Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/37—Digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/745—Bifidobacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/4816—Wall or shell material
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/12—Antidiarrhoeals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This application includes a Sequence Listing submitted electronically as a text file named 25969PCT_sequencelisting.txt, created on February 2, 2014, with a size of 4,100,000 bytes. The sequence listing is incorporated by reference.
• Mammals are colonized by microbes in the gastrointestinal (Gl) 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 Gl 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.
• resources may be food, location and the availability of space to grow or a physical structure to which the microbe may attach.
• host diet is involved in shaping the Gl 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.
• 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
• 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
• probiotics have asserted that their preparations of bacteria promote mammalian health by preserving the natural microflora in the Gl tract and reinforcing the normal controls on aberrant immune responses. See, e.g., U.S. Patent No. 8,034,601 .
• Probiotics 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 nor correct dysbioses of the Gl tract in many situations. Additionally, such probiotics are insufficient to prevent, reduce or treat a pathogenic bacterial infection of the gastrointestinal tract, such as by Clostridium difficile, by reducing the population of the gastrointestinal tract by the pathogenic bacteria.
• the present invention demonstrates effective compositions for and methods of preventing or reducing pathogenic bacterial growth, proliferation, and/or colonization, whereby bacterial compositions that contain one or more types of nonpathogenic bacteria are introduced into the gastrointestinal tract and effectively compete with pathogenic bacteria for monomeric or polymeric carbohydrate nutrients, and/or amino acid nutrients, and/or vitamin nutrients.
• organisms may prevent disease by metabolizing available germinants utilized by the pathogenic bacteria and prevent pathogenic spore germination.
• competition for overlapping nutrients among two or more types of non-pathogenic bacteria that are introduced into the gastrointestinal tract may leads to secretion of molecules inhibitory to a pathogen and/or pathobiont by one or multiple of the types of introduced non-pathogenic bacteria.
• the inhibitory compound may be a toxin, antibiotic, or a bacterocin (see Chiuchiolo et al. Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25 J Bacteriol. 2001 Mar;183(5): 1755-64).
• Figure 1 illustrates variable regions mapped onto the 16s sequence (Figure 1A) and annotated 16s sequence ( Figure 1 B) with bolded variable
• Figure 2 is a photograph of a CsCI gradient demonstrating spore separation from other material.
• Figure 3 illustrates population of fecal bacterial cell suspension (left) after CsCI separation (center) and after a CsCI and sucrose gradient (right)
• Figure 4 illustrates a prophylaxis model with the ethanol treated spore preparation and the ethanol treated, gradient-purified spore preparation.
• Figure 5 illustrates a relapse prevention model with ethanol treated spores and ethanol treated, gradient purified spores.
• Figure 6 illustrates a linear range of DPA assay compared to CFU counts/ml.
• Figure 7 illustrates bacterial diversity measured in the ethanol treated spore treatment sample and patient pre- and post-treatment samples.
• Figure 8 illustrates patient bacterial ecology is shifted by treatment with an ethanol treated spore treatment from a dysbiotic state to a state of health.
• Figure 9 illustrates Augmentation of Bacteroides species in patients.
• Figure 10 illustrates species Engrafting versus Species Augmenting in patient's microbiomes after treatment with an ethanol-treated spore preparation.
• Figure 1 1 illustrates the results of a nutrient utilization assay with Clostridium difficile and potential competitors of the pathogen. Plus (+) indicates that it is a nutrient for the isolate tested. Minus (-) indicates that it is not a nutrient for the isolate tested.
• 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:
• 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.
• 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 or microbiome of the gut or other body area, including mucosal or skin surfaces in which the normal diversity and/or function of the ecological network is disrupted. Any disruption from the preferred (e.g., ideal) state of the microbiota can be considered a dysbiosis, even if such dysbiosis does not result in a detectable decrease in health. This state of dysbiosis may be unhealthy, it may be unhealthy under only certain conditions, or it may prevent a subject from becoming healthier. Dysbiosis may be due to a decrease in diversity, the
• pathobiont refers to specific bacterial species found in healthy hosts that may trigger immune-mediated pathology and/or disease in response to certain genetic or environmental factors. Chow et al., (201 1 ) Curr Op Immunol.
• pathobionts of the intestinal microbiota and inflammatory disease. 23: 473-80 are a pathogen that is mechanistically distinct from an acquired infectious organism.
• pathogen includes both acquired infectious organisms and pathobionts.
• pathogen includes both acquired infectious organisms and pathobionts.
• pathogen includes any such organism or entity that is capable of causing or affecting a disease, disorder or condition of a host organism containing the organism or entity.
• Physical 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.
• phylogenetic reconstruction algorithms e.g. parsimony, maximum likelihood, or Bayesian
• Operational taxonomic units 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.
• the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence.
• the entire genomes of two entities are sequenced and compared.
• select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared.
• 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 MJ, Wang Q, O'Sullivan O, Greene-Diniz R, Cole JR, Ross RP, and O'Toole PW. 2010.
• OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU.
• OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., "house-keeping" genes), or a combination thereof. Such characterization employs, e.g., WGS data or a whole genome sequence.
• Residual habitat products refers to material derived from the habitat for microbiota within or on a human or animal.
• 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.
• bacterial viruses i.e., phage
• 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.
• 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 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.
• 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, 1 1 17-1 173, 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
• V1 , V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU .
• the V1 , V2, and V3 regions are used to characterize an OTU.
• the V3, V4, and V5 regions are used to characterize an OTU.
• the V4 region is used to characterize an OTU.
• 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.
• phenotype refers to a set of observable characteristics of an individual entity.
• 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.
• Network Ecology refers to a consortium of OTUs that co- occur in some number of subjects.
• a “network” is defined
• 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;
• Network Class refers 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.
• 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.
• non-Keystone OTU refers to an OTU that is observed in a Network Ecology and is not a keystone OTU.
• Physical 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.
• phylogenetic tree that represents the genetic diversity all species, genera, or taxonomic families relative to one another.
• phylogenetic diversity may be measured using the total branch length or average branch length of a phylogenetic tree.
• a "spore” or a population of “spores” includes bacteria (or other single- celled organisms) that are generally viable, more resistant to environmental influences such as heat and bacteriocidal agents than vegetative forms of the same bacteria, and typically capable of germination and out-growth.
• "Spore-formers” or bacteria “capable of forming spores” are those bacteria containing the genes and other necessary abilities to produce spores under suitable environmental conditions.
• 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.
• 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.
• 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.
• the spore preparation comprises spore forming species that are enriched over viable non-spore formers or vegetative forms of spore formers.
• spores are enriched by 2-fold, 5-fold, 10- fold, 50-fold, 100-fold, 1000-fold, 10,000-fold or greater than 10,000-fold compared to all vegetative forms of bacteria.
• 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.
• isolated encompasses a bacterium or other entity or substance that has been (1 ) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man.
• Isolated bacteria include those bacteria that are cultured, even if such cultures are not monocultures. Isolated bacteria may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
• isolated bacteria are more than about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
• a substance is "pure” if it is substantially free of other components.
• the terms "purify,” “purifying” and “purified” refer to a bacterium or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production.
• a bacterium or a bacterial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the bacterium or bacterial population, or by passage through culture , and a purified bacterium or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered "isolated.”
• purified bacteria and bacterial populations are more than about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
• composition can be independently purified from one or more other bacteria produced and/or present in the material or environment containing the bacterial type.
• Bacterial compositions and the bacterial components thereof are generally purified from residual habitat products.
• Demonstrations of pathogen inhibition such as decrease in the growth of a pathogenic bacterium or reduction in the level of colonization of a pathogenic bacterium are provided herein and otherwise recognized by one of ordinary skill in the art.
• Inhibition of a pathogenic bacterium's "growth” may include inhibiting the increase in size of the pathogenic bacterium and/or inhibiting the proliferation (or multiplication) of the pathogenic bacterium.
• Inhibition of colonization of a pathogenic bacterium may be demonstrated by measuring the amount or burden of a pathogen before and after a treatment.
• An "inhibition" or the act of “inhibiting” includes the total cessation and partial reduction of one or more activities of a pathogen, such as growth, proliferation, colonization, and function.
• 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.
• "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
• Measuring reductions of adherent pathogens may be
• a "combination" of two or more bacteria includes the physical coexistence 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.
• non-comestible products 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.
• 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.
• 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
• vitamin C essential in minute amounts for normal growth and activity of the body and obtained naturally from plant and animal foods or synthetically made, provitamins, derivatives, analogs.
• 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.
• 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.
• ROS Reactive Oxygen Species
• 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.
• “Fermentation products” are compounds that are produced by bacteria or bacterial populations through carbohydrate metabolic processes. These metabolites include but are not limited to lactate, butyrate, acetate, ethanol, succinate, formate.
• a substance that organisms use to live and grow includes but is not limited to vitamins, minerals, antioxidant, fermentation products, cofactors, proteins, amino acids, nucleotides, nucleic acids, lipids, carbohydrates and metabolic products of these molecules.
• bacteria and combinations of bacteria of the human gut microbiota with the capacity to meaningfully provide functions of a healthy microbiota when administered to mammalian hosts. Without being limited to a specific mechanism, it is thought that such compositions inhibit the growth, proliferation, germination, and/or colonization of one or a plurality of pathogenic bacteria in the dysbiotic microbiotal niche, so that a healthy, diverse and protective microbiota colonizes and populates the intestinal lumen to establish or reestablish ecological control over pathogens or potential pathogens (e.g., some bacteria are pathogenic bacteria only when present in a dysbiotic environment). Inhibition of pathogens includes those pathogens such as C. difficile, Salmonella spp., enteropathogenic E coli, multi-drug resistant bacteria such as
• VRE Enterococci
• 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.
• the present invention demonstrates effective methods of preventing or reducing pathogenic bacterial growth, proliferation, and/or colonization, whereby bacterial compositions that contain one or more types of non-pathogenic bacteria are introduced into the gastrointestinal tract and effectively compete with pathogenic bacteria for monomeric or polymeric carbohydrate nutrients, and/or amino acid nutrients, and/or vitamin nutrients.
• organisms may prevent disease by metabolizing available germinants utilized by the pathogenic bacteria and prevent pathogenic spore germination.
• competition for overlapping nutrients among two or more types of non-pathogenic bacteria that are introduced into the gastrointestinal tract may leads to secretion of molecules inhibitory to a pathogen and/or pathobiont by one or multiple of the types of introduced non-pathogenic bacteria.
• the inhibitory compound may be a toxin, antibiotic, or a bacterocin (see Chiuchiolo et al. Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25 J Bacteriol. 2001 Mar;183(5): 1755-64).
• the enteric bacterium Clostridium difficile (C. difficile) is an opportunistic pathogen whose infection frequently follows antibiotic treatment.
• FMT fecal microbiota transplantation
• C. difficile In order to maintain steady-state concentrations in the gastrointestinal tract of a mammalian subject (where continuous dilution occurs as a result of the peristaltic wave), C. difficile is dependent upon the availability of a small number of nutrients, particularly carbohydrates such as glucose, mannitol, fructose, N- acetylglucosamine (NAG) and N-acetylneuraminic acid (NAN), amino acids, and vitamins such as biotin, pantothenate and pyridoxine. Free monosaccharide levels of NAG and NAN are low in the mammalian gastrointestinal tract; instead, NAG and NAN are present in higher order carbohydrate polymers such as mucins and inulin.
• carbohydrates such as glucose, mannitol, fructose, N- acetylglucosamine (NAG) and N-acetylneuraminic acid (NAN)
• NAG and NAN are present in higher order carbohydrate polymers such as mu
• the bacterial compositions containing one or more types of non-pathogenic bacteria are introduced into the gastrointestinal tract to prevent or treat infection by utilizing the nutrients and/or metabolizing the germinants utilized by C. difficile.
• the latter will be achieved when the introduced bacterial composition depletes one or more of the nutrients (e.g., carbohydrate or vitamin) utilized by C. difficile to a concentration that inhibits further growth of C. difficile or alternatively, reduces the rate of growth such that the introduced bacteria and/or other resident bacteria outcompete C. difficile.
• compositions comprising an effective amount of a first type of bacteria and a second type of bacteria that overlap with C. difficile nutrition utilization, and/or are independently capable of proliferating in a nutrient medium having a threshold concentration of a nutrient below that concentration required for Clostridium difficile proliferation.
• the first and second type of bacteria are isolated, such as from the gastrointestinal tract of a healthy mammal, and are not identical to each other.
• Exemplary nutrients include carbohydrates, amino acids T vitamins, minerals, and cofactors such as germinants.
• Exemplary minerals include iron, phosphate, copper, nickel, and magnesium.
• Additional nutrients include nitrogen sources, such as ammonia or urea. Table XXX5 contains representative nitrogen sources.
• Exemplary fermentation products include lactate, butyrate, acetate, ethanol, succinate, and formate.
• Exemplary carbohydrate nutrients include glucose, mannitol, fructose, N-acetylglucosamine (NAG) and N-acetylneuraminic acid (NAN).
• the bacterial compositions contain bacteria that overlap with C. difficile nutrition utilization and/or and are independently capable of proliferating in a nutrient medium having a threshold concentration of at least one of the following: glucose, mannitol, fructose, NAG and NAN below that concentration required for Clostridium difficile proliferation.
• the bacterial compositions include bacteria that are independently capable of proliferating in a nutrient medium having a threshold concentration of each of glucose, mannitol, fructose, NAG and NAN below the concentrations required for Clostridium difficile proliferation.
• the threshold concentration may be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or less than about 10% of the concentration of that nutrient required for Clostridium difficile proliferation.
• the bacterial compositions contain bacteria that can proliferate faster than C. difficile at a given nutrient concentration, effectively out-competing the C. difficile for a limited resource.
• these two types are independently capable of proliferating at a rate at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more than 300% greater than the proliferation rate of Clostridium difficile in a nutrient medium having a concentration of a nutrient such as glucose, mannitol, fructose, NAG and NAN, or a combination thereof.
• a nutrient such as glucose, mannitol, fructose, NAG and NAN, or a combination thereof.
• composition having two or more types of bacteria that are capable of growing to a maximal bacterial concentration in excess of the maximal bacterial concentration capable by C. difficile, as determined, e.g., by turbidity, colony counts, total biomass, or other means described herein or otherwise known in the art.
• the bacterial composition is delivered at an efficacious dose such that the population of bacteria in the composition deplete glucose, mannitol, fructose, NAG and NAN and other carbohydrates necessary for C. difficile growth.
• Exemplary vitamin nutrients include biotin, pantothenate, folic acid and pyridoxine.
• Table XXX1 contains representative vitamins, minerals, and cofactors.
• the bacteria present in the therapeutic composition are better able to transport one or more vitamins from the medium into the bacterial cell, or they are more efficient at utilizing the vitamin(s) once it is inside the cell.
• the bacterial compositions comprise bacteria that overlap with C. difficile nutrition utilization requirements and/or are independently capable of
• the bacterial compositions contain bacteria that are independently capable of proliferating in a nutrient medium having a threshold concentration of each of biotin, pantothenate and pyridoxine below the concentrations required for Clostridium difficile proliferation.
• the threshold concentration may be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or less than about 10% of the concentration of that nutrient required for Clostridium difficile proliferation.
• the bacterial compositions contain bacteria that can proliferate faster than C. difficile at a given nutrient
• compositions comprising two or more types of bacteria, these two types are independently capable of proliferating at a rate at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more than 300% greater than the proliferation rate of Clostridium difficile in a nutrient medium having a concentration of a nutrient such as biotin, pantothenate, pyridoxine, or a combination thereof.
• a nutrient such as biotin, pantothenate, pyridoxine, or a combination thereof.
• a composition comprising two or more types of bacteria that are capable of growing to a maximal bacterial concentration in excess of the maximal bacterial concentration capable by C. difficile, as determined, e.g., by turbidity, colony counts, total biomass, or other means described herein or otherwise known in the art.
• the bacterial composition is delivered at an efficacious dose such that the population of bacteria in the composition deplete biotin, pantothenate and pyridoxine and other vitamins necessary for C. difficile growth.
• compositions comprising effective amounts of one or more types of bacteria that compete with C. difficile for nutrients, e.g., polymeric carbohydrates, glycosylated proteins and/or vitamins.
• compositions comprising effective amounts of one or more types of bacteria that have rates of saccharification and consumption of the resulting products that exceed other bacterial species in the gastrointestinal tract.
• bacterial compositions comprising first and second types of bacteria that are independently capable of saccharification at a rate at least 10% greater than Clostridium difficile. Also provided are bacterial compositions comprising first and second types of bacteria that are independently capable of saccharification at a rate at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more than 300% greater than any other bacterial species in the local environment that digests polymeric carbohydrates and releases the hydrolysis products into the media for consumption by, e.g.,
• Suitable carbohydrate polymers to target for selective digestion include a fructan, an arabinoxylan, a lactulose, and a galactooligosaccharide or a glycosylated protein such as a mucin.
• compositions comprising bacterial types capable of efficient saccharification of complex carbohydrates and metabolism of simple sugars.
• a composition comprising an effective amount of a first type of bacteria and a second type of bacteria formulated for oral administration to a mammalian subject, wherein the first type of bacteria is: (i) isolated, (ii) not identical to the second type of bacteria, and (iii) independently capable of saccharification at a rate at least 10% greater than Clostridium difficile, and wherein the second type of bacteria is: (i) isolated, (ii) not identical to the first type of bacteria, and (iii) independently capable of glycolysis of a nutrient selected from the group consisting of glucose, mannitol, fructose, NAG and NAN, at a rate at least 10% greater than Clostridium difficile or at a concentration at least 10% less than Clostridium difficile, or the combination thereof.
• a composition comprising an effective amount of a first type of bacteria and formulated for oral administration to a mammalian subject, where
• the first type of bacteria is: (i) isolated, (ii) not identical to the second type of bacteria, and (iii) independently capable of saccharification of a necessary C. difficile nutrient
• the second type of bacteria is: (i) isolated, (ii) not identical to the first type of bacteria, and (iii) independently capable of glycolysis of a nutrient selected from the group consisting of glucose, mannitol, fructose, NAG and NAN; and wherein the delivered bacteria effectively metabolize for nutrients to levels below that which is required for C. difficile growth.
• Microbial e.g., bacterial compositions may comprise two types of bacteria (termed “binary combinations” or “binary pairs") or greater than two types of bacteria.
• a bacterial composition may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 , 22, 23, 24, 25, 26, 27, 28, 29 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or at least 40, at least 50 or greater than 50 types of bacteria, as defined by species or operational taxonomic unit (OTU), or otherwise as provided herein.
• OTU operational taxonomic unit
• the number of types of bacteria present in a bacterial composition is at or below a known value.
• the bacterial composition comprises 50 or fewer types of bacteria, such as 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , or 10 or fewer, or 9 or fewer types of bacteria, 8 or fewer types of bacteria, 7 or fewer types of bacteria, 6 or fewer types of bacteria, 5 or fewer types of bacteria, 4 or fewer types of bacteria, or 3 or fewer types of bacteria.
• a bacterial composition comprises from 2 to no more than 40, from 2 to no more than 30, from 2 to no more than 20, from 2 to no more than 15, from 2 to no more than 10, or from 2 to no more than 5 types of bacteria.
• Bacterial compositions may be prepared comprising at least two types of isolated bacteria, chosen from the species in Table 1 .
• the microbial, e.g., bacterial composition comprises at least one and preferably more than one of the following: Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum,
• Clostridium ramosum Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1 109 and 1 108-1 ), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus).
• at least one of the preceding species is not substantially present in the bacterial composition.
• the bacterial composition comprises at least one and preferably more than one of the following: Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum, Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1 109 and 1 108-1 ), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus).
• Enterococcus faecalis previously known as Streptococcus faecalis
• Clostridium innocuum Clostridium ramosum
• Bacteroides ovatus Bacteroides vulgatus
• Bacteroides thetaoiotaomicron Escherichia coli (1 109 and 1 108-1
• the bacterial composition comprises at least one and preferably more than one of the following: Acidaminococcus intestinalis, Bacteroides ovatus, two strains of Bifidobacterium adolescentis, two strains of
• aerofaciens two strains of Dorea longicatena, Escherichia coli, Eubacterium desmolans, Eubacterium eligens, Eubacterium limosum, four strains of Eubacterium rectale, Eubacterium ventriosumi, Faecalibacterium prausnitzii, Lachnospira pectinoshiza, Lactobacillus casei, Lactobacillus casei/paracasei, Paracateroides distasonis, Raoultella sp., one strain of Roseburia (chosen from Roseburia faecalis or Roseburia faecis), Roseburia intestinalis, two strains of Ruminococcus torques, two strains of
• Ruminococcus obeum, and Streptococcus mitis are substantially present in the bacterial composition.
• the bacterial composition comprises at least one and preferably more than one of the following: Barnesiella intestinihominis; Lactobacillus reuteri; a species characterized as one of Enterococcus hirae,
• Enterococus faecium, or Enterococcus durans a species characterized as one of Anaerostipes caccae or Clostridium indolis; a species characterized as one of
• at least one of the preceding species is not substantially present in the bacterial composition.
• the bacterial composition comprises at least one and preferably more than one of the following: Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bartlettii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium camis, Clostridium celatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridium cochlearium, Clostridium difficile, Clostridium fallax, Clostridium felsineum, Clostridium ghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, Clostridium limosum, Clostridium absonum, Clostri
• Clostridium oroticum Clostridium paraputrificum, Clostridium perfringens, Clostridium piliforme, Clostridium putrefaciens, Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridium tertium, Clostridium tetani, Clostridium welchii, and Clostridium villosum.
• at least one of the preceding species is not substantially present in the bacterial composition.
• the bacterial composition comprises at least one and preferably more than one of the following: Clostridium innocuum, Clostridum bifermentans, Clostridium butyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides uniformis, three strains of Escherichia coli, and Lactobacillus sp. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
• the bacterial composition comprises at least one and preferably more than one of the following: Clostridium bifermentans, Clostridium innocuum, Clostridium butyricum, three strains of Escherichia coli, three strains of Bacteroides, and Blautia producta.
• Clostridium bifermentans Clostridium innocuum
• Clostridium butyricum three strains of Escherichia coli
• Bacteroides three strains of Bacteroides
• Blautia producta Blautia producta
• the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides sp., Escherichia coli, and non pathogenic Clostridia, including Clostridium innocuum, Clostridium bifermentans and Clostridium ramosum.
• Bacteroides sp. Escherichia coli
• non pathogenic Clostridia including Clostridium innocuum, Clostridium bifermentans and Clostridium ramosum.
• at least one of the preceding species is not substantially present in the bacterial composition.
• the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides species, Escherichia coli and non-pathogenic Clostridia, such as Clostridium butyricum, Clostridium bifermentans and Clostridium innocuum. In an alternative embodiment, at least one of the preceding species is not substantially present in the bacterial composition.
• the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides frag ilis-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.
• the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides, Eubacteria, Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas, and Peptostreptococcus.
• Bacteroides Eubacteria, Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas, and Peptostreptococcus.
• at least one of the preceding species is not substantially present in the bacterial composition.
• the bacterial composition comprises at least one and preferably more than one of the following: Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens, Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previously known as Peptostreptococcus productus II), Bacteroides fragilis ss.
• A Eubacterium biforme, Bifidobacterium infantis, Eubacterium rectale lll-F, Coprococcus comes, Bacteroides capillosus, Ruminococcus albus, Eubactenum formicigenerans, Eubacterium hallii, Eubacterium ventriosum I, Fusobactenum 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, Eubactenum cylindroides, Eubacterium ruminantium, Eubactenum CH-1, Staphylococcus
• Ruminococcus AT Peptococcus AU-1 , Eubacterium AG, -AK, -AL, -AL-1, -AN;
• Peptococcus magnus Peptococcus G, AU-2; Streptococcus intermedius,
• Desuifomonas pigra Bacteroides L-4, -N-i; Fusobacterium H, Lactobacillus G, and
• At least one of the preceding species is not substantially present in the bacterial composition.
• Bacterial compositions may be prepared comprising at least two types of isolated bacteria, chosen from the species in Table 1 .
• 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, 1 1 17-1 173, 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)).
• At least one of the V1 , V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU.
• the V1 , V2, and V3 regions are used to characterize an OTU.
• the V3, V4, and V5 regions are used to characterize an OTU.
• the V4 region is used to characterize an OTU .
• the bacterial composition does not comprise at least one of Enterococcus faecalis (previously known as Streptococcus faecalis), Clostridium innocuum, Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichia coli (1 109 and 1 108-1 ), Clostridum bifermentans, and Blautia producta (previously known as Peptostreptococcus productus).
• the bacterial composition does not comprise at least one of Acidaminococcus intestinalis, Bacteroides ovatus, two species of
• Bifidobacterium adolescentis two species of Bifidobacterium longum, Collinsella aerofaciens, two species of Dorea longicatena, Escherichia coli, Eubacterium eligens, Eubacterium limosum, four species of Eubacterium rectale, Eubacterium ventriosumi, Faecalibacterium prausnitzii, Lactobacillus casei, Lactobacillus paracasei,
• the bacterial composition does not comprise at least one of 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.
• the bacterial composition does not comprise at least one of Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium camis, Clostridium celatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridium cochlearium, Clostridium difficile, Clostridium fallax,
• Clostridium haemolyticum Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, Clostridium limosum, Clostridium malenominatum, Clostridium novyi, Clostridium oroticum, Clostridium paraputrificum, Clostridium perfringens, Clostridium piliforme, Clostridium putrefaciens, Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium
• the bacterial composition does not comprise at least one of Clostridium innocuum, Clostridum bifermentans, Clostridium butyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides uniformis, three strains of Escherichia coli, and Lactobacillus sp.
• the bacterial composition does not comprise at least one of Clostridium bifermentans, Clostridium innocuum, Clostridium butyricum, three strains of Escherichia coli, three strains of Bacteroides, and Blautia producta (previously known as Peptostreptococcus productus).
• the bacterial composition does not comprise at least one of Bacteroides sp., Escherichia coli, and non pathogenic Clostridia, including Clostridium innocuum, Clostridium bifermentans and Clostridium ramosum.
• the bacterial composition does not comprise at least one of more than one Bacteroides species, Escherichia coli and non-pathogenic Clostridia, such as Clostridium butyricum, Clostridium bifermentans and Clostridium innocuum.
• the bacterial composition does not comprise at least one of Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroides gracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae, Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris, Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, and Bacteroides vulgatus.
• the bacterial composition does not comprise at least one of Bacteroides, Eubacteria, Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas, and Peptostreptococcus.
• the bacterial composition does not comprise at least one of Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens, Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previously known as Peptostreptococcus productus II), Bacteroides fragilis ss. Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacterium aerofaciens III, Blautia producta (previously known as Peptostreptococcus productus I), Ruminococcus bromii, Bifidobacterium
• 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,
• 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 ska, 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.
• the bacterial composition provides a protective or therapeutic effect against infection by one or more Gl pathogens of interest.
• 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,
• CRE Enterobacteriaceae
• VRE vancomycin-resistant Enterococci
• these pathogens include, but are not limited to, Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium
• 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
• 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).
• ESBL extended spectrum beta-lactam resistant Enterococci
• the bacterial compositions comprise purified spore populations.
• Purified spore populations contain combinations of commensal bacteria of the human gut microbiota with the capacity to meaningfully provide functions of a healthy microbiota when administered to a mammalian subject. Without being limited to a specific mechanism, it is thought that such compositions inhibit the growth of a pathogen such as C. difficile, Salmonella spp., enteropathogenic E. coli, and vancomycin-resistant Enterococcus spp., so that a healthy, diverse and protective microbiota can be maintained or, in the case of pathogenic bacterial infections such as C. difficile infection, repopulate the intestinal lumen to reestablish ecological control over potential pathogens.
• yeast spores and other fungal spores are also purified and selected for therapeutic use.
• compositions containing nonpathogenic, germination-competent bacterial spores, spore forming organisms and non- spore forming organisms for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health.
• compositions are advantageous in being suitable for safe administration to humans and other mammalian subjects and are efficacious in numerous gastrointestinal diseases, disorders and conditions and in general nutritional health.
• compositions containing a purified population of bacterial spores, spore forming organisms and non-spore forming organisms are provided herein.
• purify refers to the state of a population (e.g., a plurality of known or unknown amount and/or concentration) of desired bacterial spores or bacteria, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired bacterial spore, or alternatively a removal or reduction of residual habitat products as described herein.
• a purified population has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other embodiments, a purified population has an amount and/or concentration of desired bacterial spores or bacteria at or above an acceptable amount and/or concentration. In other embodiments, the purified population of bacterial spores is enriched as compared to the starting material (e.g., liquid culture) from which the population is obtained.
• the starting material e.g., liquid culture
• This enrichment may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material.
• the purified populations have reduced or undetectable levels of one or more pathogenic activities, such as toxicity, an infection of the mammalian recipient subject, an immunomodulatory activity, an autoimmune response, a metabolic response, or an inflammatory response or a neurological response.
• pathogenic activities such as toxicity, an infection of the mammalian recipient subject, an immunomodulatory activity, an autoimmune response, a metabolic response, or an inflammatory response or a neurological response.
• Such a reduction in a pathogenic activity may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material.
• the purified populations of bacterial spores have reduced sensory components as compared to fecal material, such as reduced odor, taste, appearance, and umami.
• substantially free of residual habitat products this means that the bacterial spore composition no longer contains a substantial amount of the biological matter associated with the microbial community while living on or in the human or animal subject, and the purified population of spores may be 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contamination of the biological matter associated with the microbial community.
• Substantially free of residual habitat products may also mean that the bacterial spore composition contains no detectable cells from a human or animal, and that only microbial cells are detectable, in particular, only desired microbial cells are detectable.
• 1 x10 " 2 %, 1 x10 "3 %, 1 x10 "4 %, 1x10 "5 %, 1 x10 "6 %, 1x10 "7 %, 1 x10 “8 % of the cells in the bacterial composition are human or animal, as compared to microbial cells.
• the residual habitat product present in the purified population is reduced at least a certain level from the fecal material obtained from the mammalian donor subject, e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%.
• substantially free of residual habitat products or substantially free of a detectable level of a pathogenic material means that the bacterial composition contains no detectable viral (including bacterial viruses (i.e., phage)), fungal, or mycoplasmal or toxoplasmal contaminants, or a eukaryotic parasite such as a helminth.
• the purified spore populations are substantially free of an acellular material, e.g., DNA, viral coat material, or non-viable bacterial material.
• purified spore populations can be demonstrated by genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired bacterial spores from non-desired, contaminating materials.
• Exemplary biological materials include fecal materials such as feces or materials isolated from the various segments of the small and large intestines.
• Fecal materials are obtained from a mammalian donor subject, or can be obtained from more than one donor subject, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or from greater than 1000 donors, where such materials are then pooled prior to purification of the desired bacterial spores.
• the desired bacterial spores are purified from a single fecal material sample obtained from a single donor, and after such purification are combined with purified spore populations from other purifications, either from the same donor at a different time, or from one or more different donors, or both.
• Preferred bacterial genera include Acetonema, Alkaliphilus,
• Caldicellulosiruptor Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Clostridium, Cohnella, Coprococcus, Dendrosporobacter Desulfitobacterium,
• Desulfosporosinus Desulfotomaculum, Dorea, Eubacterium, Faecalibacterium,
• Propionispora Roseburia, Ruminococcus, Sarcina, Sporobacterium, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporosarcina, Sporotomaculum, Subdoligranulum, Symbiobacterium, Syntrophobotulus, Syntrophospora, Ter bacillus,
• Thermoanaerobacter and Thermosinus.
• Preferred bacterial species are provided at Table X4. Where specific strains of a species are provided, one of skill in the art will recognize that other strains of the species can be substituted for the named strain.
• spore-forming bacteria are identified by the presence of nucleic acid sequences that modulate sporulation.
• 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 oF, ⁇ , oG and ⁇ ), whose activities are confined to the forespore (oF and oG) or the mother cell ( ⁇ and ⁇ ).
• spore populations containing more than one type of bacterium are provided.
• 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.
• all or essentially all of the bacterial species present in a purified population are isolated or originally isolated from a fecal material treated as described herein or otherwise known in the art.
• one or more than one bacterial spores or types of bacteria are generated in culture and combined to form a purified population.
• one or more of these culture-generated populations are combined with a fecal material-derived population to generate a hybrid population.
• Bacterial compositions may contain at least two types of these preferred bacteria, including strains of the same species.
• a bacterial composition may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 or more than 20 types of bacteria, as defined by species or operational taxonomic unit (OTU) encompassing such species.
• OTU operational taxonomic unit
• compositions containing a population of bacterial spores suitable for therapeutic administration to a mammalian subject in need thereof are produced by generally following the steps of: (a) providing a fecal material obtained from a mammalian donor subject; and (b) subjecting the fecal material to at least one purification treatment or step under conditions such that a population of bacterial spores is produced from the fecal material.
• composition is formulated such that a single oral dose contains at least about 1 x10 4 colony forming units of the bacterial spores, and a single oral dose will typically contain about 1 x10 4 , 1 x10 5 , 1 x10 6 , 1 x10 7 , 1 x10 8 , 1 x10 9 , 1 x10 10 , 1 x10 11 , 1 x10 12 , 1 x10 13 , 1 x10 14 , 1x10 15 , or greater than 1 x10 15 CFUs of the bacterial spores.
• the presence and/or concentration of a given type of bacteria spore may be known or unknown in a given purified spore population.
• the concentration of spores of a given strain, or the aggregate of all strains is e.g., 1 x10 4 , 1 x10 5 , 1 x10 6 , 1 x10 7 , 1 x10 8 , 1 x10 9 , 1 x10 10 , 1 x10 1 1 , 1x10 12 , 1 x10 13 , 1 x10 14 , 1 x10 15 , or greater than 1 x10 15 viable bacterial spores per gram of composition or per administered dose.
• the composition contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% spores on a mass basis.
• the administered dose does not exceed 200, 300, 400, 500, 600, 700, 800, 900 milligrams or 1 , 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, or 1 .9 grams in mass.
• the bacterial spore compositions are generally formulated for oral or gastric administration, typically to a mammalian subject.
• the composition is formulated for oral administration as a solid, semi-solid, gel, or liquid form, such as in the form of a pill, tablet, capsule, or lozenge.
• such formulations contain or are coated by an enteric coating to protect the bacteria through the stomach and small intestine, although spores are generally resistant to the stomach and small intestines.
• the bacterial spore compositions may be formulated to be effective in a given mammalian subject in a single administration or over multiple administrations.
• a single administration is substantially effective to reduce CI. difficile and/or CI. difficile toxin content in a mammalian subject to whom the composition is administered.
• substantially effective means that CI. difficile and/or CI. difficile toxin content in the subject is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99% following administration of the composition.
• OTUs can be defined either by full 16S sequencing of the rRNA gene, by sequencing of a specific hypervariable region of this gene (i.e. V1 , V2, V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination of hypervariable regions from this gene (e.g. V1 -3 or V3-5).
• the bacterial 16S rDNA is approximately 1500 nucleotides in length and is used in reconstructing the evolutionary relationships and sequence similarity of one bacterial isolate to another using phylogenetic approaches. 16S sequences are used for phylogenetic reconstruction as they are in general highly conserved, but contain specific hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most microbes.
• 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.
• PCR polymerase chain reaction
• the sequencing method used may be, but is not limited to, Sanger sequencing.
• 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 lllumina (sequencing by synthesis) method using barcoded primers allowing for multiplex reactions.
• a next-generation sequencing method such as an lllumina (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
• Methods for producing bacterial compositions can include three main processing steps, combined with one or more mixing steps.
• the steps include organism banking, organism production, and preservation.
• 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.
• the agar or broth can contain nutrients that provide essential elements and specific factors that enable growth.
• An example would be a medium composed of 20 g/L glucose, 10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1 .5 g/L sodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/L magnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1 mg/L menadione.
• a variety of microbiological media and variations are well known in the art (e.g. R.M. Atlas, Handbook of Microbiological Media (2010) CRC Press).
• Medium can be added to the culture at the start, may be added during the culture, or may be intermittently/continuously flowed through the culture.
• the strains in the bacterial composition may be cultivated alone, as a subset of the bacterial composition, or as an entire collection comprising the bacterial composition.
• 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.
• bacterial compositions for human use this is often at 37°C temperature, pH, and other parameter with values similar to the normal human niche.
• the environment can be actively controlled, passively controlled (e.g., via buffers), or allowed to drift.
• anaerobic bacterial compositions e.g., gut microbiota
• an anoxic/reducing environment can be employed. This can be
• reducing agents such as cysteine
• a culture of a bacterial composition can be grown at 37°C, pH 7, in the medium above, pre-reduced with 1 g/L cysteine HCI.
• the culture When the culture has generated sufficient biomass, it can be preserved for banking.
• the organisms can be placed into a chemical milieu that protects from freezing (adding 'cryoprotectants'), drying ('lyoprotectants'), and/or osmotic shock ('osmoprotectants'), dispensing into multiple (optionally identical) containers to create a uniform bank, and then treating the culture for preservation.
• Containers are generally impermeable and have closures that assure isolation from the environment.
• Cryopreservation treatment is accomplished by freezing a liquid at ultra-low temperatures (e.g., at or below -80°C).
• Dried preservation removes water from the culture by evaporation (in the case of spray drying or 'cool drying') or by sublimation (e.g., for freeze drying, spray freeze drying). Removal of water improves long-term bacterial composition storage stability at temperatures elevated above cryogenic. If the bacterial composition comprises spore forming species and results in the production of spores, the final composition can be purified by additional means, such as density gradient centrifugation preserved using the techniques described above.
• Bacterial composition banking can be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank. As an example of
• a bacterial composition culture can be harvested by centrifugation to pellet the cells from the culture medium, the supernate decanted and replaced with fresh culture broth containing 15% glycerol. The culture can then be aliquoted into 1 ml_ cryotubes, sealed, and placed at -80°C for long-term viability retention. This procedure achieves acceptable viability upon recovery from frozen storage.
• Organism production can be conducted using similar culture steps to banking, including medium composition and culture conditions. It can be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there can be several subcultivations of the bacterial composition prior to the final cultivation. At the end of cultivation, the culture is harvested to enable further formulation into a dosage form for administration. This can involve concentration, removal of undesirable medium components, and/or introduction into a chemical milieu that preserves the bacterial composition and renders it acceptable for administration via the chosen route. For example, a bacterial composition can be cultivated to a concentration of 10 10 CFU/mL, then concentrated 20-fold by tangential flow
• the spent medium can be exchanged by diafiltering with a preservative medium consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphate buffer.
• 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.
• the powder can be blended to an appropriate potency, and mixed with other cultures and/or a filler such as microcrystalline cellulose for
• compositions disclosed herein are administered to a patient or a user (sometimes collectively referred to as a "subject").
• administer and “administration” encompasses embodiments in which one person directs another to consume a bacterial composition in a certain manner and/or for a certain purpose, and also situations in which a user uses a bacteria composition in a certain manner and/or for a certain purpose independently of or in variance to any instructions received from a second person.
• Non-limiting examples of embodiments in which one person directs another to consume a bacterial composition in a certain manner and/or for a certain purpose include when a physician prescribes a course of conduct and/or treatment to a patient, when a parent commands a minor user (such as a child) to consume a bacterial composition, when a trainer advises a user (such as an athlete) to follow a particular course of conduct and/or treatment, and when a manufacturer, distributer, or marketer recommends conditions of use to an end user, for example through advertisements or labeling on packaging or on other materials provided in association with the sale or marketing of a product.
• the bacterial compositions offer a protective and/or therapeutic effect against infection by one or more Gl pathogens of interest and can be administered after an acute case of infection has been resolved in order to prevent relapse, during an acute case of infection as a complement to antibiotic therapy if the bacterial composition is not sensitive to the same antibiotics as the Gl pathogen, or to prevent infection or reduce transmission from disease carriers.
• the present bacterial compositions can be useful in a variety of clinical situations.
• the bacterial compositions can be administered as a complementary treatment to antibiotics when a patient is suffering from an acute infection, to reduce the risk of recurrence after an acute infection has subsided, or when a patient will be in close proximity to others with or at risk of serious gastrointestinal infections (physicians, nurses, hospital workers, family members of those who are ill or hospitalized).
• the present bacterial compositions can be administered to animals, including humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), and household pets (e.g., dogs, cats, rodents).
• the bacterial composition can be administered enterically, in other words, by a route of access to the gastrointestinal tract. This includes oral administration, rectal administration (including enema, suppository, or colonoscopy), by an oral or nasal tube (nasogastric, nasojejunal, oral gastric, or oral jejunal), as detailed more fully herein.
• the patient Prior to administration of the bacterial composition, the patient can optionally have a pretreatment protocol to prepare the gastrointestinal tract to receive the bacterial composition.
• the pretreatment protocol is advisable, such as when a patient has an acute infection with a highly resilient pathogen.
• the pretreatment protocol is entirely optional, such as when the pathogen causing the infection is not resilient, or the patient has had an acute infection that has been successfully treated but where the physician is concerned that the infection may recur.
• the pretreatment protocol can enhance the ability of the bacterial composition to affect the patient's microbiome.
• At least one antibiotic can be administered to alter the bacteria in the patient.
• a standard colon-cleansing preparation can be administered to the patient to substantially empty the contents of the colon, such as used to prepare a patient for a colonscopy.
• substantially emptying the contents of the colon this application means removing at least 75%, at least 80%, at least 90%, at least 95%, or about 100% of the contents of the ordinary volume of colon contents.
• Antibiotic treatment can precede the colon-cleansing protocol.
• the antibiotic can be stopped in sufficient time to allow the antibiotic to be substantially reduced in concentration in the gut before the bacterial composition is administered.
• the antibiotic can be discontinued 1 , 2, or 3 days before the administration of the bacterial composition.
• the antibiotic can be discontinued 3, 4, 5, 6, or 7 antibiotic half-lives before administration of the bacterial composition.
• the antibiotic can be chosen so the constituents in the bacterial composition have an MIC50 that is higher than the concentration of the antibiotic in the gut.
• MIC50 of a bacterial composition or the elements in the composition can be determined by methods well known in the art. Reller et al., Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices, Clinical Infectious Diseases 49(1 1 ):1749-1755 (2009).
• the additional time between antibiotic administration and administration of the bacterial composition is not necessary. If the pretreatment protocol is part of treatment of an acute infection, the antibiotic can be chosen so that the infection is sensitive to the antibiotic, but the constituents in the bacterial composition are not sensitive to the antibiotic.
• the mammalian subject is a human subject who has one or more symptoms of a dysbiosis.
• the bacterial compositions described herein are suitable for treatment thereof.
• the mammalian subject has not received antibiotics in advance of treatment with the bacterial compositions.
• 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.
• the mammalian subject has not previously received an antibiotic compound in the one month prior to administration of the therapeutic composition.
• the mammalian subject has received one or more treatments with one or more different antibiotic compounds and such treatment(s) resulted in no improvement or a worsening of symptoms.
• the gastrointestinal disease, disorder or condition is diarrhea caused by C. difficile including recurrent C. difficile infection, ulcerative colitis, colitis, Crohn's disease, or irritable bowel disease.
• the therapeutic composition is administered only once prior to improvement of the disease, disorder or condition.
• 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.
• the preparation can be administered intermittently according to a set schedule, e.g., once a day, once weekly, or once monthly, or when the subject relapses from the primary illness.
• the preparation may be administered on a long-term basis to subjects who are at risk for infection with or who may be carriers of these pathogens, including subjects who will have an invasive medical procedure (such as surgery), who will be hospitalized, who live in a long-term care or rehabilitation facility, who are exposed to pathogens by virtue of their profession (livestock and animal processing workers), or who could be carriers of pathogens (including hospital workers such as physicians, nurses, and other health care professionals).
• 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).
• the bacterial composition is administered enterically. This preferentially includes oral administration, or by an oral or nasal tube (including nasogastric, nasojejunal, oral gastric, or oral jejunal). In other embodiments, administration includes rectal administration (including enema, suppository, or colonoscopy).
• the bacterial composition can be administered to at least one region of the gastrointestinal tract, including the mouth, esophagus, stomach, small intestine, large intestine, and rectum. In some embodiments, it is administered to all regions of the gastrointestinal tract.
• the bacterial compositions can be administered orally in the form of medicaments such as powders, capsules, tablets, gels or liquids.
• the bacterial compositions can also be administered in gel or liquid form by the oral route or through a nasogastric tube, or by the rectal route in a gel or liquid form, by enema or instillation through a colonoscope or by a suppository.
• the composition is administered colonoscopically and, optionally, if the bacterial composition is administered by other rectal routes (such as an enema or suppository) or even if the subject has an oral administration, the subject can have a colon-cleansing preparation.
• the colon-cleansing preparation can facilitate proper use of the colonoscope or other administration devices, but even when it does not serve a mechanical purpose, it can also maximize the proportion of the bacterial composition relative to the other organisms previously residing in the gastrointestinal tract of the subject. Any ordinarily acceptable colon-cleansing preparation may be used such as those typically provided when a subject undergoes a colonoscopy.
• the bacteria and bacterial compositions are provided in a dosage form.
• the dosage form is designed for administration of at least one OTU or combination thereof disclosed herein, wherein the total amount of bacterial composition administered is selected from 0.1 ng to 10g, 10ng to 1g, 100ng to 0.1g, 0.1 mg to 500mg, 1 mg to 100mg, or from 10-15mg.
• the bacterial composition is consumed at a rate of from 0.1 ng to 10g a day, 10ng to 1 g a day, 100ng to 0.1 g a day, 0.1 mg to 500mg a day, 1 mg to 100mg a day, or from 10-15mg a day, or more.
• the treatment period is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year.
• the treatment period is from 1 day to 1 week, from 1 week to 4 weeks, from 1 month, to 3 months, from 3 months to 6 months, from 6 months to 1 year, or for over a year.
• 10 5 and 10 12 microorganisms total can be administered to the patient in a given dosage form.
• an effective amount can be provided in from 1 to 500 ml or from 1 to 500 grams of the bacterial composition having from 10 7 to 10 11 bacteria per ml or per gram, or a capsule, tablet or suppository having from 1 mg to 1000 mg lyophilized powder having from 10 7 to 10 11 bacteria.
• Those receiving acute treatment can receive higher doses than those who are receiving chronic administration (such as hospital workers or those admitted into long- term care facilities).
• any of the preparations described herein can be administered once on a single occasion or on multiple occasions, such as once a day for several days or more than once a day on the day of administration (including twice daily, three times daily, or up to five times daily).
• the preparation can be administered intermittently according to a set schedule, e.g., once weekly, once monthly, or when the patient relapses from the primary illness.
• the preparation can be administered on a long-term basis to individuals who are at risk for infection with or who may be carriers of these pathogens, including individuals who will have an invasive medical procedure (such as surgery), who will be hospitalized, who live in a long-term care or rehabilitation facility, who are exposed to pathogens by virtue of their profession (livestock and animal processing workers), or who could be carriers of pathogens (including hospital workers such as physicians, nurses, and other health care
• compositions can be selected for individual patients or for patients with particular profiles. For example, 16S sequencing can be performed for a given patient to identify the bacteria present in his or her microbiota.
• sequencing can either profile the patient's entire microbiome using 16S sequencing (to the family, genera, or species level), a portion of the patient's microbiome using 16S sequencing, or it can be used to detect the presence or absence of specific candidate bacteria that are biomarkers for health or a particular disease state, such as markers of multi-drug resistant organisms or specific genera of concern such as Escherichia.
• composition Based on the biomarker data, a particular composition can be selected for
• patients can be screened to determine the composition of their microbiota to determine the likelihood of successful treatment.
• the bacterial compositions can be administered with other agents in a combination therapy mode, including anti-microbial agents and prebiotics.
• Administration can be sequential, over a period of hours or days, or simultaneous.
• the bacterial compositions are included in combination therapy with one or more anti-microbial agents, which include anti-bacterial agents, anti-fungal agents, anti-viral agents and anti-parasitic agents.
• Anti-bacterial agents can include cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).
• cephalosporin antibiotics cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin
• Anti-viral agents can include Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, Foscarnet, Fomivirsen, Ganciclovir, Indinavir, Idoxuridine, Lamivudine, Lopinavir Maraviroc, MK-2048,
• Nelfinavir Nevirapine, Penciclovir, Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine, Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine, Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir, Zalcitabine, Zanamivir and
• antifungal compounds include, but are not limited to polyene antifungals such as natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, and hamycin; imidazole antifungals such as miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole; triazole antifungals such as fluconazole, itraconazole, isavuconazole, ravuconazole,
• posaconazole posaconazole, voriconazole, terconazole, and albaconazole
• thiazole antifungals such as abafungin
• allylamine antifungals such as terbinafine, naftifine, and butenafine
• echinocandin antifungals such as anidulafungin, caspofungin, and micafungin.
• Other compounds that have antifungal properties include, but are not limited to polygodial, benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine or 5-fluorocytosine, griseofulvin, and haloprogin.
• the bacterial compositions are included in combination therapy with one or more corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines,
• a prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health.
• Prebiotics can include complex carbohydrates, amino acids, peptides, or other essential nutritional components for the survival of the bacterial composition.
• Prebiotics include, but are not limited to, amino acids, biotin, fructooligosaccharide, galactooligosaccharides, inulin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, oligofructose, oligodextrose, tagatose, trans-galactooligosaccharide, and xylooligosaccharides.
• Composition Populates a Subject's Gastrointestinal Tract
• an animal model such as a mouse model
• the model can begin by evaluating the microbiota of the mice. Qualitative assessments can be accomplished using 16S profiling of the microbial community in the feces of normal mice. It can also be accomplished by full genome sequencing, whole genome shotgun sequencing (WGS), or traditional microbiological techniques.
• WGS whole genome shotgun sequencing
• Quantitative assessments can be conducted using quantitative PCR (qPCR), described below, or by using traditional microbiological techniques and counting colony formation.
• the mice can receive an antibiotic treatment to mimic the condition of dysbiosis.
• Antibiotic treatment can decrease the taxonomic richness, diversity, and evenness of the community, including a reduction of abundance of a significant number of bacterial taxa. Dethlefsen et al., The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing, PLoS Biology 6(1 1 ):3280 (2008). At least one antibiotic can be used, and antibiotics are well known.
• Antibiotics can include aminoglycoside antibiotic (amikacin,
• rhodostreptomycin streptomycin, tobramycin, and apramycin
• amoxicillin ampicillin
• Augmentin an amoxicillin/clavulanate potassium combination
• cephalosporin cefaclor, defadroxil, cefazolin, cefixime, fefoxitin, cefprozil, ceftazimdime, cefuroxime
• mice can be provided with drinking water containing a mixture of the antibiotics kanamycin, colistin, gentamycin, metronidazole and vancomycin at 40 mg/kg, 4.2 mg/kg, 3.5 mg/kg, 21 .5 mg/kg, and 4.5 mg/kg (mg per average mouse body weight), respectively, for 7 days.
• mice can be administered ciprofloxacin at a dose of 15-20 mg/kg (mg per average mouse body weight), for 7 days.
• 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.
• 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 10 4 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 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , and/or 10 10 .
• mice can be evaluated using 16S sequencing, full genome sequencing, whole genome shotgun sequencing (WGS), or traditional microbiological techniques to determine whether the test bacterial composition has populated the gastrointestinal tract of the mice. For example only, one day, three days, one week, two weeks, and one month after administration of the bacterial composition to the mice, 16S profiling is conducted to determine whether the test bacterial composition has populated the gastrointestinal tract of the mice. Quantitative assessments, including qPCR and traditional microbiological techniques such as colony counting, can additionally or alternatively be performed, at the same time intervals.
• the number of sequence counts that correspond exactly to those in the bacterial composition over time can be assessed to determine specifically which components of the bacterial composition reside in the gastrointestinal tract over a particular period of time.
• the strains of the bacterial composition persist for a desired period of time.
• 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.
• a bacterial composition can be chosen for its ability to populate one or more than one region of the gastrointestinal tract, including, but not limited to the stomach, the small intestine (duodenum, jejunum, and ileum), the large intestine (the cecum, the colon (the ascending, transverse, descending, and sigmoid colon), and the rectum).
• An in vivo study can be conducted to determine which regions of the gastrointestinal tract a given bacterial composition will populate.
• a mouse model similar to the one described above can be conducted, except instead of assessing the feces produced by the mice, particular regions of the gastrointestinal tract can be removed and studied individually. For example, at least one particular region of the
• gastrointestinal tract can be removed and a qualitative or quantitative determination can be performed on the contents of that region of the gastrointestinal tract.
• the contents can optionally be removed and the qualitative or quantitative determination may be conducted on the tissue removed from the mouse.
• qPCR quantitative PCR
• qPCR can be conducted using HotMasterMix (5PRIME, Gaithersburg, MD) and primers specific for the bacterial composition of interest, and may be conducted on a MicroAmp ® Fast Optical 96-well Reaction Plate with Barcode (0.1 mL) (Life Technologies, Grand Island, NY) and performed on a BioRad C1000TM Thermal Cycler equipped with a CFX96TM Real-Time System (BioRad, Hercules, CA), with fluorescent readings of the FAM and ROX channels. The Cq value for each well on the FAM channel is determined by the CFX ManagerTM software version 2.1 .
• the logio(cfu/ml) of each experimental sample is calculated by inputting a given sample's Cq value into linear regression model generated from the standard curve comparing the Cq values of the standard curve wells to the known logio(cfu/ml) of those samples.
• the skilled artisan may employ alternative qPCR modes.
• bacterial compositions For example, the sensitivity of bacterial compositions to certain environmental variables is determined, e.g., in order to select for particular desirable characteristics in a given composition, formulation and/or use.
• the constituents in the bacterial composition can be tested for pH resistance, bile acid resistance, and/or antibiotic sensitivity, either individually on a constituent-by- constituent basis or collectively as a bacterial composition comprised of multiple bacterial constituents (collectively referred to in this section as bacterial composition).
• a bacterial composition will be administered other than to the colon or rectum (i.e., for example, an oral route), optionally testing for pH resistance enhances the selection of bacterial compositions that will survive at the highest yield possible through the varying pH environments of the distinct regions of the Gl tract.
• bacterial compositions can be prepared that survive these varying pH ranges (specifically wherein at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or as much as 100% of the bacteria can survive gut transit times through various pH ranges). This can be tested by exposing the bacterial composition to varying pH ranges for the expected gut transit times through those pH ranges.
• 18-hour cultures of bacterial compositions can be grown in standard media, such as gut microbiota medium ("GMM", see Goodman et al., Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice, PNAS 108(15):6252-6257 (201 1 )) 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.
• GMM gut microbiota medium
• PNAS 108(15):6252-6257 (201 1 ) or another animal-products-free medium
• 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.
• testing for bile-acid resistance enhances the selection of bacterial compositions that will survive exposures to bile acid during transit through the Gl tract.
• Bile acids are secreted into the small intestine and can, like pH, affect the survival of bacterial compositions. This can be tested by exposing the bacterial compositions to bile acids for the expected gut exposure time to bile acids.
• bile acid solutions can be prepared at desired concentrations using 0.05 mM Tris at pH 9 as the solvent. After the bile acid is dissolved, the pH of the solution may be adjusted to 7.2 with 10% HCI.
• Bacterial compositions can be cultured in 2.2 ml of a bile acid composition mimicking the concentration and type of bile acids in the patient, 1 .0 ml of 10% sterile-filtered feces media and 0.1 ml of an 18-hour culture of the given strain of bacteria. Incubations may be conducted for from 2.5 to 3 hours or longer.
• An alternative method for testing stability to bile acid is described in U.S. Patent No. 4,839,281 . Survival of bacteria may be determined by culturing the bacteria and counting colonies on appropriate selective or non-selective media.
• bacterial compositions can be tested for sensitivity to antibiotics.
• bacterial compositions can be chosen so that the bacterial constituents are sensitive to antibiotics such that if necessary they can be eliminated or substantially reduced from the patient's
• the bacterial compositions may optionally be tested for the ability to adhere to gastrointestinal cells.
• a method for testing adherence to gastrointestinal cells is described in U.S. Patent No. 4,839,281 . Methods for Purifying Spores
• a solvent treatment is a miscible solvent treatment (either partially miscible or fully miscible) or an immiscible solvent treatment.
• Miscibility is the ability of two liquids to mix with each to form a homogeneous solution. Water and ethanol, for example, are fully miscible such that a mixture containing water and ethanol in any ratio will show only one phase. Miscibility is provided as a wt/wt%, or weight of one solvent in 100 g of final solution. If two solvents are fully miscible in all proportions, their miscibility is 100%.
• alcohols e.g., methanol, ethanol, isopropanol, butanol, etc.
• the alcohols can be provided already combined with water; e.g., a solution containing10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 85%, 90%, 95% or greater than 95%
• Other solvents are only partially miscible, meaning that only some portion will dissolve in water.
• Diethyl ether for example, is partially miscible with water. Up to 7 grams of diethyl ether will dissolve in 93 g of water to give a 7% (wt/wt%) solution.
• miscible materials include ethers, dimethoxyethane, or tetrahydrofuran
• an oil such as an alkane and water are immiscible and form two phases.
• immiscible treatments are optionally combined with a detergent, either an ionic detergent or a non-ionic detergent.
• exemplary detergents include Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, or a polyol.
• the fecal materials are subjected to one or more chromatographic treatments, either sequentially or in parallel.
• chromatographic treatments either sequentially or in parallel.
• a solution containing the fecal material is contacted with a solid medium containing a hydrophobic interaction chromatographic (HIC) medium or an affinity chromatographic medium.
• HIC hydrophobic interaction chromatographic
• a solid medium capable of absorbing a residual habitat product present in the fecal material is contacted with a solid medium that adsorbs a residual habitat product.
• the HIC medium contains sepharose or a derivatized sepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, or butyl-s sepharose.
• the affinity chromatographic medium contains material derivatized with mucin type I, II, III, IV, V, or VI, or oligosaccharides derived from or similar to those of mucins type I, II, III, IV, V, or VI.
• the affinity chromatographic medium contains material derivatized with antibodies that recognize spore-forming bacteria.
• the physical disruption of the fecal material particularly by one or more mechanical treatment such as blending, mixing, shaking, vortexing, impact pulverization, and sonication.
• the mechanical disrupting treatment substantially disrupts a non-spore material present in the fecal material and does not substantially disrupt a spore present in the fecal material.
• Mechanical treatments optionally include filtration treatments, where the desired spore populations are retained on a filter while the undesirable (non-spore) fecal components to pass through, and the spore fraction is then recovered from the filter medium.
• undesirable particulates and eukaryotic cells may be retained on a filter while bacterial cells including spores pass through.
• the spore fraction retained on the filter medium is subjected to a diafiltration step, wherein the retained spores are contacted with a wash liquid, typically a sterile saline-containing solution or other diluent, in order to further reduce or remove the undesirable fecal components.
• the fecal material is mixed in a saline-containing solution such as phosphate- buffered saline (PBS) and subjected to a heated environment, such as a warm room, incubator, water-bath, or the like, such that efficient heat transfer occurs between the heated environment and the fecal material.
• a heated environment such as a warm room, incubator, water-bath, or the like
• the fecal material solution is mixed during the incubation to enhance thermal conductivity and disrupt particulate aggregates.
• Thermal treatments can be modulated by the temperature of the environment and/or the duration of the thermal treatment.
• the fecal material or a liquid comprising the fecal material is subjected to a heated environment, e.g., a hot water bath of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or greater than 100 degrees Celsius, for at least about 1 , 5, 10, 15, 20, 30, 45 seconds, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 hours.
• the thermal treatment occurs at two different temperatures, such as 30 seconds in a 100 degree Celsius environment followed by 10 minutes in a 50 degree Celsius environment.
• the temperature and duration of the thermal treatment are sufficient to kill or remove pathogenic materials while not substantially damaging or reducing the germination-competency of the spores.
• ionizing radiation typically gamma irradiation, ultraviolet irradiation or electron beam irradiation provided at an energy level sufficient to kill pathogenic materials while not substantially damaging the desired spore populations.
• ionizing radiation typically gamma irradiation, ultraviolet irradiation or electron beam irradiation provided at an energy level sufficient to kill pathogenic materials while not substantially damaging the desired spore populations.
• ultraviolet radiation at 254nm provided at an energy level below about 22,000 microwatt seconds per cm 2 will not generally destroy desired spores.
• a solution containing the fecal material is subjected to one or more centrifugation treatments, e.g., at about 1000 x g, 2000 x g, 3000 x g, 4000 x g, 5000 x g, 6000 x g, 7000 x g, 8000 x g or greater than 8000 x g.
• Differential centrifugation separates desired spores from undesired non-spore material; at low forces the spores are retained in solution, while at higher forces the spores are pelleted while smaller impurities (e.g., virus particles, phage) are retained in solution.
• impurities e.g., virus particles, phage
• density or mobility gradients or cushions e.g., step cushions
• Percoll, Ficoll, Nycodenz, Histodenz or sucrose gradients are used to separate desired spore populations from other materials in the fecal material.
• formulations for administration to humans and other subjects in need thereof 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.
• the composition comprises at least one carbohydrate.
• a “carbohydrate” refers to a sugar or polymer of sugars.
• saccharide 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 C n H2 n O n .
• a carbohydrate can be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide.
• the most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined
• oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more
• 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.
• the composition comprises at least one lipid.
• 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).
• 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 ),
• EPA eicosadienoic acid
• DHA docosapentaenoic acid
• the composition comprises at least one modified lipid, for example, a lipid that has been modified by cooking.
• the composition comprises at least one supplemental mineral or mineral source.
• supplemental mineral or mineral source examples 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.
• the composition comprises at least one supplemental vitamin.
• the at least one vitamin can be fat-soluble or water soluble vitamins.
• Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
• Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
• the composition comprises an excipient.
• 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.
• the excipient is a buffering agent.
• suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
• the excipient comprises a preservative.
• suitable preservatives include antioxidants, such as alpha- tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
• the composition comprises a binder as an excipient.
• suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium
• the composition comprises a lubricant as an excipient.
• 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.
• the composition comprises a dispersion enhancer as an excipient.
• 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.
• the composition comprises a disintegrant as an excipient.
• the disintegrant is a non-effervescent disintegrant.
• 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.
• the disintegrant is an effervescent disintegrant.
• suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
• the excipient comprises a flavoring agent.
• Flavoring agents can be chosen from synthetic flavor oils and flavoring aromatics;
• 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.
• the excipient comprises a sweetener.
• 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.
• 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.
• the composition comprises a coloring agent.
• 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.
• parenteral as used herein includes subcutaneous, intravenous, intramuscular, or intrasternal injection and infusion techniques.
• 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.
• the core material comprises at least one of a solid, a liquid, and an emulsion.
• 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, hydroxypropyl methyl 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
• Tablets, pills, and the like can be compressed, multiply compressed, multiply layered, and/or coated.
• the coating can be single or multiple. In one
• the coating material comprises at least one of a saccharide, a
• 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.
• the coating material comprises a protein.
• the coating material comprises at least one of a fat and an oil.
• the at least one of a fat and an oil is high temperature melting.
• the at least one of a fat and an oil is hydrogenated or partially hydrogenated.
• the at least one of a fat and an oil is derived from a plant.
• the at least one of a fat and an oil comprises at least one of glycerides, free fatty acids, and fatty acid esters.
• 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.
• powders or granules embodying the bacterial compositions disclosed herein can be incorporated into a food product.
• the food product is a drink for oral administration.
• 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.
• 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.
• the food product can be a solid foodstuff.
• 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.
• the compositions disclosed herein are incorporated into a therapeutic food.
• the therapeutic food is a ready-to-use food that optionally contains some or all essential macronutrients and micronutrients.
• the compositions disclosed herein are incorporated into a supplementary food that is designed to be blended into an existing meal.
• the supplemental food contains some or all essential macronutrients and micronutrients.
• 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.
• 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 (10 8 to 10 11 bacteria), 160 mg microcrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesium stearate.
• from 10 5 to 10 12 bacteria may be used, 10 5 to 10 7 , 10 6 to 10 7 , or 10 8 to 10 10 , with attendant adjustments of the excipients if necessary.
• an enteric-coated capsule or tablet or with a buffering or protective composition can be used.
• 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 -80C. Aliquots of the cultures can be placed into cell lysis buffer and colony PCR methods can be used to amplify and sequence the 16S rDNA gene (Example 2). Alternatively, colonies may be streaked to purity in several passages on solid media. Well separated colonies are streaked onto the fresh plates of the same kind and incubated for 48-72 hours at 37C. 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 2 & 3. 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 2 &
• the sample can be sequenced prior to germination (if appropriate DNA isolation procedures are used to Isye 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.
• MALDI-TOF-mass spec can also be used for species identification (as reviewed in Anaerobe 22:123).
• 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.
• 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.
• PCR polymerase chain reaction
• the sequencing method used may be, but is not limited to, Sanger sequencing.
• 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 lllumina (sequencing by synthesis) method using barcoded primers allowing for multiplex reactions.
• a next- generation sequencing method such as an lllumina (sequencing by synthesis) method using barcoded primers allowing for multiplex reactions.
• Escherichia coli can be distinguished using DNAs from the genes that encode heat- labile (LTI, LTIIa, and LTIIb) and heat-stable (STI and STII) toxins, verotoxin types 1 , 2, and 2e (VT1 , VT2, and VT2e, respectively), cytotoxic necrotizing factors (CNF1 and CNF2), attaching and effacing mechanisms ⁇ eaeA), enteroaggregative mechanisms (Eagg), and enteroinvasive mechanisms (Einv).
• the optimal genes to utilize for taxonomic assignment of OTUs by use of marker genes will be familiar to one with ordinary skill of the art of sequence based taxonomic identification.
• Genomic DNA is extracted from pure microbial cultures using a hot alkaline lysis method. 1 ⁇ of microbial culture is added to 9 ⁇ of Lysis Buffer (25mM NaOH, 0.2 mM EDTA) and the mixture is incubated at 95°C for 30 minutes.
• Lysis Buffer 25mM NaOH, 0.2 mM EDTA
• 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, CA) or by standard methods known to those skilled in the art.
• V1 -V9 regions refer to the first through ninth hypervariable regions of the 16S rRNA gene that are used for genetic typing of bacterial samples. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1 1 17-1 173, 1243-1294 and 1435-1465 respectively using numbering based on the E. coli system of nomenclature.
• V1 , V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize an OTU.
• the V1 , V2, and V3 regions are used to characterize an OTU.
• the V3, V4, and V5 regions are used to characterize an OTU.
• the V4 region is used to characterize an OTU.
• the PCR is performed on commercially available thermocyclers such as a BioRad MyCyclerTM Thermal Cycler (BioRad, Hercules, CA). 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.
• thermocyclers such as a BioRad MyCyclerTM Thermal Cycler (BioRad, Hercules, CA).
• 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.
• Amplification performed for downstream sequencing by short read technologies such as lllumina require amplification using primers known to those skilled in the art that additionally include a sequence-based barcoded tag.
• primers known to those skilled in the art that additionally include a sequence-based barcoded tag.
• 2 ⁇ of extracted gDNA is added to a 20 ⁇ final volume PCR reaction.
• the PCR reaction also contains 1 x HotMasterMix (5PRIME, Gaithersburg, MD), 200 nM of V4_515f_adapt
• thermocyclers such as a BioRad MyCyclerTM Thermal Cycler (BioRad, Hercules, CA).
• 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.
• 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.
• Nucleic Acid Detection The prepared library is sequenced on lllumina HiSeq or MiSeq sequencers (lllumina, San Diego, CA) 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), 16SV4SeqRev (AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT), and 16SV4lndex (ATTAGAWACCCBDGTAGTCCGGCTGACTGACT) (IDT, Coralville, IA) 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.
• 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.
• tree topologies and nodal structure are used to refine the resolution of the analysis.
• phylogenetic methods 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 CR, and Warnow T. 201 1 .
• 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.
• 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.
• 16S-V4 OTU identification 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 ).
• 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.
• 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.
• the sample is moved to the anaerobic chamber, resuspended in prereduced PBS, mixed and incubated for 1 hour at 37C 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.
• 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 e.g., dodecylamine
• 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 HCI (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.
• BHIS Brain heart infusion powder (37 g/L), yeast extract (5 g/L), L- cysteine HCI (1 g/L)
• Oxgall provides additional bile acid germinants.
• 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
• 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.
• BHIS/oxgall solution is used as a germinant and contains 0.5X BHIS medium with 0.25% oxgall (dehydrated bovine bile) where 1 x BHIS medium contains the following per L of solution: 6g Brain Heart Infusion from solids, 7g peptic digest of animal tissue, 14.5g of pancreatic digest of casein, 5g of yeast extract, 5g sodium chloride, 2g glucose, 2.5g disodium phosphate, and 1 g cysteine. Additionally, Ca-DPA is a germinant and contains 40mM CaCI2, and 40mM 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 KH 2 P0 4 , 0.53 g Na 2 HP0 4 , 0.3 g NH 4 CI, 0.3 g NaCI, 0.1 g MgC x 6H 2 O, 0.1 1 g CaC , 1 ml alkaline trace element solution, 1 ml acid trace element solution, 1 ml vitamin solution, 0.5 mg resazurin, 4 g NaHC0 3 , 0.25 g Na 2 S x 9 H 2 0.
• 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 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, 0.01 19M Phosphate Buffer.
• PBS Phosphate Buffered Saline
• 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 5mM 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 5mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mM proline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine,
• BHIS medium + DPA is a germinant mixture and contains
• EcSN 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 20,000rcf for
• Example 5 The purification and isolation of a spore forming fraction from feces
• 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 10min) is performed to remove solvent and the pellet is retained and washed.
• a homogenization device e.g., laboratory blender
• 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 10min) is performed to remove solvent and the pellet is retained and washed.
• 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 CsCI gradient, sucrose gradient or combination of the two generating a ethanol treated, gradient-purified spore preparation.
• a CsCI gradient is performed by loading a 20% volume of spore suspension on top a 80% volume of a stepwise CsCI gradient (w/v) containing the steps of 64%, 50%, 40% CsCI (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).
• the spores run roughly in the 30% and 40% sucrose fractions.
• the lower spore fraction ( Figure 2) is then removed and washed to produce a concentrated ethanol treated, gradient-purified spore preparation.
• Example 6 Bacterial compositions prevent C. difficile infection in a mouse model
• 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.
• 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 10mg/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 Iog10 cfu of C. difficile (ATCC 43255) via oral gavage.
• 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
• 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).
• Dosing for CFU counts was determined from the final ethanol treated spores and dilutions of total spores were administered at 1 x, 0.1 x, 0.01x of the spore mixture for the ethanol treated fraction and a 1 x dose for the heat treated fraction.
• Example 7 The prophylactic and relapse prevention hamster models
• Clindamycin (10mg/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.
• 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.
• the results are shown in Figure 4, illustrating the prophylaxis model with the ethanol treated spore preparation and the ethanol treated, gradient-purified spore preparation.
• 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.
• Example 8 Clinical treatment of recurrent C. difficile in patients
• test articles like bacterial composition including but not limited to a ethanol treated spore preparations (e.g. see Example 5) 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.
• blood-borne pathogens HBV, HBV, CMV, HAV and Treponema pallidum
• fecal bacterial pathogens Salmonella, Shigella, Yersinia, Campylobacter,
• 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.
• CDAD antibiotic therapy 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.
• 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 5).
• the culture was centrifuged at 10,000 x 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 -80C for long term storage.
• 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 H 2 :C0 2 :N 2 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.
• 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.
• Cultures were grown static or shaking, depending which yielded maximal cell titer.
• 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 10 6 to 10 10 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 1 st and/or 2nd freeze thaw, while the viability of others were unaffected.
• Example 12 Preparation of bacterial compositions.
• 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.
• the cfu in the final mixture was estimated based on the second freeze-thaw titer of the individual strains.
• strains may be combined at equal counts in order to deliver between 1 e4 and 1 e10 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 1 e10.
• Example 13 Provision of gut microbiome sample material.
• 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/m 2 and ⁇ 25 kg/m 2 .
• 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
• 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.
• 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 x 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.
• the suspension was then centrifuged at a low speed, e.g., 1000 x 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 x g for 10 minutes to pellet the vegetative organisms and spores.
• the pellet was then resuspended in 1 x PBS with vigorous vortexing until the sample material appears homogenous
• Example 14 Bacterial compositions populating the gut in a mouse model.
• compositions were evaluated in a mouse model to demonstrate the ability to populate the gastrointestinal tract. Bacteria were grown as described in Example 12. Compositions were pre-made under anaerobic conditions and suspended in PBS + 15% glycerol and stored at > -70°C prior to use.
• mice (10 females/group; 5 per cage) were pre-treated on Days -14 to -5 with an antibiotic cocktail consisting of 10% glucose, kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) and vancomycin (0.056 mg/ml) in their drinking water. On Day -3 they received 10 mg/kg Clindamycin by oral gavage. On Day -1 , they were dosed with a microbial composition by oral gavage in a volume of 0.2 ml. (Table ZA).
• Microbial compositions comprised approximately equal numbers of each OTU and were dosed at approximately 1 x10 9 , 1 x10 8 and 1 x10 7 per OTU for each composition (e.g. microbial composition 1 , comprising 15 strains, was dosed at approximately 1 .5x10 10 , 1 .5x10 9 , and 1 .5x10 8 total CFU).
• Fecal samples were collected from each cage on Day -1 (approximately 1 hour before dosing) and on Days 2, 3 and 4 post-dosing. Feces were stored frozen prior to processing and sequencing. Weight gain of mice treated with either microbial composition was similar to that of naive, control mice.
• Clostridium difficile (ATCC 43255) via oral gavage. Mortality for C. difficile challenged animals was assessed every day from Day 0 to Day 6 and the weight and subsequent weight change of the animal was assessed with weight loss being associated with C. difficile infection. Mortality and reduced weight loss of the test article compared to the empty vehicle was used to assess the success of the test article.
• Fecal samples were processed by isolating and sequencing DNA according to ***Example 2 and 3.
• the OTU assignment of fecal samples from Days -1 , 2, 3 and 4 was determined by analyzing 16S-V4 sequence reads and assigning OTUs as described in ***Example 2. Clades were assigned as described in ***Example 2. Total read counts were determined for each OTU or each clade by summing the results from cages of the same experimental group. Samples with 10 or fewer sequence reads for a given OTU or clade were considered to be below background and were not included in the summation process. Results are shown by OTU (Table TAB) and by clade (Table TAC).
• Table TAB Population of OTUs on Days 2, 3 and 4 following dosing with Microbial Compositions
• Table TAC Population of Clades on Days 2, 3 and 4 following dosing with Microbial Compositions
• this group includes CI . butyricum, CI. hylemonae, CI. orbiscindens, CI. symbiosum, and L.
• bacterium_5_1_57FAA. CI. disporicum is comparable to this group as it has sequence reads on Day -1 that are very close to background (10 and 29 in compositions 1 and 2, respectively), which subsequently increase by as much as 1000-fold on Days 2, 3 or 4.
• OTUs such as Co. aerofaciens, C. comes, R. bromii, B. producta, CI. bolteae, CI. mayombei, CI. innocuum, CI. tertium and R. gnavus which are not detectable at the OTU level in either the Day -1 sample or in subsequent samples.
• Co. aerofaciens C. comes, R. bromii, B. producta, CI. bolteae, CI. mayombei, CI. innocuum, CI. tertium and R. gnavus which are not detectable at the OTU level in either the Day -1 sample or in subsequent samples.
• Co. aerofaciens C
• 16S-V4 OTU identification to assign an OTU as a specific species depends in part on the resolving power of the 16S-V4 region for a particular species or group of species. Both the density of available reference 16S sequences for different regions of the tree as well as the inherent variability in the 16S gene between different species will determine the definitiveness of a taxonomic annotation. So in some cases, the population of a species can be followed using clade-based assignments when OTU based-detection is insensitive in a complex population. For instance, the clade-based analysis in Table 1 supports the case that R. bromii, B. producta, CI.
• mice challenged in parallel with C. difficile were significantly protected as shown in Table TAD.
• mice gavaged with vehicle experienced 100% mortality while microbial compositions 1 and 2 protected at all dose levels with between 0 and 10% mortality by Day 6, the last day of the experiment.
• weight loss in animals treated with microbial compositions 1 and 2 was minimal compared to animals receiving the vehicle gavage.
• Example 15 Use of Biolo assay to determine bacterial strain nutrient utilization
• a screen was performed to test the ability of Clostridium difficile and potential competitor species to utilize a panel of 190 different carbon sources.
• the screen was carried out using PM1 and PM2 MicroPlates (Biolog #121 1 1 , #121 12), IF-Oa base media (Biolog #72268) and Biolog Redox Dye Mix D (Biolog #74224).
• PM1 and PM2 MicroPlates Biolog #121 1 1 , #121 12
• IF-Oa base media Biolog #72268
• Biolog Redox Dye Mix D Biolog #74224
• a single colony was then re-streaked to a BBA plate and incubated anaerobically at 37° C for 24 hr.
• the MicroPlates were pre-reduced by incubating for at least 24 hr in a hydrogen free anaerobic environment before use. All liquid media and supplements used were pre-reduced by placing them in an anaerobic chamber with loose lids for at least 24 hr before use. Alternatively, combinations of bacteria can also be tested.
• the base media for inoculation was prepared by adding 0.029 mL of 1 M potassium ferricyanide to 0.240 mL of Dye Mix D followed by addition of 19.7 mL of IF-Oa, 4 mL sterile water and 0.024 mL 0.5 mM menadione.
• concentrations of potassium ferricyanide and menadione were adjusted to achieve the optimal redox balance or to test multiple redox conditions.
• Potassium ferricyanide was tested at a final concentration of 0.38, 0.12, 0.038 and 0.06 mM.
• Menadione was tested at a final concentration of 0.5, 0.16 and 0.05 ⁇ . In total, this yields 9 redox conditions for testing.
• Reduction of the tetrazolium dye that forms the basis for the endpoint measurement was sensitive to the redox state of each bacterial culture, and thus to the ratio of menadione to potassium ferricyanide. It was therefore important to test various ratios for each bacterial isolate and was also important in some cases to test a species at multiple menadione / potassium ferricyanide ratios in order to detect all conditions in which a possible nutrient utilization was detectable. Some species were tested beyond the 20 hr time point to detect all conditions resulting in a positive result. In these cases plates were read at 20, 44 or 96 hr.
• a similar screen can be performed to test the utilization of vitamins, amino acids, or cofactors.
• Biolog MicroPlates for screening of vitamins, amino acids or cofactors that are of interest would be used in place of the PM1 and PM2 plates, for example PM5.
• Table XXX1 contains a list of representative vitamins, minerals, and cofactors.
• a universal carbon source such as glucose will be used as a positive control to demonstrate reduction of the tetrazolium dye under the specific conditions of the assay.
• Example 16 Use of transcriptomics to determine bacterial strain nutrient utilization
• the ability of a strain to utilize a particular carbon source can be determined by a transcriptomics based approach.
• the C. difficile microarray based on the genome sequence of strain 630 can be found in ArrayExpress (accession number A-BUG- 20).
• the microarray contains 3,679 coding sequences (CDSs).
• CDSs 3,679 coding sequences
• a Genisphere 3DNA Array 900 MPX microarray kit is used for cDNA synthesis, labeling, and hybridization. Ten micrograms of starting RNA is used for the cDNA synthesis reactions. The microarray slides are hybridized competitively with each cDNA.
• microarray slides are scanned using a ScanArray 4000 instrument (Packard Instrument Co.), and fluorescence intensities are quantified using ImaGene (BioDiscovery) software.
• Raw expression data from the arrays are analyzed with R and Limma (linear model for microarray data) software from the Bioconductor project.
• RNA-seq based approach can be used to identify and quantify the relative abundance of mRNA in each sample [can we insert a reference for how to perform an RNA-seq experiment?].
• Example 17A Use of in vivo transcriptomics to determine bacterial strain nutrient utilization
• C. difficile genes for catabolism of sialic acid and fucose were found to have increased levels of expression in vivo relative to when grown in vitro in growth medium, and in Janoir et al (l&l 2013 81 :3757), expression of genes required for catabolism of glucose, sorbitol and fructose are induced in vivo relative to in vitro, suggesting their use during infection.
• RNA FlashGel RNA FlashGel
• Enriched RNA is quantified using RiboGreen (Invitogen, Carlsbad, CA). One hundred nanograms of mRNA-enriched RNA is used for library preparation using Superscript II (Invitrogen, Carlsbad, CA) and an mRNA-seq kit (lllumina, San Diego, CA). The cDNA library is sequenced using an lllumina HiSeq 2500. Reads are then mapped against the appropriate C. difficile reference genome. The 8 hr time point can be used as a reference to identify genes and pathways that are upregulated in vivo, or alternatively a control sample grown in vitro in a standard growth medium can be used as a reference. The pattern of upregulated catabolic genes defines a map of nutrients for which an effective anti-C. difficile consortium of organisms would compete.
• combinations of bacterial strains can be inoculated into a mouse and analysed simultaneously from the same cecal sample using publicly available reference genomes from the human microbiome for read mapping. Species with potential ability to compete with C. difficile are selected based on their pattern of overlap for carbon sources analyzed.
• Example 17B Use of literature search to determine bacterial strain nutrient utilization
• the nutrient utilization capabilities of a pathogen are also determined by means of a literature search.
• Compounds that can support growth of C. difficile were determined from scientific publications describing nutrients that are utilized by the organism (for example, Nakamura et al, 1982, Microbiol Immunol 26:107), from scientific papers describing a defined media that allows growth of the organism (for example, George et al, J. Clin. Microbiol. 1979, 9:214), reference manuals that compile data from the literature such as Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology: Vol. 3: The Firmicutes By Paul Vos, George Garrity, Dorothy Jones, Noel R. Krieg, Wolfgang Ludwig, Fred A.
• Genomics studies describing analysis of reference genomes can suggest nutrients that are potentially used by a pathogen, based on the presence of metabolic pathways and transport proteins for given substrates. For example, Sebaihia et al., Nature Genetics, 2006 which presents the genome for the virulent Clostridium difficile strain 630, describes a 19 gene cluster for ethanolamine degradation suggesting ethanolamine is likely a carbon source used by C. difficile.
• Transcriptomics studies in which mRNA transcript levels are determined by microarray or RNA-seq methods, can reveal metabolic pathways that are upregulated in one condition relative to another.
• genes for catabolism of sialic acid and fucose were found to have increased levels of expression in vivo relative to when grown in vitro in growth medium, and in Janoir et al. (I&l 2013 81 :3757), expression of genes required for catabolism of glucose, sorbitol and fructose are induced in vivo relative to in vitro, suggesting their use during infection.
• 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
• DPA dipicolinic acid
• 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.
• Purified spores are produced as described previously (e.g. see http://www.epa.gov/pesticides/methods/MB-28-00.pdf). Serial dilutions of purified spores from C. bifermentans, C. sporogenes, and C. butyricum cultures were prepared and measured by plating on BBA media and incubating overnight at 37C to determine CFU/ml. Figure 6 shows the linear correspondence across different spore producing bacteria across several logs demonstrating the DPA assay as means to assess spore content.
• AC shows spore content data from 3 different ethanol treated spore preparations used to successfully treat 3 patients suffering from recurrent C. difficile infection.
• the spore content of each spore preparation is characterized using the two described methods.
• 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 x 10 5 or less using the SCFU assay (e.g. Preparation 1 , Table AC). If that SCFU dose was used to normalize dosing in a clinical setting, however, then the actual spore doses given to patients would be much lower for other ethanol treated spore preparations as measured as by the DPA assay (Table AD).
• Preparation 3 would lead to a potential underdosing of more than 100-fold. This can be rectified only by setting potency specifications based on the DPA assay which better reflects total spore counts in an ethanol treated spore preparation. The unexpected finding of this work is that the DPA assay is uniquely suited to set potency and determine dosing for an ethanol treated spore preparation.
• Example 19 Enqraftment, Augmentation and reduction of Pathogen Carriage in Patients Treated with Spore Compositions
• the microbiome was characterized by 16S-V4 sequencing prior to treatment (pretreatment) with an ethanol treated spore preparation and up to 25 days after receiving treatment.
• pretreatment an ethanol treated spore preparation
• a bacterial composition in the vegetative state or a mixture of vegetative bacteria and bacterial spores.
• the treatment of patient 1 with an ethanol treated spore preparation led to the engraftment of OTUs from the spore treatment and augmentation in the microbiome of the patient ( Figure 7 and Figure 8).
• OTUs that were absent in the patient pretreatment
• These OTUs comprised both bacterial species that were capable and not capable of forming spores, and OTUs that represent multiple phylogenetic clades.
• Organisms absent in Patient 1 pre-treatment either engraft directly from the ethanol treated spore fraction or are augmented by the creation of a gut environment favoring a healthy, diverse microbiota.
• Bacteroides fragilis group species were increased by 4 and 6 logs in patients 1 and 2 ( Figure 9).
• Bacteroides species in patients Comparing the number of Bacteroides fragilis groups species per cfu/g of feces pre-treatment and in week 4 post treatment reveals an increase of 4 logs or greater.
• the ability of 16S-V4 OTU identification to assign an OTU as a specific species depends in part on the resolution of the 16S-V4 region of the 16S gene for a particular species or group of species. Both the density of available reference 16S sequences for different regions of the tree as well as the inherent variability in the 16S gene between different species will determine the definitiveness of a taxonomic annotation to a given sequence read.
• taxonomic annotations of a read can be rolled up to a higher level using a clade-based assignment procedure (Table 1 ).
• Table 1 taxonomic annotations of a read can be rolled up to a higher level using a clade-based assignment procedure (Table 1 ).
• 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
• 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.
• Klebsiella and an additional 19% were assigned to the genus Fusobacterium. These striking data are evidence of a profoundly dysbiotic microbiota associated with recurrent C. difficile infection and chronic antibiotic use.
• Klebsiella is a resident of the human microbiome in only about 2% of subjects based on an analysis of HMP database (www.hmpdacc.org), and the mean relative abundance of Klebsiella is only about 0.09% in the stool of these people. It's surprising presence at 20% relative abundance in Patient 1 before treatment is an indicator of a proinflammatory gut environment enabling a "pathobiont" to overgrow and outcompete the commensal organisms normally found in the gut. Similarly, the dramatic overgrowth of
• Fusobacterium indicates a profoundly dysbiotic gut microbiota.
• Fusobacterium, F. nucleatum an OTU phylogenetically indistinguishable from
• Fusobacterium sp. 3_1_33 based on 16S-V4 has been termed "an emerging gut pathogen" based on its association with IBD, Crohn's disease, and colorectal cancer in humans and its demonstrated causative role in the development of colorectal cancer in animal models [Allen-Vercoe, Gut Microbes (201 1 ) 2:294-8]. Importantly, neither Klebsiella nor Fusobacterium was detected in the 16S-V4 reads by Day 25 (Table 18).
• Klebsiella spp. carriage is consistent across multiple patients.
• Four separate patients were evaluated for the presence of Klebsiella spp. pre treatment and 4 weeks post treatment.
• Klebsiella spp. were detected by growth on selective Simmons Citrate Inositol media as previously described. Serial dilution and plating, followed by determining cfu/mL titers of morphologically distinct species and 16S full length sequence identification of representatives of those distinct morphological classes, allowed calculation of titers of specific species.
• the genus Bacteroides is an important member of the gastrointestinal microbiota; 100% of stool samples from the Human Microbiome Project contain at least one species of Bacteroides with total relative abundance in these samples ranging from 0.96% to 93.92% with a median relative abundance of 52.67% (www.hmpdacc.org reference data set HMSMCP). Bacteroides in the gut has been associated with amino acid fermentation and degradation of complex polysaccharides. Its presence in the gut is enhanced by diets rich in animal-derived products as found in the typical western diet [David, L. A. et al, Nature (2013) doi:10.1038/nature12820].
• Bacteroides on PFA agar was approximately 2x10 7 cfu/g due to the growth of multiple non-Bacteroides species in the pretreatment sample on that medium. Colony counts of Bacteroides species on Day 25 were up to 2xio 10 cfu/g, consistent with the 16S-V4 sequencing, demonstrating a profound reconstitution of the gut microbiota in Patient 1 (Table 20).
• Bacteroides species These organisms were most likely either present at extremely low abundance in the Gl tract of Patient 1 , or present in a reservoir in the Gl tract from which they could rebound to high titer. Those species may also be reinoculated via oral uptake from food following treatment.
• Augmentation is an important phenomenon in that it shows the ability to use an ethanol treated spore ecology or other bacterial composition to restore a healthy microbiota by seeding a diverse array or commensal organisms beyond the actual component organisms in the bacterial composition such as but not limited to an ethanol treated spore population itself; specifically the spore composition treatment itself and the engraftment of OTUs from the spore composition create a niche that enables the outgrowth of OTUs required to shift a dysbiotic microbiome to a microbial ecology that is associated with health.
• Table 21 The diversity of Bacteroides species and their approximate relative abundance in the gut of Patient 1 is shown in Table 21 , comprising at least 8 different species.
• FIG. 10 The results are shown in Figure 10;the figure illustrates Species Engrafting versus Species Augmenting in patients microbiomes after treatment with a bacterial composition such as but not limited to an ethanol-treated spore population. Relative abundance of species that engrafted or augmented as described were determined based on the number of 16S sequence reads. Each plot is from a different patient treated with the bacterial composition such as but not limited to an ethanol- treated spore population for recurrent C. difficile.
• a bacterial composition such as but not limited to an ethanol-treated spore population.
• Enterobacteriaceae was assessed by plating pretreatment and Day 28 clinical samples from Patients 2, 4 and 5 on MacConkey lactose plus 1 ug/mL of imipenem. Resistant organisms were scored by morphology, enumerated and DNA was submitted for full length 16S rDNA sequencing as described above. Isolates were identified as
• morganii isolates from pretreatment samples from Patient D had MICs of 2-4 ug/mL and P. pennerii isolates had MICs of 4-8 ug/mL.
• the bacterial composition such as but not limited to an ethanol treated spores administered to Patient 4 caused the clearance of 2 imipenem resistant organisms (Table 16). While this example specifically uses a spore preparation, the methods herein describe how one skilled in the art would use a more general bacterial composition to achieve the same effects. The specific example should not be viewed as a limitation of the scope of this disclosure.
• Example 20 Identifying the Core Ecology from the bacterial combination.
• OTUs that engraft in a treated patients and the percentage of patients in which they engraft are denoted, as are the clades, spore forming status, and Keystone OTU status.
• Bolded OTUs occur in >80% of the ethanol preps and engraft in ⁇ 50% of the treated patients.
• OTUs detected by a minimum of ten 16S-V4 sequence reads in at least a one ethanol treated spore preparation (pan- microbiome).
• Lachnospiraceae_bacterium_6_l_63 clade_309 100 60% Y N FAA %
• Coprococcus_catus clade_393 100 70% Y Y
• Subdoligranulum variabile clade_478 100 80% Y Y
• OTUs detected by sequencing a bacterial composition (as in may include non-viable cells or other contaminant DNA molecules not associated with the composition.
• OTUs that are present in a large percentage of the bacterial composition e.g. ethanol spore preparations analyzed and that engraft in a large number of patients represent a subset of the Core Ecology that are highly likely to catalyze the shift from a dysbiotic disease ecology to a healthy microbiome.
• a third lens was applied to further refine insights into the Core Ecology of the bacterial composition e.g. spore preparation.
• Computational-based, network analysis has enabled the description of microbial ecologies that are present in the microbiota of a broad population of healthy individuals. These network ecologies are comprised of multiple OTUs, some of which are defined as Keystone OTUs. Keystone OTUs are computationally defined as described herein. Keystone OTUs form a foundation to the microbially ecologies in that they are found and as such are central to the function of network ecologies in healthy subjects. Keystone OTUs associated with microbial ecologies associated with healthy subjects are often are missing or exist at reduced levels in subjects with disease. Keystone OTUs may exist in low, moderate, or high abundance in subjects.11111111 further notes which of the OTUs in the bacterial composition e.g. spore preparation are Keystone OTUs exclusively associated with individuals that are healthy and do not harbor disease.
• Bilophila wadsworthia a gram negative anaerobe, is present in 9 of 10 donations, yet it does not engraft in any patient, indicating that it is likely a non-viable contaminant in the ethanol treated spore preparation. Finally, it is worth noting the high preponderance of previously defined Keystone OTUs among the most frequent OTUs in the spore preparations.
• CES Core Ecology Score
• the CES has a maximum possible score of 5 and a minimum possible score of 0.8.
• an OTU found in 8 of the 10 bacterial composition such as but not limited to a spore preparations that engrafted in 3 patients and was a Keystone OTU would be assigned the follow CES:
• Table GC ranks the top 20 OTUs by CES with the further requirement that an OTU must be shown to engraft to be a considered an element of a core ecology.
• Another aspect of functional redundancy is that evolutionarily related organisms (i.e. those close to one another on the phylogenetic tree, e.g. those grouped into a single clade) will also be effective substitutes in the Core Ecology or a subset thereof for treating C. difficile.
• Subsets may be selected by picking any 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 OTUs from
• OTUs can be selected as substitutes for OTUs with acceptable CES values.
• These organisms can be cultured anaerobically in vitro using the appropriate media (selected from those described in Example 5 above), and then combined in a desired ratio.
• a typical experiment in the mouse C. difficile model utilizes at least 10 4 and preferably at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or more than 10 9 colony forming units of a each microbe in the composition. Variations in the culture yields may sometimes mean that organisms are combined in unequal ratios, e.g.
• each strain be provided in a minimum amount so that the strain's contribution to the efficacy of the Core Ecology subset can be measured.
• Table GB describes the clades for each OTU detected in a spore preparation and Table 1 describes the OTUs that can be used for substitutions based on clade relationships.
• Example 22 Prophylactic use and treatment in a Mouse model of vancomycin resistant enterococcus (VRE) colonization.
• VRE vancomycin resistant enterococcus
• VRE vancomycin-resistant Enterococcus
• 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.
• VRE infection mouse model is used as previously described (Ubeda et al, Infectious Immunity 2013, Ubeda et al, Journal of clinical investigation, 2010). Briefly, experiments are done with 7-week-old C57BL/6J female mice purchased from Jackson Laboratory, housed with irradiated food, and provided with acidified water. Mice are individually housed to avoid contamination between mice due to coprophagia. For experimental infections with VRE, mice are treated with ampicillin (0.5 g/liter) in their drinking water, which is changed every 3 days.
• mice are infected by means of oral gavage with 10 8 CFU of the vancomycin-resistant Enterococcus faecium strain purchased from ATCC (ATCC 700221 ).
• antibiotic treatment is stopped and VRE levels are determined at different time points by plating serial dilutions of fecal pellets on Enterococcosel agar plates (Difco) with vancomycin (8 ug/ml; Sigma).
• VRE colonies are identified by appearance and confirmed by Gram staining or other methods previously described (e.g. see example 1 , 2 and 3).
• 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 2 and 3).
• ampicillin is administered as described above for day -7 to day 1 , treatment with the test article or vehicle control is
• Example 23 Prophylactic use and treatment of a Mouse model of Carbapenem resistant klebsiella (CRKB) colonization
• KPC K. pneumoniae carbapenemase
• KPC-Kp KPC-Kp
• MDR multidrug-resistant organisms
• a treatment protocol in a mouse model as previously described e.g. Perez et al, Antimicrobial Agents Chemotherapy, 201 1
• Perez et al Antimicrobial Agents Chemotherapy, 201 1
• test article for treating carbapenem resistant Klebsiella and reducing carriage in the Gl tract.
• Female CF1 mice Harlan Sprague-Dawley, Indianapolis, IN
• AST Antibiotic susceptibility testing
• CCSI Clinical and Laboratory Standards Institute
• mice (10 per group) are assigned to either a bacterial composition (test article), ethanol treated, spore preparation (e.g. as described herein), 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 (Per Stammum; Popper & Sons, New Hyde Park, NY).
• PBS phosphate-buffered saline
• Stool samples were collected 1 , 4, 6, and 1 1 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
• MacConkey agar with and without 0.5 ug/ml of imipenem, and the number of CFU per gram of stool was determined.
• 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.
• 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.
• 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.
• 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
• 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
• 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.
• 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.
• 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.
• 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.
• 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.”
• a Keystone Node is defined as one that is shown to be significantly different from what is observed using permuted test datasets to measure significance.
• Example 25 Method of Preparing the Bacterial Composition for Administration to a Patient
• strains for the bacterial composition are independently cultured and mixed together before administration. Both strains are independently be grown at 37°C, pH 7, in a GMM or other animal-products-free medium, pre-reduced with 1 g/L cysteineYHCI. After each strain reaches a sufficient biomass, it is preserved for banking by adding 15% glycerol and then frozen at -80°C in 1 ml cryotubes.
• Each strain is then be cultivated to a concentration of 10 10 CFU/mL, then concentrated 20-fold by tangential flow microfiltration; the spent medium is exchanged by diafiltering with a preservative medium consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphate buffer, or other suitable preservative medium.
• the suspension is freeze-dried to a powder and titrated.
• the powder is blended with microcrystalline cellulose and magnesium stearate and formulated into a 250 mg gelatin capsule containing 10 mg of lyophilized powder (10 8 to 10 1 1 bacteria), 160 mg microcrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesium stearate.
• Example 26 Use of microbiological assay to determine bacterial strain germinant utilization!
• C. difficile which is highly infectious when in the dormant, spore phase, but must germinate and grow in order to cause the symptoms of
• CDAD Clostridium diffici ⁇ 'e-associated disease
• One method to isolate and identify single strains or sets of strains that utilize similar nutrients and germinants to a given pathogen involves selecting for such microbes from complex communities of bacteria. Plating a complex community of bacteria on plates that are selective for the pathogenic vegetative or spore of interest can lead to the isolation of species with similar nutrient and germinant utilization profiles.
• CDSA Clostridium difficile selective agar
• Treatment 3 consists of 15 organisms that comprise C. disporicum, C. mayombei, C. tertium, C. innocuum, and Collineslla aerofaciens each of which was shown to utilized similar nutrients and germinants as C. difficile in the present example (Table X).
• This treatment prevented C. c /ff/c//e-associated mortality, weight loss, and clinical symptoms upon challenge with the pathogen.
• Table X Species isolated from a fecal suspension after ethanol treatment and plating on media aqars that are selective for C. difficile
• Example 27 Use of Bioloq assay to determine bacterial strain qerminant utilization
• Bacterial species that inhibit the germination of spores of a pathogen can be determined by finding a competitor species or combination of competitor species that overlap for germination requirements. As an example, the method described in Example 15 can be used to find a list of germination requirements for a spore forming pathogen. Potential competitors can be tested in parallel and chosen based on the level of overlap for germination requirements when the bacteria are grown from spores. For this assay, a culture consisting of pure spores is used as the inoculating material. A pure spore sample is prepared by treating a culture of a spore forming species with 50% ethanol for 1 hr with mixing. After incubation in ethanol the suspension is pelleted in a centrifuge at 12,000 rpm for 5 min. The spores are then resuspended in PBS.
• Germination from spores are tested in parallel with and without added germinants. After incubation for 1 hr to allow germination, each sample is serially diluted and plated for titer. Nutrients are scored as germinants if titer is higher in the presence of the nutrient. A combination of competitors which overlap sufficiently with the germination
• Example 28 Inhibition of a pathogen by identifying compositions that modify its qerminants required for sporulationl
• C. difficile is a spore former whose germination is induced by primary bile salts, including cholate, taurocholate, glycocholate and other taurocholate derivatives [Sorg and Sonnenshein (2008) J Bad 190: 2505-2512; Theriot, CM. et al., (2013) Nat Communications DOI: 10.1038/ncomms41 14].
• Many commensal organisms encode enzymes that dehydroxylate primary bile salts to secondary bile salts, including lithocholate, deoxycholate isomers, and various conjugated bile salts.
• An overnight culture of the commensal of interest is incubated in a growth media containing added taurocholate or cholate.
• Concentrations of between 0.001 % and 0.1 % are tested with each growth media in a 96 well measuring growth by monitoring ⁇ in order to find a concentration of each bile salt that is not inhibitory to growth of the commensal organism of interest. For each condition in which growth was observed, an aliquot is analyzed. The microbes are removed by centrifugation, and the supernatant tested using LC- electrospray-triple quadrupole mass spectrometry, or an equivalent method, to detect the production of secondary bile acids from taurocholate and cholate. A set of reference standards, including commercially available primary and secondary bile acids, is prepared and run in parallel. A microbial composition is then selected by combining two or more commensals capable of metabolizing taurocholate and cholate and tested for the ability to prevent C. difficile infection in the mouse as described in the examples herein.
• Example 29 Determination of nutrient utilization bv a pathogen
• the specific nutrient utilization capabilities of a pathogen can be determined by use of one, or a combination of methods described above. In one instance, a list of compounds which allow, enhance or are necessary for the growth of Clostridium difficile was determined using the method described in Example 15 above. Wells that had a final value of 1 .5x the negative control well under any of the conditions tested were scored as a growth requirement. Three strains of the same pathogen species were tested using a variety of redox buffer conditions, and compounds that supported growth of any of the strains were considered a growth substrate for the pathogen species.
• Example 30 Determination of nutrient utilization by a potential competitor
• the specific nutrient utilization capabilities of a bacterial species which is a potential pathogen competitor can be determined by use of one or, a combination of methods described above. In one instance, a list of compounds which allow, enhance or are necessary for the growth of a potential pathogen competitor species was determined by using the method described in Example 15 above. Wells that had a final absorbance value of at least 1 .5x the negative control well under any of the conditions tested were scored as a nutrient that the competitor is capable of utilizing.
• a potential competitor of the pathogen or combination of competitors can be chosen based on the level of overlap with nutrient utilization capabilities of the pathogen. As a general principle, the greater the overlap, the greater potential the isolate or set of isolates has as a competitor to the pathogen.
• Example 31 Use of minimum nutrient threshold quantification to identify potential competitors!
• the minimum concentration of a nutrient or other growth requirement necessary for growth can be determined by growth in a minimal defined media assay.
• a minimal defined media is constructed that lacks at least one constituent necessary for growth.
• the specific missing nutrient is titrated into the defined media by two fold dilutions along a row of a 96-well plate.
• the potential competitor of a particular pathogen is then inoculated into all wells of the plate, and the growth rate is determined as a function of the test nutrient concentration.
• the plate is incubated for 48 hr and growth is measured by optical density (OD) at 600 nm.
• OD optical density
• the well with the lowest concentration of nutrient that allows growth is determined to be the minimum threshold concentration.
• Potential competitors of the pathogen can be chosen by virtue of having a minimum threshold concentration below that of the pathogen.
• the proliferation rate of a bacterial species can be determined by inoculating the species being tested into a defined media and measuring the titer at several points over a time course.
• a pathogen or potential competitor of the pathogen is inoculated into a minimal defined media containing the nutrient being tested.
• Clostridium difficile growth rates can be measured in defined media containing a single carbon source (Theriot et al., 2013 (Nature Communications)). A variety of carbon sources are tested, with glucose as a positive control and no carbohydrate media as negative controls.
• the defined media used for the assay contains 0.5% (wt/vol) of the test carbohydrate, no other carbon source, and 0.125 mg/l biotin, 1 mg/l pyridoxine and pantothenate, 75 mg/l histidine, glycine and tryptophan, 150 mg/l arginine, methionine and threonine, 225 mg/l valine and isoleucine, 300 mg/l leucine, 400 mg/l cysteine, and 450 mg/l proline.
• progress of growth can be determined by taking a small aliquot at each timepoint, serially diluting and plating to a nutrient rich solid media to count colonies.
• Example 33 Use of in vitro assay to test potential bacterial competitor consortia for functionality
• An in vitro assay is performed to test the ability of a chosen species or combination of species to inhibit the growth of a pathogen such as Clostridium difficile in media that is otherwise suitable for growth of the pathogen.
• a liquid media suitable for growth of the pathogen is chosen, such as Brain Heart Infusion Broth (BHI) for C.
• the potential competitor species or a combination of competitor species are inoculated into 3 ml. of the media and incubated anaerobically for 24 hr at 37° C. After incubation the cells are pelleted in a centrifuge at 10,000 rcf for 5 min. Supernatant is removed and filtered through a 0.22 m filter to remove all cells. C. difficile or another pathogen of interest is then inoculated into the filtered spent supernatant and grown anaerobically at 37° C for 24 hr. A control culture in fresh media is incubated in parallel. After incubation, the titer of C. difficile is determined by serially diluting and plating to Brucella Blood Agar (BBA) plates and incubated anaerobically for 24 hr at 37° C.
• BBA Brucella Blood Agar
• Colonies are counted to determine the final titer of the pathogen after incubation in competitor conditioned media and control media. The percent reduction in final titer is calculated and considered inhibitory if a statistically significant reduction in growth is measured. Alternatively, the inhibition of pathogen growth is monitored by OD 6 oo measurement of the test and control cultures.
• Example 34 Selection of species to test in vivo for pathogen inhibition
• the bacterial combinations can then be prepared as described and tested in vivo in an appropriate host model as described herein.
• Example 35 Use of in vivo mouse model to test potential bacterial competitor consortia for functionality
• Clostridium difficile associated disease were chosen by use of a screen for nutrient utilization of both the pathogen and 17 potential competitors of the pathogen using the methods described herein.
• the use of nutrients used by each of the potential competitors was cross-referenced to the list of nutrients used by any of the Clostridium difficile strains tested under any condition tested.
• the 17 strains were rank-ordered for overlap with the list of Clostridium difficile nutrients.
• Clostridium hylemonae Blautia producta, Clostridium butyricum
• Clostridium difficile nutrients Four species combinations were chosen from species that ranked in the top 50% when rank ordered for overlap with Clostridium difficile nutrients.
• a three species combination with reduced overlap was also chosen based on a rank ordering of nutrient utilization by Clostridium difficile.
• 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 10mg/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 Iog10 cfu of C. difficile (ATCC 43255) via oral gavage.
• a positive control group receives Vancomycin from day -1 through day 3 in addition to the antibiotic protocol and C.
• 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 empty 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. Symptom scoring was based on Appearance (0-2 pts based on normal, hunched, piloerection, or lethargic), Respiration (0-2 pts based on normal, rapid or shallow, with abdominal breathing), Clinical Signs (0-2 points based on normal, wet tail, cold-to-the-touch, or isolation from other animals).
• the average minimum relative weight is calculated as the mean of each mouse's minimum weight relative to Day -1 and the average maximum clinical score is calculated as the mean of each mouse's maximum combined clinical score with a score of 4 assigned in the case of death.
• the treatments are specified in Table W and the results are reported in Table Y.
• Table W Treatment compositions tested C. difficile mouse model.
• Table Y Results of bacterial compositions tested in a C. difficile mouse model.
• Liquid media include (can be adapted to solid media by addition of 1 .5% agar):
• Gifu Anaerobic Medium without dextrose supplemented with fructooligosaccharides/inulin (0.4%), mannitol (0.4%), inulin (0.4%), or fructose (0.4%), or a combination thereof.
• fructooligosaccharides/inulin, mannitol and sodium lactate
• PEA sheep blood (Anaerobe Systems; 5% Sheep Blood with Phenylethyl Alcohol)
• PY-salicin peptone-yeast extract agar supplemented with salicin
• M-BHI Modified Brain Heart Infusion
• thioglycolate 10mg hemin, 2g soluble starch, 2g FOS/lnulin, 1g cellobiose, 1g L-arabinose, 1 g mannitol, 1 Na-lactate, 1 ml_ Tween 80, 0.6g MgS04x7H20, 0.6g CaCI2, 6g (NH4)2S04, 3g KH2P04, 0.5g K2HP04, 33mM Acetic acid, 9mM propionic acid, 1 mM Isobutyric acid, 1 mM isovaleric acid, and after autoclaving add 50ml_ of 8% NaHC03 solution and 50ml_ 1 M MOPS-KOH (pH 7).
• Clostridium difficile was grown under anaerobic conditions in SweetB-Fosln or other suitable media for the growth of C. difficile.
• SweetB-Fosln is a complex media composed of brain heart infusion, yeast extract, cysteine, cellobiose, maltose, soluble starch, and fructooligosaccharides/inulin, and hemin, and is buffered with MOPs. After 24 hr of growth the culture was diluted 100,000 fold into a complex media such as SweetB-Fosln which is suitable for the growth of a wide variety of anaerobic bacterial species. The diluted C. difficile mixture was then aliquoted to wells of a 96-well plate (180 uL to each well).
• 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 -80C before quantifying C. difficile by qPCR assay
• 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.
• macromolecules e.g., bacteriocins, cell-surface proteins, or polysaccharides
• 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.
• C. difficile selective media such as CCFA (cycloserine cefoxitin fructose agar, Anaerobe Systems), CDSA (Clostridium difficile selective agar, which is cycloserine cefoxitin mannitol agar, Becton Dickinson).
• C. difficile selective media such as CCFA (cycloserine cefoxitin fructose agar, Anaerobe Systems), CDSA (Clostridium difficile selective agar, which is cycloserine cefoxitin mannitol agar, Becton Dickinson).
• the standard curve was generated from a well on each assay plate containing only pathogenic C. difficile grown in SweetB + Fosln 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 was extracted from 5 ⁇ of each sample using a dilution, freeze/thaw, and heat lysis protocol. 5 ⁇ _ of thawed samples were added to 45 ⁇ _ of UltraPure water (Life Technologies, Carlsbad, CA) 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.
• the genomic DNA could be isolated using the Mo Bio Powersoil ® -htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories,
• the qPCR reaction mixture contained 1 x SsoAdvanced Universal Probes Supermix, 900 nM of Wr-tcdB-F primer
• 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.
• the qPCR could be performed with other standard methods known to those skilled in the art.
• the Cq value for each well on the FAM channel was determined by the CFX ManagerTM 3.0 software.
• the logio(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 logio(cfu/ml_) of those samples.
• the log inhibition was calculated for each sample by subtracting the logio(cfu/ml_) of C. difficile in the sample from the
• 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.
• 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 5. Equally important, the In vitro inhibition assay describes binary pairs that do not effectively inhibit C.
• 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 6 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.
• Table 1 List of Operational Taxonomic Units (OTU) with taxonomic assignments made to Genus, Species, and Phyloqenetic 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 ⁇ '.
• 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.

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