001177
clade_535
N
OP


Campylobacter jejuni
465
AL139074
clade_535
N
Category-B


Campylobacter upsaliensis
473
AEPU01000040
clade_535
N
OP


Atopobium minutum
183
HM007583
clade_539
N
N


Atopobium parvulum
184
CP001721
clade_539
N
N


Atopobium rimae
185
ACFE01000007
clade_539
N
N


Atopobium sp. BS2
186
HQ616367
clade_539
N
N


Atopobium sp. F0209
187
EU592966
clade_539
N
N


Atopobium sp. ICM42b10
188
HQ616393
clade_539
N
N


Atopobium sp. ICM57
189
HQ616400
clade_539
N
N


Atopobium vaginae
190
AEDQ01000024
clade_539
N
N


Coriobacteriaceae bacterium BV3Ac1
677
JN809768
clade_539
N
N


Actinomyces naeslundii
63
X81062
clade_54
N
N


Actinomyces oricola
67
NR_025559
clade_54
N
N


Actinomyces oris
69
BABV01000070
clade_54
N
N


Actinomyces sp. 7400942
70
EU484334
clade_54
N
N


Actinomyces sp. ChDC B197
72
AF543275
clade_54
N
N


Actinomyces sp. GEJ15
73
GU561313
clade_54
N
N


Actinomyces sp. M2231_94_1
79
AJ234063
clade_54
N
N


Actinomyces sp. oral clone GU067
83
AY349362
clade_54
N
N


Actinomyces sp. oral clone IO077
85
AY349364
clade_54
N
N


Actinomyces sp. oral clone IP073
86
AY349365
clade_54
N
N


Actinomyces sp. oral clone JA063
88
AY349367
clade_54
N
N


Actinomyces sp. oral taxon 170
89
AFBL01000010
clade_54
N
N


Actinomyces sp. oral taxon 171
90
AECW01000034
clade_54
N
N


Actinomyces urogenitalis
95
ACFH01000038
clade_54
N
N


Actinomyces viscosus
96
ACRE01000096
clade_54
N
N


Orientia tsutsugamushi
1383
AP008981
clade_541
N
OP


Megamonas funiformis
1198
AB300988
clade_542
N
N


Megamonas hypermegale
1199
AJ420107
clade_542
N
N


Aeromicrobium marinum
102
NR_025681
clade_544
N
N


Aeromicrobium sp. JC14
103
JF824798
clade_544
N
N


Luteococcus sanguinis
1190
NR_025507
clade_544
N
N


Propionibacteriaceae bacterium NML 02_0265
1568
EF599122
clade_544
N
N


Rhodococcus corynebacterioides
1622
X80615
clade_546
N
N


Rhodococcus erythropolis
1624
ACNO01000030
clade_546
N
N


Rhodococcus fascians
1625
NR_037021
clade_546
N
N


Segniliparus rotundus
1690
CP001958
clade_546
N
N


Segniliparus rugosus
1691
ACZI01000025
clade_546
N
N


Exiguobacterium acetylicum
878
FJ970034
clade_547
N
N


Macrococcus caseolyticus
1194
NR_074941
clade_547
N
N


Streptomyces sp. 1 AIP_2009
1890
FJ176782
clade_548
N
N


Streptomyces sp. SD 524
1892
EU544234
clade_548
N
N


Streptomyces sp. SD 528
1893
EU544233
clade_548
N
N


Streptomyces thermoviolaceus
1895
NR_027616
clade_548
N
N


Borrelia afzelii
388
ABCU01000001
clade_549
N
OP


Borrelia crocidurae
390
DQ057990
clade_549
N
OP


Borrelia duttonii
391
NC_011229
clade_549
N
OP


Borrelia hermsii
393
AY597657
clade_549
N
OP


Borrelia hispanica
394
DQ057988
clade_549
N
OP


Borrelia persica
395
HM161645
clade_549
N
OP


Borrelia recurrentis
396
AF107367
clade_549
N
OP


Borrelia spielmanii
398
ABKB01000002
clade_549
N
OP


Borrelia turicatae
399
NC_008710
clade_549
N
OP


Borrelia valaisiana
400
ABCY01000002
clade_549
N
OP


Providencia alcalifaciens
1586
ABXW01000071
clade_55
N
N


Providencia rettgeri
1587
AM040492
clade_55
N
N


Providencia rustigianii
1588
AM040489
clade_55
N
N


Providencia stuartii
1589
AF008581
clade_55
N
N


Treponema pallidum
1932
CP001752
clade_550
N
OP


Treponema phagedenis
1934
AEFH01000172
clade_550
N
N


Treponema sp. clone DDKL_4
1939
Y08894
clade_550
N
N


Acholeplasma laidlawii
17
NR_074448
clade_551
N
N


Mycoplasma putrefaciens
1323
U26055
clade_551
N
N


Mycoplasmataceae genomosp. P1 oral clone MB1_G23
1325
DQ003614
clade_551
N
N


Spiroplasma insolitum
1750
NR_025705
clade_551
N
N


Collinsella intestinalis
660
ABXH02000037
clade_553
N
N


Collinsella stercoris
661
ABXJ01000150
clade_553
N
N


Collinsella tanakaei
662
AB490807
clade_553
N
N


Caminicella sporogenes
458
NR_025485
clade_554
N
N


Acidaminococcus fermentans
21
CP001859
clade_556
N
N


Acidaminococcus intestini
22
CP003058
clade_556
N
N


Acidaminococcus sp. D21
23
ACGB01000071
clade_556
N
N


Phascolarctobacterium faecium
1462
NR_026111
clade_556
N
N


Phascolarctobacterium sp. YIT 12068
1463
AB490812
clade_556
N
N


Phascolarctobacterium succinatutens
1464
AB490811
clade_556
N
N


Acidithiobacillus ferrivorans
25
NR_074660
clade_557
N
N


Xanthomonadaceae bacterium NML 03_0222
2015
EU313791
clade_557
N
N


Catabacter hongkongensis
494
AB671763
clade_558
N
N


Christensenella minuta
512
AB490809
clade_558
N
N


Clostridiales bacterium oral clone P4PA_66 P1
536
AY207065
clade_558
N
N


Clostridiales bacterium oral taxon 093
537
GQ422712
clade_558
N
N


Heliobacterium modesticaldum
1000
NR_074517
clade_560
N
N


Alistipes indistinctus
130
AB490804
clade_561
N
N


Bacteroidales bacterium ph8
257
JN837494
clade_561
N
N


Candidatus Sulcia muelleri
475
CP002163
clade_561
N
N


Cytophaga xylanolytica
742
FR733683
clade_561
N
N


Flavobacteriaceae genomosp. C1
884
AY278614
clade_561
N
N


Gramella forsetii
958
NR_074707
clade_561
N
N


Sphingobacterium faecium
1740
NR_025537
clade_562
N
N


Sphingobacterium mizutaii
1741
JF708889
clade_562
N
N


Sphingobacterium multivorum
1742
NR_040953
clade_562
N
N


Sphingobacterium spiritivorum
1743
ACHA02000013
clade_562
N
N


Jonquetella anthropi
1017
ACOO02000004
clade_563
N
N


Pyramidobacter piscolens
1614
AY207056
clade_563
N
N


Synergistes genomosp. C1
1904
AY278615
clade_563
N
N


Synergistes sp. RMA 14551
1905
DQ412722
clade_563
N
N


Synergistetes bacterium ADV897
1906
GQ258968
clade_563
N
N


Candidatus Arthromitus sp. SFB_mouse_Yit
474
NR_074460
clade_564
N
N


Gracilibacter thermotolerans
957
NR_043559
clade_564
N
N


Brachyspira aalborgi
404
FM178386
clade_565
N
N


Brachyspira sp. HIS3
406
FM178387
clade_565
N
N


Brachyspira sp. HIS4
407
FM178388
clade_565
N
N


Brachyspira sp. HIS5
408
FM178389
clade_565
N
N


Adlercreutzia equolifaciens
97
AB306661
clade_566
N
N


Coriobacteriaceae bacterium JC110
678
CAEM01000062
clade_566
N
N


Coriobacteriaceae bacterium phl
679
JN837493
clade_566
N
N


Cryptobacterium curtum
740
GQ422741
clade_566
N
N


Eggerthella sinensis
779
AY321958
clade_566
N
N


Eggerthella sp. 1_3_56FAA
780
ACWN01000099
clade_566
N
N


Eggerthella sp. HGA1
781
AEXR01000021
clade_566
N
N


Eggerthella sp. YY7918
782
AP012211
clade_566
N
N


Gordonibacter pamelaeae
680
AM886059
clade_566
N
N


Gordonibacter pamelaeae
956
FP929047
clade_566
N
N


Slackia equolifaciens
1732
EU377663
clade_566
N
N


Slackia exigua
1733
ACUX01000029
clade_566
N
N


Slackia faecicanis
1734
NR_042220
clade_566
N
N


Slackia heliotrinireducens
1735
NR_074439
clade_566
N
N


Slackia isoflavoniconvertens
1736
AB566418
clade_566
N
N


Slackia piriformis
1737
AB490806
clade_566
N
N


Slackia sp. NATTS
1738
AB505075
clade_566
N
N


Chlamydiales bacterium NS13
506
JN606075
clade_567
N
N


Victivallaceae bacterium NML 080035
2003
FJ394915
clade_567
N
N


Victivallis vadensis
2004
ABDE02000010
clade_567
N
N


Streptomyces griseus
1889
NR_074787
clade_573
N
N


Streptomyces sp. SD 511
1891
EU544231
clade_573
N
N


Streptomyces sp. SD 534
1894
EU544232
clade_573
N
N


Cloacibacillus evryensis
530
GQ258966
clade_575
N
N


Deferribacteres sp. oral clone JV001
743
AY349370
clade_575
N
N


Deferribacteres sp. oral clone JV023
745
AY349372
clade_575
N
N


Synergistetes bacterium LBVCM1157
1907
GQ258969
clade_575
N
N


Synergistetes bacterium oral taxon 362
1909
GU410752
clade_575
N
N


Synergistetes bacterium oral taxon D48
1910
GU430992
clade_575
N
N


Peptococcus sp. oral clone JM048
1439
AY349389
clade_576
N
N


Helicobacter winghamensis
999
ACDO01000013
clade_577
N
N


Wolinella succinogenes
2014
BX571657
clade_577
N
N


Olsenella genomosp. C1
1368
AY278623
clade_578
N
N


Olsenella profusa
1369
FN178466
clade_578
N
N


Olsenella sp. F0004
1370
EU592964
clade_578
N
N


Olsenella sp. oral taxon 809
1371
ACVE01000002
clade_578
N
N


Olsenella uli
1372
CP002106
clade_578
N
N


Nocardiopsis dassonvillei
1356
CP002041
clade_579
N
N


Peptococcus niger
1438
NR_029221
clade_580
N
N


Peptococcus sp. oral taxon 167
1440
GQ422727
clade_580
N
N


Akkermansia muciniphila
118
CP001071
clade_583
N
N


Opitutus terrae
1373
NR_074978
clade_583
N
N


Clostridiales bacterium oral taxon F32
538
HM099644
clade_584
N
N


Leptospira borgpetersenii
1161
NC_008508
clade_585
N
OP


Leptospira broomii
1162
NR_043200
clade_585
N
OP


Leptospira interrogans
1163
NC_005823
clade_585
N
OP


Methanobrevibacter gottschalkii
1213
NR_044789
clade_587
N
N


Methanobrevibacter millerae
1214
NR_042785
clade_587
N
N


Methanobrevibacter oralis
1216
HE654003
clade_587
N
N


Methanobrevibacter thaueri
1219
NR_044787
clade_587
N
N


Methanobrevibacter smithii
1218
ABYV02000002
clade_588
N
N


Deinococcus radiodurans
746
AE000513
clade_589
N
N


Deinococcus sp. R_43890
747
FR682752
clade_589
N
N


Thermus aquaticus
1923
NR_025900
clade_589
N
N


Actinomyces sp. c109
81
AB167239
clade_590
N
N


Syntrophomonadaceae genomosp. P1
1912
AY341821
clade_590
N
N


Anaerobaculum hydrogeniformans
141
ACJX02000009
clade_591
N
N


Microcystis aeruginosa
1246
NC_010296
clade_592
N
N


Prochlorococcus marinus
1567
CP000551
clade_592
N
N


Methanobrevibacter acididurans
1208
NR_028779
clade_593
N
N


Methanobrevibacter arboriphilus
1209
NR_042783
clade_593
N
N


Methanobrevibacter curvatus
1210
NR_044796
clade_593
N
N


Methanobrevibacter cuticularis
1211
NR_044776
clade_593
N
N


Methanobrevibacter filiformis
1212
NR_044801
clade_593
N
N


Methanobrevibacter woesei
1220
NR_044788
clade_593
N
N


Roseiflexus castenholzii
1642
CP000804
clade_594
N
N


Methanobrevibacter olleyae
1215
NR_043024
clade_595
N
N


Methanobrevibacter ruminantium
1217
NR_042784
clade_595
N
N


Methanobrevibacter wolinii
1221
NR_044790
clade_595
N
N


Methanosphaera stadtmanae
1222
AY196684
clade_595
N
N


Chloroflexi genomosp. P1
511
AY331414
clade_596
N
N


Halorubrum lipolyticum
992
AB477978
clade_597
N
N


Methanobacterium formicicum
1207
NR_025028
clade_597
N
N


Acidilobus saccharovorans
24
AY350586
clade_598
N
N


Hyperthermus butylicus
1006
CP000493
clade_598
N
N


Ignicoccus islandicus
1011
X99562
clade_598
N
N


Metallosphaera sedula
1206
D26491
clade_598
N
N


Thermofilum pendens
1922
X14835
clade_598
N
N


Prevotella melaninogenica
1506
CP002122
clade_6
N
N


Prevotella sp. ICM1
1520
HQ616385
clade_6
N
N


Prevotella sp. oral clone FU048
1535
AY349393
clade_6
N
N


Prevotella sp. oral clone GI030
1537
AY349395
clade_6
N
N


Prevotella sp. SEQ116
1526
JN867246
clade_6
N
N


Streptococcus anginosus
1787
AECT01000011
clade_60
N
N


Streptococcus milleri
1812
X81023
clade_60
N
N


Streptococcus sp. 16362
1829
JN590019
clade_60
N
N


Streptococcus sp. 69130
1832
X78825
clade_60
N
N


Streptococcus sp. AC15
1833
HQ616356
clade_60
N
N


Streptococcus sp. CM7
1839
HQ616373
clade_60
N
N


Streptococcus sp. OBRC6
1847
HQ616352
clade_60
N
N


Burkholderia ambifaria
442
AAUZ01000009
clade_61
N
OP


Burkholderia cenocepacia
443
AAHI01000060
clade_61
N
OP


Burkholderia cepacia
444
NR_041719
clade_61
N
OP


Burkholderia mallei
445
CP000547
clade_61
N
Category-B


Burkholderia multivorans
446
NC_010086
clade_61
N
OP


Burkholderia oklahomensis
447
DQ108388
clade_61
N
OP


Burkholderia pseudomallei
448
CP001408
clade_61
N
Category-B


Burkholderia rhizoxinica
449
HQ005410
clade_61
N
OP


Burkholderia sp. 383
450
CP000151
clade_61
N
OP


Burkholderia xenovorans
451
U86373
clade_61
N
OP


Prevotella buccae
1488
ACRB01000001
clade_62
N
N


Prevotella genomosp. P8 oral clone MB3_P13
1498
DQ003622
clade_62
N
N


Prevotella sp. oral clone FW035
1536
AY349394
clade_62
N
N


Prevotella bivia
1486
ADFO01000096
clade_63
N
N


Prevotella disiens
1494
AEDO01000026
clade_64
N
N


Bacteroides faecis
276
GQ496624
clade_65
N
N


Bacteroides fragilis
279
AP006841
clade_65
N
N


Bacteroides nordii
285
NR_043017
clade_65
N
N


Bacteroides salyersiae
292
EU136690
clade_65
N
N


Bacteroides sp. 1_1_14
293
ACRP01000155
clade_65
N
N


Bacteroides sp. 1_1_6
295
ACIC01000215
clade_65
N
N


Bacteroides sp. 2_1_56FAA
298
ACWI01000065
clade_65
N
N


Bacteroides sp. AR29
316
AF139525
clade_65
N
N


Bacteroides sp. B2
317
EU722733
clade_65
N
N


Bacteroides thetaiotaomicron
328
NR_074277
clade_65
N
N


Actinobacillus minor
45
ACFT01000025
clade_69
N
N


Haemophilus parasuis
978
GU226366
clade_69
N
N


Vibrio cholerae
1996
AAUR01000095
clade_71
N
Category-B


Vibrio fluvialis
1997
X76335
clade_71
N
Category-B


Vibrio furnissii
1998
CP002377
clade_71
N
Category-B


Vibrio mimicus
1999
ADAF01000001
clade_71
N
Category-B


Vibrio parahaemolyticus
2000
AAWQ01000116
clade_71
N
Category-B


Vibrio sp. RC341
2001
ACZT01000024
clade_71
N
Category-B


Vibrio vulnificus
2002
AE016796
clade_71
N
Category-B


Lactobacillus acidophilus
1067
CP000033
clade_72
N
N


Lactobacillus amylolyticus
1069
ADNY01000006
clade_72
N
N


Lactobacillus amylovorus
1070
CP002338
clade_72
N
N


Lactobacillus crispatus
1078
ACOG01000151
clade_72
N
N


Lactobacillus delbrueckii
1080
CP002341
clade_72
N
N


Lactobacillus helveticus
1088
ACLM01000202
clade_72
N
N


Lactobacillus kalixensis
1094
NR_029083
clade_72
N
N


Lactobacillus kefiranofaciens
1095
NR_042440
clade_72
N
N


Lactobacillus leichmannii
1098
JX986966
clade_72
N
N


Lactobacillus sp. 66c
1120
FR681900
clade_72
N
N


Lactobacillus sp. KLDS 1.0701
1122
EU600905
clade_72
N
N


Lactobacillus sp. KLDS 1.0712
1130
EU600916
clade_72
N
N


Lactobacillus sp. oral clone HT070
1136
AY349383
clade_72
N
N


Lactobacillus ultunensis
1139
ACGU01000081
clade_72
N
N


Prevotella intermedia
1502
AF414829
clade_81
N
N


Prevotella nigrescens
1511
AFPX01000069
clade_81
N
N


Prevotella pallens
1515
AFPY01000135
clade_81
N
N


Prevotella sp. oral taxon 310
1551
GQ422737
clade_81
N
N


Prevotella genomosp. C1
1495
AY278624
clade_82
N
N


Prevotella sp. CM38
1519
HQ610181
clade_82
N
N


Prevotella sp. oral taxon 317
1552
ACQH01000158
clade_82
N
N


Prevotella sp. SG12
1527
GU561343
clade_82
N
N


Prevotella denticola
1493
CP002589
clade_83
N
N


Prevotella genomosp. P7 oral clone MB2_P31
1497
DQ003620
clade_83
N
N


Prevotella histicola
1501
JN867315
clade_83
N
N


Prevotella multiformis
1508
AEWX01000054
clade_83
N
N


Prevotella sp. JCM 6330
1522
AB547699
clade_83
N
N


Prevotella sp. oral clone GI059
1539
AY349397
clade_83
N
N


Prevotella sp. oral taxon 782
1555
GQ422745
clade_83
N
N


Prevotella sp. oral taxon G71
1559
GU432180
clade_83
N
N


Prevotella sp. SEQ065
1524
JN867234
clade_83
N
N


Prevotella veroralis
1565
ACVA01000027
clade_83
N
N


Bacteroides acidifaciens
266
NR_028607
clade_85
N
N


Bacteroides cellulosilyticus
269
ACCH01000108
clade_85
N
N


Bacteroides clarus
270
AFBM01000011
clade_85
N
N


Bacteroides eggerthii
275
ACWG01000065
clade_85
N
N


Bacteroides oleiciplenus
286
AB547644
clade_85
N
N


Bacteroides pyogenes
290
NR_041280
clade_85
N
N


Bacteroides sp. 315_5
300
FJ848547
clade_85
N
N


Bacteroides sp. 31SF15
301
AJ583248
clade_85
N
N


Bacteroides sp. 31SF18
302
AJ583249
clade_85
N
N


Bacteroides sp. 35AE31
303
AJ583244
clade_85
N
N


Bacteroides sp. 35AE37
304
AJ583245
clade_85
N
N


Bacteroides sp. 35BE34
305
AJ583246
clade_85
N
N


Bacteroides sp. 35BE35
306
AJ583247
clade_85
N
N


Bacteroides sp. WH2
324
AY895180
clade_85
N
N


Bacteroides sp. XB12B
325
AM230648
clade_85
N
N


Bacteroides stercoris
327
ABFZ02000022
clade_85
N
N


Actinobacillus pleuropneumoniae
46
NR_074857
clade_88
N
N


Actinobacillus ureae
48
AEVG01000167
clade_88
N
N


Haemophilus aegyptius
969
AFBC01000053
clade_88
N
N


Haemophilus ducreyi
970
AE017143
clade_88
N
OP


Haemophilus haemolyticus
973
JN175335
clade_88
N
N


Haemophilus influenzae
974
AADP01000001
clade_88
N
OP


Haemophilus parahaemolyticus
975
GU561425
clade_88
N
N


Haemophilus parainfluenzae
976
AEWU01000024
clade_88
N
N


Haemophilus paraphrophaemolyticus
977
M75076
clade_88
N
N


Haemophilus somnus
979
NC_008309
clade_88
N
N


Haemophilus sp. 70334
980
HQ680854
clade_88
N
N


Haemophilus sp. HK445
981
FJ685624
clade_88
N
N


Haemophilus sp. oral clone ASCA07
982
AY923117
clade_88
N
N


Haemophilus sp. oral clone ASCG06
983
AY923147
clade_88
N
N


Haemophilus sp. oral clone BJ021
984
AY005034
clade_88
N
N


Haemophilus sp. oral clone BJ095
985
AY005033
clade_88
N
N


Haemophilus sp. oral taxon 851
987
AGRK01000004
clade_88
N
N


Haemophilus sputorum
988
AFNK01000005
clade_88
N
N


Histophilus somni
1003
AF549387
clade_88
N
N


Mannheimia haemolytica
1195
ACZX01000102
clade_88
N
N


Pasteurella bettyae
1433
L06088
clade_88
N
N


Moellerella wisconsensis
1253
JN175344
clade_89
N
N


Morganella morganii
1265
AJ301681
clade_89
N
N


Morganella sp. JB_T16
1266
AJ781005
clade_89
N
N


Proteus mirabilis
1582
ACLE01000013
clade_89
N
N


Proteus penneri
1583
ABVP01000020
clade_89
N
N


Proteus sp. HS7514
1584
DQ512963
clade_89
N
N


Proteus vulgaris
1585
AJ233425
clade_89
N
N


Oribacterium sinus
1374
ACKX01000142
clade_90
N
N


Oribacterium sp. ACB1
1375
HM120210
clade_90
N
N


Oribacterium sp. ACB7
1376
HM120211
clade_90
N
N


Oribacterium sp. CM12
1377
HQ616374
clade_90
N
N


Oribacterium sp. ICM51
1378
HQ616397
clade_90
N
N


Oribacterium sp. OBRC12
1379
HQ616355
clade_90
N
N


Oribacterium sp. oral taxon 108
1382
AFIH01000001
clade_90
N
N


Actinobacillus actinomycetemcomitans
44
AY362885
clade_92
N
N


Actinobacillus succinogenes
47
CP000746
clade_92
N
N


Aggregatibacter actinomycetemcomitans
112
CP001733
clade_92
N
N


Aggregatibacter aphrophilus
113
CP001607
clade_92
N
N


Aggregatibacter segnis
114
AEPS01000017
clade_92
N
N


Averyella dalhousiensis
194
DQ481464
clade_92
N
N


Bisgaard Taxon
368
AY683487
clade_92
N
N


Bisgaard Taxon
369
AY683489
clade_92
N
N


Bisgaard Taxon
370
AY683491
clade_92
N
N


Bisgaard Taxon
371
AY683492
clade_92
N
N


Buchnera aphidicola
440
NR_074609
clade_92
N
N


Cedecea davisae
499
AF493976
clade_92
N
N


Citrobacter amalonaticus
517
FR870441
clade_92
N
N


Citrobacter braakii
518
NR_028687
clade_92
N
N


Citrobacter farmeri
519
AF025371
clade_92
N
N


Citrobacter freundii
520
NR_028894
clade_92
N
N


Citrobacter gillenii
521
AF025367
clade_92
N
N


Citrobacter koseri
522
NC_009792
clade_92
N
N


Citrobacter murliniae
523
AF025369
clade_92
N
N


Citrobacter rodentium
524
NR_074903
clade_92
N
N


Citrobacter sedlakii
525
AF025364
clade_92
N
N


Citrobacter sp. 30_2
526
ACDJ01000053
clade_92
N
N


Citrobacter sp. KMSI_3
527
GQ468398
clade_92
N
N


Citrobacter werkmanii
528
AF025373
clade_92
N
N


Citrobacter youngae
529
ABWL02000011
clade_92
N
N


Cronobacter malonaticus
737
GU122174
clade_92
N
N


Cronobacter sakazakii
738
NC_009778
clade_92
N
N


Cronobacter turicensis
739
FN543093
clade_92
N
N


Enterobacter aerogenes
786
AJ251468
clade_92
N
N


Enterobacter asburiae
787
NR_024640
clade_92
N
N


Enterobacter cancerogenus
788
Z96078
clade_92
N
N


Enterobacter cloacae
789
FP929040
clade_92
N
N


Enterobacter cowanii
790
NR_025566
clade_92
N
N


Enterobacter hormaechei
791
AFHR01000079
clade_92
N
N


Enterobacter sp. 247BMC
792
HQ122932
clade_92
N
N


Enterobacter sp. 638
793
NR_074777
clade_92
N
N


Enterobacter sp. JC163
794
JN657217
clade_92
N
N


Enterobacter sp. SCSS
795
HM007811
clade_92
N
N


Enterobacter sp. TSE38
796
HM156134
clade_92
N
N


Enterobacteriaceae bacterium 9_2_54FAA
797
ADCU01000033
clade_92
N
N


Enterobacteriaceae bacterium CF01Ent_1
798
AJ489826
clade_92
N
N


Enterobacteriaceae bacterium Smarlab 3302238
799
AY538694
clade_92
N
N


Escherichia albertii
824
ABKX01000012
clade_92
N
N


Escherichia coli
825
NC_008563
clade_92
N
Category-B


Escherichia fergusonii
826
CU928158
clade_92
N
N


Escherichia hermannii
827
HQ407266
clade_92
N
N


Escherichia sp. 1_1_43
828
ACID01000033
clade_92
N
N


Escherichia sp. 4_1_40B
829
ACDM02000056
clade_92
N
N


Escherichia sp. B4
830
EU722735
clade_92
N
N


Escherichia vulneris
831
NR_041927
clade_92
N
N


Ewingella americana
877
JN175329
clade_92
N
N


Haemophilus genomosp. P2 oral clone MB3_C24
971
DQ003621
clade_92
N
N


Haemophilus genomosp. P3 oral clone MB3_C38
972
DQ003635
clade_92
N
N


Haemophilus sp. oral clone JM053
986
AY349380
clade_92
N
N


Hafnia alvei
989
DQ412565
clade_92
N
N


Klebsiella oxytoca
1024
AY292871
clade_92
N
OP


Klebsiella pneumoniae
1025
CP000647
clade_92
N
OP


Klebsiella sp. AS10
1026
HQ616362
clade_92
N
N


Klebsiella sp. Co9935
1027
DQ068764
clade_92
N
N


Klebsiella sp. enrichment culture clone SRC_DSD25
1036
HM195210
clade_92
N
N


Klebsiella sp. OBRC7
1028
HQ616353
clade_92
N
N


Klebsiella sp. SP_BA
1029
FJ999767
clade_92
N
N


Klebsiella sp. SRC_DSD1
1033
GU797254
clade_92
N
N


Klebsiella sp. SRC_DSD11
1030
GU797263
clade_92
N
N


Klebsiella sp. SRC_DSD12
1031
GU797264
clade_92
N
N


Klebsiella sp. SRC_DSD15
1032
GU797267
clade_92
N
N


Klebsiella sp. SRC_DSD2
1034
GU797253
clade_92
N
N


Klebsiella sp. SRC_DSD6
1035
GU797258
clade_92
N
N


Klebsiella variicola
1037
CP001891
clade_92
N
N


Kluyvera ascorbata
1038
NR_028677
clade_92
N
N


Kluyvera cryocrescens
1039
NR_028803
clade_92
N
N


Leminorella grimontii
1159
AJ233421
clade_92
N
N


Leminorella richardii
1160
HF558368
clade_92
N
N


Pantoea agglomerans
1409
AY335552
clade_92
N
N


Pantoea ananatis
1410
CP001875
clade_92
N
N


Pantoea brenneri
1411
EU216735
clade_92
N
N


Pantoea citrea
1412
EF688008
clade_92
N
N


Pantoea conspicua
1413
EU216737
clade_92
N
N


Pantoea septica
1414
EU216734
clade_92
N
N


Pasteurella dagmatis
1434
ACZR01000003
clade_92
N
N


Pasteurella multocida
1435
NC_002663
clade_92
N
N


Plesiomonas shigelloides
1469
X60418
clade_92
N
N


Raoultella ornithinolytica
1617
AB364958
clade_92
N
N


Raoultella planticola
1618
AF129443
clade_92
N
N


Raoultella terrigena
1619
NR_037085
clade_92
N
N


Salmonella bongori
1683
NR_041699
clade_92
N
Category-B


Salmonella enterica
1672
NC_011149
clade_92
N
Category-B


Salmonella enterica
1673
NC_011205
clade_92
N
Category-B


Salmonella enterica
1674
DQ344532
clade_92
N
Category-B


Salmonella enterica
1675
ABEH02000004
clade_92
N
Category-B


Salmonella enterica
1676
ABAK02000001
clade_92
N
Category-B


Salmonella enterica
1677
NC_011080
clade_92
N
Category-B


Salmonella enterica
1678
EU118094
clade_92
N
Category-B


Salmonella enterica
1679
NC_011094
clade_92
N
Category-B


Salmonella enterica
1680
AE014613
clade_92
N
Category-B


Salmonella enterica
1682
ABFH02000001
clade_92
N
Category-B


Salmonella enterica
1684
ABEM01000001
clade_92
N
Category-B


Salmonella enterica
1685
ABAM02000001
clade_92
N
Category-B


Salmonella typhimurium
1681
DQ344533
clade_92
N
Category-B


Salmonella typhimurium
1686
AF170176
clade_92
N
Category-B


Serratia fonticola
1718
NR_025339
clade_92
N
N


Serratia liquefaciens
1719
NR_042062
clade_92
N
N


Serratia marcescens
1720
GU826157
clade_92
N
N


Serratia odorifera
1721
ADBY01000001
clade_92
N
N


Serratia proteamaculans
1722
AAUN01000015
clade_92
N
N


Shigella boydii
1724
AAKA01000007
clade_92
N
Category-B


Shigella dysenteriae
1725
NC_007606
clade_92
N
Category-B


Shigella flexneri
1726
AE005674
clade_92
N
Category-B


Shigella sonnei
1727
NC_007384
clade_92
N
Category-B


Tatumella ptyseos
1916
NR_025342
clade_92
N
N


Trabulsiella guamensis
1925
AY373830
clade_92
N
N


Yersinia aldovae
2019
AJ871363
clade_92
N
OP


Yersinia aleksiciae
2020
AJ627597
clade_92
N
OP


Yersinia bercovieri
2021
AF366377
clade_92
N
OP


Yersinia enterocolitica
2022
FR729477
clade_92
N
Category-B


Yersinia frederiksenii
2023
AF366379
clade_92
N
OP


Yersinia intermedia
2024
AF366380
clade_92
N
OP


Yersinia kristensenii
2025
ACCA01000078
clade_92
N
OP


Yersinia mollaretii
2026
NR_027546
clade_92
N
OP


Yersinia pestis
2027
AE013632
clade_92
N
Category-A


Yersinia pseudotuberculosis
2028
NC_009708
clade_92
N
OP


Yersinia rohdei
2029
ACCD01000071
clade_92
N
OP


Yokenella regensburgei
2030
AB273739
clade_92
N
N


Conchiformibius kuhniae
669
NR_041821
clade_94
N
N


Morococcus cerebrosus
1267
JN175352
clade_94
N
N


Neisseria bacilliformis
1328
AFAY01000058
clade_94
N
N


Neisseria cinerea
1329
ACDY01000037
clade_94
N
N


Neisseria flavescens
1331
ACQV01000025
clade_94
N
N


Neisseria gonorrhoeae
1333
CP002440
clade_94
N
OP


Neisseria lactamica
1334
ACEQ01000095
clade_94
N
N


Neisseria macacae
1335
AFQE01000146
clade_94
N
N


Neisseria meningitidis
1336
NC_003112
clade_94
N
OP


Neisseria mucosa
1337
ACDX01000110
clade_94
N
N


Neisseria pharyngis
1338
AJ239281
clade_94
N
N


Neisseria polysaccharea
1339
ADBE01000137
clade_94
N
N


Neisseria sicca
1340
ACKO02000016
clade_94
N
N


Neisseria sp. KEM232
1341
GQ203291
clade_94
N
N


Neisseria sp. oral clone AP132
1344
AY005027
clade_94
N
N


Neisseria sp. oral strain B33KA
1346
AY005028
clade_94
N
N


Neisseria sp. oral taxon 014
1347
ADEA01000039
clade_94
N
N


Neisseria sp. TM10_1
1343
DQ279352
clade_94
N
N


Neisseria subflava
1348
ACEO01000067
clade_94
N
N


Okadaella gastrococcus
1365
HQ699465
clade_98
N
N


Streptococcus agalactiae
1785
AAJO01000130
clade_98
N
N


Streptococcus alactolyticus
1786
NR_041781
clade_98
N
N


Streptococcus australis
1788
AEQR01000024
clade_98
N
N


Streptococcus bovis
1789
AEEL01000030
clade_98
N
N


Streptococcus canis
1790
AJ413203
clade_98
N
N


Streptococcus constellatus
1791
AY277942
clade_98
N
N


Streptococcus cristatus
1792
AEVC01000028
clade_98
N
N


Streptococcus dysgalactiae
1794
AP010935
clade_98
N
N


Streptococcus equi
1795
CP001129
clade_98
N
N


Streptococcus equinus
1796
AEVB01000043
clade_98
N
N


Streptococcus gallolyticus
1797
FR824043
clade_98
N
N


Streptococcus genomosp. C1
1798
AY278629
clade_98
N
N


Streptococcus genomosp. C2
1799
AY278630
clade_98
N
N


Streptococcus genomosp. C3
1800
AY278631
clade_98
N
N


Streptococcus genomosp. C4
1801
AY278632
clade_98
N
N


Streptococcus genomosp. C5
1802
AY278633
clade_98
N
N


Streptococcus genomosp. C6
1803
AY278634
clade_98
N
N


Streptococcus genomosp. C7
1804
AY278635
clade_98
N
N


Streptococcus genomosp. C8
1805
AY278609
clade_98
N
N


Streptococcus gordonii
1806
NC_009785
clade_98
N
N


Streptococcus infantarius
1807
ABJK02000017
clade_98
N
N


Streptococcus infantis
1808
AFNN01000024
clade_98
N
N


Streptococcus intermedius
1809
NR_028736
clade_98
N
N


Streptococcus lutetiensis
1810
NR_037096
clade_98
N
N


Streptococcus massiliensis
1811
AY769997
clade_98
N
N


Streptococcus mitis
1813
AM157420
clade_98
N
N


Streptococcus oligofermentans
1815
AY099095
clade_98
N
N


Streptococcus oralis
1816
ADMV01000001
clade_98
N
N


Streptococcus parasanguinis
1817
AEKM01000012
clade_98
N
N


Streptococcus pasteurianus
1818
AP012054
clade_98
N
N


Streptococcus peroris
1819
AEVF01000016
clade_98
N
N


Streptococcus pneumoniae
1820
AE008537
clade_98
N
N


Streptococcus porcinus
1821
EF121439
clade_98
N
N


Streptococcus pseudopneumoniae
1822
FJ827123
clade_98
N
N


Streptococcus pseudoporcinus
1823
AENS01000003
clade_98
N
N


Streptococcus pyogenes
1824
AE006496
clade_98
N
OP


Streptococcus ratti
1825
X58304
clade_98
N
N


Streptococcus sanguinis
1827
NR_074974
clade_98
N
N


Streptococcus sinensis
1828
AF432857
clade_98
N
N


Streptococcus sp. 2_1_36FAA
1831
ACOI01000028
clade_98
N
N


Streptococcus sp. 2285_97
1830
AJ131965
clade_98
N
N


Streptococcus sp. ACS2
1834
HQ616360
clade_98
N
N


Streptococcus sp. AS20
1835
HQ616366
clade_98
N
N


Streptococcus sp. BS35a
1836
HQ616369
clade_98
N
N


Streptococcus sp. C150
1837
ACRI01000045
clade_98
N
N


Streptococcus sp. CM6
1838
HQ616372
clade_98
N
N


Streptococcus sp. ICM10
1840
HQ616389
clade_98
N
N


Streptococcus sp. ICM12
1841
HQ616390
clade_98
N
N


Streptococcus sp. ICM2
1842
HQ616386
clade_98
N
N


Streptococcus sp. ICM4
1844
HQ616387
clade_98
N
N


Streptococcus sp. ICM45
1843
HQ616394
clade_98
N
N


Streptococcus sp. M143
1845
ACRK01000025
clade_98
N
N


Streptococcus sp. M334
1846
ACRL01000052
clade_98
N
N


Streptococcus sp. oral clone ASB02
1849
AY923121
clade_98
N
N


Streptococcus sp. oral clone ASCA03
1850
DQ272504
clade_98
N
N


Streptococcus sp. oral clone ASCA04
1851
AY923116
clade_98
N
N


Streptococcus sp. oral clone ASCA09
1852
AY923119
clade_98
N
N


Streptococcus sp. oral clone ASCB04
1853
AY923123
clade_98
N
N


Streptococcus sp. oral clone ASCB06
1854
AY923124
clade_98
N
N


Streptococcus sp. oral clone ASCC04
1855
AY923127
clade_98
N
N


Streptococcus sp. oral clone ASCC05
1856
AY923128
clade_98
N
N


Streptococcus sp. oral clone ASCC12
1857
DQ272507
clade_98
N
N


Streptococcus sp. oral clone ASCD01
1858
AY923129
clade_98
N
N


Streptococcus sp. oral clone ASCD09
1859
AY923130
clade_98
N
N


Streptococcus sp. oral clone ASCD10
1860
DQ272509
clade_98
N
N


Streptococcus sp. oral clone ASCE03
1861
AY923134
clade_98
N
N


Streptococcus sp. oral clone ASCE04
1862
AY953253
clade_98
N
N


Streptococcus sp. oral clone ASCE05
1863
DQ272510
clade_98
N
N


Streptococcus sp. oral clone ASCE06
1864
AY923135
clade_98
N
N


Streptococcus sp. oral clone ASCE09
1865
AY923136
clade_98
N
N


Streptococcus sp. oral clone ASCE10
1866
AY923137
clade_98
N
N


Streptococcus sp. oral clone ASCE12
1867
AY923138
clade_98
N
N


Streptococcus sp. oral clone ASCF05
1868
AY923140
clade_98
N
N


Streptococcus sp. oral clone ASCF07
1869
AY953255
clade_98
N
N


Streptococcus sp. oral clone ASCF09
1870
AY923142
clade_98
N
N


Streptococcus sp. oral clone ASCG04
1871
AY923145
clade_98
N
N


Streptococcus sp. oral clone BW009
1872
AY005042
clade_98
N
N


Streptococcus sp. oral clone CH016
1873
AY005044
clade_98
N
N


Streptococcus sp. oral clone GK051
1874
AY349413
clade_98
N
N


Streptococcus sp. oral clone GM006
1875
AY349414
clade_98
N
N


Streptococcus sp. oral clone P2PA_41 P2
1876
AY207051
clade_98
N
N


Streptococcus sp. oral clone P4PA_30 P4
1877
AY207064
clade_98
N
N


Streptococcus sp. oral taxon 071
1878
AEEP01000019
clade_98
N
N


Streptococcus sp. oral taxon G59
1879
GU432132
clade_98
N
N


Streptococcus sp. oral taxon G62
1880
GU432146
clade_98
N
N


Streptococcus sp. oral taxon G63
1881
GU432150
clade_98
N
N


Streptococcus suis
1882
FM252032
clade_98
N
N


Streptococcus thermophilus
1883
CP000419
clade_98
N
N


Streptococcus salivarius
1826
AGBV01000001
clade_98
N
N


Streptococcus uberis
1884
HQ391900
clade_98
N
N


Streptococcus urinalis
1885
DQ303194
clade_98
N
N


Streptococcus vestibularis
1886
AEKO01000008
clade_98
N
N


Streptococcus viridans
1887
AF076036
clade_98
N
N


Synergistetes bacterium oral clone 03 5 D05
1908
GU227192
clade_98
N
N









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.







TABLE X4





Spore-forming Bacterial Species

















Alkaliphilus metalliredigens



Ammonifex degensii



Anaerofustis stercorihominis



Anaerostipes caccae



Anaerotruncus colihominis



Bacillus amyloliquefaciens



Bacillus anthracis



Bacillus cellulosilyticus



Bacillus cereus



Bacillus clausii



Bacillus coagulans



Bacillus cytotoxicus



Bacillus halodurans



Bacillus licheniformis



Bacillus pumilus



Bacillus subtilis



Bacillus thuringiensis



Bacillus weihenstephanensis



Blautia hansenii



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 hathewayi



Clostridium hylemonae



Clostridium kluyveri



Clostridium leptum



Clostridium methylpentosum



Clostridium 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 obeum



Ruminococcus torques



Subdoligranulum variabile



Symbiobacterium thermophilum



Thermoanaerobacter italicus



Thermoanaerobacter tengcongensis



Thermoanaerobacterium thermosaccharolyticum



Thermosinus carboxydivorans

















TABLE 2







Species isolated from ethanol treated spore preparation


preparation before (left) and after (right) CsCI gradient step











ethanol treated,



ethanol treated spore
gradient purified


Isolates
preparation
spore preparation












Bacillus coagulans
7
2


Blautia luti
1
1


Blautia sp
14
13


Blautia wexlerae
3
1


Ruminococcus obeum
4
2


Clostridiales sp
1
2


Clostridium aerotolerans
1
2


Clostridium disporicum
0
1


Clostridium sp
1
1


Clostridium symbiosum
0
1


Dorea longicatena
8
6


Eubacterium cellulosolvens
1
0


Eubacterium ventriosum
2
2


Gemmiger formicilis
0
1


Robinsoniella peoriensis
0
1


Roseburia hominis
3
6


Roseburia intestinalis
9
7


Ruminococcus sp
5
2


Syntrophococcus sucromutans
1
1


Turicibacter sanguinis
3
4


Clostridiales sp
7
9


Clostridium bartlettii
8
11


Clostridium irregulare
0
1


Clostridium sordellii
4
6


Lachnospiraceae sp
1
0
















TABLE 3







Mortality and weight change in mice challenged with C.


difficile with or without ethanol treated, spore product treatment.











% weight change


Test article
mortality (n = 10)
on Day 3





vehicle (negative control)
20%
−10.5% 


Donor feces (positive
0
−0.1% 


control)


EtOH-treated feces 1x
0
2.3%


EtOH-treated feces 0.1x
0
2.4%


EtOH-treated feces 0.01x
0
 −3%


heat-treated feces
0
0.1%
















TABLE 4







16S rDNA identified spore forming species from picked


colony plates.









Treatment
Species
No. isolates





70deg 1 h
Clostridium_celatum
4


70deg 1 h
Clostridium_clostridioform
1


70deg 1 h
Clostridium_hylemonae
1


70deg 1 h
Clostridium_paraputrificum
3


70deg 1 h
Clostridium_sp_D5
1


70deg 1 h
Clostridium_symbiosum
1


80deg 1 h
Clostridium_bartlettii
6


80deg 1 h
Clostridium_butyricum
1


80deg 1 h
Clostridium_paraputrificum
5


80deg 1 h
Coprobacillus_sp_D7
1


80deg 1 h
Eubacterium_sp_WAL_14571
1


80deg 1 h
Ruminococcus_bromii
1


90deg 1 h
Clostridium_butyricum
1


90deg 10 min
Ruminococcus_bromii
1


90deg 10 min
Anaerotruncus_colihominis
2


90deg 10 min
Clostridium_bartlettii
1


100deg 10 min
Ruminococcus_bromii
1
















TABLE 5







Spore-forming species identified in ethanol treated or


heat treated samples and not identified in untreated samples













isolated



isolated

from



from
isolated from
heat-


Species
untreated
EtOH-treated
treated





Acetivibrio ethanolgignens

X



Anaerofustis stercorihominis

X


Bacillus anthracis

X


Bacillus horti

X


Bacillus licheniformis


X


Bacillus nealsonii

X


Bacillus pumilus


X


Bacillus sp. BT1B_CT2

X


Bacillus thuringiensis

X


Bacteroides galacturonicus

X


(phylogenetically in Clostridiales)


Bacteroides pectinophilus

X


(phylogenetically in Clostridiales)


Blautia wexlerae

X
X


Brachyspira pilosicoli

X


Brevibacillus parabrevis


X


Clostridium aldenense

X


Clostridium beijerinckii

X


Clostridium carnis

X


Clostridium celatum

X


Clostridium favososporum

X


Clostridium hylemonae


X


Clostridium irregulare

X


Clostridium methylpentosum


X


Clostridium sp. D5

X
X


Clostridium sp. L2-50

X


Clostridium sp. MT4 E

X


Clostridium sp. NML 04A032

X


Clostridium sp. SS2/1

X


Clostridium sp. YIT 12069


X


Clostridium stercorarium

X


Clostridium xylanolyticum

X


Coprococcus sp. ART55/1

X


Deferribacteres sp. oral clone JV006

X


Desulfitobacterium frappieri

X


Eubacterium callanderi

X


Eubacterium siraeum

X


Exiguobacterium acetylicum

X


Gemmiger formicilis

X


Lachnospira multipara

X


Lachnospira pectinoschiza

X


Roseburia faecalis

X


Ruminococcus albus

X
















TABLE 6





Donor A, 45 species in 374 EtOH-resistant colonies


sequenced


OTU

















Anaerostipes_sp_3_2_56FAA



Bacillus_anthracis



Bacillus_cereus



Bacillus_thuringiensis



Blautia_producta



Blautia_sp_M25



Clostridiales_sp_SSC_2



Clostridium_aldenense



Clostridium_bartlettii



Clostridium_bolteae



Clostridium_celatum



Clostridium_disporicum



Clostridium_ghonii



Clostridium_hathewayi



Clostridium_lactatifermentans



Clostridium_mayombei



Clostridium_orbiscindens



Clostridium_paraputrificum



Clostridium_perfringens



Clostridium_sordellii



Clostridium_stercorarium



Clostridium_straminisolvens



Clostridium_tertium



Coprobacillus_sp_D7



Coprococcus_catus



Deferribacteres_sp_oral_clone_JV006



Dorea_formicigenerans



Eubacterium_rectale



Eubacterium_siraeum



Eubacterium_sp_WAL_14571



Eubacterium_ventriosum



Flexistipes_sinusarabici



Fulvimonas_sp_NML_060897



Lachnospiraceae_bacterium_2_1_58FAA



Lachnospiraceae_bacterium_3_1_57FAA



Lachnospiraceae_bacterium_A4



Lachnospiraceae_bacterium_oral_taxon_F15



Moorella_thermoacetica



Roseburia_faecalis



Roseburia_hominis



Ruminococcus_albus



Ruminococcus_bromii



Ruminococcus_gnavus



Ruminococcus_sp_5_1_39BFAA



Ruminococcus_torques

















TABLE 7





Donor B, 26 species in 195 EtOH-resistant colonies sequenced


OTU

















Bacillus_horti



Blautia_wexlerae



Chlamydiales_bacterium_NS11



Clostridiales_sp_SSC_2



Clostridium_bartlettii



Clostridium_celatum



Clostridium_disporicum



Clostridium_ghonii



Clostridium_oroticum



Clostridium_paraputrificum



Clostridium_perfringens



Clostridium_sordellii



Clostridium_sp_L2_50



Clostridium_sp_MT4_E



Clostridium_straminisolvens



Coprococcus_sp_ART55_1



Eubacterium_callanderi



Eubacterium_rectale



Eubacterium_ruminantium



Gemmiger_formicilis



Lachnospira_pectinoschiza



Ruminococcus_albus



Ruminococcus_gnavus



Ruminococcus_obeum



Ruminococcus_sp_5_1_39BFAA



Ruminococcus_sp_K_1

















TABLE 8





Donor C, 39 species in 416 EtOH-resistant colonies sequenced


OTU

















Bacteroides_galacturonicus



Bacteroides_pectinophilus



Blautia_producta



Blautia_sp_M25



Blautia_wexlerae



Clostridiales_sp_SS3_4



Clostridiales_sp_SSC_2



Clostridium_bartlettii



Clostridium_citroniae



Clostridium_disporicum



Clostridium_indolis



Clostridium_orbiscindens



Clostridium_paraputrificum



Clostridium_sordellii



Clostridium_sp_NML_04A032



Clostridium_sp_SS2_1



Clostridium_straminisolvens



Clostridium_viride



Clostridium_xylanolyticum



Coprobacillus_sp_D7



Dorea_longicatena



Eubacterium_rectale



Eubacterium_ventriosum



Hydrogenoanaerobacterium_saccharovorans



Lachnospira_multipara



Lachnospira_pectinoschiza



Lachnospiraceae_bacterium_A4



Oscillibacter_sp_G2



Pseudoflavonifractor_capillosus



Roseburia_hominis



Roseburia_intestinalis



Ruminococcus_albus



Ruminococcus_lactaris



Ruminococcus_obeum



Ruminococcus_sp_5_1_39BFAA



Ruminococcus_sp_K_1



Ruminococcus_torques



Syntrophococcus_sucromutans

















TABLE 9





Donor D, 12 species in 118 EtOH-resistant colonies sequenced


OTU

















Blautia_luti



Blautia_wexlerae



Brachyspira_pilosicoli



Clostridium_paraputrificum



Collinsella_aerofaciens



Coprobacillus_sp_D7



Desulfitobacterium_frappieri



Eubacterium_rectale



Moorella_thermoacetica



Ruminococcus_gnavus



Ruminococcus_obeum



Ruminococcus_sp_K_1

















TABLE 10





Donor E, 11 species in 118 EtOH-resistant colonies sequenced


OTU

















Blautia_luti



Blautia_wexlerae



Brachyspira_pilosicoli



Clostridium_paraputrificum



Coprobacillus_sp_D7



Desulfitobacterium_frappieri



Eubacterium_rectale



Moorella_thermoacetica



Ruminococcus_gnavus



Ruminococcus_obeum



Ruminococcus_sp_K_1

















TABLE 11





Donor F, 54 OTUs in 768 EtOH-resistant colonies sequenced


OTU

















Anaerofustis_stercorihominis



Anaerostipes_sp_3_2_56FAA



Bacillus_nealsonii



Bacillus_sp_BT1B_CT2



Blautia_producta



Butyrivibrio_crossotus



Clostridiales_bacterium_SY8519



Clostridiales_sp_1_7_47



Clostridium_aldenense



Clostridium_bartlettii



Clostridium_bolteae



Clostridium_butyricum



Clostridium_citroniae



Clostridium_clostridioforme



Clostridium_disporicum



Clostridium_favososporum



Clostridium_glycolicum



Clostridium_hathewayi



Clostridium_indolis



Clostridium_leptum



Clostridium_mayombei



Clostridium_nexile



Clostridium_orbiscindens



Clostridium_sordellii



Clostridium_sp_7_2_43FAA



Clostridium_sp_D5



Clostridium_sp_M62_1



Clostridium_sp_NML_04A032



Clostridium_spiroforme



Clostridium_symbiosum



Clostridium_tertium



Coprobacillus_sp_29_1



Coprobacillus_sp_D7



Eubacterium_contortum



Eubacterium_desmolans



Eubacterium_ramulus



Exiguobacterium_acetylicum



Faecalibacterium_prausnitzii



Lachnospiraceae_bacterium_2_1_58FAA



Lachnospiraceae_bacterium_3_1_57FAA



Lachnospiraceae_bacterium_5_1_57FAA



Lachnospiraceae_bacterium_6_1_63FAA



Lachnospiraceae_bacterium_oral_taxon_F15



Marvinbryantia_formatexigens



Mycoplasma_amphoriforme



Oscillibacter_sp_G2



Pseudoflavonifractor_capillosus



Ruminococcus_gnavus



Ruminococcus_hansenii



Ruminococcus_obeum



Ruminococcus_sp_5_1_39BFAA



Ruminococcus_sp_ID8



Turicibacter_sanguinis

















TABLE 12







Organisms grown from ethanol treated spore population


on various media (See Example 5 for full media names and references).











total number
unique
% unique


Media
reads
OTUs
OTUs













M2GSC
93
33
0.35


M-BHI
66
26
0.39


Sweet B
74
23
0.31


GAM fructose
44
18
0.41


M2 mannitol
39
17
0.44


M2 soluble starch
62
16
0.26


M2 lactate
43
14
0.33


GAM FOS/Inulin
52
14
0.27


EYA
29
13
0.45


Mucin
19
12
0.63


M2 lactose
32
12
0.38


BHIS az1/ge2
35
12
0.34


BHIS CInM az1/ge2
24
11
0.46


GAM mannitol
41
11
0.27


BBA
29
10
0.34


Sulfite-polymyxin milk
48
9
0.19


Noack-Blaut Eubacterium agar
12
4
0.33



742 total analyzed
















TABLE 13







Species identified as germinable and sporulatable by colony picking















GAM +

Sweet B +
Sweet



OTU
BBA
FOS/inulin
M2GSC
FOS/Inulin
GAM
Total
















Blautia producta
1




1


Clostridium bartlettii
4

1


5


Clostridium bolteae
2


5
1
8


Clostridium botulinum



5

5


Clostridium butyricum
37
43
8
1
33
122


Clostridium celatum
4



1
5


Clostridium clostridioforme
1



1
2


Clostridium disporicum
26
26
22
33
50
157


Clostridium glycolicum
4
9
14


27


Clostridium mayombei
2
2



4


Clostridium paraputrificum
8
8
33
16
6
71


Clostridium sordellii


14


14


Clostridium sp. 7_2_43FAA

1



1


Clostridium symbiosum
3




3


Clostridium tertium

1

1

2


(blank)

2

31

33


Totals
92
92
92
92
92
460
















TABLE 15







Results of the prophylaxis mouse model and dosing


information for the germinable, and sporulatable fractions.













#
Average
Average




Deaths
Weight on Day
Clinical




by
3 Relative to
Score on


Test Article
Dose
Day 6
Day −1
Day 3














Vehicle
NA
10
0.72
NA


Naive
NA
0
1.03
0


Donor B fecal
0.2 mL of 10%
1
0.91
0.11


suspension
suspension


Donor A Spore
8.99 * 10{circumflex over ( )}7 Spore
0
1.02
0


Prep
Equivalents/dose


germinable


Donor A Spore
7.46 * 10{circumflex over ( )}7 Spore
0
0.99
0


Prep
Equivalents/dose


Sporulatable









Clinical score is based on a combined phenotypic assessment of the mouse's health on a scale of 0-4 in several areas including appearance (0-2 pts based on normal, hunched, piloerection, or lethargic), and clinical signs (0-2 points based on normal, wet tail, cold-to-the-touch, or isolation from other animals).







TABLE 16







Bacterial OTUs associated with engraftment and


ecological augmentation and establishment of a more diverse


microbial ecology in patients treated with an ethanol treated spore preparation.












Spore
Dominant OTU



Phylogenetic
Forming
in Augmented


OTU
Clade
OTU
Ecology





Bacteroides sp. 2_1_22
clade38
N
Y


Streptococcus anginosus
clade60
N


Prevotella intermedia
clade81
N


Prevotella nigrescens
clade81
N


Oribacterium sp. ACB7
clade90
N


Prevotella salivae
clade104
N


Bacteroides intestinalis
clade171
N
Y


Bifidobacterium dentium
clade172
N


Alcaligenes faecalis
clade183
N


Rothia dentocariosa
clade194
N


Peptoniphilus lacrimalis
clade291
N


Anaerococcus sp. gpac155
clade294
N


Sutterella stercoricanis
clade302
N
Y


Bacteroides sp. 3_1_19
clade335
N
Y


Parabacteroides goldsteinii
clade335
N


Bacteroides dorei
clade378
N
Y


Bacteroides massiliensis
clade378
N


Lactobacillus iners
clade398
N


Granulicatella adiacens
clade460
N


Eggerthella sp. 1_3_56FAA
clade477
N


Gordonibacter pamelaeae
clade477
N


Finegoldia magna
clade509
N


Actinomyces nasicola
clade523
N


Streptobacillus moniliformis
clade532
N


Oscillospira guilliermondii
clade540
N


Orientia tsutsugamushi
clade541
N


Christensenella minuta
clade558
N


Clostridium oroticum
clade96
Y


Clostridium sp. D5
clade96
Y


Clostridium glycyrrhizinilyticum
clade147
Y


Coprococcus comes
clade147
Y


Ruminococcus lactaris
clade147
Y


Ruminococcus torques
clade147
Y
Y


Clostridiales sp. SS3/4
clade246
Y


Clostridium hylemonae
clade260
Y


Clostridium aerotolerans
clade269
Y


Clostridium asparagiforme
clade300
Y
Y


Clostridium sp. M62/1
clade300
Y


Clostridium symbiosum
clade300
Y


Lachnospiraceae genomo sp. C1
clade300
Y


Blautia sp. M25
clade304
Y
Y


Blautia stercoris
clade304
Y


Ruminococcus hansenii
clade304
Y


Ruminococcus obeum
clade304
Y


Ruminococcus sp. 5_1_39BFAA
clade304
Y


Bryantella formatexigens
clade309
Y


Eubacterium cellulosolvens
clade309
Y


Clostridium sp. HGF2
clade351
Y


Clostridium bartlettii
clade354
Y


Clostridium bifermentans
clade354
Y


Clostridium glycolicum
clade354
Y


Eubacterium tenue
clade354
Y


Dorea formicigenerans
clade360
Y


Dorea longicatena
clade360
Y


Lachnospiraceae bacterium 2_1_46FAA
clade360
Y


Lachnospiraceae bacterium 9_1_43BFAA
clade360
Y
Y


Ruminococcus gnavus
clade360
Y


Clostridium hathewayi
clade362
Y


Blautia hydrogenotrophica
clade368
Y


Clostridiaceae bacterium END-2
clade368
Y


Roseburia faecis
clade369
Y


Roseburia hominis
clade370
Y


Roseburia intestinalis
clade370
Y


Eubacterium sp. WAL 14571
clade384
Y


Erysipelotrichaceae bacterium 5_2_54FAA
clade385
Y


Eubacterium biforme
clade385
Y


Eubacterium dolichum
clade385
Y


Coprococcus catus
clade393
Y


Acetivibrio ethanolgignens
clade396
Y


Anaerosporobacter mobilis
clade396
Y


Bacteroides pectinophilus
clade396
Y


Eubacterium hallii
clade396
Y


Eubacterium xylanophilum
clade396
Y


Anaerostipes caccae
clade408
Y


Clostridiales bacterium 1_7_47FAA
clade408
Y


Clostridium aldenense
clade408
Y


Clostridium citroniae
clade408
Y


Eubacterium hadrum
clade408
Y
Y


Acetanaerobacterium elongatum
clade439
Y


Faecalibacterium prausnitzii
clade478
Y


Gemmiger formicilis
clade478
Y
Y


Eubacterium ramulus
clade482
Y


Lachnospiraceae bacterium 3_1_57FAA_CT1
clade483
Y


Lachnospiraceae bacterium A4
clade483
Y
Y


Lachnospiraceae bacterium DJF VP30
clade483
Y


Holdemania filiformis
clade485
Y


Clostridium orbiscindens
clade494
Y


Pseudoflavonifractor capillosus
clade494
Y


Ruminococcaceae bacterium D16
clade494
Y


Acetivibrio cellulolyticus
clade495
Y


Eubacterium limosum
clade512
Y


Anaerotruncus colihominis
clade516
Y


Clostridium methylpentosum
clade516
Y


Clostridium sp. YIT 12070
clade516
Y


Hydrogenoanaerobacterium saccharovorans
clade516
Y


Eubacterium ventriosum
clade519
Y


Eubacterium eligens
clade522
Y


Lachnospira pectinoschiza
clade522
Y


Lactobacillus rogosae
clade522
Y
Y


Clostridium leptum
clade537
Y


Eubacterium coprostanoligenes
clade537
Y


Ruminococcus bromii
clade537
Y


Clostridium viride
clade540
Y


Butyrivibrio crossotus
clade543
Y


Coprococcus eutactus
clade543
Y


Eubacterium ruminantium
clade543
Y


Eubacterium rectale
clade568
Y
Y


Roseburia inulinivorans
clade568
Y


Butyricicoccus pullicaecorum
clade572
Y


Eubacterium desmolans
clade572
Y


Papillibacter cinnamivorans
clade572
Y


Sporobacter termitidis
clade572
Y


Clostridium lactatifermentans
clade576
Y
















TABLE 18







Reduction in the opportunistic pathogen or pathobiont


load by ethanol treated spores.












Pretreatment
Day 5
Day 14
Day 25















Klebsiella (% of total reads)
20.27%
1.32%
7.62%
0.00%


Fusobacterium (% total
19.14%
3.01%
0.01%
0.00%


of reads)
















TABLE 19







Changes in Enterobacteria as a function of treatment


measured on Simmons Citrate Agar












Pretreatment titer



Patient
Organism
(cfu/g)
Day 25 titer (cfu/g)





1
Klebsiella pneumoniae
9 × 106
1 × 103


1
Klebsiella sp. Co9935
4 × 106
1 × 103


1
Escherichia coli
7 × 106
1 × 106


2
Klebsiella sp. Co9935
4 × 106
1 × 103


4
Klebsiella pneumoniae
3 × 108
<1 × 104 


4
Klebsiella sp. Co9935
6 × 107
<1 × 104 


5
Klebsiella pneumoniae
1 × 106
<1 × 104 
















TABLE 20







Augmentation of Bacteroides as a function of bacterial


composition treatment of Patient 1












Pretreatment titer



Media
Bacteroides species
(cfu/g)
Day 25 titer (cfu/g)





BBE
B. fragilis group
<2 × 104
3 × 108 


PFA
All Bacteroides
<2 × 107
2 × 1010
















TABLE 21







Bacteroides spp. post-treatment with the ethanol treated spore


preparation based full-length 16S rDNA sequences of isolated strains








Species
% of total Bacteroides cfu (1.58E10 cfu/g)





Bacteroides sp. 4_1_36
  63%


Bacteroides cellulosilyticus
  14%


Bacteroides sp. 1_1_30
  14%


Bacteroides uniformis
 4.8%


Bacteroides ovatus
 1.7%


Bacteroides dorei
0.91%


Bacteroides xylanisolvens
0.83%


Bacteroides sp. 3_1_19
0.23%
















TABLE 22







Titers (in cfu/g) of imipenem-resistant M. morganii, P. rettgeri


and P. pennerii from Patients B, D & E












Patient
Organism
Pretreatment titer
Day 28 titer*







Patient 2
M. morganii
1 × 104
 6 × 102



Patient 2
P. rettgeri
9 × 103
<5 × 101



Patient 4
M. morganii
2 × 104
<5 × 101



Patient 4
P. pennerii
2 × 104
<5 × 101



Patient 5
M. morganii
5 × 103
<5 × 101







*Limit of detection based on plating 200 uL of 10% wt/vol suspension is 5 × 101













TABLE YYY





Species identified as germinable by 16S colony pick approach

















Clostridium_paraputrificum



Clostridium_disporicum



Clostridium_glycolicum



Clostridium_bartlettii



Clostridium_butyricum



Ruminococcus_bromii



Lachnospiraceae_bacterium_2_1_58FAA



Eubacterium_hadrum



Turicibacter_sanguinis



Lachnospiraceae_bacterium_oral_taxon_F15



Clostridium_perfringens



Clostridium_bifermentans



Roseburia_sp_11SE37



Clostridium_quinii



Ruminococcus_lactaris



Clostridium_botulinum



Clostridium_tyrobutyricum



Blautia_hansenii



Clostridium_kluyveri



Clostridium_sp_JC122



Clostridium_hylemonae



Clostridium_celatum



Clostridium_straminisolvens



Clostridium_orbiscindens



Roseburia_cecicola



Eubacterium_tenue



Clostridium_sp_7_2_43FAA



Lachnospiraceae_bacterium_4_1_37FAA



Eubacterium_rectale



Clostridium_viride



Ruminococcus_sp_K_1



Clostridium_symbiosum



Ruminococcus_torques



Clostridium_algidicarnis

















TABLE ZZZ





Species identified as sporulatable by 16S NGS approach

















Clostridium_paraputrificum



Clostridium_bartlettii



Lachnospiraceae_bacterium_2_1_58FAA



Clostridium_disporicum



Ruminococcus_bromii



Eubacterium_hadrum



Clostridium_butyricum



Roseburia_sp_11SE37



Clostridium_perfringens



Clostridium_glycolicum



Clostridium_hylemonae



Clostridium_orbiscindens



Ruminococcus_lactaris



Clostridium_symbiosum



Lachnospiraceae_bacterium_oral_taxon_F15



Blautia_hansenii



Turicibacter_sanguinis



Clostridium_straminisolvens



Clostridium_botulinum



Lachnospiraceae_bacterium_4_1_37FAA



Roseburia_cecicola



Ruminococcus_sp_K_1



Clostridium_bifermentans



Eubacterium_rectale



Clostridium_quinii



Clostridium_viride



Clostridium_kluyveri



Clostridium_tyrobutyricum



Oscillibacter_sp_G2



Clostridium_sp_JC122



Lachnospiraceae_bacterium_3_1_57FAA



Clostridium_aldenense



Ruminococcus_torques



Clostridium_sp_7_2_43FAA



Clostridium_celatum



Eubacterium_sp_WAL_14571



Eubacterium_tenue



Lachnospiraceae_bacterium_5_1_57FAA



Clostridium_clostridioforme



Clostridium_sp_YIT_12070



Blautia_sp_M25



Anaerostipes_caccae



Roseburia_inulinivorans



Clostridium_sp_D5



Clostridium_asparagiforme



Coprobacillus_sp_D7



Clostridium_sp_HGF2



Clostridium_citroniae



Clostridium_difficile



Oscillibacter_valericigenes



Clostridium_algidicarnis

## Claims

1. A method of treating or reducing a severity of at least one symptom of a gastrointestinal disease associated with a dysbiosis, the method comprising
administering to a human subject a composition consisting essentially of a purified population of germinable bacterial spores in an amount effective to populate at least one region of a gastrointestinal tract in the subject and augment growth of at least one type of bacteria not detectably present in the composition or in the gastrointestinal tract prior to administration to the subject,
wherein the composition comprises at least 1×104 colony forming units of germinable bacterial spores per dose of the composition,
and the germinable bacterial spores comprise at least two different bacterial entities, each bacterial entity comprising a 16S rDNA at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO. 673, SEQ ID NO. 674, SEQ ID NO. 774, SEQ ID NO. 845, SEQ ID NO. 847, SEQ ID NO. 848, SEQ ID NO. 856, and SEQ ID NO. 1670,
thereby treating the gastrointestinal disease or reduce a severity of at least one symptom of the gastrointestinal disease in the subject.

2. The method of claim 1, wherein populating the gastrointestinal tract by the germinable bacterial spores is determined by 16S profiling, qPCR, or colony counting.

3. The method of claim 1, wherein the at least one region of the gastrointestinal tract is selected from the group consisting of the stomach, the small intestine, the large intestine, and the rectum.

4. The method of claim 1, wherein the at least one type of bacteria not detectably present in the composition or in the gastrointestinal tract of the subject prior to treatment is selected from the group consisting of Bacteroides sp. 2_1_22, Streptococcus anginosus, Prevotella intermedia, Prevotella nigrescens, Oribacterium sp. ACB7, Prevotella salivae, Bacteroides intestinalis, Bifidobacterium dentium, Alcaligenes faecalis, Rothia dentocariosa, Peptoniphilus lacrimalis, Anaerococcus sp. gpac155, Sutterella stercoricanis, Bacteroides sp. 3_1_19, Parabacteroides goldsteinii, Bacteroides dorei, Bacteroides massiliensis, Lactobacillus iners, Granulicatella adiacens, Eggerthella sp. 1_3_56FAA, Gordonibacter pamelaeae, Finegoldia magna, Actinomyces nasicola, Streptobacillus moniliformis, Oscillospira guilliermondii, Orientia tsutsugamushi, Christensenella minuta, Clostridium oroticum, Clostridium sp. D5,Clostridium glycyrrhizinilyticum, Coprococcus comes, Ruminococcus lactaris, Ruminococcus torques, Clostridiales sp. SS3/4, Clostridium hylemonae, Clostridium aerotolerans, Clostridium asparagiforme, Clostridium sp. M62/1, Clostridium symbiosum, Lachnospiraceae genomosp. Cl. Blautia sp. M25, Blautia stercoris, Ruminococcus hansenii, Ruminococcus obeum, Ruminococcus sp. 5_1_39BFAA, Bryantella formatexigens, Eubacterium cellulosolvens, Clostridium sp. HGF2, Clostridium bartlettii, Clostridium bifermentans, Clostridium glycolicum, Eubacterium tenue, Dorea formicigenerans, Dorea longicatena, Lachnospiraceae bacterium 2_1_46FAA, Lachnospiraceae bacterium 9_1_43BFAA, Ruminococcus gnavus, Clostridium hathewayi, Blautia hydrogenotrophica, Clostridiaceae bacterium END-2, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, Eubacterium sp. WAL 14571, Erysipelotrichaceae bacterium 5_2_54FAA, Eubacterium biforme, Eubacterium dolichum, Coprococcus catus, Acetivibrio ethanolgignens, Anaerosporobacter mobilis, Bacteroides pectinophilus, Eubacterium hallii, Eubacterium xylanophilum, Anaerostipes caccae, Clostridiales bacterium 1_7_47FAA, Clostridium aldenense, Clostridium citroniae, Eubacterium hadrum, Acetanaerobacterium elongatum, Faecalibacterium prausnitzii, Gemmiger formicilis, Eubacterium ramulus, Lachnospiraceae bacterium 3_1_57FAA CT1, Lachnospiraceae bacterium A4, Lachnospiraceae bacterium DJF VP30, Holdemania filiformis, Clostridium orbiscindens, Pseudoflavonifractor capillosus, Ruminococcaceae bacterium D16, Acetivibrio cellulolyticus, Eubacterium limosum, Anaerotruncus colihominis, Clostridium methylpentosum, Clostridium sp. YIT 12070, Hydrogenoanaerobacterium saccharovorans, Eubacterium ventriosum, Eubacterium eligens, Lachnospira pectinoschiza, Lactobacillus rogosae, Clostridium leptum, Eubacterium coprostanoligenes, Ruminococcus bromii, Clostridium viride, Butyrivibrio crossotus, Coprococcus eutactus, Eubacterium ruminantium, Eubacterium rectale, Roseburia inulinivorans, Butyricicoccus pullicaecorum, Eubacterium desmolans, Papillibacter cinnamivorans, Sporobacter termitidis, and Clostridium lactatifermentans.

5. The method of claim 1, wherein the dose is provided in a capsule.

6. The method of claim 5, wherein the capsule is a hypromellose capsule.

7. The method of claim 6, wherein the capsule is overencapsulated with a second capsule.

8. The method of claim 1, wherein the dose is provided in more than one capsule.

9. The method of claim 8, wherein the capsule is a hypromellose capsule.

10. The method of claim 9, wherein the capsule is overencapsulated with a second capsule.

11. The method of claim 1, each bacterial entity comprising a 16S rDNA selected from the group consisting of SEQ ID NO. 673, SEQ ID NO. 674, SEQ ID NO. 774, SEQ ID NO. 845, SEQ ID NO. 847, SEQ ID NO. 848, SEQ ID NO. 856, and SEQ ID NO. 1670.

12. The method of claim 1, each bacterial entity comprising a 16S rDNA at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 673, 674, 774, 848, and 1670.

13. The method of claim 1, wherein each bacterial entity comprises a 16S rDNA selected from the group consisting of SEQ ID NOs: 673, 674, 774, 848, and 1670.

14. The method of claim 1, wherein administration of the composition modulates the microbiota diversity present in the gastrointestinal tract of the subject.

15. The method of claim 1, wherein the gastrointestinal disease, disorder or condition is selected from the group consisting of Clostridium difficile-induced diarrhea, irritable bowel syndrome (IBS), infection or colonization with a pathogen or pathobiont including a drug resistant pathogen or pathobiont, colitis, a metabolic disorder, and Crohn's disease.

16. The method of claim 1, wherein the gastrointestinal disease, disorder or condition is Clostridium difficile-induced diarrhea.

17. The method of claim 1, wherein a pathogenic material in the purified population is depleted or inactive.

18. The method of claim 17, wherein the purified population is substantially depleted of a detectable level of a first pathogenic material.

19. The method of claim 18, wherein the detectable level is detected by qPCR.

20. The method of claim 1, wherein the purified population of germinable spores is derived from a fecal material after reduction of a residual habitat product of the fecal material.

21. The method of claim 20, wherein the residual habitat product is a virus, fungus, or mycoplasma.

22. The method of claim 20, wherein the fecal material is obtained from a validated mammalian donor subject not having a detectable level of a pathogen or a pathobiont prior to production of the fecal material.

23. The method of claim 22, wherein the detectable level is detected by qPCR.

24. The method of claim 1, wherein administering comprises oral administration.

25. The method of claim 1, wherein administering the composition results in i) a reduction or an elimination of at least one pathogen and/or pathobiont present in the gastrointestinal tract of the subject; ii) engraftment in the gastrointestinal tract of the subject of at least one type of spore-forming bacteria present in the composition; or iii) a combination of i) and ii).

26. The method of claim 1, wherein the human subject has received one or more doses of an antibiotic therapy.

27. The method of claim 1, wherein the composition is administered as a complement to an antibiotic therapy.

28. The method of claim 1, wherein
administration of the composition modulates the microbiota diversity present in the gastrointestinal tract of the subject;
the gastrointestinal disease, disorder or condition is Clostridium difficile-induced diarrhea;
administering comprises oral administration; and
administering the composition results in i) a reduction or an elimination of at least one pathogen and/or pathobiont present in the gastrointestinal tract of the subject; ii) engraftment in the gastrointestinal tract of the subject of at least one type of spore-forming bacteria present in the composition; or iii) a combination of i) and ii).

29. The method of claim 28, each entity comprising a 16S rDNA at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 673, 674, 774, 848, and 1670.

30. The method of claim 28, wherein the dose is provided in more than one hypromellose capsule each overencapsulated with a second capsule.