Patent Grant
10973861
This application includes a Sequence Listing submitted electronically as a text file named 25968US_CRF_sequencelisting.txt, created on Oct. 15, 2015, with a size of 4,165,632 bytes. The sequence listing is incorporated by reference.
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, there is 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. 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, Applicants address these and other shortcomings of the art by providing compositions and methods for treating subjects.
Disclosed herein are therapeutic compositions comprising a bacterial population comprising at least three but fewer than nine bacterial strains selected from the group consisting of Escherichia coli, Enterococcus faecalis, Clostridium innocuum, Clostridium ramosum, Clostridium bifermentans, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Blautia producta, wherein the composition is formulated for oral or gastric administration to a mammalian subject in an effective amount for prevention or treatment of a gastrointestinal disease, disorder or condition. In some embodiments, the bacterial strains are not a colonic bacterium and/or are not obtained from a fecal culture. In some embodiments, at least one Bacteroides species is detectably present in the mammalian subject prior to administration of the composition or, in other embodiments at least one Bacteroides species is not detectably present in the mammalian subject prior to administration of the composition, but is detectably present in the mammalian subject at least one hour after administration of the composition. In some embodiments, the mammalian subject has not received at least two doses of vancomycin, metronidazole and/or or similar antibiotic compound within one week prior to administration of the therapeutic composition. In some embodiments, a single administration is substantially effective to reduce C. difficile and/or C. difficile toxin content in a mammalian subject to whom the composition is administered. In some embodiments, one strain of E. coli is present in amounts at least 2, 5, 10, 50, 100 or more than 100 times greater than any other strain of E. coli present in the composition. In some embodiments, at least three bacterial strains are not present in the composition in equal ratios or are present in a ratio equivalent to the ratio of the bacterial strains in a reference mammalian subject. In some embodiments, at least one of the bacterial strains is provided in a concentration of greater than 1×102 viable bacteria per gram of composition or is provided in a concentration of less than 1×108 viable bacteria per gram of composition. In some embodiments, a plurality of the bacterial strains is provided in a concentration of less than 1×108 viable bacteria per gram of composition. In some embodiments, no more than nine different bacterial strains are used, wherein at least two strains are from the group selected from: Escherichia coli, Enterococcus faecalis, Clostridium innocuum, Clostridium ramosum, Clostridium bifermentans, Bacteroided ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Blautia producta. In some embodiments, the composition is formulated for oral administration as a solid, semi-solid, gel, or liquid form; in the form of a pill, tablet, capsule, or lozenge; the bacterial strains are substantially encapsulated in an enteric coating; and/or the bacterial strains are substantially inactive prior to localization in the gastrointestinal tract of a mammalian subject to whom the composition is administered. In some embodiments, no more than two of the bacterial strains are strains whose growth is substantially inhibited by an equivalent amount of C. difficile bacteria.
Also disclosed is a method of treating a mammalian subject suffering from or at risk of developing a gastrointestinal disease, disorder or condition, comprising the step of administering to the mammalian subject a therapeutic composition comprising a bacterial population as described herein, wherein the composition is formulated for oral or gastric administration. In some embodiments, the gastrointestinal disease, disorder or condition is selected from the group consisting of relapsing diarrhea caused by C. difficile, ulcerative colitis, colitis, Crohn's disease, and irritable bowel disease. In some embodiments, the mammalian subject is an agricultural mammal. In some embodiments, the bacterial population comprises at least six bacterial strains or comprises at least six but fewer than ten bacterial strains. In some embodiments, the therapeutic composition is administered only once prior to improvement of the disease, disorder or condition; wherein the therapeutic composition is administered at intervals greater than two days; the mammalian subject has not received at least two doses of vancomycin, metronidazole and/or or similar antibiotic compound within one week prior to administration of the therapeutic composition; the mammalian subject is not suffering from relapsing diarrhea caused by C. difficile; and/or the mammalian subject has not previously received an antibiotic compound in the one month prior to administration of the therapeutic composition.
Also disclosed is a method of treating a mammalian subject suffering from or at risk of developing a gastrointestinal disease, disorder or condition, comprising the step of administering to the mammalian subject any therapeutic composition disclosed herein.
Also disclosed is a method of treating a mammalian subject suffering from or at risk of developing a gastrointestinal disease, disorder or condition, or a metabolic disease, disorder or condition selected from the group consisting of diabetes and autism, comprising the step of administering to the mammalian subject a therapeutic composition comprising a bacterial population comprising at least three bacterial strains, wherein no greater than nine of the bacterial strains are members of a bacterial species selected from the group consisting of Escherichia coli, Enterococcus faecalis, Clostridium innocuum, Clostridium ramosum, Clostridium bifermentans, Bacteroided ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Blautia producta, wherein the composition is formulated for oral or gastric administration. In some embodiments, the method further comprises the step of detecting one or more of the administered bacterial strains in the mammalian subject subsequent to administration, for example, detecting a 16S nucleic acid sequence in at least one administered bacterial strain.
Also disclosed is a therapeutic composition comprising a bacterial population comprising at least three but fewer than nine bacterial strains, wherein the bacterial strains comprise 16S nucleic acid sequences at least 97% identical to reference 16S nucleic acid sequences in bacterial strains selected from the group consisting of Escherichia coli, Enterococcus faecalis, Clostridium innocuum, Clostridium ramosum, Clostridium bifermentans, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Blautia producta, wherein the composition is formulated for oral or gastric administration to a mammalian subject in an effective amount for prevention or treatment of a gastrointestinal disease, disorder or condition.
Also disclosed is a therapeutic composition comprising a bacterial population comprising at least three but fewer than nine bacterial strains, wherein the bacterial strains comprise Clostridium innocuum, Clostridium ramosum, and Clostridium bifermentans, wherein the composition is formulated for oral or gastric administration to a mammalian subject in an effective amount for prevention or treatment of a gastrointestinal disease, disorder or condition.
Also disclosed is a therapeutic composition comprising a bacterial population comprising at least three but fewer than nine bacterial strains, wherein the bacterial strains do not comprise detectable amounts of Bacteroides ovatus, Bacteroides vulgatus, or Bacteroides thetaiotaomicron, wherein the composition is formulated for oral or gastric administration to a mammalian subject in an effective amount for prevention or treatment of a gastrointestinal disease, disorder or condition.
Also disclosed is a method of increasing diversity of a gastrointestinal microbiota in a mammalian subject in need thereof, comprising administering to the mammalian subject a therapeutic composition comprising a bacterial population comprising at least three but fewer than nine bacterial strains, wherein the bacterial strains comprise 16S nucleic acid sequences at least 97% identical to reference 16S nucleic acid sequences in bacterial strains selected from the group consisting of Escherichia coli, Enterococcus faecalis, Clostridium innocuum, Clostridium ramosum, Clostridium bifermentans, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Blautia producta, wherein the composition is formulated for oral or gastric administration to a mammalian subject in an effective amount for increasing the diversity of the gastrointestinal microbiota of the mammalian subject subsequent to administration. In some embodiments, the diversity is increased for at least 1 day after administration or for at least 4 days after administration or for at least 7 days after administration or for at least 14 days after administration or for at least 21 days after administration. In some embodiments, wherein the mammalian subject is suffering from or at risk of developing a disease, disorder or condition other than a gastrointestinal disease, disorder or condition.
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.
“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, Ross 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.
Table 1 below shows a List of Operational Taxonomic Units (OTU) with taxonomic assignments made to Genus, Species, and Phylogenetic Clade. Clade membership of bacterial OTUs is based on 16S sequence data. Clades are defined based on the topology of a phylogenetic tree that is constructed from full-length 16S sequences using maximum likelihood methods familiar to individuals with ordinary skill in the art of phylogenetics. Clades are constructed to ensure that all OTUs in a given clade are: (i) within a specified number of bootstrap supported nodes from one another, and (ii) within 5% genetic similarity. OTUs that are within the same clade can be distinguished as genetically and phylogenetically distinct from OTUs in a different clade based on 16S-V4 sequence data, while OTUs falling within the same clade are closely related. OTUs falling within the same clade are evolutionarily closely related and may or may not be distinguishable from one another using 16S-V4 sequence data. Members of the same clade, due to their evolutionary relatedness, play similar functional roles in a microbial ecology such as that found in the human gut. Compositions substituting one species with another from the same clade are likely to have conserved ecological function and therefore are useful in the present invention. All OTUs are denoted as to their putative capacity to form spores and whether they are a Pathogen or Pathobiont (see Definitions for description of “Pathobiont”). NIAID Priority Pathogens are denoted as ‘Category-A’, ‘Category-B’, or ‘Category-C’, and Opportunistic Pathogens are denoted as ‘OP’. OTUs that are not pathogenic or for which their ability to exist as a pathogen is unknown are denoted as ‘N’. The ‘SEQ ID Number’ denotes the identifier of the OTU in the Sequence Listing File and ‘Public DB Accession’ denotes the identifier of the OTU in a public sequence repository.
“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.
16s Sequencing, 16s, 16s-rRNA, 16s-NGS: 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; www.ncbi.nlm.nih.gov/books/NBK21090/), or KEGG pathways (www.genome.jp/kegg/).
Network Class, Core Network, Core Network Ecology: 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). 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 ompared 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.
Bacterial Compositions
We have identified combinations of commensal 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 of a pathogen such as C. difficile, Salmonella spp., enteropathogenic E. coli, and vancomycin-resistant Enterococcus spp. and other pathobionts, so that a healthy, diverse and protective microbiota can be maintained or, in the case of pathogenic bacterial infections such as recurrent C. difficile infection, repopulate the intestinal lumen to reestablish ecological control over potential pathogens. Furthermore, the bacterial compositions have the ability to shift the state of the gut microbiota toward a healthy state allowing for augmentation of the microbiota with commensal bacteria found in healthy microbiomes. The bacterial compositions may also engraft in the host themselves and remain present in the gut for 1 day, 1 week, 1 month, 1 year, or longer than 1 year.
Preferred bacterial species include Escherichia coli, Streptococcus faecalis, Clostridium Innocuum, Clostridium ramosum, Clostridium bifermentans, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Blautia producta. 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. Preferred bacterial species also include species that share 97% homology in the 16s rDNA region to the species of Escherichia coli, Streptococcus faecalis, Clostridium Innocuum, Clostridium ramosum, Clostridium bifermentans, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Blautia producta as defined by the provided 16s sequences (See Table 1).
Bacterial compositions may consist essentially of no greater than a number of types of these preferred bacteria. For instance, a bacterial composition may comprise no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 types of bacteria, as defined by above species or operational taxonomic unit (OTU) encompassing such species.
Bacterial compositions may consist essentially of a range of numbers of species of these preferred bacteria, but the precise number of species in a given composition is not known. For instance, a bacterial composition may consist essentially of between 2 and 10, 3 and 10, 4 and 10, 5 and 10, 6 and 10, 7 and 10, 8 and 10, or 9 and 10; or 2 and 9, 3 and 9, 4 and 9, 5 and 9, 6 and 9, 7 and 8 or 8 and 9; or 2 and 8, 3 and 8, 4 and 8, 5 and 8, 6 and 8 or 7 and 8; or 2 and 7, 3 and 7, 4 and 7, 5 and 7, or 6 and 7; or 2 and 6, 3 and 6, 4 and 6 or 5 and 6; or 2 and 5, 3 and 5 or 4 and 5; or 2 and 4 or 3 and 4; or 2 and 3, as defined by above species or operational taxonomic unit (OTU) encompassing such species.
Bacterial compositions containing a plurality of species may be provided such that the relative concentration of a given species in the composition to any other species in the composition is known or unknown. Such relative concentrations of any two species, or OTUs, may be expressed as a ratio, where the ratio of a first species or OTU to a second species or OTU is 1:1 or any ratio other than 1:1, such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25; 1:50; 1:75, 1:100, 1:200, 1:500; 1:1000, 1:10,000, 1:100,000 or greater than 1:100,000. The ratio of bacterial strains present in a bacterial composition may be determined by the ratio of the bacterial strains in a reference mammalian subject, e.g., a healthy human not suffering from or at known risk of developing a dysbiosis.
Bacterial compositions comprising a plurality of species may be provided such that the concentration of a given strain, or the aggregate of all strains, is between 1×104 and 1×1015 viable bacteria per gram of composition or per administered dose. For example the concentration 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 bacteria per gram of composition or per administered dose. Alternatively, the concentration of a given strain, or the aggregate of all strains, is below a given concentration e.g., below 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 below 1×1015 viable bacteria per gram of composition or per administered dose.
In certain embodiments, the bacterial compositions comprise a plurality of strains of the same species of bacteria. In certain embodiments, the strain of bacteria is present in amounts at least 2, 5, 10, 50, 100 or more than 100 times greater than any other strain of that species present in the composition. For example, provided are compositions containing one strain of E. coli, where this strain is present in amounts at least 2, 5, 10, 50, 100 or more than 100 times greater than any other strain of E. coli present in the composition.
Because pathogenic bacteria such as C. difficile bacteria may inhibit growth of the bacterial strains present in the composition, it is generally beneficial such that no more than two of the bacterial strains in the bacterial composition are strains whose growth is substantially inhibited, e.g., in vitro, by an equivalent amount of C. difficile bacteria.
In one embodiment, the bacterial composition comprises at least one and preferably more than one of the following species or a species that is 97% identical based on the 16s rDNA: 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 one embodiment, the bacterial composition comprises at least one and preferably more than one of the following species or a species that is 97% identical based on the 16s rDNA: Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium camis, Clostridium celaturn, 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 malenominaturn, 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 sordeffii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridium tedium, 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 species or a species that is 97% identical based on the 16s rDNA: 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 species or a species that is 97% identical based on the 16s rDNA: 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 species or a species that is 97% identical based on the 16s rDNA: 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 species or a species that is 97% identical based on the 16s rDNA: 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 species or a species that is 97% identical based on the 16s rDNA: 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 species or a species that is 97% identical based on the 16s rDNA: 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 species or a species that is 97% identical based on the 16s rDNA: 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 haffii, 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.
A bacterial composition may be prepared comprising at least two types of isolated bacteria, wherein a first type is a first OTU comprising a species of E. coli, S. faecalis, Cl. Innocuum, Cl. ramosum, Cl. bifermentans, Bact. ovatus, Bact. vulgatus, Bact. thetaiotaomicron, or Blautia producta and the type is a second OTU is characterized by, i.e., at least 95%, 96%, 97%, 98%, 99% or including 100% sequence identity to, the first OTU. In some embodiments, two types of bacteria are provided in a composition, and the first bacteria and the second bacteria are not the same OTU.
OTUs can be defined either by full 16S sequencing of the rRNA gene (Table 1), 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.
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.
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 and pathobionts is provided in Table 1.
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).
Generation and Formulation of Bacterial Compositions
The bacterial compositions are generally formulated for oral or gastric administration, typically to a mammalian subject. Preferably, such formulations contain or are coated by an enteric coating to protect the bacteria through the stomach and small intestine. In particular embodiments, the composition is formulated for oral administration as a solid, semi-solid, powder, gel, or liquid form, such as in the form of a pill, tablet, capsule, or lozenge.
The bacterial strains may, individually or together, be substantially inactive prior to localization in the gastrointestinal tract of the mammalian subject to whom the composition is administered. The bacterial strains may be lyophilized or otherwise acted upon to increase long-term storage viability.
Preferential species include E. coli, S. faecalis, Cl. Innocuum, Cl. ramosum, Cl. bifermentans, Bact. ovatus, Bact. vulgatus, Bact. thetaiotaomicron, and Blautia producta, or species 97% identical to the 16s rDNA sequences of these species and are generally provided in bacterial compositions containing at least three strains. In certain embodiments, at least one of the bacterial strains is not a colonic bacterium. In another embodiment, at least one of the bacterial strains is not obtained from a fecal culture, e.g., the bacterial strain is obtained from culturing a non-fecal biological material from a subject, from an environmental source, or from repeatedly streaking and purifying strains from any source. Alternatively, at least one bacterial strain is genetically engineered. In another embodiment, at least one species is present in the composition in spore form. In an alternative embodiment, all of the species are present as vegetative forms, substantially free of spores.
The bacterial 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 C. difficile and/or C. difficile toxin content in a mammalian subject to whom the composition is administered. Alternatively, a single administration is substantially effective to reduce vancomycin resistant Enterococci, carbapenem resistant bacteria, or another pathobiont in a mammalian subject to whom the composition is administered (See Table 1). Substantially effective means that C. difficile and/or C. difficile toxin and/or pathobiont 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.
In some embodiments, the bacterial compositions comprise purified spore populations. Purified spore populations comprise one or more commensal bacteria of the human gut microbiota present in the form of a spore. 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, 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 bacterial compositions comprising more than one type of spore forming 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.
Pharmaceutical Compositions and Formulations of the Invention
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. In one embodiment, 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.
Administration of Bacterial Compositions
The bacterial compositions of the invention are suitable for administration to mammals and non-mammalian animals in need thereof. The 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 certain embodiments, the mammalian subject is a human subject who has one or more symptoms of a dysbiosis.
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.
The bacterial compositions are useful in methods of treating a mammalian subject suffering from or at risk of developing a gastrointestinal disease, disorder or condition. Therefore, a therapeutic benefit is provided by orally administering to the mammalian subject a therapeutic composition containing a bacterial population comprising at least three bacterial strains, wherein each bacterial strain is a member of a bacterial species selected from the group consisting of E. coli, S. faecalis, C. ramosum, C. bifermentans, Bact. ovatus, Bact. vulgatus, Bact. thetaiotaomicron, and Blautia producta, wherein the therapeutic composition is formulated for oral administration. In some embodiments, the gastrointestinal disease, disorder or condition is relapsing diarrhea caused by C. difficile, 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.
Also provided are methods of treating or preventing a mammalian subject suffering from or at risk of developing a metabolic disease, disorder or condition selected from the group consisting of diabetes, metabolic syndrome, obesity, and autism using the therapeutic compositions provided herein. 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).
An In Vivo Assay Establishing that 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, may be used. The model may begin by evaluating the microbiota of the mice. Qualitative assessments may be accomplished using 16S profiling of the microbial community in the feces of normal mice. It may also be accomplished by full genome sequencing, whole genome shotgun sequencing (WGS), or traditional microbiological techniques. Quantitative assessments may be conducted using quantitative PCR (qPCR), described in section below, or by using traditional microbiological techniques and counting colony formation.
Optionally, the mice may 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 may be used and antibiotics are well known. Antibiotics may include aminoglycoside antibiotics (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 may 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 may 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 may 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, may 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 may 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 cCompositions to Populate Different Regions of the Gastrointestinal Tract
The present bacterial compositions may also be assessed for their ability to populate different regions on the gastrointestinal tract. In one embodiment, a bacterial composition may 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 may 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 in section II.A may be conducted, except instead of assessing the feces produced by the mice, particular regions of the gastrointestinal tract may be removed and studied individually. For example, at least one particular region of the gastrointestinal tract may be removed and a qualitative or quantitative determination may be performed on the contents of that region of the gastrointestinal tract. In another embodiment, the contents may optionally be removed and the qualitative or quantitative determination may be conducted on the tissue removed from the mouse.
Methods for Testing Sensitivity of Bacterial Composition
In certain embodiments, methods for testing the sensitivity of bacterial compositions in order to select for particular desirable characteristics may be employed. For example, the constituents in the bacterial composition may 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).
1. pH Sensitivity Testing
For oral administration of the bacterial compositions, 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 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 may 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 may 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.
Alternatively, bacterial cells may be grown in a standard media, such as the GMM media described above, and adjusted to pH 2.5 with 1M HCl. The cells may be incubated anaerobically at 37° C. and their survival measured at intervals of 0, 30, 60, 120, 240, and/or 360 minutes.
A further 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.
2. 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 may be tested by exposing the bacterial compositions to bile acids for the expected gut exposure time to bile acids. For example, bile acid solutions may 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 may be cultured in 2.2 ml of a bile acid composition mimicking the concentration and type of bile acids in the subject, 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. Survival of bacteria may be determined by culturing the bacteria and counting colonies on appropriate selective or non-selective media.
As another alternative, bacterial strains may be streaked onto agar plates supplemented with porcine bile (Sigma) at 0.5%, 1%, and 5% (w/v). Plates may be incubated at 37° C. under anaerobic conditions and the growth recorded after 48 hours. Growth may be compared with control plates by an experienced observer and the growth of colonies scored as: 0=no growth, 1=hazy translucent growth (<33% control plates with 0% bile), 2=definite growth but not as good as controls (>33% but <66%), 3=growth equivalent to controls (>66%).
A further 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.
3. Antibiotic Sensitivity Testing
As a further optional sensitivity test, bacterial compositions may be tested for sensitivity to antibiotics. In one embodiment, bacterial compositions may be chosen so that the bacterial constituents are sensitive to antibiotics such that if necessary they can be eliminated or substantially reduced from the subject's gastrointestinal tract by at least one antibiotic targeting the bacterial composition.
4. Adherence to Gastrointestinal Cells
The bacterial compositions may optionally be tested for the ability to adhere to gastrointestinal cells. Without being bound by theory, in some instances, adherence can enhance the ability to repopulate a subject's gastrointestinal tract and thus may be used as a criterion in some embodiments. As a first embodiment, this may be conducted in a tissue culture model, where gastrointestinal epithelial cells, such as but not limited to CACO-2 cells (ATCC HTB-37), are grown in tissue culture flasks to differentiation in an antibiotic containing media, grown for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 days in an antibiotic-free medium, incubated with bacterial cells for 30 minutes, 60 minutes, 90 minutes, or 120 minutes, and washed three times. After washing, the number of adherent bacteria may be assessed by lysing the epithelial cells and enumerating the bacteria using the plate count method or qPCR, as described herein.
As another mode, bacterial adherence may be evaluated using an engineered tissue model of the lining of the gastrointestinal tract. Viney et al., “Co-culture of Intestinal Epithelial and Stromal Cells in 3D Collagen-based Environments,” Regen Med 4(3):397-406 (2009). After preparation of the engineered tissue model, the bacterial cells may be incubated, washed, and adherence enumerated, as described above.
Furthermore, bacterial adherence may be evaluated using a tissue explant from the gastrointestinal tract of a subject. Gastrointestinal tract tissue may be surgically removed from a healthy donor. Alternatively, gastrointestinal tract tissue may be surgically removed from a donor who has a gastrointestinal disease, such as from an unused portion of a biopsy. After surgical excision, the bacterial cells may be incubated, washed, and adherence enumerated, as described above.
As an alternative, one technique involves the collection of the effluent from a subject with a well functioning ileostomy by saline lavage, as described in U.S. Pat. No. 4,839,281.
Assessment of Microbiota Diversity
Microbiota diversity, as assessed by deep 16S rRNA sequencing or metagenomic sequencing, may be evaluated at a variety of time points to assess the effect of microbial compositions on restoring microbiota diversity. Microbial diversity may optionally be assessed before administration of an antibiotic or removal of microbiota by a colon-cleansing preparation. Microbial diversity may also be assessed before administration of a microbial composition and after administration of a microbial composition at any frequency (e.g., more frequently than 1 day, 1 day, 3 days, 1 week, 2 weeks, one month, or more than one month). One may provide for any temporal resolution that has more or less frequent sampling intervals, and/or samples taken for a longer time period (e.g., at least 1 week, at least 1 month, at least three months, at least six months, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or over more than 10 years.
Diversity may be measured according to known sequencing methods, including, but not limited to 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). Sequence data used for diversity analysis may comprise 16S rRNA, whole genome sequence, any subset of a genome as well as whole genome shotgun metagenomic sequence (WGS). Sequence data may be generated using any sequencing technology including, but not limited to Sanger, Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Bioscienses, and/or Oxford Nanopore. Subjects may be assessed for a variety of diversity metrics, including, but not limited to, observed taxon richness, Shannon diversity index, Shannon equitability index, alpha diversity, beta diversity, Chao1 index, Simpson diversity index, normalized abundance over time, and/or phylogenetic breadth.
In another embodiment, subjects may be assessed for a variety of diversity metrics, including, but not limited to, Simpson diversity or by plotting rarefaction curve comparisons of reference OTUs for subjects before and after treatment with microbial compositions. Phylogenetic diversity may be plotted against the number of sequence reads (full 16S sequence or one or any combination of more hypervariable regions of the 16S sequence, whole genome sequence, or specific marker loci). Shahinas, et al., Toward an understanding of changes in diversity associated with fecal microbiome transplantation based on 16S rRNA Gene Deep Sequencing, mBio 3(5):e00338-12 (2012).
In another embodiment, diversity may be shown by graphing a bar plot of abundance at the family level and showing an increase in either the number of families or the balance of abundance between families.
In another embodiment, diversity may be shown by plotting heatmaps of the abundance of an OTU, species, genus, and/or higher taxonomic assignment showing sets of samples that are enriched or depleted for specific constituents.
In another embodiment, diversity may be shown by generation of a phylogenetic tree of OTUs, species, genera, and/or higher or lower clade assignments. In this embodiment genetic distances may be computed between all OTUs, and summary statistics including, but not limited to average genetic distance and number of unique clades may be computed.
In another embodiment, beta-diversity may be computed using diversity metrics familiar to those with ordinary skill in the art that include but are not limited to Bray-Curtis Dissimilarity Indices or Jaccard Distances and plotted using Principal Coordinates Analysis.
Beneficial bacterial compositions may result in an increase in diversity after their administration that is 10%, 20%, 30%, 40%, or 50% higher than diversity before their administration. In another embodiment bacterial compositions may result in restoration of diversity to at least 70%, 80%, 90%, 95%, or up to 100% of the diversity either measured before administration of an antibiotic or removal of microbiota by a colon-cleansing preparation, or alternatively, as compared to the diversity of a reference mammalian subject.
Methods for Preparing a Bacterial Composition for Administration to a Subject
Methods for producing bacterial compositions may include three main processing steps, combined with one or more mixing steps. The steps are: 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 using a culturing step, the agar or broth may 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. 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 may be actively controlled, passively controlled (e.g., via buffers), or allowed to drift. For example, for anaerobic bacterial compositions (e.g., gut applications), an anoxic/reducing environment may 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 may be grown at 37° C., pH 7, in the medium above, pre-reduced with 1 g/L cysteineŸHCl.
When the culture has generated sufficient biomass, it may be preserved for banking. The organisms may 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. Bacterial composition banking may 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 may be harvested by centrifugation to pellet the cells from the culture medium, the supernatant 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 may be conducted using similar culture steps to banking, including medium composition and culture conditions. It may be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there may 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 may be cultivated to a concentration of 1010 CFU/mL, then concentrated 20-fold by tangential flow microfiltration; the spent medium may 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 may 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 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 one embodiment, the number of bacteria of each type may be present in the same amount or in different amounts. For example, in a bacterial composition with two types of bacteria, the bacteria may be present in from a 1:10,000 ratio to a 1:1 ratio, from a 1:10,000 ratio to a 1:1,000 ratio, from a 1:1,000 ratio to a 1:100 ratio, from a 1:100 ratio to a 1:50 ratio, from a 1:50 ratio to a 1:20 ratio, from a 1:20 ratio to a 1:10 ratio, from a 1:10 ratio to a 1:1 ratio. For bacterial compositions comprising at least three types of bacteria, the ratio of type of bacteria may be chosen pairwise from ratios for bacterial compositions with two types of bacteria. For example, in a bacterial composition comprising bacteria A, B, and C, at least one of the ratio between bacteria A and B, the ratio between bacteria B and C, and the ratio between bacteria A and C may be chosen, independently, from the pairwise combinations above.
Methods of Treating a Subject
A. Overview of Method
The bacterial compositions may be used to populate the gastrointestinal tract of a subject, resulting in one or more of a changed constitution of a subject's microbiota and improvement or correction of a dysbiosis. Without being bound by theory, bacterial compositions can promote mammalian health by restoring the natural microflora in the GI tract and reinforcing the normal controls on aberrant immune responses.
Bacterial compositions can also improve fiber and protein digestion. Improving fiber and protein digestion is desirable as it promotes the growth of microbiota. A probiotic composition with multiple species has been shown to reduce production of toxic metabolites from protein fermentation. Rehman, Effects of Probiotics and antibiotics on intestinal homeostasis in a computer controlled model of the large intestine, BMC Microbiology 12:47 (2012). Carbohydrate fermentation is, for the most part, believed to be a beneficial process in the large gut, because the growth of saccharolytic bacteria stimulates their requirements for toxic products associated with putrefaction, for incorporation into cellular proteins, thereby protecting the host. However, as digestive materials move along the gut, carbohydrates become depleted, which may be linked to the increased prevalence of colonic disease in the distal bowel. Macfarlane, et al., Bacteria, colonic fermentation, and gastrointestinal health, J AOAC Int. 95(1):50-60 (2012). Thus, restoring microbiota, including, but not limited to, restoring microbiota in the distal bowel can provide health benefits.
Fiber digestion may be determined using the method described in Vickers et al., Comparison of fermentation of selected fructooligosaccharides and other fiber substrates by canine colonic microflora, Am. J. Vet. Res. 61 (4), 609-615 (2001), with the exception that instead of inoculating using diluted fecal samples each experiment may use the bacterial compositions of interest.
In one embodiment, the pathogen may be Clostridium difficile, Salmonella spp., pathogenic Escherichia coli, or vancomycin-resistant Enterococcus spp. In yet another embodiment, the pathogen may be Clostridium difficile, or other pathogen or pathobiont listed in Table 1.
The present bacterial compositions may be useful in a variety of clinical situations. For example, the bacterial compositions may be administered as a complementary treatment to antibiotics when a subject is suffering from an acute infection, to reduce the risk of recurrence after an acute infection has subsided, or when a subject 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 may 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 is 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 in the section below.
B. Pretreatment Protocols
Prior to administration of the bacterial composition, the subject may 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 subject has an acute infection with a highly resilient pathogen or when the microbiota resident in the subject's gastrointestinal tract is likely to resist colonization by the bacterial composition. In other embodiments, the pretreatment protocol is entirely optional, such as when the dysbiosis is not associated with a pathogenic infection; when, if an infection is present, the pathogen causing the infection is not resilient, or when the subject 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 may enhance the ability of the bacterial composition to affect the subject's microbiome.
As one way of preparing the subject for administration of the microbial ecosystem, a standard colon-cleansing preparation may be administered to the subject to substantially empty the contents of the colon, such as used to prepare a subject 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.
If a subject has received an antibiotic for treatment of an infection, or if a subject has received an antibiotic as part of a specific pretreatment protocol, in one embodiment the antibiotic should 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 may be discontinued 1, 2, or 3 days before the administration of the bacterial composition. In one embodiment, the antibiotic may be discontinued 3, 4, 5, 6, or 7 antibiotic half-lives before administration of the bacterial composition. In another embodiment, the antibiotic may 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 may 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 may be chosen so that the infection is sensitive to the antibiotic, but the constituents in the bacterial composition are not sensitive to the antibiotic.
C. Routes of Administration
In the present method, the bacterial composition is administered enterically, in other words by a route of access to the gastrointestinal tract. 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.
D. Dosages and Schedule for Administration
In one embodiment, from 108 and 1011 microorganisms total may be administered to the subject in a given dosage form. In one mode, an effective amount may be provided in from 1 to 500 ml or from 1 to 500 grams of the bacterial composition having from 108 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 108 to 1015 bacteria. Those receiving acute treatment may 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 may 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). 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 health care professionals).
E. Subject Selection
Particular bacterial compositions may be selected for individual subjects or for subjects with particular profiles. For example, 16S sequencing may be performed for a given subject to identify the bacteria present in his or her microbiota. The sequencing may either profile the subject's entire microbiome using 16S sequencing (to the family, genera, or species level), a portion of the subject's microbiome using 16S sequencing, or it may be used to detect the presence or absence of specific candidate bacteria that are biomarkers for health or a particular disease state. Based on the biomarker data, a particular composition may be selected for administration to a subject to supplement or complement a subject's microbiota in order to restore health or treat or prevent disease.
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.
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.
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).
An in vivo mouse model was employed to demonstrate the protective effect of the bacterial compositions against C. difficile. In this model (based on Chen, et al., A mouse model of Clostridium difficile associated disease, Gastroenterology 135(6):1984-1992 (2008)), mice were made susceptible to C. difficile by a 7 or 9 day treatment (days −14 or −12 until −5 of experiment) with 5 to 7 antibiotics (kanamycin, colistin, gentamycin, metronidazole and vancomycin, and optionally including ampicillin and ciprofloxacin) delivered via their drinking water, followed by a single dose with clindamycin on day −3. The mice were then challenged three days later (on day 0) with 104 spores of C. difficile via oral gavage (i.e., oro-gastric lavage). The outcomes assessed each day from day 0 to day 6 were weight, clinical signs, mortality and shedding of C. difficile in the feces. Weight, clinical signs and mortality were also evaluated on day −1. Weight loss, clinical signs of disease, and C. difficile shedding were typically observed without treatment. Vancomycin treatment protects against these outcomes and serves as a positive control when provided by oral gavage on days −1 to 4. Clinical signs were subjective, and scored each day by the same experienced observer. Mortality includes euthanasia of animals that lose greater than or equal to 25% of their body weight or that display severe morbidity as assessed by clinical signs. Feces were gathered from the mouse cages (5 mice per cage) each day, and suspended in 5 mL of PBS by vortexing. The shedding of C. difficile spores is detected in the feces using a selective plating assay as described for the in vitro assay above, after a 1 hour treatment with 50% ethanol at room temperature to kill C. difficile vegetative cells. C. difficile may also be detected in the mouse fecal suspension via qPCR for the toxin gene as described herein. The effects of various administrations, including 10% suspension of human feces in PBS (as a positive control), microbial compositions, or PBS (as a negative vehicle control), were determined by introducing the bacterial composition in a 0.2 mL volume into the mice via oral gavage on day −1, one day prior to C. difficile challenge. Vancomycin, as discussed above, is given on days −1 to −4 as another positive control. Alternative dosing schedules can be employed, including multiple doses of bacterial composition, and 103 to 1010 of a given organism or composition may be delivered.
Exemplary bacterial compositions as described herein were administered as follows.
In a first demonstration, the results of which are provided in tabular form as Table 3B, Treatment 1 is the vehicle control of phosphate buffered saline (PBS), Treatment 2 is a positive control of 10% fecal suspension in PBS which has total anaerobic cfu/ml of 4.5×109, Treatment 3 is a positive control of 10% fecal suspension in PBS which has total anaerobic cfu/ml of 6.2×108, Treatment 4 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 7.1×109. Treatment 5 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 7.1×108. Treatment 6 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family EnterococcaceaeATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 7.1×107. Treatment 7 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 3.7×109. Treatment 8 is a bacterial composition comprising Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483) with a total anaerobic cfu/ml of 3.5×109. Treatment 9 is a bacterial composition comprising Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 6.7×109. Treatment 10 is a bacterial composition comprising Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), and Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 1.2×109. Treatment 11 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium celatum (family Clostridiaceae, SPC21278) with a total anaerobic cfu/ml of 6.1×109. Treatment 12 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 1×1010.
In a second demonstration, the results of which are provided in tabular form as Table 2, Treatment 1 is the vehicle control of phosphate buffered saline (PBS), Treatment 2 is a positive control of 10% fecal suspension in PBS which has total anaerobic cfu/ml of 5×109, Treatment 3 is a positive control of 10% fecal suspension in PBS which has total anaerobic cfu/ml of 7×108.
Treatment 4 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 2.2×109, Treatment 5 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 2.2×108. Treatment 6 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 2.2×107, Treatment 7 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, ATCC 35320), Enterococcus faecalis (family Enterococcaceae ATCC, 19433), Blautia producta (family Lachnospiraceae, DSM 14466), Bacteroides vulgatus (family Bacteroidaceae, ATCC 8482), Bacteroides thetaiotaomicron (family Bacteroidaceae, ATCC 29148), Bacteroides ovatus (family Bacteroidaceae, ATCC 8483), Clostridium innocuum (family Erysipelotrichaceae, DSM 22910) with a total anaerobic cfu/ml of 5×109, Treatment 8 is a bacterial composition comprising Escherichia coli (family, Enterobacteriaceae, SPC21221), Escherichia coli (family, Enterobacteriaceae, SPC21248), Enterococcus faecalis (family, Enterococcaceae, SPC21240), Enterococcus faecalis (family, Enterococcaceae, SPC21252), Blautia producta (family, Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family, Bacteroidaceae, SPC21122), Bacteroides vulgatus (family, Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides thetaiotaomicron, (family Bacteroidaceae, SPC21133), Bacteroides ovatus (family, Bacteroidaceae, SPC21141), Bacteroides ovatus (family, Bacteroidaceae, SPC21181), Clostridium innocuum (family, Erysipelotrichaceae, SPC21112), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), with a total anaerobic cfu/ml of 1.7×1010, Treatment 9 is a bacterial composition comprising Escherichia coli (family, Enterobacteriaceae, SPC21221), Escherichia coli (family, Enterobacteriaceae, SPC21248), Enterococcus faecalis (family, Enterococcaceae, SPC21240), Enterococcus faecalis (family, Enterococcaceae, SPC21252), Blautia producta (family, Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family, Bacteroidaceae, SPC21122), Bacteroides vulgatus (family, Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides thetaiotaomicron, (family Bacteroidaceae, SPC21133), Bacteroides ovatus (family, Bacteroidaceae, SPC21141), Bacteroides ovatus (family, Bacteroidaceae, SPC21181), Clostridium innocuum (family, Erysipelotrichaceae, SPC21112), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), with a total anaerobic cfu/ml of 1.7×109, Treatment 10 is a bacterial composition comprising Escherichia coli (family, Enterobacteriaceae, SPC21221), Escherichia coli (family, Enterobacteriaceae, SPC21248), Enterococcus faecalis (family, Enterococcaceae, SPC21240), Enterococcus faecalis (family, Enterococcaceae, SPC21252), Blautia producta (family, Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family, Bacteroidaceae, SPC21122), Bacteroides vulgatus (family, Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides thetaiotaomicron, (family Bacteroidaceae, SPC21133), Bacteroides ovatus (family, Bacteroidaceae, SPC21141), Bacteroides ovatus (family, Bacteroidaceae, SPC21181), Clostridium innocuum (family, Erysipelotrichaceae, SPC21112), Clostridium bifermentans (family Peptostreptococcaceae, ATCC 638), and Clostridium ramosum (family Erysipelotrichaceae, ATCC 25582), with a total anaerobic cfu/ml of 1.7×108, Treatment 11 is a bacterial composition comprising Escherichia coli (family, Enterobacteriaceae, SPC21221), Escherichia coli (family, Enterobacteriaceae, SPC21248), Enterococcus faecalis (family, Enterococcaceae, SPC21240), Enterococcus faecalis (family, Enterococcaceae, SPC21252), Blautia producta (family, Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family, Bacteroidaceae, SPC21122), Bacteroides vulgatus (family, Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides thetaiotaomicron, (family Bacteroidaceae, SPC21133), Bacteroides ovatus (family, Bacteroidaceae, SPC21141), Bacteroides ovatus (family, Bacteroidaceae, SPC21181), and Clostridium innocuum (family, Erysipelotrichaceae, SPC21112) with a total anaerobic cfu/ml of 1.2×1010, Treatment 12 is a bacterial composition comprising Escherichia coli (family, Enterobacteriaceae, SPC21221), Escherichia coli (family, Enterobacteriaceae, SPC21248), Enterococcus faecalis (family, Enterococcaceae, SPC21240), Enterococcus faecalis (family, Enterococcaceae, SPC21252), Blautia producta (family, Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family, Bacteroidaceae, SPC21122), Bacteroides vulgatus (family, Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides thetaiotaomicron, (family Bacteroidaceae, SPC21133), Bacteroides ovatus (family, Bacteroidaceae, SPC21141), Bacteroides ovatus (family, Bacteroidaceae, SPC21181), Clostridium innocuum (family, Erysipelotrichaceae, SPC21112), and Clostridium celatum (family Clostridiaceae, SPC21278) with a total anaerobic cfu/ml of 9×109, Treatment 13 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21122), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides ovatus (family Bacteroidaceae, SPC21141), and Clostridium innocuum (family Erysipelotrichaceae, SPC21112) with a total anaerobic cfu/ml of 6×109. Treatment 14 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21248), Enterococcus faecalis (family Enterococcaceae, SPC21252), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family, Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21133), Bacteroides ovatus (family Bacteroidaceae, SPC21181), and Clostridium innocuum (family Erysipelotrichaceae, SPC21112) with a total anaerobic cfu/ml of 5×109.
In a third demonstration, the results of which are provided in tabular form as Table 3A, Treatment 1 is the vehicle control of phosphate buffered saline (PBS), Treatment 2 is a positive control of 10% fecal suspension in PBS which has total anaerobic cfu/ml of 2.3×1011,
Treatment 3 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides Bacteroides ovatus (family Bacteroidaceae, SPC21141), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), and Clostridium celatum (family Clostridiaceae, SPC21278) with a total anaerobic cfu/ml of 2.5×109, Treatment 4 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides ovatus (family Bacteroidaceae, SPC21141), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), and Clostridium celatum (family Clostridiaceae, SPC21278) with a total anaerobic cfu/ml of 2.5×108, Treatment 5 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides ovatus (family Bacteroidaceae, SPC21141), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), and Clostridium celatum (family Clostridiaceae, SPC21278) with a total anaerobic cfu/ml of 2.5×107, Treatment 6 is a bacterial composition comprising Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides ovatus (family Bacteroidaceae, SPC21141), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), and Clostridium celatum (family Clostridiaceae, SPC21278) with a total anaerobic cfu/ml of 2.8×109, Treatment 7 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides ovatus (family Bacteroidaceae, SPC21141), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), and Clostridium celatum (family Clostridiaceae, SPC21278) with a total anaerobic cfu/ml of 6×109, Treatment 8 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), and Clostridium celatum (family Clostridiaceae, SPC21278) with a total anaerobic cfu/ml of 1.8×109, Treatment 9 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), and Bacteroides ovatus (family Bacteroidaceae, SPC21141) with a total anaerobic cfu/ml of 2×109, Treatment 10 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides ovatus (family Bacteroidaceae, SPC21141), Clostridium innocuum (family Erysipelotrichaceae, SPC21112),
Clostridium celatum (family Clostridiaceae, SPC21278), Clostridium butyricum (family Clostridiaceae, SPC21367), and Clostridium glycolicum (family Peptostreptococcaceae, SPC21349) with a total anaerobic cfu/ml of 8×109, Treatment 11 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides Bacteroides ovatus (family Bacteroidaceae, SPC21141), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), Clostridium celatum (family Clostridiaceae, SPC21278), Clostridium butyricum (family Clostridiaceae, SPC21367), Clostridium glycolicum (family Peptostreptococcaceae, SPC21349), Dorea formicigerans (family Lachnospiraceae, SPC21308), Ruminococcus torques (family Ruminococcaceae, SPC21344), Eubacterium tenue (family Peptostreptococcaceae, SPC21391) and Eubacterium cylindroides (family Erysipelotrichacea, SPC21300) with a total anaerobic cfu/ml of 2.5×109. Treatment 12 is a bacterial composition comprising Blautia producta (family Lachnospiraceae, SPC2115104), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), and Clostridium celatum (family Clostridiaceae, SPC21278), with a total anaerobic cfu/ml of 2.1×107. Treatment 13 is a bacterial composition comprising Blautia producta (family Lachnospiraceae, SPC2115104), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), Clostridium celatum (family Clostridiaceae, SPC21278), Clostridium butyricum (family Clostridiaceae, SPC21367), and Clostridium glycolicum (family Peptostreptococcaceae, SPC21349) with a total anaerobic cfu/ml of 1.4×108. Treatment 14 is a bacterial composition comprising Blautia producta (family Lachnospiraceae, SPC2115104), Clostridium innocuum (family Erysipelotrichaceae,
SPC21112), Clostridium celatum (family Clostridiaceae, SPC21278), Clostridium butyricum (family Clostridiaceae, SPC21367), Clostridium glycolicum (family Peptostreptococcaceae, SPC21349), Dorea formicigerans (family Lachnospiraceae, SPC21308), Ruminococcus torques (family Ruminococcaceae, SPC21344), Eubacterium tenue (family Peptostreptococcaceae, SPC21391) and Eubacterium cylindroides (family Erysipelotrichacea, SPC21300) with a total anaerobic cfu/ml of 3.1×107. Treatment 15 is a bacterial composition comprising Escherichia coli (family Enterobacteriaceae, SPC21221), Enterococcus faecalis (family Enterococcaceae, SPC21240), Blautia producta (family Lachnospiraceae, SPC2115104), Bacteroides vulgatus (family Bacteroidaceae, SPC21192), Bacteroides thetaiotaomicron (family Bacteroidaceae, SPC21132), Bacteroides ovatus (family Bacteroidaceae, SPC21141), Clostridium innocuum (family Erysipelotrichaceae, SPC21112), Clostridium celatum (family Clostridiaceae, SPC21278), Clostridium butyricum (family Clostridiaceae, SPC21367), Clostridium glycolicum (family Peptostreptococcaceae, SPC21349), Dorea formicigerans (family Lachnospiraceae, SPC21308), Ruminococcus torques (family Ruminococcaceae, SPC21344), Eubacterium tenue (family Peptostreptococcaceae, SPC21391) and Eubacterium cylindroides (family Erysipelotrichacea, SPC21300) that was treated with 50% EtOH for 1 hour at 37C, centrifuged to pellet the bacterial cells and resuspended in sterile PBS at the equivalent original volume and having a total anaerobic cfu/ml of 1.3×107.
For each of the three demonstrations, scores for each treatment range from 0 to 9. Scores were calculated as follows. Groups of animals (N=10/group) were scored for % mortality. Mortality was normalized in a given demonstration by setting the observed mortality of the PBS vehicle control group=3. A treatment group was then scored based on the observed mortality for that group divided by the mortality of the PBS vehicle control times 3. Thus, if the mortality of the PBS vehicle group was 60% (score=3), then a treatment group with mortality of 30% would receive a score of 1.5. Similarly, mean weight loss on Day 3 was normalized to a score of 3 for the PBS vehicle control group. A score for each treatment was calculated by dividing the actual mean weight loss by the mean weight loss for the PBS vehicle control group and multiplying by 3. A similar procedure was used for the clinical scores evaluation. Finally, the individual values for mortality, mean weight loss on Day 3 and clinical scores were summed to give a final value of 0-9 for each treatment, with 0 being best (no death, no weight loss, no clinical symptoms) and 9 being equivalent to the vehicle control.
The identity of the bacterial 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 (described below in Example 3). 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 3 and 4. 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 3 & 4 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).
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 and may include, but will not be limited, to highly conserved “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.
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 (
The PCR is performed on commercially available thermocyclers such as a BioRad MyCycler™ 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 lx HotMasterMix (5PRIME, Gaithersburg, Md.), 200 nM of V4 515f adapt (AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCG GTAA (SEQ ID NO: 2035), IDT, Coralville, Iowa), and 200 nM of barcoded 806rbc (CAAGCAGAAGACGGCATACGAGAT 12bpGolayBarcode AGTCAGTCAGCCGGACTAC HVGGGTWTCTAAT (SEQ ID NOS 2036 and 2037, respectively, in order of appearance), 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 MyCycler™ 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-iT™ PicoGreen® 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.
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.
Mixtures of bacteria can include species that are in spore form. Germinating a spore fraction increases the number of viable bacteria 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.
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:
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 (
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 5 and 6). Individual colonies were picked and DNA isolated for full-length 16S sequencing to identify the species composition (e.g. see Examples 3 and 4). Analysis revealed that 22 species were observed in total (Table 4) 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.
To test the therapeutic potential of the bacterial composition such as but not limited to a spore population, 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 7) 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 5). 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.
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 bacterial composition such as but not limited to a spore population, spore preparation, vegetative cell population, 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 bacterial composition 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.
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.
To assess the efficacy of test articles like bacterial compositions including but not limited to a ethanol treated spore preparations (e.g. see Example 7) 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 AAAB and AAAC). 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 CU recurrence and no requirement for resumption of antibiotics (total of 8 patients). Additionally, there were no treatment-related serious adverse events.
The above protocol could be modified to deliver other bacterial compositions e.g. vegetative cells, spore preparations, combinations thereof.
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 6). 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.
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.
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.
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.
For sourcing of microbial cultures and for use as a positive control in in vivo studies, fresh gut microbiome samples, e.g. 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. Gut microbiome samples e.g. 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 microbiome sample was suspended in a solution, and/or fibrous and/or particulate materials were removed. A frozen aliquot containing a known weight of sample 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 sample until the material appeared homogeneous. The sample 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 sample material appears homogenous.
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 (Fichtel 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 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-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 37C to determine CFU/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 6 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.
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 7). 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 7).
It becomes immediately obvious 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.
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.
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, 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/6 J 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 examples 2, 3 and 4). 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 bacterial composition test article such as but not limited to an 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 3 and 4).
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 3 and 4).
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 bacterial composition test article to induce a healthy microbiome.
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 bacterial composition (test article) 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, blaSHV-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 were 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 bacterial composition (test article), ethanol treated, spore preparation (e.g. see example 7), 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. per, 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 AAAB and AAAC). Additional experiments are performed to examine whether the administration the bacterial composition (test article) 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.
Construction of Binary Pairs in a High-Throughput 96-Well Format.
To allow high-throughput screening of binary pairs, vials of −80° C. glycerol stock banks were thawed and diluted to 1e8 CFU/mL. 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, plates were removed from −80° C. and thawed at room temperature under anaerobic conditions when testing in an In vitro inhibition assay with Clostridium difficile.
Construction of Ternary Combinations in a High-Throughput 96-Well Format
To allow high-throughput screening of ternary combinations, vials of −80° C. glycerol stock banks were thawed and diluted to 1e8 CFU/mL. 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 an In vitro inhibition assay with Clostridium difficile.
Construction of an In Vitro Inhibition Assay to Screen for Ecobiotic™ Compositions Inhibitory to the Growth of Clostridium difficile
An overnight culture of Clostridium difficile was grown under anaerobic conditions in SweetB-FosIn or other suitable media for the growth of C. difficile. SweetB-FosIn 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 was diluted 100,000 fold into a complex media such as SweetB-FosIn which is suitable for the growth of a wide variety of anaerobic bacterial species. The diluted C. difficile mixture was then aliquoted to wells of a 96-well plate (180 uL to each well). 20 uL of a unique binary pair of potential inhibitory species was then added to each well at a final concentration of 1e6 CFU/mL of each species. Alternatively the assay can be tested with binary pairs 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 were included for a comparison to the growth of C. difficile without inhibition. Additional wells were used for controls that either inhibit or do not inhibit the growth of C. difficile. One example of a positive control that inhibits growth was a combination of Blautia producta, Clostridium bifermentans and Escherichia coli. One example of a control that shows reduced inhibition of C. difficile growth as a combination of Bacteroides thetaiotaomicron, Bacteroides ovatus and Bacteroides vulgatus. Plates were wrapped with parafilm and incubated for 24 hr at 37° C. under anaerobic conditions. After 24 hr the wells containing C. difficile alone were serially diluted and plated to determine titer. The 96-well plate was then frozen at −80 C before quantifying C. difficile by qPCR assay.
Construction of an In Vitro Inhibition Assay to Screen for Bacterial Compositions that Produce Diffusible Products Inhibitory to the Growth of Clostridium difficile Using a Filter Insert.
The In vitro inhibition assay described above was modified by using a 0.22 uM filter insert (Millipore™ MultiScreen™ 96-Well Assay Plates—Item MAGVS2210) in 96-well format to physically separate C. difficile from the bacterial compositions. The C. difficile was aliquoted into the 96-well plate while the bacterial compositions were aliquoted into media on the filter overlay. The nutrient media as in contact on both sides of the 0.22 uM filter, allowing exchange of nutrients, small molecules and many macromolecules (e.g., bacteriocins, cell-surface proteins, or polysaccharides) by diffusion. In this embodiment, after 24 hr incubation, the filter insert containing the bacterial compositions was removed. The plate containing C. difficile was then transferred to a 96-well plate reader suitable for measuring optical density (OD) at 600 nm. The growth of C. difficile in the presence of different bacterial compositions was compared based on the OD measurement.
Construction of an In Vitro Inhibition Assay to Screen for Bacterial Compositions Inhibitory to the Growth of Clostridium difficile Using Clostridium difficile Selective Media for Quantification
The In vitro inhibition assay described above can be modified to determine final C. difficile titer by serially diluting and plating to C. difficile selective media (Bloedt et al 2009) such as CCFA (cycloserine cefoxitin fructose agar, Anaerobe Systems), CDSA (Clostridium difficile selective agar, which is cycloserine cefoxitin mannitol agar, Becton Dickinson).
Quantification of C. difficile Using Quantitative PCR (qPCR) Standard Curve Preparation
The standard curve was generated from a well on each assay plate containing only pathogenic C. difficile grown in SweetB+FosIn media as provided herein and quantified by selective spot plating. Serial dilutions of the culture were performed in sterile phosphate-buffered saline. Genomic DNA was extracted from the standard curve samples along with the other wells.
Genomic DNA Extraction
Genomic DNA was extracted from 5 μl of each sample using a dilution, freeze/thaw, and heat lysis protocol. 5 μL of thawed samples were added to 45 μL of UltraPure water (Life Technologies, Carlsbad, Calif.) and mixed by pipetting. The plates with diluted samples were frozen at −20° C. until use for qPCR which includes a heated lysis step prior to amplification. Alternatively the genomic DNA could be 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.
qPCR Composition and Conditions
The qPCR reaction mixture contained lx 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 was 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 were added and the plate sealed with a Microseal ‘13’ Adhesive Seal (BioRad, Hercules, Calif.). The qPCR was performed on a BioRad C1000™ Thermal Cycler equipped with a CFX96®Real-Time System (BioRad, Hercules, Calif.). The thermocycling conditions were 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 could be performed with other standard methods known to those skilled in the art.
Data Analysis
The Cq value for each well on the FAM channel was determined by the CFX Manager™ 3.0 software. The log10(cfu/mL) of C. difficile each experimental sample was 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 was 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 was calculated for all replicates for each composition.
A histogram of the range and standard deviation of each composition was plotted. Ranges or standard deviations of the log inhibitions that were distinct from the overall distribution were examined as possible outliers. If the removal of a single log inhibition datum from one of the binary pairs that were identified in the histograms would bring the range or standard deviation in line with those from the majority of the samples, that datum was removed as an outlier, and the mean log inhibition was recalculated.
The pooled variance of all samples evaluated in the assay was estimated as the average of the sample variances weighted by the sample's degrees of freedom. The pooled standard error was 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 were determined by multiplying the pooled standard error to the z score corresponding to a given percentage threshold. Mean log inhibitions outside the confidence interval were considered to be inhibitory if positive or stimulatory if negative with the percent confidence corresponding to the interval used. Samples with mean log inhibition greater than the 99% confidence interval (C.I) of the null hypothesis are reported as ++++, those with a 95%<C.I. <99% as +++, those with a 90%<C.I. <95% as ++, those with a 80%<C.I. <90% as + while samples with mean log inhibition less than than the 99% confidence interval (C.I) of the null hypothesis are reported as −−−−, those with a 95%<C.I. <99% as −−−, those with a 90%<C.I. <95% as −−, those with a 80%<C.I. <90% as −.
Many binary pairs inhibit C. difficile Table 8. 622 of 989 combinations show inhibition with a confidence interval >80%; 545 of 989 with a C.I. >90%; 507 of 989 with a C.I. >95%; 430 of 989 with a C.I. of >99%. Non-limiting but exemplary binary pairs include those with mean log reduction greater than 0.366, e.g. Allistipes shahii paired with Blautia producta, Clostridium hathaweyi, or Colinsella aerofaciens, or Clostidium mayombei paired with C. innocuum, C. tertium, Colinsella aerofaciens, or any of the other 424 combinations shown in Table 8. Equally important, the In vitro inhibition assay describes binary pairs that do not effectively inhibit C. difficile. 188 of 989 combinations promote growth with >80% confidence; 52 of 989 show a lack of inhibition with >90% confidence; 22 of 989 show a lack of inhibition with >95% confidence; 3 of 989, including B. producta combined with Coprococcus catus, Alistipes shahii combined with Dorea formicigenerans, and Eubacterium rectale combined with Roseburia intestinalis, show a lack of inhibition with >99% confidence. 249 of 989 combinations are neutral in the assay, meaning they neither promote nor inhibit C. difficile growth to the limit of measurement.
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.).
The In vitro inhibition assay 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 9 with a score of ++++, such as Colinsella aerofaciens, Coprococcus comes, and Blautia producta. Equally important, the In vitro inhibition 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.
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.
Eubacterium saburreum
Eubacterium sp. oral clone IR009
Alicyclobacillus acidocaldarius
Clostridium baratii
Clostridium colicanis
Clostridium paraputrificum
Clostridium sardiniense
Eubacterium budayi
Eubacterium moniliforme
Eubacterium multiforme
Eubacterium nitritogenes
Anoxybacillus flavithermus
Bacillus aerophilus
Bacillus aestuarii
Bacillus amyloliquefaciens
Bacillus anthracis
Bacillus atrophaeus
Bacillus badius
Bacillus cereus
Bacillus circulans
Bacillus firmus
Bacillus flexus
Bacillus fordii
Bacillus halmapalus
Bacillus herbersteinensis
Bacillus idriensis
Bacillus lentus
Bacillus licheniformis
Bacillus megaterium
Bacillus nealsonii
Bacillus niabensis
Bacillus niacini
Bacillus pocheonensis
Bacillus pumilus
Bacillus safensis
Bacillus simplex
Bacillus sonorensis
Bacillus sp. 10403023
Bacillus sp. 2_A_57_CT2
Bacillus sp. 2008724126
Bacillus sp. 2008724139
Bacillus sp. 7_16AIA
Bacillus sp. AP8
Bacillus sp. B27(2008)
Bacillus sp. BT1B_CT2
Bacillus sp. GB1.1
Bacillus sp. GB9
Bacillus sp. HU19.1
Bacillus sp. HU29
Bacillus sp. HU33.1
Bacillus sp. JC6
Bacillus sp. oral taxon F79
Bacillus sp. SRC_DSF1
Bacillus sp. SRC_DSF10
Bacillus sp. SRC_DSF2
Bacillus sp. SRC_DSF6
Bacillus sp. tc09
Bacillus sp. zh168
Bacillus sphaericus
Bacillus sporothermodurans
Bacillus subtilis
Bacillus thermoamylovorans
Bacillus thuringiensis
Bacillus weihenstephanensis
Geobacillus kaustophilus
Geobacillus stearothermophilus
Geobacillus thermodenitrificans
Geobacillus thermoglucosidasius
Lysinibacillus sphaericus
Clostridium beijerinckii
Clostridium botulinum
Clostridium butyricum
Clostridium chauvoei
Clostridium favososporum
Clostridium histolyticum
Clostridium isatidis
Clostridium limosum
Clostridium sartagoforme
Clostridium septicum
Clostridium sp. 7_2_43FAA
Clostridium sporogenes
Clostridium tertium
Clostridium carnis
Clostridium celatum
Clostridium disporicum
Clostridium gasigenes
Clostridium quinii
Clostridium hylemonae
Clostridium scindens
Clostridium glycyrrhizinilyticum
Clostridium nexile
Coprococcus comes
Ruminococcus lactaris
Ruminococcus torques
Paenibacillus lautus
Paenibacillus polymyxa
Paenibacillus sp. HGF5
Paenibacillus sp. HGF7
Eubacterium sp. oral clone JI012
Alicyclobacillus contaminans
Alicyclobacillus herbarius
Alicyclobacillus pomorum
Blautia coccoides
Blautia glucerasea
Blautia glucerasei
Blautia hansenii
Blautia luti
Blautia producta
Blautia schinkii
Blautia sp. M25
Blautia stercoris
Blautia wexlerae
Bryantella formatexigens
Clostridium coccoides
Eubacterium cellulosolvens
Ruminococcus hansenii
Ruminococcus obeum
Ruminococcus sp. 5_1_39BFAA
Ruminococcus sp. K_1
Syntrophococcus sucromutans
Bacillus alcalophilus
Bacillus clausii
Bacillus gelatini
Bacillus halodurans
Bacillus sp. oral taxon F26
Clostridium innocuum
Clostridium sp. HGF2
Clostridium perfringens
Sarcina ventriculi
Clostridium bartlettii
Clostridium bifermentans
Clostridium ghonii
Clostridium glycolicum
Clostridium mayombei
Clostridium sordellii
Clostridium sp. MT4 E
Eubacterium tenue
Clostridium argentinense
Clostridium sp. JC122
Clostridium sp. NMBHI_1
Clostridium subterminale
Clostridium sulfidigenes
Dorea formicigenerans
Dorea longicatena
Ruminococcus gnavus
Ruminococcus sp. ID8
Blautia hydrogenotrophica
Lactonifactor longoviformis
Robinsoniella peoriensis
Eubacterium infirmum
Eubacterium sp. WAL 14571
Eubacterium biforme
Eubacterium cylindroides
Eubacterium dolichum
Eubacterium sp. 3_1_31
Eubacterium tortuosum
Bulleidia extructa
Solobacterium moorei
Coprococcus catus
Clostridium cochlearium
Clostridium malenominatum
Clostridium tetani
Acetivibrio ethanolgignens
Anaerosporobacter mobilis
Bacteroides pectinophilus
Clostridium aminovalericum
Clostridium phytofermentans
Eubacterium hallii
Eubacterium xylanophilum
Ruminococcus callidus
Ruminococcus champanellensis
Ruminococcus sp. 18P13
Ruminococcus sp. 9SE51
Anaerostipes caccae
Anaerostipes sp. 3_2_56FAA
Clostridium aerotolerans
Clostridium aldenense
Clostridium algidixylanolyticum
Clostridium amygdalinum
Clostridium asparagiforme
Clostridium bolteae
Clostridium celerecrescens
Clostridium citroniae
Clostridium clostridiiformes
Clostridium clostridioforme
Clostridium hathewayi
Clostridium indolis
Clostridium lavalense
Clostridium saccharolyticum
Clostridium sp. M62_1
Clostridium sp. SS2_1
Clostridium sphenoides
Clostridium symbiosum
Clostridium xylanolyticum
Eubacterium hadrum
Clostridium difficile
Eubacterium sp. AS15b
Eubacterium sp. OBRC9
Eubacterium sp. oral clone OH3A
Eubacterium yurii
Clostridium acetobutylicum
Clostridium algidicarnis
Clostridium cadaveris
Clostridium carboxidivorans
Clostridium estertheticum
Clostridium fallax
Clostridium felsineum
Clostridium frigidicarnis
Clostridium kluyveri
Clostridium magnum
Clostridium putrefaciens
Clostridium sp. HPB_46
Clostridium tyrobutyricum
Sutterella parvirubra
Acetanaerobacterium elongatum
Clostridium cellulosi
Ethanoligenens harbinense
Eubacterium rectale
Eubacterium sp. oral clone GI038
Lachnobacterium bovis
Roseburia cecicola
Roseburia faecalis
Roseburia faecis
Roseburia hominis
Roseburia intestinalis
Roseburia inulinivorans
Brevibacillus brevis
Brevibacillus laterosporus
Bacillus coagulans
Sporolactobacillus inulinus
Kocuria palustris
Nocardia farcinica
Bacillus sp. oral taxon F28
Catenibacterium mitsuokai
Clostridium sp. TM_40
Coprobacillus cateniformis
Coprobacillus sp. 29_1
Clostridium rectum
Eubacterium nodatum
Eubacterium saphenum
Eubacterium sp. oral clone JH012
Eubacterium sp. oral clone JS001
Faecalibacterium prausnitzii
Gemmiger formicilis
Subdoligranulum variabile
Clostridium sp. MLG055
Clostridium cocleatum
Clostridium ramosum
Clostridium saccharogumia
Clostridium spiroforme
Coprobacillus sp. D7
Clostridium sp. SY8519
Eubacterium ramulus
Erysipelothrix inopinata
Erysipelothrix rhusiopathiae
Erysipelothrix tonsillarum
Holdemania filiformis
Coxiella burnetii
Clostridium hiranonis
Clostridium irregulare
Clostridium orbiscindens
Clostridium sp. NML 04A032
Flavonifractor plautii
Pseudoflavonifractor capillosus
Acetivibrio cellulolyticus
Clostridium aldrichii
Clostridium clariflavum
Clostridium stercorarium
Clostridium straminisolvens
Clostridium thermocellum
Fusobacterium nucleatum
Eubacterium barkeri
Eubacterium callanderi
Eubacterium limosum
Anaerotruncus colihominis
Clostridium methylpentosum
Clostridium sp. YIT 12070
Hydrogenoanaerobacterium
saccharovorans
Ruminococcus albus
Ruminococcus flavefaciens
Clostridium haemolyticum
Clostridium novyi
Clostridium sp. LMG 16094
Eubacterium ventriosum
Bacteroides galacturonicus
Eubacterium eligens
Lachnospira multipara
Lachnospira pectinoschiza
Lactobacillus rogosae
Bacillus horti
Bacillus sp. 9_3AIA
Eubacterium brachy
Filifactor alocis
Filifactor villosus
Clostridium leptum
Clostridium sp. YIT 12069
Clostridium sporosphaeroides
Eubacterium coprostanoligenes
Ruminococcus bromii
Eubacterium siraeum
Clostridium viride
Oscillibacter sp. G2
Oscillibacter valericigenes
Oscillospira guilliermondii
Butyrivibrio crossotus
Clostridium sp. L2_50
Coprococcus eutactus
Coprococcus sp. ART55_1
Eubacterium ruminantium
Collinsella aerofaciens
Alkaliphilus metalliredigenes
Alkaliphilus oremlandii
Clostridium sticklandii
Turicibacter sanguinis
Fulvimonas sp. NML 060897
Desulfitobacterium frappieri
Desulfitobacterium hafniense
Desulfotomaculum nigrificans
Lutispora thermophila
Brachyspira pilosicoli
Eggerthella lenta
Streptomyces albus
Anaerofustis stercorihominis
Butyricicoccus pullicaecorum
Eubacterium desmolans
Papillibacter cinnamivorans
Sporobacter termitidis
Clostridium colinum
Clostridium lactatifermentans
Clostridium piliforme
Saccharomonospora viridis
Thermobifida fusca
Leptospira licerasiae
Moorella thermoacetica
Thermoanaerobacter
pseudethanolicus
Flexistipes sinusarabici
Gloeobacter violaceus
Eubacterium sp. oral clone JN088
Clostridium oroticum
Clostridium sp. D5
Eubacterium contortum
Eubacterium fissicatena
Corynebacterium coyleae
Corynebacterium mucifaciens
Corynebacterium ureicelerivorans
Corynebacterium appendicis
Corynebacterium genitalium
Corynebacterium glaucum
Corynebacterium imitans
Corynebacterium riegelii
Corynebacterium sp. L_2012475
Corynebacterium sp. NML
Corynebacterium sundsvallense
Corynebacterium tuscaniae
Prevotella maculosa
Prevotella oris
Prevotella salivae
Prevotella sp. ICM55
Prevotella sp. oral clone AA020
Prevotella sp. oral clone GI032
Prevotella sp. oral taxon G70
Prevotella corporis
Bacteroides sp. 4_1_36
Bacteroides sp. AR20
Bacteroides sp. D20
Bacteroides sp. F_4
Bacteroides uniformis
Prevotella nanceiensis
Prevotella sp. oral taxon 299
Prevotella bergensis
Prevotella buccalis
Prevotella timonensis
Prevotella oralis
Prevotella sp. SEQ072
Leuconostoc carnosum
Leuconostoc gasicomitatum
Leuconostoc inhae
Leuconostoc kimchii
Edwardsiella tarda
Photorhabdus asymbiotica
Psychrobacter arcticus
Psychrobacter cibarius
Psychrobacter cryohalolentis
Psychrobacter faecalis
Psychrobacter nivimaris
Psychrobacter pulmonis
Pseudomonas aeruginosa
Pseudomonas sp. 2_1_26
Corynebacterium confusum
Corynebacterium propinquum
Corynebacterium
pseudodiphtheriticum
Bartonella bacilliformis
Bartonella grahamii
Bartonella henselae
Bartonella quintana
Bartonella tamiae
Bartonella washoensis
Brucella abortus
Brucella canis
Brucella ceti
Brucella melitensis
Brucella microti
Brucella ovis
Brucella sp. 83_13
Brucella sp. BO1
Brucella suis
Ochrobactrum anthropi
Ochrobactrum intermedium
Ochrobactrum pseudintermedium
Prevotella genomosp. C2
Prevotella multisaccharivorax
Prevotella sp. oral clone
Prevotella sp. oral taxon 292
Prevotella sp. oral taxon 300
Prevotella marshii
Prevotella sp. oral clone IK053
Prevotella sp. oral taxon 781
Prevotella stercorea
Prevotella brevis
Prevotella ruminicola
Prevotella sp. sp24
Prevotella sp. sp34
Prevotella albensis
Prevotella copri
Prevotella oulorum
Prevotella sp. BI_42
Prevotella sp. oral clone P4PB_83
Prevotella sp. oral taxon G60
Prevotella amnii
Bacteroides caccae
Bacteroides finegoldii
Bacteroides intestinalis
Bacteroides sp. XB44A
Bifidobacterium adolescentis
Bifidobacterium angulatum
Bifidobacterium animalis
Bifidobacterium breve
Bifidobacterium catenulatum
Bifidobacterium dentium
Bifidobacterium gallicum
Bifidobacterium infantis
Bifidobacterium kashiwanohense
Bifidobacterium longum
Bifidobacterium
pseudocatenulatum
Bifidobacterium pseudolongum
Bifidobacterium scardovii
Bifidobacterium sp. HM2
Bifidobacterium sp. HMLN12
Bifidobacterium sp. M45
Bifidobacterium sp. MSX5B
Bifidobacterium sp. TM_7
Bifidobacterium thermophilum
Leuconostoc citreum
Leuconostoc lactis
Alicyclobacillus acidoterrestris
Alicyclobacillus cycloheptanicus
Acinetobacter baumannii
Acinetobacter calcoaceticus
Acinetobacter genomosp. C1
Acinetobacter haemolyticus
Acinetobacter johnsonii
Acinetobacter junii
Acinetobacter lwoffii
Acinetobacter parvus
Acinetobacter schindleri
Acinetobacter sp. 56A1
Acinetobacter sp. CIP 101934
Acinetobacter sp. CIP 102143
Acinetobacter sp. M16_22
Acinetobacter sp. RUH2624
Acinetobacter sp. SH024
Lactobacillus jensenii
Alcaligenes faecalis
Alcaligenes sp. CO14
Alcaligenes sp. S3
Oligella ureolytica
Oligella urethralis
Eikenella corrodens
Kingella denitrificans
Kingella genomosp. P1 oral cone
Kingella kingae
Kingella oralis
Kingella sp. oral clone ID059
Neisseria elongata
Neisseria genomosp. P2 oral clone
Neisseria sp. oral clone JC012
Neisseria sp. SMC_A9199
Simonsiella muelleri
Corynebacterium glucuronolyticum
Corynebacterium
pyruviciproducens
Rothia aeria
Rothia dentocariosa
Rothia sp. oral taxon 188
Corynebacterium accolens
Corynebacterium macginleyi
Corynebacterium
pseudogenitalium
Corynebacterium
tuberculostearicum
Lactobacillus casei
Lactobacillus paracasei
Lactobacillus zeae
Prevotella dentalis
Prevotella sp. oral clone ASCG10
Prevotella sp. oral clone HF050
Prevotella sp. oral clone ID019
Prevotella sp. oral clone IK062
Prevotella genomosp. P9 oral
Prevotella sp. oral clone AU069
Prevotella sp. oral clone CY006
Prevotella sp. oral clone FL019
Actinomyces genomosp. C1
Actinomyces genomosp. C2
Actinomyces genomosp. P1 oral
Actinomyces georgiae
Actinomyces israelii
Actinomyces massiliensis
Actinomyces meyeri
Actinomyces odontolyticus
Actinomyces orihominis
Actinomyces sp. CCUG 37290
Actinomyces sp. ICM34
Actinomyces sp. ICM41
Actinomyces sp. ICM47
Actinomyces sp. ICM54
Actinomyces sp. oral clone IP081
Actinomyces sp. oral taxon 178
Actinomyces sp. oral taxon 180
Actinomyces sp. TeJ5
Haematobacter sp. BC14248
Paracoccus denitrificans
Paracoccus marcusii
Grimontia hollisae
Shewanella putrefaciens
Afipia genomosp. 4
Rhodopseudomonas palustris
Methylobacterium extorquens
Methylobacterium podarium
Methylobacterium radiotolerans
Methylobacterium sp. 1sub
Methylobacterium sp. MM4
Achromobacter denitrificans
Achromobacter piechaudii
Achromobacter xylosoxidans
Bordetella bronchiseptica
Bordetella holmesii
Bordetella parapertussis
Bordetella pertussis
Microbacterium chocolatum
Microbacterium flavescens
Microbacterium lacticum
Microbacterium oleivorans
Microbacterium oxydans
Microbacterium paraoxydans
Microbacterium phyllosphaerae
Microbacterium schleiferi
Microbacterium sp. 768
Microbacterium sp. oral strain
Microbacterium testaceum
Corynebacterium atypicum
Corynebacterium mastitidis
Corynebacterium sp. NML
Mycobacterium elephantis
Mycobacterium paraterrae
Mycobacterium phlei
Mycobacterium sp. 1776
Mycobacterium sp. 1781
Mycobacterium sp. AQ1GA4
Mycobacterium sp. GN_10546
Mycobacterium sp. GN_10827
Mycobacterium sp. GN_11124
Mycobacterium sp. GN_9188
Mycobacterium sp. GR_2007_210
Anoxybacillus contaminans
Bacillus aeolius
Brevibacterium frigoritolerans
Geobacillus sp. E263
Geobacillus sp. WCH70
Geobacillus thermocatenulatus
Geobacillus thermoleovorans
Lysinibacillus fusiformis
Planomicrobium koreense
Sporosarcina newyorkensis
Sporosarcina sp. 2681
Ureibacillus composti
Ureibacillus suwonensis
Ureibacillus terrenus
Ureibacillus thermophilus
Ureibacillus thermosphaericus
Prevotella micans
Prevotella sp. oral clone DA058
Prevotella sp. SEQ053
Treponema socranskii
Treponema sp. 6:H:D15A_4
Treponema sp. oral taxon 265
Treponema sp. oral taxon G85
Porphyromonas endodontalis
Porphyromonas sp. oral clone
Porphyromonas sp. oral clone
Porphyromonas sp. oral clone
Porphyromonas sp. oral clone
Acidovorax sp. 98_63833
Comamonas sp. NSP5
Delftia acidovorans
Xenophilus aerolatus
Oribacterium sp. oral taxon 078
Oribacterium sp. oral taxon 102
Weissella cibaria
Weissella confusa
Weissella hellenica
Weissella kandleri
Weissella koreensis
Weissella paramesenteroides
Weissella sp. KLDS 7.0701
Mobiluncus curtisii
Enhydrobacter aerosaccus
Moraxella osloensis
Moraxella sp. GM2
Brevibacterium casei
Brevibacterium epidermidis
Brevibacterium sanguinis
Brevibacterium sp. H15
Acinetobacter radioresistens
Lactobacillus alimentarius
Lactobacillus farciminis
Lactobacillus kimchii
Lactobacillus nodensis
Lactobacillus tucceti
Pseudomonas mendocina
Pseudomonas pseudoalcaligenes
Pseudomonas sp. NP522b
Pseudomonas stutzeri
Paenibacillus barcinonensis
Paenibacillus barengoltzii
Paenibacillus chibensis
Paenibacillus cookii
Paenibacillus durus
Paenibacillus glucanolyticus
Paenibacillus lactis
Paenibacillus pabuli
Paenibacillus popilliae
Paenibacillus sp. CIP 101062
Paenibacillus sp. JC66
Paenibacillus sp. R_27413
Paenibacillus sp. R_27422
Paenibacillus timonensis
Rothia mucilaginosa
Rothia nasimurium
Prevotella sp. oral taxon 302
Prevotella sp. oral taxon F68
Prevotella tannerae
Porphyromonas asaccharolytica
Porphyromonas gingivalis
Porphyromonas macacae
Porphyromonas sp. UQD 301
Porphyromonas uenonis
Leptotrichia buccalis
Leptotrichia hofstadii
Leptotrichia sp. oral clone HE012
Leptotrichia sp. oral taxon 223
Bacteroides fluxus
Bacteroides helcogenes
Parabacteroides johnsonii
Parabacteroides merdae
Treponema denticola
Treponema genomosp. P5 oral
Treponema putidum
Treponema sp. oral clone
Treponema sp. oral taxon 247
Treponema sp. oral taxon 250
Treponema sp. oral taxon 251
Anaerococcus hydrogenalis
Anaerococcus sp. 8404299
Anaerococcus sp. gpac215
Anaerococcus vaginalis
Propionibacterium acidipropionici
Propionibacterium avidum
Propionibacterium granulosum
Propionibacterium jensenii
Propionibacterium propionicum
Propionibacterium sp. H456
Propionibacterium thoenii
Bifidobacterium bifidum
Leuconostoc mesenteroides
Leuconostoc
pseudomesenteroides
Johnsonella ignava
Propionibacterium acnes
Propionibacterium sp. 434_HC2
Propionibacterium sp. LG
Propionibacterium sp. S555a
Alicyclobacillus sp. CCUG 53762
Actinomyces cardiffensis
Actinomyces funkei
Actinomyces sp. HKU31
Actinomyces sp. oral taxon C55
Kerstersia gyiorum
Pigmentiphaga daeguensis
Aeromonas allosaccharophila
Aeromonas enteropelogenes
Aeromonas hydrophila
Aeromonas jandaei
Aeromonas salmonicida
Aeromonas trota
Aeromonas veronii
Marvinbryantia formatexigens
Rhodobacter sp. oral taxon C30
Rhodobacter sphaeroides
Lactobacillus antri
Lactobacillus coleohominis
Lactobacillus fermentum
Lactobacillus gastricus
Lactobacillus mucosae
Lactobacillus oris
Lactobacillus pontis
Lactobacillus reuteri
Lactobacillus sp. KLDS 1.0707
Lactobacillus sp. KLDS 1.0709
Lactobacillus sp. KLDS 1.0711
Lactobacillus sp. KLDS 1.0713
Lactobacillus sp. KLDS 1.0716
Lactobacillus sp. KLDS 1.0718
Lactobacillus sp. oral taxon 052
Lactobacillus vaginalis
Brevibacterium aurantiacum
Brevibacterium linens
Lactobacillus pentosus
Lactobacillus plantarum
Lactobacillus sp. KLDS 1.0702
Lactobacillus sp. KLDS 1.0703
Lactobacillus sp. KLDS 1.0704
Lactobacillus sp. KLDS 1.0705
Agrobacterium radiobacter
Agrobacterium tumefaciens
Corynebacterium argentoratense
Corynebacterium diphtheriae
Corynebacterium
pseudotuberculosis
Corynebacterium renale
Corynebacterium ulcerans
Aurantimonas coralicida
Aureimonas altamirensis
Lactobacillus acidipiscis
Lactobacillus salivarius
Lactobacillus sp. KLDS 1.0719
Lactobacillus buchneri
Lactobacillus genomosp. C1
Lactobacillus genomosp. C2
Lactobacillus hilgardii
Lactobacillus kefiri
Lactobacillus parabuchneri
Lactobacillus parakefiri
Lactobacillus curvatus
Lactobacillus sakei
Aneurinibacillus aneurinilyticus
Aneurinibacillus danicus
Aneurinibacillus migulanus
Aneurinibacillus terranovensis
Staphylococcus aureus
Staphylococcus auricularis
Staphylococcus capitis
Staphylococcus caprae
Staphylococcus carnosus
Staphylococcus cohnii
Staphylococcus condimenti
Staphylococcus epidermidis
Staphylococcus equorum
Staphylococcus haemolyticus
Staphylococcus hominis
Staphylococcus lugdunensis
Staphylococcus pasteuri
Staphylococcus pseudintermedius
Staphylococcus saccharolyticus
Staphylococcus saprophyticus
Staphylococcus sp. clone bottae7
Staphylococcus sp. H292
Staphylococcus sp. H780
Staphylococcus succinus
Staphylococcus warneri
Staphylococcus xylosus
Cardiobacterium hominis
Cardiobacterium valvarum
Pseudomonas fluorescens
Pseudomonas gessardii
Pseudomonas monteilii
Pseudomonas poae
Pseudomonas putida
Pseudomonas sp. G1229
Pseudomonas tolaasii
Pseudomonas viridiflava
Listeria grayi
Listeria innocua
Listeria ivanovii
Listeria monocytogenes
Listeria welshimeri
Capnocytophaga sp. oral clone
Capnocytophaga sputigena
Leptotrichia genomosp. C1
Leptotrichia shahii
Leptotrichia sp. neutropenic Patient
Leptotrichia sp. oral clone GT018
Leptotrichia sp. oral clone GT020
Bacteroides sp. 20_3
Bacteroides sp. 3_1_19
Bacteroides sp. 3_2_5
Parabacteroides distasonis
Parabacteroides goldsteinii
Parabacteroides gordonii
Parabacteroides sp. D13
Capnocytophaga genomosp. C1
Capnocytophaga ochracea
Capnocytophaga sp. GEJ8
Capnocytophaga sp. oral strain
Capnocytophaga sp. S1b
Paraprevotella clara
Bacteroides heparinolyticus
Prevotella heparinolytica
Treponema genomosp. P4 oral
Treponema genomosp. P6 oral
Treponema sp. oral taxon 254
Treponema sp. oral taxon 508
Treponema sp. oral taxon 518
Chlamydia muridarum
Chlamydia trachomatis
Chlamydia psittaci
Chlamydophila pneumoniae
Chlamydophila psittaci
Anaerococcus octavius
Anaerococcus sp. 8405254
Anaerococcus sp. 9401487
Anaerococcus sp. 9403502
Gardnerella vaginalis
Campylobacter lari
Anaerobiospirillum
succiniciproducens
Anaerobiospirillum thomasii
Ruminobacter amylophilus
Succinatimonas hippei
Actinomyces europaeus
Actinomyces sp. oral clone GU009
Moraxella catarrhalis
Moraxella lincolnii
Moraxella sp. 16285
Psychrobacter sp. 13983
Actinobaculum massiliae
Actinobaculum schaalii
Actinobaculum sp. BM#101342
Actinobaculum sp. P2P_19 P1
Actinomyces sp. oral clone IO076
Actinomyces sp. oral taxon 848
Actinomyces neuii
Mobiluncus mulieris
Blastomonas natatoria
Novosphingobium aromaticivorans
Sphingomonas sp. oral clone
Sphingopyxis alaskensis
Oxalobacter formigenes
Veillonella atypica
Veillonella dispar
Veillonella genomosp. P1 oral
Veillonella parvula
Veillonella sp. 3_1_44
Veillonella sp. 6_1_27
Veillonella sp. ACP1
Veillonella sp. AS16
Veillonella sp. BS32b
Veillonella sp. ICM51a
Veillonella sp. MSA12
Veillonella sp. NVG 100cf
Veillonella sp. OK11
Veillonella sp. oral clone ASCG01
Veillonella sp. oral clone ASCG02
Veillonella sp. oral clone OH1A
Veillonella sp. oral taxon 158
Kocuria marina
Kocuria rhizophila
Kocuria rosea
Kocuria varians
Micrococcus antarcticus
Micrococcus luteus
Micrococcus lylae
Micrococcus sp. 185
Lactobacillus brevis
Lactobacillus parabrevis
Pediococcus acidilactici
Pediococcus pentosaceus
Lactobacillus dextrinicus
Lactobacillus perolens
Lactobacillus rhamnosus
Lactobacillus saniviri
Lactobacillus sp. BT6
Mycobacterium mageritense
Mycobacterium neoaurum
Mycobacterium smegmatis
Mycobacterium sp. HE5
Dysgonomonas gadei
Dysgonomonas mossii
Porphyromonas levii
Porphyromonas somerae
Bacteroides barnesiae
Bacteroides coprocola
Bacteroides coprophilus
Bacteroides dorei
Bacteroides massiliensis
Bacteroides plebeius
Bacteroides sp. 3_1_33FAA
Bacteroides sp. 3_1_40A
Bacteroides sp. 4_3_47FAA
Bacteroides sp. 9_1_42FAA
Bacteroides sp. NB_8
Bacteroides vulgatus
Bacteroides ovatus
Bacteroides sp. 1_1_30
Bacteroides sp. 2_1_22
Bacteroides sp. 2_2_4
Bacteroides sp. 3_1_23
Bacteroides sp. D1
Bacteroides sp. D2
Bacteroides sp. D22
Bacteroides xylanisolvens
Treponema lecithinolyticum
Treponema parvum
Treponema sp. oral clone JU025
Treponema sp. oral taxon 270
Parascardovia denticolens
Scardovia inopinata
Scardovia wiggsiae
Mogibacterium diversum
Mogibacterium neglectum
Mogibacterium pumilum
Mogibacterium timidum
Borrelia burgdorferi
Borrelia garinii
Borrelia sp. NE49
Caldimonas manganoxidans
Lautropia mirabilis
Lautropia sp. oral clone AP009
Peptoniphilus asaccharolyticus
Peptoniphilus duerdenii
Peptoniphilus harei
Peptoniphilus indolicus
Peptoniphilus lacrimalis
Peptoniphilus sp. gpac077
Peptoniphilus sp. JC140
Peptoniphilus sp. oral taxon 386
Peptoniphilus sp. oral taxon 836
Dialister pneumosintes
Dialister sp. oral taxon 502
Cupriavidus metallidurans
Herbaspirillum seropedicae
Herbaspirillum sp. JC206
Janthinobacterium sp. SY12
Massilia sp. CCUG 43427A
Ralstonia pickettii
Ralstonia sp. 5_7_47FAA
Francisella novicida
Francisella philomiragia
Francisella tularensis
Ignatzschineria indica
Ignatzschineria sp. NML 95_0260
Streptococcus mutans
Lactobacillus gasseri
Lactobacillus hominis
Lactobacillus iners
Lactobacillus johnsonii
Lactobacillus senioris
Lactobacillus sp. oral clone HT002
Weissella beninensis
Sphingomonas echinoides
Sphingomonas sp. oral taxon A09
Sphingomonas sp. oral taxon F71
Zymomonas mobilis
Arcanobacterium haemolyticum
Arcanobacterium pyogenes
Trueperella pyogenes
Lactococcus garvieae
Lactococcus lactis
Brevibacterium mcbrellneri
Brevibacterium paucivorans
Brevibacterium sp. JC43
Selenomonas artemidis
Selenomonas sp. FOBRC9
Selenomonas sp. oral taxon 137
Desmospora activa
Desmospora sp. 8437
Paenibacillus sp. oral taxon F45
Corynebacterium ammoniagenes
Corynebacterium aurimucosum
Corynebacterium bovis
Corynebacterium canis
Corynebacterium casei
Corynebacterium durum
Corynebacterium efficiens
Corynebacterium falsenii
Corynebacterium flavescens
Corynebacterium glutamicum
Corynebacterium jeikeium
Corynebacterium kroppenstedtii
Corynebacterium lipophiloflavum
Corynebacterium matruchotii
Corynebacterium minutissimum
Corynebacterium resistens
Corynebacterium simulans
Corynebacterium singulare
Corynebacterium sp. 1 ex sheep
Corynebacterium sp. NML
Corynebacterium striatum
Corynebacterium urealyticum
Corynebacterium variabile
Aerococcus sanguinicola
Aerococcus urinae
Aerococcus urinaeequi
Aerococcus viridans
Fusobacterium naviforme
Moryella indoligenes
Selenomonas genomosp. P5
Selenomonas sp. oral clone IQ048
Selenomonas sputigena
Hyphomicrobium sulfonivorans
Methylocella silvestris
Legionella pneumophila
Lactobacillus coryniformis
Arthrobacter agilis
Arthrobacter arilaitensis
Arthrobacter bergerei
Arthrobacter globiformis
Arthrobacter nicotianae
Mycobacterium abscessus
Mycobacterium chelonae
Bacteroides salanitronis
Paraprevotella xylaniphila
Barnesiella intestinihominis
Barnesiella viscericola
Parabacteroides sp. NS31_3
Tannerella forsythia
Tannerella sp. 6_1_58FAA_CT1
Mycoplasma amphoriforme
Mycoplasma genitalium
Mycoplasma pneumoniae
Mycoplasma penetrans
Ureaplasma parvum
Ureaplasma urealyticum
Treponema genomosp. P1
Treponema sp. oral taxon 228
Treponema sp. oral taxon 230
Treponema sp. oral taxon 231
Treponema sp. oral taxon 232
Treponema sp. oral taxon 235
Treponema sp. ovine footrot
Treponema vincentii
Parasutterella excrementihominis
Parasutterella secunda
Sutterella morbirenis
Sutterella sanguinus
Sutterella sp. YIT 12072
Sutterella stercoricanis
Sutterella wadsworthensis
Propionibacterium freudenreichii
Propionibacterium sp. oral taxon
Tessaracoccus sp. oral taxon F04
Peptoniphilus ivorii
Peptoniphilus sp. gpac007
Peptoniphilus sp. gpac018A
Peptoniphilus sp. gpac148
Flexispira rappini
Helicobacter bilis
Helicobacter cinaedi
Helicobacter sp. None
Brevundimonas subvibrioides
Hyphomonas neptunium
Phenylobacterium zucineum
Streptococcus downei
Streptococcus sp. SHV515
Acinetobacter sp. CIP 53.82
Halomonas elongata
Halomonas johnsoniae
Butyrivibrio fibrisolvens
Roseburia sp. 11SE37
Roseburia sp. 11SE38
Shuttleworthia satelles
Shuttleworthia sp. MSX8B
Shuttleworthia sp. oral taxon G69
Bdellovibrio sp. MPA
Desulfobulbus sp. oral clone
Desulfovibrio desulfuricans
Desulfovibrio fairfieldensis
Desulfovibrio piger
Desulfovibrio sp. 3_1_syn3
Geobacter bemidjiensis
Brachybacterium alimentarium
Brachybacterium conglomeratum
Brachybacterium tyrofermentans
Dermabacter hominis
Aneurinibacillus thermoaerophilus
Brevibacillus agri
Brevibacillus centrosporus
Brevibacillus choshinensis
Brevibacillus invocatus
Brevibacillus parabrevis
Brevibacillus reuszeri
Brevibacillus sp. phR
Brevibacillus thermoruber
Lactobacillus murinus
Lactobacillus oeni
Lactobacillus ruminis
Lactobacillus vini
Gemella haemolysans
Gemella morbillorum
Gemella morbillorum
Gemella sanguinis
Gemella sp. oral clone ASCE02
Gemella sp. oral clone ASCF04
Gemella sp. oral clone ASCF12
Gemella sp. WAL 1945J
Sporolactobacillus nakayamae
Gluconacetobacter entanii
Gluconacetobacter europaeus
Gluconacetobacter hansenii
Gluconacetobacter oboediens
Gluconacetobacter xylinus
Auritibacter ignavus
Dermacoccus sp. Ellin185
Janibacter limosus
Janibacter melonis
Acetobacter aceti
Acetobacter fabarum
Acetobacter lovaniensis
Acetobacter malorum
Acetobacter orientalis
Acetobacter pasteurianus
Acetobacter pomorum
Acetobacter syzygii
Acetobacter tropicalis
Gluconacetobacter azotocaptans
Gluconacetobacter diazotrophicus
Gluconacetobacter johannae
Nocardia brasiliensis
Nocardia cyriacigeorgica
Nocardia puris
Nocardia sp. 01_Je_025
Rhodococcus equi
Oceanobacillus caeni
Oceanobacillus sp. Ndiop
Ornithinibacillus bavariensis
Ornithinibacillus sp. 7_10AIA
Virgibacillus proomii
Corynebacterium amycolatum
Corynebacterium hansenii
Corynebacterium xerosis
Staphylococcus fleurettii
Staphylococcus sciuri
Staphylococcus vitulinus
Stenotrophomonas maltophilia
Stenotrophomonas sp. FG_6
Mycobacterium africanum
Mycobacterium alsiensis
Mycobacterium avium
Mycobacterium colombiense
Mycobacterium gordonae
Mycobacterium intracellulare
Mycobacterium kansasii
Mycobacterium lacus
Mycobacterium leprae
Mycobacterium lepromatosis
Mycobacterium mantenii
Mycobacterium marinum
Mycobacterium microti
Mycobacterium parascrofulaceum
Mycobacterium seoulense
Mycobacterium sp. 1761
Mycobacterium sp. 1791
Mycobacterium sp. 1797
Mycobacterium sp.
Mycobacterium sp. NLA001000736
Mycobacterium sp. W
Mycobacterium tuberculosis
Mycobacterium ulcerans
Mycobacterium vulneris
Xanthomonas campestris
Xanthomonas sp. kmd_489
Dietzia natronolimnaea
Dietzia sp. BBDP51
Dietzia sp. CA149
Dietzia timorensis
Gordonia bronchialis
Gordonia polyisoprenivorans
Gordonia sp. KTR9
Gordonia sputi
Gordonia terrae
Leptotrichia goodfellowii
Leptotrichia sp. oral clone IK040
Leptotrichia sp. oral clone
Butyricimonas virosa
Odoribacter laneus
Odoribacter splanchnicus
Capnocytophaga gingivalis
Capnocytophaga granulosa
Capnocytophaga sp. oral clone
Capnocytophaga sp. oral strain S3
Capnocytophaga sp. oral taxon
Capnocytophaga canimorsus
Capnocytophaga sp. oral clone
Lactobacillus catenaformis
Lactobacillus vitulinus
Cetobacterium somerae
Fusobacterium gonidiaformans
Fusobacterium mortiferum
Fusobacterium necrogenes
Fusobacterium necrophorum
Fusobacterium sp. 12_1B
Fusobacterium sp. 3_1_5R
Fusobacterium sp. D12
Fusobacterium ulcerans
Fusobacterium varium
Mycoplasma arthritidis
Mycoplasma faucium
Mycoplasma hominis
Mycoplasma orale
Mycoplasma salivarium
Mitsuokella jalaludinii
Mitsuokella multacida
Mitsuokella sp. oral taxon 521
Mitsuokella sp. oral taxon G68
Selenomonas genomosp. C1
Selenomonas genomosp. P8 oral
Selenomonas ruminantium
Alloscardovia omnicolens
Alloscardovia sp. OB7196
Bifidobacterium urinalis
Prevotella loescheii
Prevotella sp. oral clone ASCG12
Prevotella sp. oral clone GU027
Prevotella sp. oral taxon 472
Selenomonas dianae
Selenomonas flueggei
Selenomonas genomosp. C2
Selenomonas genomosp. P6 oral
Selenomonas genomosp. P7 oral
Selenomonas infelix
Selenomonas noxia
Selenomonas sp. oral clone FT050
Selenomonas sp. oral clone GI064
Selenomonas sp. oral clone
Selenomonas sp. oral clone
Selenomonas sp. oral clone IK004
Selenomonas sp. oral clone JI021
Selenomonas sp. oral clone JS031
Selenomonas sp. oral clone OH4A
Selenomonas sp. oral clone
Selenomonas sp. oral taxon 149
Agrococcus jenensis
Microbacterium gubbeenense
Pseudoclavibacter sp. Timone
Tropheryma whipplei
Zimmermannella bifida
Legionella hackeliae
Legionella longbeachae
Legionella sp. D3923
Legionella sp. D4088
Legionella sp. H63
Legionella sp. NML 93L054
Legionella steelei
Tatlockia micdadei
Helicobacter pullorum
Roseomonas cervicalis
Roseomonas mucosa
Roseomonas sp. NML94_0193
Roseomonas sp. NML97_0121
Roseomonas sp. NML98_0009
Roseomonas sp. NML98_0157
Rickettsia akari
Rickettsia conorii
Rickettsia prowazekii
Rickettsia rickettsii
Rickettsia slovaca
Rickettsia typhi
Anaeroglobus geminatus
Megasphaera genomosp. C1
Megasphaera micronuciformis
Tsukamurella paurometabola
Tsukamurella tyrosinosolvens
Abiotrophia para_adiacens
Carnobacterium divergens
Carnobacterium maltaromaticum
Enterococcus avium
Enterococcus caccae
Enterococcus casseliflavus
Enterococcus durans
Enterococcus faecalis
Enterococcus faecium
Enterococcus gallinarum
Enterococcus gilvus
Enterococcus hawaiiensis
Enterococcus hirae
Enterococcus italicus
Enterococcus mundtii
Enterococcus raffinosus
Enterococcus sp. BV2CASA2
Enterococcus sp. CCRI_16620
Enterococcus sp. F95
Enterococcus sp. RfL6
Enterococcus thailandicus
Fusobacterium canifelinum
Fusobacterium genomosp. C1
Fusobacterium genomosp. C2
Fusobacterium periodonticum
Fusobacterium sp. 1_1_41FAA
Fusobacterium sp. 11_3_2
Fusobacterium sp. 2_1_31
Fusobacterium sp. 3_1_27
Fusobacterium sp. 3_1_33
Fusobacterium sp. 3_1_36A2
Fusobacterium sp. AC18
Fusobacterium sp. ACB2
Fusobacterium sp. AS2
Fusobacterium sp. CM1
Fusobacterium sp. CM21
Fusobacterium sp. CM22
Fusobacterium sp. oral clone
Fusobacterium sp. oral clone
Granulicatella adiacens
Granulicatella elegans
Granulicatella paradiacens
Granulicatella sp. oral clone
Granulicatella sp. oral clone
Granulicatella sp. oral clone
Granulicatella sp. oral clone
Tetragenococcus halophilus
Tetragenococcus koreensis
Vagococcus fluvialis
Chryseobacterium anthropi
Chryseobacterium gleum
Chryseobacterium hominis
Treponema refringens
Treponema sp. oral clone JU031
Treponema sp. oral taxon 239
Treponema sp. oral taxon 271
Alistipes finegoldii
Alistipes onderdonkii
Alistipes putredinis
Alistipes shahii
Alistipes sp. HGB5
Alistipes sp. JC50
Alistipes sp. RMA 9912
Mycoplasma agalactiae
Mycoplasma bovoculi
Mycoplasma fermentans
Mycoplasma flocculare
Mycoplasma ovipneumoniae
Arcobacter butzleri
Arcobacter cryaerophilus
Campylobacter curvus
Campylobacter rectus
Campylobacter showae
Campylobacter sp. FOBRC14
Campylobacter sp. FOBRC15
Campylobacter sp. oral clone
Campylobacter sputorum
Bacteroides ureolyticus
Campylobacter gracilis
Campylobacter hominis
Dialister invisus
Dialister micraerophilus
Dialister microaerophilus
Dialister propionicifaciens
Dialister succinatiphilus
Megasphaera elsdenii
Megasphaera genomosp. type_1
Megasphaera sp. BLPYG_07
Megasphaera sp. UPII 199_6
Chromobacterium violaceum
Laribacter hongkongensis
Methylophilus sp. ECd5
Finegoldia magna
Parvimonas micra
Parvimonas sp. oral taxon 110
Peptostreptococcus micros
Peptostreptococcus sp. oral clone
Peptostreptococcus sp. P4P_31
Helicobacter pylori
Anaplasma marginale
Anaplasma phagocytophilum
Ehrlichia chaffeensis
Neorickettsia risticii
Neorickettsia sennetsu
Pseudoramibacter alactolyticus
Veillonella montpellierensis
Veillonella sp. oral clone ASCA08
Veillonella sp. oral clone ASCB03
Inquilinus limosus
Sphingomonas sp. oral clone
Anaerococcus lactolyticus
Anaerococcus prevotii
Anaerococcus sp. gpac104
Anaerococcus sp. gpac126
Anaerococcus sp. gpac155
Anaerococcus sp. gpac199
Anaerococcus tetradius
Bacteroides coagulans
Peptostreptococcus sp. 9succ1
Peptostreptococcus sp. oral clone
Tissierella praeacuta
Helicobacter canadensis
Peptostreptococcus anaerobius
Peptostreptococcus stomatis
Bilophila wadsworthia
Desulfovibrio vulgaris
Actinomyces nasicola
Cellulosimicrobium funkei
Lactococcus raffinolactis
Flavobacterium sp. NF2_1
Myroides odoratimimus
Myroides sp. MY15
Chlamydophila pecorum
Parachlamydia sp. UWE25
Fusobacterium russii
Streptobacillus moniliformis
Abiotrophia defectiva
Abiotrophia sp. oral clone
Catonella genomosp. P1 oral clone
Catonella morbi
Catonella sp. oral clone FL037
Eremococcus coleocola
Facklamia hominis
Granulicatella sp. M658_99_3
Campylobacter coli
Campylobacter concisus
Campylobacter fetus
Campylobacter jejuni
Campylobacter upsaliensis
Atopobium minutum
Atopobium parvulum
Atopobium rimae
Atopobium sp. BS2
Atopobium sp. F0209
Atopobium sp. ICM42b10
Atopobium sp. ICM57
Atopobium vaginae
Actinomyces naeslundii
Actinomyces oricola
Actinomyces oris
Actinomyces sp. 7400942
Actinomyces sp. ChDC B197
Actinomyces sp. GEJ15
Actinomyces sp. M2231_94_1
Actinomyces sp. oral clone GU067
Actinomyces sp. oral clone IO077
Actinomyces sp. oral clone IP073
Actinomyces sp. oral clone JA063
Actinomyces sp. oral taxon 170
Actinomyces sp. oral taxon 171
Actinomyces urogenitalis
Actinomyces viscosus
Orientia tsutsugamushi
Megamonas funiformis
Megamonas hypermegale
Aeromicrobium marinum
Aeromicrobium sp. JC14
Luteococcus sanguinis
Rhodococcus corynebacterioides
Rhodococcus erythropolis
Rhodococcus fascians
Segniliparus rotundus
Segniliparus rugosus
Exiguobacterium acetylicum
Macrococcus caseolyticus
Streptomyces sp. 1 AIP_2009
Streptomyces sp. SD 524
Streptomyces sp. SD 528
Streptomyces thermoviolaceus
Borrelia afzelii
Borrelia crocidurae
Borrelia duttonii
Borrelia hermsii
Borrelia hispanica
Borrelia persica
Borrelia recurrentis
Borrelia spielmanii
Borrelia turicatae
Borrelia valaisiana
Providencia alcalifaciens
Providencia rettgeri
Providencia rustigianii
Providencia stuartii
Treponema pallidum
Treponema phagedenis
Treponema sp. clone DDKL_4
Acholeplasma laidlawii
Mycoplasma putrefaciens
Spiroplasma insolitum
Collinsella intestinalis
Collinsella stercoris
Collinsella tanakaei
Caminicella sporogenes
Acidaminococcus fermentans
Acidaminococcus intestini
Acidaminococcus sp. D21
Phascolarctobacterium faecium
Phascolarctobacterium sp. YIT
Phascolarctobacterium
Acidithiobacillus ferrivorans
Catabacter hongkongensis
Christensenella minuta
Heliobacterium modesticaldum
Alistipes indistinctus
Candidatus Sulcia muelleri
Cytophaga xylanolytica
Gramella forsetii
Sphingobacterium faecium
Sphingobacterium mizutaii
Sphingobacterium multivorum
Sphingobacterium spiritivorum
Jonquetella anthropi
Pyramidobacter piscolens
Candidatus Arthromitus sp.
Gracilibacter thermotolerans
Brachyspira aalborgi
Brachyspira sp. HIS3
Brachyspira sp. HIS4
Brachyspira sp. HIS5
Adlercreutzia equolifaciens
Cryptobacterium curtum
Eggerthella sinensis
Eggerthella sp. 1_3_56FAA
Eggerthella sp. HGA1
Eggerthella sp. YY7918
Gordonibacter pamelaeae
Gordonibacter pamelaeae
Slackia equolifaciens
Slackia exigua
Slackia faecicanis
Slackia heliotrinireducens
Slackia isoflavoniconvertens
Slackia piriformis
Slackia sp. NATTS
Victivallis vadensis
Streptomyces griseus
Streptomyces sp. SD 511
Streptomyces sp. SD 534
Cloacibacillus evryensis
Deferribacteres sp. oral clone
Deferribacteres sp. oral clone
Peptococcus sp. oral clone JM048
Helicobacter winghamensis
Wolinella succinogenes
Olsenella genomosp. C1
Olsenella profusa
Olsenella sp. F0004
Olsenella sp. oral taxon 809
Olsenella uli
Nocardiopsis dassonvillei
Peptococcus niger
Peptococcus sp. oral taxon 167
Akkermansia muciniphila
Opitutus terrae
Leptospira borgpetersenii
Leptospira broomii
Leptospira interrogans
Methanobrevibacter gottschalkii
Methanobrevibacter millerae
Methanobrevibacter oralis
Methanobrevibacter thaueri
Methanobrevibacter smithii
Deinococcus radiodurans
Deinococcus sp. R_43890
Thermus aquaticus
Actinomyces sp. c109
Anaerobaculum hydrogeniformans
Microcystis aeruginosa
Prochlorococcus marinus
Methanobrevibacter acididurans
Methanobrevibacter arboriphilus
Methanobrevibacter curvatus
Methanobrevibacter cuticularis
Methanobrevibacter filiformis
Methanobrevibacter woesei
Roseiflexus castenholzii
Methanobrevibacter olleyae
Methanobrevibacter ruminantium
Methanobrevibacter wolinii
Methanosphaera stadtmanae
Halorubrum lipolyticum
Methanobacterium formicicum
Acidilobus saccharovorans
Hyperthermus butylicus
Ignicoccus islandicus
Metallosphaera sedula
Thermofilum pendens
Prevotella melaninogenica
Prevotella sp. ICM1
Prevotella sp. oral clone FU048
Prevotella sp. oral clone GI030
Prevotella sp. SEQ116
Streptococcus anginosus
Streptococcus milleri
Streptococcus sp. 16362
Streptococcus sp. 69130
Streptococcus sp. AC15
Streptococcus sp. CM7
Streptococcus sp. OBRC6
Burkholderia ambifaria
Burkholderia cenocepacia
Burkholderia cepacia
Burkholderia mallei
Burkholderia multivorans
Burkholderia oklahomensis
Burkholderia pseudomallei
Burkholderia rhizoxinica
Burkholderia sp. 383
Burkholderia xenovorans
Prevotella buccae
Prevotella genomosp. P8 oral
Prevotella sp. oral clone FW035
Prevotella bivia
Prevotella disiens
Bacteroides faecis
Bacteroides fragilis
Bacteroides nordii
Bacteroides salyersiae
Bacteroides sp. 1_1_14
Bacteroides sp. 1_1_6
Bacteroides sp. 2_1_56FAA
Bacteroides sp. AR29
Bacteroides sp. B2
Bacteroides thetaiotaomicron
Actinobacillus minor
Haemophilus parasuis
Vibrio cholerae
Vibrio fluvialis
Vibrio furnissii
Vibrio mimicus
Vibrio parahaemolyticus
Vibrio sp. RC341
Vibrio vulnificus
Lactobacillus acidophilus
Lactobacillus amylolyticus
Lactobacillus amylovorus
Lactobacillus crispatus
Lactobacillus delbrueckii
Lactobacillus helveticus
Lactobacillus kalixensis
Lactobacillus kefiranofaciens
Lactobacillus leichmannii
Lactobacillus sp. 66c
Lactobacillus sp. KLDS 1.0701
Lactobacillus sp. KLDS 1.0712
Lactobacillus sp. oral clone HT070
Lactobacillus ultunensis
Prevotella intermedia
Prevotella nigrescens
Prevotella pallens
Prevotella sp. oral taxon 310
Prevotella genomosp. C1
Prevotella sp. CM38
Prevotella sp. oral taxon 317
Prevotella sp. SG12
Prevotella denticola
Prevotella genomosp. P7 oral
Prevotella histicola
Prevotella multiformis
Prevotella sp. JCM 6330
Prevotella sp. oral clone GI059
Prevotella sp. oral taxon 782
Prevotella sp. oral taxon G71
Prevotella sp. SEQ065
Prevotella veroralis
Bacteroides acidifaciens
Bacteroides cellulosilyticus
Bacteroides clarus
Bacteroides eggerthii
Bacteroides oleiciplenus
Bacteroides pyogenes
Bacteroides sp. 315_5
Bacteroides sp. 31SF15
Bacteroides sp. 31SF18
Bacteroides sp. 35AE31
Bacteroides sp. 35AE37
Bacteroides sp. 35BE34
Bacteroides sp. 35BE35
Bacteroides sp. WH2
Bacteroides sp. XB12B
Bacteroides stercoris
Actinobacillus pleuropneumoniae
Actinobacillus ureae
Haemophilus aegyptius
Haemophilus ducreyi
Haemophilus haemolyticus
Haemophilus influenzae
Haemophilus parahaemolyticus
Haemophilus parainfluenzae
Haemophilus
paraphrophaemolyticus
Haemophilus somnus
Haemophilus sp. 70334
Haemophilus sp. HK445
Haemophilus sp. oral clone
Haemophilus sp. oral clone
Haemophilus sp. oral clone BJ021
Haemophilus sp. oral clone BJ095
Haemophilus sp. oral taxon 851
Haemophilus sputorum
Histophilus somni
Mannheimia haemolytica
Pasteurella bettyae
Moellerella wisconsensis
Morganella morganii
Morganella sp. JB_T16
Proteus mirabilis
Proteus penneri
Proteus sp. HS7514
Proteus vulgaris
Oribacterium sinus
Oribacterium sp. ACB1
Oribacterium sp. ACB7
Oribacterium sp. CM12
Oribacterium sp. ICM51
Oribacterium sp. OBRC12
Oribacterium sp. oral taxon 108
Actinobacillus
actinomycetemcomitans
Actinobacillus succinogenes
Aggregatibacter
actinomycetemcomitans
Aggregatibacter aphrophilus
Aggregatibacter segnis
Averyella dalhousiensis
Bisgaard Taxon
Bisgaard Taxon
Bisgaard Taxon
Bisgaard Taxon
Buchnera aphidicola
Cedecea davisae
Citrobacter amalonaticus
Citrobacter braakii
Citrobacter farmeri
Citrobacter freundii
Citrobacter gillenii
Citrobacter koseri
Citrobacter murliniae
Citrobacter rodentium
Citrobacter sedlakii
Citrobacter sp. 30_2
Citrobacter sp. KMSI_3
Citrobacter werkmanii
Citrobacter youngae
Cronobacter malonaticus
Cronobacter sakazakii
Cronobacter turicensis
Enterobacter aerogenes
Enterobacter asburiae
Enterobacter cancerogenus
Enterobacter cloacae
Enterobacter cowanii
Enterobacter hormaechei
Enterobacter sp. 247BMC
Enterobacter sp. 638
Enterobacter sp. JC163
Enterobacter sp. SCSS
Enterobacter sp. TSE38
Escherichia albertii
Escherichia coli
Escherichia fergusonii
Escherichia hermannii
Escherichia sp. 1_1_43
Escherichia sp. 4_1_40B
Escherichia sp. B4
Escherichia vulneris
Ewingella americana
Haemophilus genomosp. P2 oral
Haemophilus genomosp. P3 oral
Haemophilus sp. oral clone JM053
Hafnia alvei
Klebsiella oxytoca
Klebsiella pneumoniae
Klebsiella sp. AS10
Klebsiella sp. Co9935
Klebsiella sp. enrichment culture
Klebsiella sp. OBRC7
Klebsiella sp. SP_BA
Klebsiella sp. SRC_DSD1
Klebsiella sp. SRC_DSD11
Klebsiella sp. SRC_DSD12
Klebsiella sp. SRC_DSD15
Klebsiella sp. SRC_DSD2
Klebsiella sp. SRC_DSD6
Klebsiella variicola
Kluyvera ascorbata
Kluyvera cryocrescens
Leminorella grimontii
Leminorella richardii
Pantoea agglomerans
Pantoea ananatis
Pantoea brenneri
Pantoea citrea
Pantoea conspicua
Pantoea septica
Pasteurella dagmatis
Pasteurella multocida
Plesiomonas shigelloides
Raoultella ornithinolytica
Raoultella planticola
Raoultella terrigena
Salmonella bongori
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella enterica
Salmonella typhimurium
Salmonella typhimurium
Serratia fonticola
Serratia liquefaciens
Serratia marcescens
Serratia odorifera
Serratia proteamaculans
Shigella boydii
Shigella dysenteriae
Shigella flexneri
Shigella sonnei
Tatumella ptyseos
Trabulsiella guamensis
Yersinia aldovae
Yersinia aleksiciae
Yersinia bercovieri
Yersinia enterocolitica
Yersinia frederiksenii
Yersinia intermedia
Yersinia kristensenii
Yersinia mollaretii
Yersinia pestis
Yersinia pseudotuberculosis
Yersinia rohdei
Yokenella regensburgei
Conchiformibius kuhniae
Morococcus cerebrosus
Neisseria bacilliformis
Neisseria cinerea
Neisseria flavescens
Neisseria gonorrhoeae
Neisseria lactamica
Neisseria macacae
Neisseria meningitidis
Neisseria mucosa
Neisseria pharyngis
Neisseria polysaccharea
Neisseria sicca
Neisseria sp. KEM232
Neisseria sp. oral clone AP132
Neisseria sp. oral strain B33KA
Neisseria sp. oral taxon 014
Neisseria sp. TM10_1
Neisseria subflava
Okadaella gastrococcus
Streptococcus agalactiae
Streptococcus alactolyticus
Streptococcus australis
Streptococcus bovis
Streptococcus canis
Streptococcus constellatus
Streptococcus cristatus
Streptococcus dysgalactiae
Streptococcus equi
Streptococcus equinus
Streptococcus gallolyticus
Streptococcus genomosp. C1
Streptococcus genomosp. C2
Streptococcus genomosp. C3
Streptococcus genomosp. C4
Streptococcus genomosp. C5
Streptococcus genomosp. C6
Streptococcus genomosp. C7
Streptococcus genomosp. C8
Streptococcus gordonii
Streptococcus infantarius
Streptococcus infantis
Streptococcus intermedius
Streptococcus lutetiensis
Streptococcus massiliensis
Streptococcus mitis
Streptococcus oligofermentans
Streptococcus oralis
Streptococcus parasanguinis
Streptococcus pasteurianus
Streptococcus peroris
Streptococcus pneumoniae
Streptococcus porcinus
Streptococcus pseudopneumoniae
Streptococcus pseudoporcinus
Streptococcus pyogenes
Streptococcus ratti
Streptococcus sanguinis
Streptococcus sinensis
Streptococcus sp. 2_1_36FAA
Streptococcus sp. 2285_97
Streptococcus sp. ACS2
Streptococcus sp. AS20
Streptococcus sp. BS35a
Streptococcus sp. C150
Streptococcus sp. CM6
Streptococcus sp. ICM10
Streptococcus sp. ICM12
Streptococcus sp. ICM2
Streptococcus sp. ICM4
Streptococcus sp. ICM45
Streptococcus sp. M143
Streptococcus sp. M334
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral clone
Streptococcus sp. oral taxon 071
Streptococcus sp. oral taxon G59
Streptococcus sp. oral taxon G62
Streptococcus sp. oral taxon G63
Streptococcus suis
Streptococcus thermophilus
Streptococcus salivarius
Streptococcus uberis
Streptococcus urinalis
Streptococcus vestibularis
Streptococcus viridans
Bacillus coagulans
Blautia luti
Blautia sp
Blautia wexlerae
Ruminococcus obeum
Clostridiales sp
Clostridium aerotolerans
Clostridium disporicum
Clostridium sp
Clostridium symbiosum
Dorea longicatena
Eubacterium cellulosolvens
Eubacterium ventriosum
Gemmiger formicilis
Robinsoniella peoriensis
Roseburia hominis
Roseburia intestinalis
Ruminococcus sp
Syntrophococcus
sucromutans
Turicibacter sanguinis
Clostridiales sp
Clostridium bartlettii
Clostridium irregulare
Clostridium sordellii
Lachnospiraceae sp
Table 6 shows spore quantitation for ethanol treated spore preparations using spore CFU (SCFU) assay and DPA assay.
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
Blautia producta
This application is the National Stage of International Application No. PCT/US14/14747, filed Feb. 4, 2014, which claims the benefit of U.S. Provisional Application No. 61/760,584, filed on Feb. 4, 2013, and to U.S. Provisional Application No. 61/760,585, filed on Feb. 4, 2013, and to U.S. Provisional Application No. 61/760,574, filed on Feb. 4, 2013, and to U.S. Provisional Application No. 61/760,606, filed on Feb. 4, 2013, and to U.S. Provisional Application No. 61/798,606, filed on Mar. 15, 2013, and to U.S. Provisional Application No. 61/926,928 61,926,918, filed on Jan. 13, 2014, each of which is incorporated herein by reference in its entirety for all purposes.
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| Number | Date | Country | |
|---|---|---|---|
| 20160158295 A1 | Jun 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61760574 | Feb 2013 | US | |
| 61760585 | Feb 2013 | US | |
| 61760606 | Feb 2013 | US | |
| 61760584 | Feb 2013 | US | |
| 61798606 | Mar 2013 | US | |
| 61926918 | Jan 2014 | US |
