Compositions for modulating gut microflora populations, for treating dysbiosis, and for disease prevention, and methods for making and using same

Abstract

In alternative embodiments, compositions, e.g., articles of manufacture and kits, and methods are provided that include combinations or formulations of microorganisms, e.g., live non-pathogenic bacteria and / or bacterial spores, e.g., as probiotics, for the control, amelioration, prevention, and treatment of diseases or conditions, e.g., dysbiosis, or to increase the health or developmental capacity of an individual. In alternative embodiments, compositions or formulations, e.g., articles of manufacture and kits, and methods are provided that include at least one live non-pathogenic bacteria and / or bacterial spore and at least one probiotic or synbiotic. In alternative embodiments, these live non-pathogenic bacteria and / or bacterial spores (and optionally probiotics) are administered to an individual in need thereof, thereby resulting in the alteration or modulation of the individual's intestinal microflora population.

Description

[Technical Field]

[0001] Related Applications This Patent Cooperation Treaty (PCT) international application claims the benefit of priority under § 119(e) of U.S. Provisional Patent Application No. (USSN) 63 / 448,752, filed February 28, 2023, and USSN 63 / 545,116, filed October 20, 2023. The foregoing applications are expressly incorporated herein by reference in their entirety for all purposes. All publications, patents, patent applications, and GenBank and NCBI RefSeq assembly sequences and sequence references cited herein are hereby expressly incorporated herein by reference for all purposes.

[0002] Reference to Electronic Sequence Listing

[0001] This application contains a Sequence Listing that has been submitted electronically in .XML format, which is hereby incorporated by reference in its entirety. The .XML copy, created on February 27, 2024, is named "6411.154262PCT.xml" and is 346,666 bytes in size. The Sequence Listing contained in this .XML file is a part of the present specification and is hereby incorporated by reference in its entirety.

[0003] Technical Field This invention relates generally to microbiology, medicine, and pharmacology. In alternative embodiments, compositions or formulations, e.g., articles of manufacture and kits, and methods, are provided that include a combination or mix (or consortium) of microorganisms, e.g., live non-pathogenic bacteria and / or bacterial spores, e.g., as probiotics, for the control, amelioration, prevention, and treatment of a disease or condition, e.g., dysbiosis, or for increasing the health or developmental capacity of an individual. In alternative embodiments, compositions or formulations, e.g., articles of manufacture and kits, and methods, are provided that include at least one live non-pathogenic bacteria and / or bacterial spore and at least one probiotic. In alternative embodiments, these live non-pathogenic bacteria and / or bacterial spores (and optionally the probiotic) are administered to an individual in need thereof, thereby resulting in the alteration or modulation of the individual's intestinal microflora population. [Background technology]

[0004] background Vaginally born infants inherit their gut microbiome primarily from their mothers during passage through the birth canal and then via breastfeeding, a process called vertical transmission. This includes microorganisms such as Bifidobacterium species, bacteria that modulate the infant immune system, help prevent pathogen invasion by acidifying the intestinal environment, and act as keystone strains supporting other commensal bacterial species.

[0005] Conversely, cesarean section (C-section) birth bypasses the birth canal, thereby interrupting the inheritance of Bifidobacterium species and other important commensal organisms and allowing inflammatory microorganisms, such as Enterococcus, Enterobacter, Clostridial, and Klebsiella species, to predominate. This microbial dysbiosis leads to chronic inflammation that can cause asthma, environmental allergies, childhood obesity, immune disorders such as type 1 diabetes (T1D), inflammatory bowel disease, and a wide range of cancers. C-section birth, along with the increasing prevalence of formula feeding over breastfeeding, contributes to the significant loss of B. infantis and other important Bifidobacterium species from the general population. Furthermore, mothers who did not inherit Bifidobacterium species as infants are unable to pass them on to their children.

[0006] Dysbiotic infants tend to grow into dysbiotic adults, resulting in a higher incidence of inflammatory diseases, such as cancer. Inflammatory conditions in the gut can also lead to a high failure rate (greater than 50%) of antibody-based checkpoint inhibitor anticancer immunotherapies, which block T cell inhibitory signals, enhancing their ability to recognize and kill cancer cells. Examples of checkpoint inhibitors that target key T cell regulatory / inhibitory functions include cytotoxic T lymphocyte-associated protein 4 (CTLA-4, optionally ipilimumab or YERVOY®), programmed cell death protein 1 (PD-1, optionally pembrolizumab or KEYTRUDA®, nivolumab or OPDIVO®), and its ligand (PD-L1, optionally atezolizumab or TECENTRIQ®, avelumab or BAVENCIO®, and durvalumab or IMFINZI®). It was hypothesized that a healthier, anti-inflammatory gut microbiome better primed T cells in response to activation by checkpoint inhibition, while the chronic inflammatory state brought about by a dysbiotic microbiota led to T cell exhaustion and checkpoint inhibitor ineffectiveness, so the likelihood of response or non-response to checkpoint inhibitors directly correlates with the state of the gut microbiome and its contribution to gastrointestinal immune function.

[0007] The negative impact of both infant and adult microbiome dysbiosis highlights the need and opportunity to ameliorate and repair adverse inflammatory responses by reintroducing key commensal microorganisms that may help restore the proper regulatory immunological effects of the gut microbiome. In both infants and adults, dysbiosis manifests, at least in part, through loss of gut wall integrity due to degradation of the intestinal epithelium, either due to the toxic effects of invasive pathogens and / or the loss of supportive commensal short-chain fatty acid (SCFA)-producing bacterial species. Probiotic microorganisms, such as Bifidobacterium, have been shown to help re-tighten and restore intestinal epithelial integrity by stimulating Toll-like receptors, which act to increase the formation of tight junctions between intestinal epithelial cells. Probiotic Bifidobacterium may also help improve epithelial cell survival and health by supporting beneficial SCFA-producing microorganisms, such as Faecalibacteria, Anaerostipes, Eubacterium, and Roseburia species. There is a clear need to develop new and effective microbiome restoration therapies to address these conditions and pathologies. Summary of the Invention [Means for solving the problem]

[0008] overview In an alternative embodiment, - for controlling, ameliorating, reducing or preventing the symptoms or mortality of dysbiosis or infection in an individual in need thereof; Optionally, the infection is a bacterial or viral infection; optionally, the dysbiosis causes or exacerbates Failure to Thrive (FTT) in the individual, optionally, the dysbiosis is in an infant, a child, a pregnant woman, or a mother (material dysbiosis), optionally, the infant is 0-36 months of age; Optionally, the dysbiosis can be the presence of pathogenic bacteria, or a bacteria or mix of bacteria that is not normally present in the microbiome of an individual, infant or child; Optionally, high levels of pathogenic bacteria, or bacteria or mix of bacteria not normally present in the microbiome, are present in dysbiosis; Alternatively, dysbiosis may be caused by a high level of antibiotic resistance, or a skewed metabolic balance that differs from that of a healthy population, or a skewed immunological state that differs from that of a healthy population, or a loss of metabolic function associated with a healthy population, or an increase in bacteria associated with adverse events for the mother and her child. - modulates the microbiome of an individual, Optionally, the dysbiosis being treated or the condition being treated or ameliorated includes dysbiosis caused or exacerbated by preterm birth, prolonged stay in a neonatal intensive care unit, drug or antibiotic treatment, substance abuse by the pregnant woman, nutritional or environmental stress experienced by the pregnant woman, maternal drug or antibiotic treatment before or after birth, birth by Caesarean section, formula or nutritional supplement intake, and / or known dysbiosis in the mother; Optionally, the individual is a human, and optionally the human is a human child or human infant, and optionally the infant is 0-36 months of age, or 1 week to 30 months of age; Optionally, the microbiome of an individual is modulated to positively affect the growth, development or health of the individual (or to increase the individual's ability to develop), or to enhance the effectiveness of a treatment in an individual in need thereof, optionally wherein the treatment is a drug treatment, or a treatment for a disease or condition, optionally wherein the disease is cancer, a genetic disorder, a psychiatric or neurological disorder, or an autoimmune disease; - for treating, ameliorating, reducing the symptoms or severity of, or preventing a disease or condition caused by dysbiosis in an individual in need thereof, or for treating, ameliorating, or reducing or preventing a disease or condition whose treatment can be augmented by administration of the biotherapeutics (also called probiotics) provided herein; Optionally, the disease or condition is failure to thrive (FTT), or a condition in an individual (e.g., an infant or child) in which the individual has slowed or stopped physical growth, e.g., when height or weight measurements fall below the 3rd or 5th percentile (i.e., a downward change in growth across two major growth percentiles); Optionally, the disease or condition is an infectious disease, cancer or autoimmune disease, a genetic or hereditary disease, or a neurological condition; 1. A method comprising: The method is (a)(i) at least two different species or genera (or types) of non-pathogenic bacteria (also called probiotics) and / or non-pathogenic bacterial spores; or (ii) at least one live non-pathogenic bacteria and / or non-pathogenic bacterial spores and at least one probiotic (also called a synbiotic, or a combination of a probiotic and a prebiotic); administering or having administered to an individual in need thereof a composition or formulation comprising each of the non-pathogenic bacteria comprises (or is in the form of) a plurality of live non-pathogenic colony-forming bacteria, a plurality of non-pathogenic germinating bacterial spores, or a combination or mix thereof; Step; or (b)(i)(1) Non-pathogenic bacteria of at least two different species or genera (or types), each of which comprises (or is in the form of) a plurality of non-pathogenic colony-forming live bacteria, a plurality of non-pathogenic germinating bacterial spores, or a combination thereof; or (2) at least one live non-pathogenic bacteria and / or non-pathogenic bacterial spores and at least one probiotic (also called a synbiotic, or a combination of a probiotic and a prebiotic); providing a composition or formulation comprising: Optionally, the at least two different species or genera (or types) of non-pathogenic bacteria of (b)(i)(1), or at least one live non-pathogenic bacterium and / or non-pathogenic bacterial spore of (b)(i)(2), are genetically engineered to contain or express a new or heterologous trait or phenotype; and (ii) administering or maintaining the composition or formulation to an individual in need thereof. Includes; Optionally, the composition or formulation comprises one or any combination or mix (or consortium) of live non-pathogenic bacteria (or spores, if the bacteria form spores) of one or at least two different species or genera set forth in Table 1 or Table 4, or a live biotherapeutic (also called probiotic) composition or combination of bacteria set forth in Table 2 or Table 30, or wherein at least one live non-pathogenic bacterium and / or non-pathogenic bacterial spore and at least one probiotic (or synbiotic) comprises a combination set forth in Table 8 or Table 32, Optionally, at least one of the bacteria in the synbiotic provided herein, or the combination, mix (or consortium) provided herein, is a Bifidobacterium or Bacillus species, optionally Bifidobacterium infantis species; Optionally, the different species or genera (or types) of live non-pathogenic bacteria are present in approximately equal amounts, or each of the different species or genera (or types) of live non-pathogenic bacteria or non-pathogenic germinable bacterial spores is at least about 1%, 5%, 10%, 20%, 30%, 40%, or 50% or more, or between about 1% and 75% of the total amount of live non-pathogenic bacteria and non-pathogenic germinable bacterial spores in the formulation; Optionally, only live non-pathogenic bacteria or substantially only live non-pathogenic bacteria are present in the formulation, or only non-pathogenic germinable bacterial spores or substantially only non-pathogenic germinable bacterial spores are present in the formulation, or approximately equal amounts of live non-pathogenic bacteria and non-pathogenic germinable bacterial spores are present in the formulation. A method is provided.

[0009] In alternative embodiments of the compositions, formulations, or pharmaceutical preparations provided herein, or used in the methods provided herein, the composition or formulation further comprises at least one prebiotic (e.g., as in Table 8 or the synbiotics shown in Table 32), nutrient, metabolite, or drug, optionally wherein the drug comprises an antibiotic or Alternatively, optionally, the method further comprises administering a prebiotic, a synbiotic (e.g., as in the synbiotics shown in Table 8 or Table 32), a nutrient, a metabolite, or a drug, optionally wherein the drug comprises an antibiotic; optionally, at least one dose of a prebiotic, synbiotic (e.g., as in the synbiotics shown in Table 8 or Table 32), nutrient, metabolite, or drug is administered prior to the first administration of the bacterial formulation, mix, or consortium, and optionally, at least one dose of an antibiotic is administered 1 or 2 days or more prior to the first administration of the formulation; - the composition, formulation or pharmaceutical preparation comprises an inner core surrounded by one (or at least one) outer layer of a polymeric material encapsulating the inner core, wherein the non-pathogenic bacteria or non-pathogenic germinable bacterial spores or prebiotics are substantially in the inner core and, optionally, the non-pathogenic bacteria or non-pathogenic germinable bacterial spores or prebiotics are in the outer layer (or in an outer layer); Optionally, the polymeric material comprises a natural polymeric material; - The composition, formulation or pharmaceutical preparation comprises or further comprises a live biotherapeutic (also called a probiotic) and at least one prebiotic (e.g., as listed in Table 3), synbiotic (e.g., a combination of a probiotic and a prebiotic shown in Table 8 or Table 32), or drug, which can be prepared by mixing the two components together, as appropriate.

[0010] In alternative embodiments, the harvested and / or dried activated microbial cells can be combined with at least one prebiotic or drug, such as a powdered or lyophilized form of a prebiotic, a synbiotic (e.g., a probiotic and prebiotic combination shown in Table 8 or Table 32), or a drug. The harvested and / or dried microbial cells and the powdered form of the prebiotic or synbiotic can be in a single-dose packet, which can contain about 1 million to about 100 billion colony-forming units (cfu) of bacteria, optionally about 0.1 grams (g) to about 20 g of the prebiotic or synbiotic, or about 0.1 mg to 1 gram of the drug. The composition, formulation, or pharmaceutical preparation may contain or further contain nutrients designed to produce metabolic benefits, such as tryptophan, or secondary metabolites. Any of the compositions, formulations, or pharmaceutical preparations provided herein may further contain secondary metabolites. The secondary metabolites may be short-chain fatty acids, such as acetate, lactate, or a combination thereof. The composition, formulation, or pharmaceutical preparation may include or further include a stabilizer, such as a flow agent. Flow agents may include starch, silicon dioxide, tricalcium phosphate, powdered cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, bone phosphate, sodium silicate, calcium silicate, magnesium trisilicate, sodium aluminosilicate, potassium aluminum silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, and polydimethylsiloxane. The stabilizer may be milk protein or another suitable pharmaceutical-grade or infant formula-grade diluent (e.g., lactose). The milk protein may include the protein fraction of nonfat dry milk. The composition, formulation, or pharmaceutical preparation may comprise or further comprise a surface carbohydrate-binding protein (e.g., a solute-binding protein). The surface carbohydrate-binding protein may bind to the cell surface glycosylation of the intestinal mucosa and / or mucosal layer, thereby enabling more effective binding and interaction with the intestinal mucosa. This surface carbohydrate binding may then eliminate the binding of pathogenic bacteria.

[0011] In alternative embodiments, the compositions, formulations, or pharmaceutical preparations provided herein are dried (e.g., by spray drying or freeze drying) and formulated into unit dose pharmaceuticals, such as packets, sachets, orally disintegrating tablets, foodstuffs, capsules, lozenges, effervescent tablets, etc. The unit dose pharmaceuticals can be formed from a variety of materials, including, but not limited to, plastic or paper. In some embodiments, the unit dose pharmaceuticals include a moisture barrier layer and / or an oxygen barrier layer.

[0012] In various embodiments, the compositions, formulations or pharmaceutical preparations provided herein are in the form of anal delivery, such as suppositories or enemas.In alternative embodiments, the compositions are packaged in sachets made of moisture- and / or oxygen-impermeable polymers.These sachets can be filled with protective gas, such as nitrogen or argon.

[0013] In alternative embodiments, the compositions, formulations or pharmaceutical preparations provided herein are provided or formulated in the form of dry powder, liquid, suspension, or tablet or capsule format, with or without enteric coating.Dry powder can be freeze-dried or spray-dried.Freeze-dried compositions are preferably frozen in the presence of a suitable cryoprotectant.The cryoprotectant can be, for example, glucose, lactose, raffinose, sucrose, trehalose, adonitol, glycerol, mannitol, methanol, polyethylene glycol, propylene glycol, ribitol, alginate, bovine serum albumin, carnitine, citrate, cysteine, dextran, dimethyl sulfoxide, sodium glutamate, glycine betaine, glycogen, hypotaurine, peptone, polyvinylpyrrolidone or taurine. Enteric coatings include, but are not limited to, fatty acids, waxes, shellac, plastics, vegetable fibers, methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, cellulose acetate trimellitate, sodium alginate, and zein.

[0014] In alternative embodiments, the microorganisms used in the compositions, formulations, or pharmaceutical preparations provided herein, or the methods provided herein, are mixed with a cryopreservative, such as trehalose or glycerol, optionally under anaerobic conditions, and optionally frozen by a process such as, but not limited to, rapid freezing (cooling with liquid nitrogen) or by controlled temperature reduction in a cryopreservation freezing system. Once frozen, the microorganisms can be dehydrated under vacuum using a process that best preserves the integrity of the microbial cells. The concentration of the microorganisms in the dry powder can be between 1 million and 500 billion cfu / g. In some embodiments, the dry powder can be between 1 billion and 100 billion cfu / g, and in the most preferred embodiments, the dry powder can be between 1 billion and 50 billion cfu / g.

[0015] In an alternative embodiment, the powdered microorganisms are resuspended in an edible oil, exemplary edible oils including, but not limited to, triglyceride oils (e.g., vegetable oils, olive oil, and medium chain triglycerides), diglyceride oils, monoglyceride oils, and / or silicone oils.

[0016] In alternative embodiments, the prebiotic or synbiotic compositions, nutrients or drugs provided herein can be dissolved in a polar liquid, such as, but not limited to, water, saline, mammalian milk (such as human breast milk), or infant formula, and provided in liquid form, while the microorganisms are provided separately as a suspension in a carrier liquid, which may include a powder or a solution containing the prebiotics or synbiotics provided herein.

[0017] In alternative embodiments, the microorganism and oligosaccharide compositions used in the compositions, formulations, or pharmaceutical preparations provided herein, or the methods provided herein, are in a combined form or formulation, or are provided separately. In some embodiments, the microorganism is combined with the oligosaccharide in a single-dose packet containing about 1 billion to about 100 billion cfu of the microorganism and about 0.1 to about 20 g of prebiotics or synbiotics.

[0018] In an alternative embodiment of the compositions, formulations or pharmaceutical preparations provided herein, or the methods provided herein, - the composition, formulation or pharmaceutical preparation is formulated or manufactured as or in a nanosuspension delivery system; an encochleated formulation; or a multi-layered crystalline helical structure without an internal aqueous space; - the composition, formulation or pharmaceutical preparation is formulated or manufactured as a delayed or slow enteric release composition or preparation, optionally the formulation comprises a gastro-resistant coating designed to dissolve at a pH of 7 in the terminal ileum, optionally the active ingredient is coated with an acrylic resin or equivalent, optionally a poly(meth)acrylate, optionally methacrylic acid copolymer B, NF, optionally EUDRAGIT S™ (Evonik Industries AG, Essen, Germany), which dissolves at or above a pH of 7, optionally comprises a multi-matrix (MMX) formulation, optionally manufactured with an enteric coating to avoid stomach acid and bile in the duodenum; - the composition, formulation or pharmaceutical preparation is formulated or manufactured as a delayed-release, extended-release or slow enteric release composition or preparation, optionally formulated using CAPSUGEL™ (Lonza); - the plurality of live non-pathogenic colony-forming bacteria used in the compositions, formulations or pharmaceutical preparations provided herein, or the methods provided herein, are substantially dormant live colony-forming bacteria, or the plurality of live non-pathogenic colony-forming bacteria or the plurality of non-pathogenic germinable bacterial spores are lyophilized, and optionally the dormant live colony-forming bacteria comprise live vegetative bacterial cells that have been made dormant by lyophilization, spray drying, or freeze-drying; - the composition, formulation or pharmaceutical preparation contains at least about 1 x 10 4 Colony forming units (CFU), or approximately 1 × 10 1~1×10 13 CFU, 1 × 10 2 ~1×10 10 CFU, 1 × 10 2 ~1×10 8 CFU, 1 × 10 3 ~1×10 7 CFU, or 1 x 10 4 ~1×10 6 Contains CFU of non-pathogenic live bacteria and / or non-pathogenic germinating bacterial spores; - Tax ID: Agathobaculum (TaxID:20) 48137)、Alistipes(TaxID:239759)、Anaeromassilibacil lus(TaxID:1924093)、Anaerostipes(TaxID:207244)、Asaccharobacter(TaxID:553372)、Bacteroides(TaxID:816) 、Barnesiella(TaxID:397864)、Bifidobacterium(TaxID:1678)、Blautia(TaxID:572511)、Butyricicoccus(TaxID: 580596)、Clostridium(TaxID:1485)、Collinsella(TaxID:102106)、Coprococcus(TaxID:33042)、Dorea(TaxID:189) 330)、Eubacterium(TaxID:1730)、Faecalibacterium(TaxID:216851)、Fusicatenibacter(TaxID:1407607)、Gemmig er(TaxID:204475)、Gordonibacter(TaxID:644652)、Lachnoclostridium(TaxID:1506553)、Methanobrevibacter(T axID:2172)、Parabacteroides(TaxID:375288)、Romboutsia(TaxID:1501226)、Roseburia(TaxID:841)、Ruminococc us(TaxID:1263)、Erysipelotrichaceae(TaxID:128827)、Coprobacillus(TaxID:100883)、Erysipelatoclostridium sp.SNUG30099(TaxID:1982626)、Erysipelatoclostridium(TaxID:1505663)、Acetatifactor(TaxID:1427378)、Adlercre utzia(TaxID:447020)、Agathobacter(TaxID:1766253)、Anaerotruncus(TaxID:244127)、Bariatricus(TaxID:1924081)、Butyrivibrio(TaxID:830), Christensenellaceae(TaxID:990719), Clostridiales(TaxID:186802), Dialister(TaxID:39948), Dranco urtella(TaxID:1903506), Eggerthella(TaxID:84111), Eisenbergiella(TaxID:1432051), Enterocloster(TaxID:2719313), Enteroco ccus(TaxID:1350), Intestinibacter(TaxID:1505657), Lachnospira(TaxID:28050), Lachnospiraceae(TaxID:186803), Mediterranei bacter(TaxID:2316020), Negativibacillus(TaxID:1980693), Oscillibacter(TaxID:459786), Phocaeicola(TaxID:909656), Pseudobacter utyrivibrio(TaxID:46205), Pseudoflavonifractor(TaxID:1017280), Ruminococcaceae(TaxID:541000), Sellimonas(TaxID:1769710 ), Solobacterium(TaxID:123375), Terrisporobacter(TaxID:1505652), Tidjanibacter(TaxID:1929083), Veillonella(TaxID:29465) , Lacticaseibacillus (TaxID: 2759736), Limosilactobacillus (TaxID: 2742598), or combinations or mixes (or consortia) thereof (optionally a synbiotic, or a combination of one species and a probiotic, optionally as in the synbiotic combinations shown in Table 8 or Table 32) (or any one, some, or all), - the composition, formulation or pharmaceutical preparation comprises at least one (or any one, some, or all) of the non-pathogenic bacteria or spore forms thereof set forth in Table 1 or Table 4, or a live biotherapeutic composition (also called a probiotic) or a combination or mix (or consortium) of the bacteria set forth in Table 2 or Table 30; - the composition, formulation or pharmaceutical preparation comprises a combination of at least one non-pathogenic bacterium and / or its spores (or spores derived therefrom) as set forth in Table 1 or Table 4, or a live biotherapeutic composition (also called a probiotic) or a combination or mix (or consortium) of bacteria as set forth in Table 2 or Table 30; and / or - The composition, formulation or pharmaceutical preparation comprises water, sterile water, saline, sterile saline, a pharmaceutically acceptable preservative, carrier, buffer, diluent, adjuvant or combinations thereof.

[0019] In alternative embodiments, the methods further include administering to the subject a prebiotic or synbiotic (e.g., a mixture of prebiotics and probiotics shown in Table 8 or Table 32), a nutrient, an infant formula, or a drug, such as an antibiotic or an anti-cancer agent. In alternative embodiments, the compositions, formulations, and pharmaceutical compounds provided herein comprise, are mixed with, or are formulated with a prebiotic or synbiotic (e.g., a mixture of prebiotics and probiotics shown in Table 8 or Table 32), a nutrient, or a drug, such as an antibiotic.

[0020] In alternative embodiments, the prebiotic or synbiotic (e.g., a mixture of prebiotics and probiotics shown in Table 8 or Table 32) increases the growth of the anti-inflammatory bacterial population present in the probiotic composition. In alternative embodiments, the prebiotic or synbiotic increases the growth of or promotes the restoration of a healthy gut microbiome.

[0021] In alternative embodiments, the prebiotic or synbiotic (e.g., a mixture of prebiotics and probiotics shown in Table 8 or Table 32) comprises a monomer or polymer selected from the group consisting of arabinoxylan, xylose, soluble fiber dextran, soluble corn fiber, polydextrose, lactose, N-acetyl-lactosamine, glucose, and combinations thereof. In one embodiment of the foregoing aspect, the prebiotic or synbiotic comprises a monomer or polymer selected from the group consisting of galactose, glucose, lactose, fructose, rhamnose, mannose, uronic acid, fucose, sialic acid, N-acetylglucosamine, 2'-fucosyllactose, lacto-N-tetraose, 3'-fucosyllactose, 3'sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, 2',3-di-fucosyllactose, and combinations thereof. In one embodiment of the foregoing aspect, the prebiotic or synbiotic comprises a monosaccharide selected from the group consisting of arabinose, fructose, fucose, lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose, and combinations thereof. In one embodiment of the foregoing aspect, the prebiotic or synbiotic comprises a disaccharide selected from the group consisting of xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose, and combinations thereof. In one embodiment of the foregoing aspect, the prebiotic or synbiotic comprises a polysaccharide, and the polysaccharide is a xylooligosaccharide. In one embodiment of the foregoing aspect, the prebiotic or synbiotic comprises a sugar selected from the group consisting of arabinose, fructose, fucose, lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose, xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose, xylooligosaccharides, and combinations thereof.

[0022] In an alternative embodiment, the compositions, formulations, or pharmaceutical compositions provided herein or used in the methods provided herein comprise: - administered orally, topically, by aerosol, sublingually, or rectally, or formulated for oral, topical, aerosol, sublingual, or rectal administration, or formulated and / or administered as a freeze-dried composition, liposome, liquid, food, gel, supplement, gummy, candy, ice, lozenge, tablet, pill, or capsule, or suppository, or as an enema preparation, or the formulation is administered or in a form for rectal or colonic administration; - formulated or mixed into infant or pediatric foods, beverages, nutritional supplements or drinks, for example, the compositions, formulations, or pharmaceutical compositions provided herein are formulated or mixed into milk (e.g., human milk, cow's milk or soy protein, optionally fortified with vitamins, minerals, and other nutrients), infant formula, soy-based formula, amino acid-based formula, hydrolyzed infant formula (made from cow's milk or soy protein that has been broken down into smaller proteins that are easier for infants to digest), supplemented (collected) human breast milk, and the like, which may be supplemented with DHA or docosahexaenoic acid, or any omega-3 fatty acid, or iron drops; - administered to an individual in need thereof in 1, 2, 3, or 4, or more doses, wherein 1, 2, 3, 4, or 5, or more doses are administered daily (as needed, once daily, bid or tid, or more often), every other day, every third day, or about once a week, as needed, wherein 2, 3, 4, or more doses are administered at least 1 week apart (or the doses are about 1 week apart); - the composition, formulation, or pharmaceutical composition provided herein, or used in the methods provided herein, further comprises a drug, e.g., an antibiotic, or the method further comprises administration of a drug (e.g., an antibiotic), optionally wherein at least one dose of the drug (e.g., an antibiotic) is administered prior to the first administration of the composition, formulation, or pharmaceutical composition provided herein, optionally wherein at least one dose of the antibiotic is administered 1 or 2 days or more prior to the first administration of the composition, formulation, or pharmaceutical composition provided herein; - the composition, formulation, or pharmaceutical composition provided herein, or used in the methods provided herein, further comprises a drug, e.g., an inhibitor of an inhibitory immune checkpoint molecule, which may comprise a protein or polypeptide that binds to the inhibitory immune checkpoint protein, and optionally, the inhibitor of the inhibitory immune checkpoint protein is an antibody or antigen-binding fragment thereof that specifically binds to the inhibitory immune checkpoint protein; - optionally, the inhibitor of an inhibitory immune checkpoint molecule targets a compound or protein comprising CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4, CD152 or cluster of differentiation 152); also known as programmed cell death protein 1, PD-1 or CD279; also known as programmed death-ligand 1 (PD-L1), cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1); PD-L2; A2AR (adenosine A2A receptor, also known as ADORA2A); B7-H3; B7-H4; BTLA (B and T lymphocyte attenuator protein); KIR (killer cell immunoglobulin-like receptor); IDO (indoleamine-pyrrole 2,3-dioxygenase); LAG3 (lymphocyte activation gene 3 protein); TIM-3; VISTA (V domain Ig suppressor of T-cell activation protein); or any combination thereof; - optionally, the inhibitor of an inhibitory immune checkpoint molecule is ipilimumab or YERVOY®; pembrolizumab or KEYTRUDA®; nivolumab or OPDIVO®; atezolizumab or TECENTRIQ®; avelumab or BAVENCIO®; durvalumab or IMFINZI®; AMP-224 (MedImmune), AMP-514 (anti-programmed cell death 1 (PD-1) monoclonal antibody (mAb) (MedImmune)), PDR001 (a humanized mAb targeting PD-1), STI-A1110 or STI-A1010 (Sorrento Therapeutics), BMS-936559 (Bristol-Myers Squibb), BMS-986016 (Bristol-Myers Squibb), TSR-042 (Tesaro), JNJ-61610588 (Janssen Research & Development), MSB-0020718C, AUR-012, enoblituzumab (also known as MGA271) (MacroGenics, Inc.), MBG453, LAG525 (Novartis), BMS-986015 (Bristol-Myers Squibb), cemiplimab (or LIBTAYO®) (Regeneron), or any combination thereof; - optionally, the stimulatory immune checkpoint molecule comprises a member of the tumor necrosis factor (TNF) receptor superfamily, optionally CD27, CD40, OX40, GITR (glucocorticoid-induced TNFR family related gene protein) or CD137, or a member of the B7-CD28 superfamily, optionally CD28 or inducible T cell costimulator (ICOS);

[0023] - optionally, the drug, nutrient, or prebiotic or synbiotic is administered by aerosol, spray, intravenous (IV), intramuscular (IM), intratumoral, or subcutaneous injection; or is administered orally or by suppository; or the formulation further comprises at least one immune checkpoint inhibitor; - optionally, the compositions, formulations, or pharmaceutical compositions provided herein are administered to treat or ameliorate a condition or disease for maldevelopment, e.g., cancer, or to augment a drug or therapy administered to an individual in need thereof for the treatment of a condition or disease; Optionally, the cancer is melanoma, advanced melanoma, cutaneous or intraocular melanoma, primary neuroendocrine carcinoma of the skin, breast cancer, cancer of the head and neck, uterine cancer, rectal and colorectal cancer, cancer of the head and neck, cancer of the small intestine, colon cancer, cancer of the anal region, stomach cancer, lung cancer, brain cancer, non-small cell lung cancer, ovarian cancer, angiosarcoma, bone cancer, osteosarcoma, prostate cancer; cancer of the bladder; cancer of the kidney or ureter, or renal cell carcinoma, or carcinoma of the renal pelvis; neoplasm of the central nervous system (CNS), or renal cell carcinoma; Optionally, the disease or condition is necrotizing enterocolitis (NEC); Optionally, the disease or condition is irritable bowel disease, irritable bowel syndrome (IBD), celiac disease, gastroesophageal reflux disease (GERD) or Crohn's disease; Optionally, the disease or condition is an autoimmune disease, and optionally the autoimmune disorder is an allergy, e.g., a food, pollen, or drug allergy, asthma, diabetes, Crohn's disease, type 1 diabetes, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, lupus, scleroderma, and / or psoriasis; Optionally, the disease or condition is obesity, metabolic syndrome, type I or type II diabetes, or pre-diabetes; Optionally, the disease or condition is arthritis, inflammatory arthritis or gout; Optionally, the disease or condition is a dermatological disorder, e.g., psoriasis, urticaria, or angioedema; Optionally, the disease or condition is a neurological disease, such as anxiety, autism, stress, bipolar syndrome, depression, psychosis, essential tremor, Tourette's syndrome, Huntington's disease, multiple sclerosis or other demyelinating disease, organic psychosis, obsessive-compulsive disorder, Alzheimer's disease or Parkinson's disease; Optionally, the method comprises or further comprises administering, administering or delivering genetically (or recombinantly) engineered cells, which optionally are microorganisms or spores derived from microorganisms used in the methods of any of the preceding claims or methods provided herein; or are live biotherapeutic compositions (also called probiotics) or combinations or mixes (or consortia) of non-pathogenic bacteria or spores derived therefrom, or bacteria set forth in Table 1 or Table 4, or bacteria set forth in Table 2 or Table 30, Optionally, the disease or condition is an infection or a symptom or long-term sequelae of an infection (e.g., Long COVID), and optionally the infection is a viral, protozoal, fungal, or bacterial infection; Optionally, the microorganism is genetically engineered to express or secrete a heterologous immunomodulatory molecule or to overexpress an endogenous immunomodulatory molecule, optionally the immunomodulatory molecule is an immunomodulatory protein or peptide, optionally the immunomodulatory molecule is an immunostimulatory molecule; Optionally, the microorganism is genetically engineered to overexpress a pathway for the production of at least one short chain fatty acid (SCFA), optionally the SCFA comprising butyrate or butyric acid, propionate or acetate; Optionally, the microorganisms are genetically engineered to express catabolic pathways that provide an environmental niche, such as human milk oligosaccharide consumption, Optionally, the microorganisms are genetically engineered to express catabolic pathways that shift the SCFA balance either through the production or consumption of SCFAs, Optionally, the microorganism is engineered to consume prebiotics or synbiotics and produce postbiotics, e.g., to consume ellagic acid to produce urolithin A or tryptophan to produce indole-3-lactate; Optionally, the microorganism is genetically engineered by inserting heterologous nucleic acid into the microorganism, and optionally the heterologous nucleic acid encodes an exogenous membrane protein; Optionally, the immune stimulatory molecule, protein, or peptide comprises a non-specific immune stimulatory protein, optionally, the non-specific immune stimulatory protein comprises a cytokine, optionally, the cytokine comprises an interferon (optionally, IFN-α2a, IFN-α2b), and an interleukin (optionally, IL-2, IL-4, IL-7, IL-12), interferon (IFN), TNF-α, granulocyte colony stimulating factor (also known as G-CSF, filgrastim, lenograstim, or Neupogen®), granulocyte monocyte colony stimulating factor (also known as GM-CSF, molgramostim, sargramostim, LEUKOMAX®, MIELOGEN®, or LEUKINE®), or any combination thereof; Optionally, the immunostimulatory molecule, protein, or peptide comprises a specific immunostimulatory protein or peptide, and optionally, the specific immunostimulatory protein or peptide comprises an immunogen capable of generating a specific humoral or cellular immune response, or an immune response against a cancer antigen; Optionally, the engineered cell is a lymphocyte, optionally the engineered cell expresses a chimeric antigen receptor (CAR), optionally the lymphocyte is a B cell or a T cell (CAR-T cell), optionally the lymphocyte is a tumor infiltrating lymphocyte (TIL); Optionally, the microorganism is genetically engineered to substantially reduce, reduce or eliminate the toxicity of the microorganism; Optionally, the microorganism is genetically engineered to contain a kill switch so that the microorganism can be inactivated after administration of an appropriate trigger or signal; Optionally, the microorganism is genetically engineered to secrete an anti-inflammatory composition or have an anti-inflammatory effect; Optionally, the genetically engineered cells are administered or delivered before, simultaneously with, and / or after the administration or delivery of the formulation.

[0024] In an alternative embodiment, (a) a combination or mix (or consortium) of microorganisms set forth in Table 1 or Table 4, or a live biotherapeutic composition (also called a probiotic) or combination of bacteria set forth in Table 2 or Table 30; (b) a combination or mix (or consortium) of microorganisms used in the methods provided herein or provided herein; or / and (c) A formulation or pharmaceutical composition is provided that includes one (e.g., as in a synbiotic, or a combination of one and a probiotic, e.g., a synbiotic combination, as set forth in Table 8 or Table 32) or at least two different species or genera (or types) of non-pathogenic bacteria, each of which includes (or is in the form of) a plurality of live non-pathogenic colony-forming bacteria, a plurality of non-pathogenic, germinating, non-pathogenic bacterial spores, or a combination or mix (or consortium) thereof, and the formulation includes at least one (or any one, some, or all) of the non-pathogenic bacteria or spores of a family or genus (or class) set forth in Table 1 or Table 4, or a live biotherapeutic composition (also called a probiotic), or a combination, mix, or consortium of bacteria set forth in Table 2 or Table 30; or Agathobaculum(TaxID:2048137)、Alistipes(TaxID:239759)、Anaeromassilibacillus(TaxID:1924093)、Anae rostipes(TaxID:207244)、Asaccharobacter(TaxID:553372)、Bacteroides(TaxID:816)、Barnesiella(TaxID:3 97864)、Bifidobacterium(TaxID:1678)、Blautia(TaxID:572511)、Butyricicoccus(TaxID:580596)、Clostridi um(TaxID:1485)、Collinsella(TaxID:102106)、Coprococcus(TaxID:33042)、Dorea(TaxID:189330)、Eubacteri um(TaxID:1730)、Faecalibacterium(TaxID:216851)、Fusicatenibacter(TaxID:1407607)、Gemmiger(TaxID:2 04475)、Gordonibacter(TaxID:644652)、Lachnoclostridium(TaxID:1506553)、Methanobrevibacter(TaxID:21) 72)、Parabacteroides(TaxID:375288)、Romboutsia(TaxID:1501226)、Roseburia(TaxID:841)、Ruminococcus(T axID:1263)、Erysipelotrichaceae(TaxID:128827)、Coprobacillus(TaxID:100883)、Erysipelatoclostridium sp.SNUG30099 (TaxID: 1982626), Erysipelatoclostridium (TaxID: 1505663), Acetatifactor (TaxID: 1427378), Adlercreutzia (TaxID: 447020), Agathobacter (TaxID: 1766253), Anaerotruncus (TaxID: 244127), Bariatricus (TaxID: 1924081), Butyrivibrio (TaxID: 830), Christensenellaceae (TaxID: 990719), Clostridiales (TaxID: 186802), Dialister (TaxID: 39948), Drancourtella (TaxID: 1903506), Eggerthella (TaxID: 84111), Eisenbergiella (TaxID: 1432051), Enterocloster (TaxID: 2719313), Enterococcus (TaxID: 1350), Intestinibacter (TaxID: 1505657), Lachnospira (TaxID: 28050), Lachnospiraceae (TaxID: 186803), Mediterraneibacter (TaxID: 2316020), Negativibacillus (TaxID: 1980693), Oscillibacter (TaxID: 459786), Phocaeicola (TaxID: 909656), Pseudobutyrivibrio (TaxID: 46205), Pseudoflavonifractor (TaxID: 1017280), Ruminococcaceae (TaxID: 541000), Sellimonas (TaxID: 1769710), Solobacterium (TaxID: 123375), Terrisporobacter (TaxID: 1505652), Tidjanibacter (TaxID: 1929083), Veillonella (TaxID: 29465), Lacticaseibacillus (TaxID: 2759736), Limosilactobacillus (TaxID: 2742598), or a combination thereof.

[0025] In an alternative embodiment of the compositions, formulations or pharmaceutical compositions provided herein, or the methods provided herein, - the composition, formulation or pharmaceutical composition comprises at least one (or any one, some, or all) of the non-pathogenic bacteria or spore forms thereof set forth in Table 1 or Table 4, or a live biotherapeutic composition (also called a probiotic) or a combination or mix (or consortium) of bacteria set forth in Table 2 or Table 30, optionally formulated or mixed with prebiotics or synbiotics (e.g., as listed in Table 3), nutrients and / or drugs; - the composition, formulation or pharmaceutical composition comprises an inner core surrounded by an outer layer of a polymeric material that encapsulates the inner core, wherein the non-pathogenic bacteria or non-pathogenic germinable bacterial spores are substantially within the inner core, and optionally the polymeric material comprises a natural polymeric material; - the plurality of live non-pathogenic colony-forming bacteria are substantially dormant live colony-forming bacteria, or the plurality of live non-pathogenic colony-forming bacteria or the plurality of non-pathogenic germinable bacterial spores are lyophilized, and optionally the dormant live non-pathogenic colony-forming bacteria comprise live vegetative bacterial cells that have been made dormant by lyophilization or freeze-drying; - the composition, formulation or pharmaceutical composition contains at least 1 x 10 4 Colony forming units (CFU), or approximately 1 × 10 3 ~1×10 10 CFU, or approximately 1 x 10 2 ~1×10 8 CFU, 1 × 10 3 ~1×10 7 CFU, or 1 x 10 4 ~1×10 6 Contains CFU of live non-pathogenic bacteria and / or non-pathogenic germinating bacterial spores; - the composition, formulation or pharmaceutical composition comprises water, saline, a pharmaceutically acceptable preservative, carrier, buffer, diluent, adjuvant or combinations thereof; - the composition, formulation or pharmaceutical composition is formulated for oral or rectal administration or is formulated as a liquid, aerosol, spray, powder, food, supplement, nutraceutical, medical food, gel, gel tab, candy (e.g., lollipop), lozenge, tablet, pill or capsule, or suppository; - the composition, formulation or pharmaceutical composition further comprises a biofilm disrupting or lysing agent, an antibiotic, an inhibitor of an inhibitory immune checkpoint molecule and / or a stimulatory immune checkpoint molecule (or any composition for use in checkpoint blockade immunotherapy), Optionally, the inhibitor of an inhibitory immune checkpoint molecule comprises a protein or polypeptide that binds to the inhibitory immune checkpoint protein, and optionally, the inhibitor of an inhibitory immune checkpoint molecule is an antibody or antigen-binding fragment thereof that binds to the inhibitory immune checkpoint protein, as described above.

[0026] In an alternative embodiment, a kit or article of manufacture is provided that includes a formulation or pharmaceutical composition provided herein, and optionally the article of manufacture is an implant.

[0027] In an alternative embodiment, there is provided a use of a formulation or pharmaceutical composition provided herein, or a kit or article of manufacture provided herein, for controlling, ameliorating, preventing, or treating cancer in an individual in need thereof.

[0028] In an alternative embodiment, there is provided a use of a composition, formulation, or pharmaceutical composition provided herein in the manufacture of a medicament for controlling, ameliorating, preventing, or treating cancer in an individual in need thereof.

[0029] In alternative embodiments, provided are compositions, formulations, or pharmaceutical compositions provided herein, or kits provided herein, for use in controlling, ameliorating, preventing, or treating dysbiosis in infants, which may lead to diseases, including, but not limited to, diabetes, obesity, allergies, asthma, autism, and eczema.

[0030] In alternative embodiments, provided are compositions, formulations, or pharmaceutical compositions provided herein, or kits provided herein, for use in controlling, ameliorating, preventing, or treating dysbiosis in adults that may lead to disease. Diseases in adults that are associated with dysbiosis include, but are not limited to, cancer, diabetes, obesity, allergies, asthma, gout, Alzheimer's disease, and Parkinson's disease.

[0031] In alternative embodiments, there is provided a composition, formulation or pharmaceutical composition provided herein, or a kit provided herein, for use in controlling, ameliorating, preventing or treating a dysbiosis that may affect health outcomes for a pregnant woman and her child, e.g., her infant, and optionally the composition, formulation or pharmaceutical composition is administered to treat poor growth in the infant or child, or to a healthy infant or child to increase or enhance their health or capacity to develop.

[0032] In alternative embodiments, there is provided a composition, formulation, or pharmaceutical composition provided herein, or a kit provided herein, for use in the control, amelioration, prevention, or treatment of a dysbiosis that may affect the efficacy of pharmaceutical treatment.

[0033] In alternative embodiments, the compositions, formulations, or pharmaceutical compositions provided herein, or the kits provided herein, are provided for use in controlling, ameliorating, preventing, or treating cancer in an individual in need thereof. In alternative embodiments, the cancer is melanoma, advanced melanoma, cutaneous or intraocular melanoma, primary cutaneous neuroendocrine carcinoma, breast cancer, head and neck cancer, uterine cancer, rectal and colorectal cancer, head and neck cancer, cancer of the small intestine, colon cancer, cancer of the anal region, stomach cancer, lung cancer, brain cancer, non-small cell lung cancer, ovarian cancer, angiosarcoma, bone cancer, osteosarcoma, prostate cancer; cancer of the bladder; cancer of the kidney or ureter, or renal cell carcinoma, or carcinoma of the renal pelvis; neoplasm of the central nervous system (CNS), or renal cell carcinoma.

[0034] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0035] All publications, patents, patent applications, and GenBank and NCBI RefSeq assembly sequences and sequence references cited herein are hereby expressly incorporated by reference for all purposes.

[0036] DESCRIPTION OF THE DRAWINGS The drawings presented herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims. [Brief explanation of the drawings]

[0037] [Figure 1]Figure 1 graphically illustrates sample and cluster relationships from the MY BABY BIOME™ study. Distances were measured between all pairs of 289 infant gut microbiome samples using gUniFrac. Hierarchical clustering was performed using these distances. Hierarchical clustering resulted in three clusters (C1, C2, and C3), each containing microbiomes with broad similarities. Principal coordinate analysis was performed to visualize how samples and clusters related to each other.

[0038] [Figure 2] Figure 2 shows the average abundance of six phyla in each of three clusters for 289 infant gut samples. C1 is enriched in Actinobacteriota, the phylum that contains Bifidobacterium, and represents the expected infant microbiome. C2 has enrichment in Bacteroidota (the phylum that includes Bacteroides), which is typical of more mature gut microbiomes that are dysbiotic for infants. C3 is enriched in Firmicutes and Proteobacteria and is classified as dysbiotic.

[0039] [Figure 3] Figure 3 illustrates a ternary plot created by representing 287 infant gut samples as a two-dimensional vector by aggregating the relative abundances of Actinobacteriota, Bacteroidota, and various Firmicutes and Proteobacteria phyla combinations. Two samples were excluded because their composition was less than 90% of this set of phyla. The dysbiotic C3 state is primarily in the upper corner, while the typical infant microbiome is located mostly in the lower right among the C1 samples.

[0040] [Figure 4]Figure 4 reproduces the PCoA plot from Figure 1, with symbols indicating grouping of samples by mode of delivery. The upper right lobe, where C3 was located in Figure 1, also shows enrichment in C-section infants, while the C2 region (lower right), which is typical of a more mature gut microbiome, shows enrichment in vaginally delivered infants, consistent with the observation that vaginal delivery frequently results in vertical transmission of the microbiota.

[0041] [Figure 5] Figure 5 reproduces the ternary plot from Figure 3, with symbols indicating method of delivery. It can be seen that infants born by C-section typically have very low levels of Bacteroidota; a striking example of the lack of vertical transmission from mother to child during C-section delivery.

[0042] [Figure 6] Figure 6 reproduces the PCoA plot from Figure 1, with symbols indicating the feeding regimen for the infants. It can be seen that the leftmost lobe (where C1 is located) has abundances in samples from breast-fed and mixed-fed infants. This result is consistent with the observation that Bifidobacterium has an increased ability to metabolize HMOs found in breast milk.

[0043] [Figure 7] Figure 7 illustrates a phylogenetic tree created by hierarchical clustering of 289 infant gut microbiome samples. This was generated by measuring the distance between samples using gUniFrac and hierarchical clustering using the Ward method. The C3 cluster at the top, with enrichment for C-section-born infants, is seen, followed by the C2 cluster, with enrichment for vaginally-born infants, and finally the C1 cluster.

[0044] [Figure 8]Figure 8 reproduces the dendrogram from Figure 7, but instead labels samples by the predominant feeding regime for the infant: breast milk, mixed milk, or formula. The dendrogram was generated by measuring the distance between samples using gUniFrac and by performing hierarchical clustering using the Ward method.

[0045] [Figure 9] Figure 9 reproduces the PCoA from Figure 1 with samples shaded by Bifidobacterium abundance. It can be seen that the leftmost lobe (location C1) is highest in Bifidobacterium.

[0046] [Figure 10] Figure 10 graphically illustrates a volcano plot showing which taxa were enriched in the C1 cluster. We measure the average fold-change and Mann-Whitney-U p-value for each taxon across 289 infant gut microbiome samples (all species, genera, families, ..., phyla in our data) for C1 samples versus other samples. The plot shows -log10(p-value) versus log2(fold-change). We randomized the data to find an appropriate significance threshold and ignored taxa below that threshold (circles). We ignored taxa above the significance threshold if the group had an abundance less than 0.5% in more than 10% of the samples (diamonds). Using bootstrapping, we removed samples that did not have consistent enrichment in resampling the data, which removes outlier-biased taxa (non-robust "x" symbols). The remaining taxa are considered enriched (squares) or depleted (plus).

[0047] [Figure 11]Figure 11 graphically illustrates strip plots of the relative abundance of select taxa enriched in C1 measured across 289 infant gut microbiome samples. Taxon selection followed the procedure for Figure 10, and from the enriched taxa, we selected 10 representative groups. We plotted all samples and separated them by cluster. Nine of the 10 taxa are at the species level, and the tenth is the genus Collinsella. Of the nine species-level enriched taxa shown, eight are Bifidobacterium.

[0048] [Figure 12] Figure 12 graphically illustrates strip plots of selected species depleted in C2 compared to C1. Taxon selection followed the procedure for Figure 10, and from the enriched taxa, we selected eight representative species.

[0049] [Figure 13] Figure 13 graphically illustrates strip plots of selected species depleted in C3 compared to C1. Taxon selection followed the procedure for Figure 10, and from the enriched taxa we selected 11 representative species.

[0050] [Figure 14] Figure 14 graphically illustrates a strip plot of select taxa enriched in C3 compared to C1. Taxon selection followed the procedure described in Figure 10, and representative taxa are shown here.

[0051] [Figure 15] Figure 15 graphically illustrates the combined distribution of B. infantis, B. longum, B. bifidum, and B. breve across 289 infant gut microbiomes. It can be seen that the largest fraction of the population is in the 0-10% abundance category of the total consortium abundance.

[0052] [Figure 16]Figure 16 graphically illustrates the data from Figure 15, with infants separated into two cohorts according to mode of delivery. It can be seen that Bifidobacterium consortium abundance is lower in infants born by C-section.

[0053] [Figure 17] Figure 17 graphically illustrates the data from Figure 15, with infants separated into three cohorts according to feeding method. A decreased probability of high consortium abundance for formula-fed infants and a bimodal distribution of consortium abundance in breast-fed babies is observed, suggesting higher levels of Bifidobacterium consortium, when present.

[0054] [Figure 18] Figure 18 graphically illustrates a clustergram showing a representative subset of 73 infant gut microbiome samples compared to the bacterial species found at 5% or greater prevalence in these samples. Species are ordered by their taxonomic organization consistent with the Newick tree of the 207 GTDB releases.

[0055] [Figure 19] Figure 19 graphically illustrates a heatmap showing gene ortholog membership for HMO metabolism genes across many representative species and the novel PB-STR-093 strain found in the infant gut microbiome. Representative species genomes were downloaded from GTDB release 207. Genes are grouped into H1-H5 and urease clusters. Strain PB-STR-093 (B. infantis subspecies) is shown under GTDB r207 B. infantis.

[0056] [Figure 20]Figure 20 graphically demonstrates that feeding method is a significant driver of metabolism. Differentially expressed metabolites are plotted (ANOVA statistics, FDR p-value <0.05). Metabolite abundance is compared between breastfed (squares), breast- and formula-fed (circles), and formula-fed (triangles) infants using median centered log ratio (CLR) values.

[0057] [Figure 21] Figure 21 graphically illustrates the network analysis of B. infantis, B. breve, B. longum, B. bifidum, immune markers, and metabolites, revealing significant interactions with anti-inflammatory markers in the infant gut. Each node (circle) represents a trait. Nodes are shaded based on the multi-omics dataset from which they are derived (circle outline for bacteria, shaded circle without outline for metabolites, shaded circle with outline for immune markers). Lines connecting nodes indicate both statistical significance and strength of association (shorter = larger absolute correlation coefficient), with solid lines representing positive associations and dashed lines indicating negative associations.

[0058] [Figure 22] Figure 22 graphically illustrates the network analysis of all Bifidobacterium species, immune markers, and metabolites, revealing that our core Bifidobacterium consortium (B. infantis, B. bifidum, B. breve, B. longum) forms a tight cluster with other Bifidobacterium in the infant gut. Each node (circle) represents a trait. Nodes are shaded based on the multi-omics dataset from which they are derived (circle outline for bacteria, shaded circle without outline for metabolites, shaded with outline for immune markers). The lines connecting the nodes indicate both statistical significance and strength of association (shorter = larger absolute correlation coefficient), with solid lines representing positive associations and dashed lines indicating negative associations.

[0059] [Figure 23] Figure 23 graphically illustrates a network analysis of all microbiome, immune markers, and metabolites, revealing that Proteobacteria is significantly positively associated with the pro-inflammatory chemokine MCP-1. For this analysis, non-Bifidobacterium taxa had all leads summed at the phylum level. Here, we place special emphasis on the network module containing the Proteobacteria phylum. Each node (circle) represents a feature. The lines connecting the nodes indicate both statistical significance and the strength of the association (shorter for larger absolute correlation coefficients), with solid lines representing positive associations and dashed lines representing negative associations.

[0060] [Figure 24] Figure 24 graphically illustrates a pan-genome comparison of B. infantis strains. Ten NCBI B. infantis reference strains and one new isolate are shown with co-occurring genes highlighted. Strains were observed to group into two distinct clades (C1 and C2), with C1 having the highest degree of intra-clade similarity. NCBI GCA accession numbers for the depicted strains are: C2-L5:GCA 001281305, C2-L4:GCA_017299595, C2-L3:GCA_017378625, C2-L2:GCA_015102215, C2-L1:GCA_018140675, C1-L5:GCA_000020425, C1-L4:GCA_902381625, C1-L3:GCA_900637215, C1-L2:GCA_000269965, C1-L1:GCA_902167885.

[0061] [Figure 25]Figure 25 demonstrates, via Krona charts, the development of a C1 gut environment in the context of human milk oligosaccharides versus formula. When grown on human milk oligosaccharides, the gut environment maintains a C1 community structure dominated by Bifidobacterium, whereas when grown on formula, the community structure diverges and shifts to a C3 community structure dominated by Firmicutes and Proteobacteria. Each Krona chart represents the overall community composition in the simulated gut environment.

[0062] [Figure 26] Figure 26 demonstrates through Krona charts that the introduction of Bifidobacterium infantis shifts the community structure in the simulated gut environment. Comparing the first two Krona charts, the introduction of Bifidobacterium infantis dramatically shifts the simulated gut environment from a C3 community structure to a C1 community structure. By comparing the second and third Krona charts, it can be seen that the introduction of human milk oligosaccharide LNT further boosts the abundance of Bifidobacterium infantis in the samples.

[0063] [Figure 27]Figure 27 graphically demonstrates the ability of Bifidobacterium infantis to reduce the presence of pathogens or other harmful bacteria. Upon introduction of Bifidobacterium infantis to a simulated intestinal environment, a reduction in harmful or pathogenic bacteria is observed. Shown here are the levels of three different bacteria in a simulated intestinal environment. Four sample groups show the levels of Escherichia coli, Streptococcus vestibularis, and Bifidobacterium infantis (two replicates each) with the indicated carbon source and introduction of B. infantis or a control. While Bifidobacterium infantis can reduce the levels of these bacteria on its own, importantly, introduction of human milk oligosaccharides, e.g., LNT, further reduces the presence of these bacteria, demonstrating the ability of Bifidobacterium infantis to suppress pathogens and other unwanted bacteria in a prebiotic-dependent manner.

[0064] [Figure 28] Figure 28 graphically demonstrates cytokine expression with and without B. infantis. Cytokine induction was assessed using supernatant from the simulated intestinal environment compared to background medium. When the simulated intestinal environment was created with additional Bifidobacterium infantis (+spike), a significant reduction in the induction of pro-inflammatory cytokines was observed, demonstrating the anti-inflammatory properties of the microorganisms in the simulated intestinal environment.

[0065] [Figure 29] Figure 29 graphically illustrates the growth of C3 fecal samples with and without probiotics in a ternary plot. Probiotic inoculation and Bifidobacterium growth were performed to investigate the restoration of an in vitro simulated infant gut microbiome. Probiotic inoculation results in a shift toward more Actinobacteriota and a more typical infant gut microbiome.

[0066] [Figure 30] Figure 30 graphically depicts the relative abundance of Bifidobacterium in in vitro growth of the probiotic-simulated C3 infant gut microbiome via boxplot. Combo_15 is a control with no Bifidobacterium species in the inoculum, while the other combinations have approximately equivalent Bifidobacterium colony-forming units (CFU). Combo_14 had the highest Bifidobacterium growth.

[0067] [Figure 31] Figure 31 shows by strip plot the relative abundance of Bifidobacterium in an in vitro simulation of the C3 infant gut microbiome after probiotic stimulation. Three different C3 samples are shown.

[0068] [Figure 32] Figure 32 shows a pan-genome analysis of B. infantis. The genome of the Persephone biosciences B. infantis strain was analyzed in combination with the publicly available B. infantis genome to determine distinguishing characteristics of the strain.

[0069] [Figure 33] Figure 33 shows a pan-genome analysis of B. longum. The genome of the Persephone biosciences B. longum strain was analyzed in combination with the publicly available B. longum genome to determine distinguishing characteristics of the strain.

[0070] [Figure 34] Figure 34 shows a pan-genome analysis of B. breve. The genome of the Persephone biosciences B. breve strain was analyzed in combination with publicly available B. breve genomes to determine distinguishing characteristics of the strain.

[0071] [Figure 35]Figure 35 shows a pan-genome analysis of B. bifidum. The genome of the Persephone biosciences B. bifidum strain was analyzed in combination with the publicly available B. bifidum genome to determine distinguishing characteristics of the strain.

[0072] [Figure 36] Figure 36 graphically illustrates differential metabolite abundance in different Bifidobacterium combinations by central logarithmic ratio (CLR) analysis. Box plots represent the distribution of CLR values ​​for three key metabolites: indole-3-lactate, 4-hydroxyphenyllactate, and arginine across various Bifidobacterium combinations (e.g., "Combo_1," "Combo_2," "Combo_4") introduced into a simulated in vitro gut environment. Notably, Combo_15 served as our control and contained no additional Bifidobacterium. The y-axis represents CLR values, providing insight into the relative abundance of each metabolite, while the x-axis represents specific Bifidobacterium combinations. The figure illustrates the variation in metabolite abundance across different Bifidobacterium compositions, aiding in a deeper understanding of their metabolic profiles.

[0073] [Figure 37] Figure 37 graphs the fold change versus -loglO(p) (Mann-Whitney U) for metadata variables in the DIABIMMUNE study, comparing mean Bifidobacterium abundance for fecal samples obtained from individuals aged 110 days to 1 year with and without the metadata flag. High significance is seen for reduced Bifidobacterium abundance in formula-fed infants and in infants who had a milk or birch allergy by the time they were 3 years old.

[0074] [Figure 38]FIG. 38 shows a scatter plot demonstrating an inverse relationship between Bifidobacterium abundance and total IGE (Spearman r=−0.185, p-value=0.013).

[0075] [Figure 39] Figure 39 shows a ternary plot depicting the "three-country cohort" data from the DIABIMMUNE study. Industrialized Finland exhibits lower Actinobacteriota abundance compared to its neighboring region in Russia.

[0076] [Figure 40] Figure 40 graphically depicts a 6-month medical history follow-up from MY BABY BIOME™ revealing that 11 individuals had adverse skin conditions, either eczema or dermatitis. These events were less prevalent in the high Bifidobacterium region of the PCoA region (top left).

[0077] [Figure 41] Figure 41 demonstrates the differences in abundance for selected Bifidobacterium and Bifidobacterium combinations for samples from infants who developed either eczema or dermatitis versus those who did not by the 6-month study time point. A statistically significant trend (Mann-Whitney U) is seen for B. bifidum.

[0078] [Figure 42]Figure 42 graphically illustrates the network analysis of all Bifidobacterium species, immune markers, and metabolites, revealing that our core Bifidobacterium consortium (B. infantis, B. bifidum, B. breve, B. longum) clusters tightly with other Bifidobacterium species in the infant gut. Each node (circle) represents a trait. Nodes are shaded based on the multi-omics dataset from which they are derived (circle outline for bacteria, shaded circle without outline for metabolites, shaded with outline for immune markers). Lines connecting nodes indicate both statistical significance and strength of association (shorter = larger absolute correlation coefficient), with solid lines representing positive associations and dashed lines indicating negative associations. [Figure 43] Figure 43 shows flow cytometry data for four different Bifidobacterium strains produced at a 7 liter fermentation scale, with cells binned into three categories: dead, live, and injured.

[0079] [Figure 44] Figure 44 shows the relationships between GUNIFRAC™ (gUniFrac) (Jun Chen et al.) clusters created from KRAKEN2™ classified samples (C1, C2, and C3) and DIRICHLET MULTINOMIAL MIXTURE™ models (DMM1, DMM2, and DMM3). The DMM clusters are constructed without knowledge of the phylogenetic tree.

[0080] [Figure 45]Figure 45 is a basic example of the differences between the GUNIFRAC™ (gUniFrac) and DMM clusters. C1 is characterized by high Bifidobacteria abundance, and therefore samples high in B. dentium are classified as C1. In the DMM cluster, there is only an estimated joint probability distribution, and no knowledge of the phylogenetic tree used to group the samples. The B. dentium-high sample can now be seen to be a member of DMM1, rather than DMM3, which is considered the healthy infant gut DMM cluster.

[0081] [Figure 46] Figure 46 shows the differential abundance between DMM3 (healthy infant intestine) and the combination of DMM1 and DMM2. Similar analyses were performed for DMM2 and DMM1. The enriched taxa for each DMM cluster are listed in Table 39.

[0082] [Figure 47] Figure 47 shows the differences in the distribution of antibiotic resistance hits between gUniFrac clusters. Samples classified as C1 (healthy infant gut) tend to have a lower number of antibiotic resistance markers.

[0083] [Figure 48] Figure 48 shows the differences in the distribution of antibiotic resistance hits between Dirichlet multinomial mixture clusters. Samples classified as DMM3 (healthy infant gut) tend to have a lower number of antibiotic resistance markers. [Figure 49] Inverse relationship between antibiotic resistance markers and Bifidobacterium abundance. [Figure 50] Distribution of antibiotic resistance genes found in each sample separated by feeding style. Breast-fed babies have significantly fewer antibiotic resistance markers.

[0084] [Figure 51]Distribution of consortium relative abundance (sum of relative abundances of B. infantis, B. bifidum, B. longum, and B. breve) separated by mode of feeding and mode of delivery. Vaginally born, breast-fed infants had the highest median consortium abundance, while formula-fed, C-section-born infants had higher consortium abundance than formula-fed, vaginally born infants. DETAILED DESCRIPTION OF THE INVENTION

[0085] Like reference symbols in the various drawings indicate like elements.

[0086] Detailed Description In alternative embodiments, compositions and methods are provided, e.g., articles of manufacture and kits, and methods for their use, that include novel combinations or mixes (or consortia) of microorganisms, also referred to as live biotherapeutic compositions (also referred to as probiotics), e.g., non-pathogenic live (optionally dormant) bacteria and / or bacterial spores, e.g., the exemplary combinations or mixes (or consortia) of microorganisms listed in Table 1 or Table 4, or live biotherapeutic compositions or combinations or mixes (or consortia) of bacteria set forth in Table 2 or Table 30.

[0087] In an alternative embodiment, - for controlling, ameliorating, reducing or preventing the symptoms or mortality of dysbiosis or infection in an individual in need thereof; Optionally, the infection is a bacterial or viral infection; Optionally, the dysbiosis causes or exacerbates failure to thrive (FTT) in the individual, and optionally, the dysbiosis is in a newborn, infant, or mother (maternal dysbiosis), and optionally, the newborn or infant is 0 to 36 months of age. - for modulating or altering the microbiome of an individual, Optionally, the individual is a human, and optionally the human is a human child or a human infant or newborn, and optionally the infant or newborn is 0-36 months of age; If desired, the individual's microbiome is modulated to positively affect the growth, development, or health of the individual (or to increase the individual's ability to develop); - optionally, the individual's microbiome is modulated to enhance the effectiveness of a treatment in the individual in need thereof, optionally the treatment is a drug treatment or administration, optionally the drug treatment is for cancer; - for treating, ameliorating, reducing or preventing a disease or condition caused by dysbiosis in an individual in need thereof, wherein optionally the individual is a human, and optionally the human is a human child or a human infant or newborn, and optionally the infant or newborn is 0-36 months of age, and optionally the disease or condition is failure to thrive (FTT), Compositions, e.g., articles of manufacture and kits, and methods for using same, comprising: The method is (a) administering or maintaining administered to an individual, e.g., a child or infant, in need thereof, a formulation comprising one (e.g., as in a synbiotic, or one and probiotic combination, e.g., a synbiotic combination, as set forth in Table 8 or Table 32) or at least two different species or genera (or types) of non-pathogenic bacteria, wherein each of the non-pathogenic bacteria comprises (or is in the form of) a plurality of live non-pathogenic colonizing bacteria, a plurality of non-pathogenic germinating bacterial spores, or a combination thereof; Including, Compositions and methods are provided.

[0088] In alternative embodiments, the compositions, articles of manufacture, kits, and methods provided herein are used as therapies (e.g., as monotherapy or as cotherapy or cotreatment) for the control, amelioration, prevention, and / or treatment of a disease or condition, e.g., cancer.

[0089] In alternative embodiments, the compositions, articles of manufacture, kits and / or methods provided herein are administered to an individual receiving a drug or therapy, e.g., cancer therapy, thereby resulting in the alteration or modulation of the patient's gut microflora population, thus resulting in the enhancement of the drug or other therapy, e.g., reducing the dosage or amount of the drug required for effective therapy, or the frequency with which the drug must be administered to be effective.

[0090] In alternative embodiments, the compositions, articles of manufacture, and methods provided herein can be used to modulate or modify an individual's gut microbial population, thereby altering the pharmacodynamics of a drug administered to the patient, e.g., enhancing the pharmacodynamics of the drug, e.g., altering (e.g., speeding up or slowing down, or enhancing) the individual's ability to absorb the drug, or increasing the dose effectiveness of the drug (e.g., resulting in a lower dose of the drug being required for the intended effect), or the gut microbes acting orthogonally to the drug target (e.g., resulting in the presence of the microbes being essential for the drug to have the intended effect). For example, in alternative embodiments, the compositions, articles of manufacture, and methods provided herein can be used to modulate or modify a patient's gut microbial population, thereby increasing the dose effectiveness of a cancer drug, thereby enhancing the control or treatment of the cancer.

[0091] In alternative embodiments, the amount, identity, presence, and / or ratio of gut microbiota in a subject is manipulated to facilitate monotherapy or one or more co-treatments; for example, in alternative embodiments, a combination or mix (or consortium) of microorganisms provided herein is administered in conjunction with (e.g., simultaneously with, or before and / or after) chemotherapy, radiation therapy, immune checkpoint inhibitors, chimeric antigen receptor (CAR) T-cell therapy (CAR-T), or other immunotherapy or cancer treatment.

[0092] Described herein for the first time are novel combinations or mixes (or consortia) of specific microorganisms, e.g., bacteria, e.g., Bifidobacterium or Bacillus species, optionally Bifidobacterium infantis species, including, for example, microorganisms (or bacteria) found in the human gut or recombinantly engineered or cultivated microorganisms, which can be administered as monotherapy or as cotherapy, in alternative embodiments, to infants or newborns, for example, to increase their ability to develop or grow, or to resist infection or disease, or to cancer or autoimmune patients, where in alternative embodiments the cancer patient is undergoing immune checkpoint inhibitor treatment, or is undergoing chemotherapy, radiation therapy, immune checkpoint inhibitor, chimeric antigen receptor (CAR) T-cell therapy (CAR-T), or other immunotherapy or cancer treatment.

[0093] The inventors have demonstrated a correlation between these microbial combinations and their associated metabolic functions, as well as the efficacy of treatment in both human patients and mouse cancer models. In an alternative embodiment, administering the microbial combinations provided herein to cancerous mice increases the percentage of animals that exhibit significant tumor size reduction compared to mice that do not have their gut microbiome altered using the compositions or methods provided herein but are given the same drugs.

[0094] In alternative embodiments, chemotherapy, radiation therapy, chimeric antigen receptor (CAR) T-cell therapy (CAR-T), or other immunotherapy or cancer treatment, e.g., an immune checkpoint inhibitor (or an inhibitor of an inhibitory immune checkpoint molecule) and / or a stimulatory immune checkpoint molecule (or more correctly, a stimulatory immune molecule), is administered (e.g., administered simultaneously or sequentially) or formulated with a combination of microorganisms provided herein, e.g., administered or formulated with a non-pathogenic bacterium and / or a non-pathogenic, germination-competent bacterial spore provided herein.

[0095] Immune checkpoint inhibitors (also described as inhibitors of inhibitory immune checkpoint molecules) can function by interfering with naturally occurring regulatory pathways to prevent T cell proliferation. In the tumor microenvironment, these inhibitory pathways are highly active, resulting in T cells often being driven into an ineffective state. Checkpoint inhibitors bind to specific proteins in these regulatory pathways associated with inhibiting T cell activation, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed cell death-ligand 1 (PD-L1), thereby enabling an excitatory T cell response to tumor antigens. Thus, in an alternative embodiment, inhibitors of inhibitory immune checkpoint molecules are molecules that can directly (or specifically) bind to CTLA-4, PD-1, PD-L1, or other components of inhibitory immune checkpoints, preventing them from properly binding to their natural corresponding receptors or ligands.

[0096] In alternative embodiments, stimulatory immune checkpoint molecules, which may also be, or more correctly described as, stimulatory immune molecules, enhance T cell excitation and activation, either by potentiating the action of checkpoint inhibitors or by an independent mechanism.

[0097] In alternative embodiments, therapeutic compositions, e.g., formulations and pharmaceutical compositions, comprising live, non-pathogenic (optionally dormant) microorganisms, e.g., bacteria and / or germination-competent bacterial spores, are provided, which can be used for the prevention or treatment of cancer or side effects of cancer therapy, e.g., drug therapy, or can be used or administered in conjunction with chemotherapy, radiation therapy, immune checkpoint inhibitors, chimeric antigen receptor (CAR) T-cell therapy (CAR-T), or other immunotherapy or cancer treatment.

[0098] In alternative embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions provided herein or used to practice the methods provided herein comprise live colonizing (optionally dormant) bacteria and / or germinating bacterial spores that can be used in monotherapy or cotherapy, e.g., as an adjuvant to antineoplastic drug treatment administered to cancer patients, or can be administered in conjunction with or as a supplement to chemotherapy, radiation therapy, immune checkpoint inhibitors, chimeric antigen receptor (CAR) T-cell therapy (CAR-T), or other immunotherapy or cancer treatment.

[0099] In some embodiments, the therapeutic compositions provided herein act or are used as probiotic compositions that can be administered in conjunction with, before, and / or after chemotherapy, radiation therapy, immune checkpoint inhibitors, chimeric antigen receptor (CAR) T-cell therapy (CAR-T), or other immunotherapy or cancer treatment. In alternative embodiments, the therapeutic compositions (e.g., formulations) provided herein comprise bacteria and / or spores and an anti-neoplastic active agent, e.g., an immune checkpoint inhibitor.

[0100] In alternative embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions provided herein or used to practice the methods provided herein comprise live colonizing (optionally dormant) bacteria and / or germinating bacterial spores for use as monotherapy or in combination (e.g., as cotherapy) or as supplementation to a drug (which may be a small molecule or protein, e.g., a therapeutic antibody) that blocks an immune checkpoint to induce immune stimulation in cancer patients. The therapeutic compositions and drugs (e.g., antibodies) provided herein can be administered separately or together, at different or the same time points, or can be administered sequentially or simultaneously.

[0101] In alternative embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions provided herein comprise live colonizing (optionally dormant) bacteria and / or germinating bacterial spores that can be used as adjuvants to anti-cancer or antineoplastic drug treatments, e.g., immune checkpoint treatments, administered to cancer patients. In alternative embodiments, the therapeutic composition comprises an antineoplastic drug or immune checkpoint active agent. In alternative embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions provided herein are administered concomitantly with, or after, or both with and after, administration of an antineoplastic drug or immune checkpoint active agent.

[0102] In an alternative embodiment, the formulation or pharmaceutical composition further comprises or is made with an outer layer of a polymeric material (e.g., a natural polymeric material) that encapsulates or surrounds a core comprising the microbial combinations provided herein.

[0103] In alternative embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions provided herein or used to practice the methods provided herein can include a pharmaceutically acceptable carrier, diluent, and / or adjuvant. In other embodiments, a pharmaceutically acceptable preservative is present. In yet other embodiments, a pharmaceutically acceptable germinate is present. In still other embodiments, the therapeutic compositions contain or further comprise an effective dose of about 0.005, 0.05, 0.5, 5.0 milligrams (mg) per kilogram (kg) of body weight, or about 0.005-10 mg per kilogram of body weight of a prebiotic or synbiotic nutrient.

[0104] In alternative embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions provided herein or used to practice the methods provided herein are in the form of tablets, gel tabs, or capsules, e.g., polymer capsules, e.g., gelatin or hydroxypropyl methylcellulose (HPMC, or hypromellose) capsules (e.g., VCAPS PLUS™ (Capsugel, Lonza)). In other embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions are in or manufactured as a food or beverage, e.g., ice cream, candy, confectionery, or lozenge, or any liquid, e.g., a drink.

[0105] In alternative embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions provided herein or used to practice the methods provided herein comprise at least one bacterial type that is not detectable, has low natural abundance, or is not naturally found in the gastrointestinal tract of a healthy or normal subject (e.g., a human). In alternative embodiments, the gastrointestinal tract refers to the stomach, small intestine, large intestine, and rectum, or a combination thereof.

[0106] In an alternative embodiment, a method of ameliorating, preventing, or treating at least one symptom of cancer, and / or a gastrointestinal condition resulting from cancer therapy is provided.

[0107] In alternative embodiments, the microbiome population or composition of a subject is modulated or altered by administering to the subject a therapeutic composition, formulation, or pharmaceutical composition provided herein or by practicing a method provided herein.

[0108] In an alternative embodiment, the term "microbiome" encompasses the community of microorganisms that can live persistently and / or transiently in and on a subject's body, e.g., the human gut, including bacteria, viruses and bacterial viruses, archaea, and eukaryotes. In an alternative embodiment, the term "microbiome" encompasses the "genetic content" of that microbial community, which includes genomic DNA, RNA (ribosomal RNA, messenger RNA, and transfer RNA), epigenome, plasmids, and all other types of genetic information.

[0109] In alternative embodiments, the term "subject" refers to any animal subject, including humans, laboratory animals (e.g., primates, rats, mice), farm animals (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 disease, such as cancer, and an autoimmune disease or condition, or a developmental disorder.

[0110] In an alternative embodiment, the term "type(s)" when used in conjunction with "bacteria" or "bacterial" refers to bacteria identified at the genus level, species level, subspecies level, strain level, or by any other taxonomic method known in the art.

[0111] In an alternative embodiment, the phrase "dormant live bacteria" refers to biotrophic bacterial cells that have been rendered dormant by lyophilization or freeze-drying. Such dormant biotrophic bacterial cells are capable of immediately resuming growth and reproduction upon resuscitation.

[0112] In alternative embodiments, the term "spore" also includes "endospore," which terms may refer to any bacterial entity in a dormant, non-vegetative, non-reproductive stage, including spores, that are resistant to environmental stresses, such as desiccation, temperature fluctuations, nutrient depletion, radiation, and chemical disinfectants. In alternative embodiments, "spore germination" refers to the dormant spore initiating active metabolism and developing into a fully functional vegetative bacterial cell capable of reproduction and colonization. In alternative embodiments, a "germinant" is a material, composition, and / or physicochemical process that can either directly or indirectly induce the vegetative growth of dormant bacterial spores in a host organism or in vitro.

[0113] In alternative embodiments, the term "colonizing" refers to vegetative bacteria capable of forming viable bacterial colonies or spores capable of germinating and forming viable bacterial colonies.

[0114] In an alternative embodiment, the term "natural polymeric material" includes naturally occurring polymers that are not readily digested by human enzymes and, as a result, pass essentially intact through most of the human digestive system until they reach the large or small intestine.

[0115] In alternative embodiments, the therapeutic compositions, formulations, or pharmaceutical compositions provided herein comprise a population of live, dormant, non-pathogenic bacteria and / or bacterial spores. The dormant live bacteria may be capable of colonization, and in the case of spores, germination and colonization. Thus, in alternative embodiments, the compositions are useful for altering a subject's gastrointestinal biome, for example, by increasing the population of that bacterial type or microorganism, or by altering the microenvironment of the gastrointestinal biome, for example, by altering the chemical microenvironment or disrupting or disintegrating intestinal mucin or biofilm, thereby providing treatment for symptoms resulting from cancer, gastrointestinal conditions, and cancer therapy, and ultimately improving the health of the subjects to whom they are administered.

[0116] In alternative embodiments, the terms "purify," "purified," and "purifying" are used interchangeably to describe the known or unknown composition of a population of bacterial types, the amount of that bacterial type, and / or the concentration of the bacterial type; a purified population may be free of any undesirable traits or activities, or if present, they may be below an acceptable amount or level. In alternative embodiments, various populations of bacterial types are purified, and the terms "purified," "purify," and "purifying" refer to a population of desired bacteria and / or bacterial spores that have undergone at least one process of purification; for example, the process includes screening individual colonies derived from fecal material for a desired phenotype, e.g., their effectiveness in enhancing the pharmacodynamics of a drug (such as a cancer drug, e.g., a drug inhibitory to an immune checkpoint), where, for example, the individual's ability to absorb the drug is altered (e.g., accelerated or decelerated, or enhanced), or the dose effectiveness of the drug is increased (e.g., resulting in a lower dose of the drug being required for the intended effect), or the immune system is primed for improved drug effectiveness or selection or enrichment of desired bacterial types.

[0117] Enrichment can be achieved by increasing the amount and / or concentration of bacterial types, for example, by culturing in a medium that selectively favors the growth of certain types of microorganisms, by screening pure microbial isolates for desired genotypes, or by removing or reducing undesired bacterial types.

[0118] In an alternative embodiment, the bacteria used to practice the compositions and methods provided herein are derived from fecal material donors who are in good health, have a microbial biome associated with good health, and typically have not received antibiotics during or prior to the collection period, so that antibiotics do not remain in the donor's system. In an alternative embodiment, the donor subject does not suffer from, or have a family history of, kidney cancer, bladder cancer, breast cancer, prostate cancer, lymphoma, leukemia, or autoimmune disease. In an alternative embodiment, the donor subject does not suffer from, or have a family history of, irritable bowel disease, irritable bowel syndrome, celiac disease, Crohn's disease, colorectal cancer, anal cancer, gastric cancer, sarcoma, any other type of cancer, or a family history of these diseases. In an alternative embodiment, the donor subject does not suffer from, or have a family history of, a psychiatric illness, such as anxiety disorder, depression, bipolar disorder, autism spectrum disorder, panic disorder, obsessive-compulsive disorder, attention deficit disorder, eating disorders (e.g., bulimia, anorexia), mood disorders, or schizophrenia. In yet other embodiments, the donor subject has no known or known history of food allergies or intolerances.

[0119] In alternative embodiments, the fecal material donor's health is screened prior to collection of the fecal material, e.g., 1, 2, 3, 4, 8, 16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 weeks prior to collection. In alternative embodiments, the fecal material donor is also screened after collection, e.g., 1, 2, 3, 4, 8, 16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 weeks after collection. Pre-screening and post-screening can be performed daily, weekly, biweekly, monthly, or yearly. In alternative embodiments, individuals who have not tested positive for pathogenic bacteria and / or viruses (e.g., HIV, hepatitis, polio, adeno-associated virus, pox, coxsackievirus, etc.) before and after collection are considered verified donors.

[0120] In an alternative embodiment, to purify bacteria and / or bacterial spores, fecal material is collected from a donor subject and placed in an anaerobic chamber a short time after defecation, for example, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes or less after defecation, or longer. In an alternative embodiment, the fecal material sample collected from the donor subject is placed in the anaerobic chamber within about 1 minute to 48 hours or more after defecation from the donor.

[0121] Bacteria from a sample of collected fecal material can be collected in several ways. For example, the sample can be mixed with anaerobic nutrient broth, a dilution of the resulting mixture can be performed, and bacteria present in the dilution can be grown on solid anaerobic media. Alternatively, bacteria can be isolated by directly streaking a sample of the collected material onto solid anaerobic media for growth of isolated colonies. In an alternative embodiment, to increase the ease of isolating bacteria from a fecal sample mixed with anaerobic nutrient broth, the resulting mixture can be shaken, vortexed, blended, filtered, and centrifuged to disrupt and / or remove large non-bacterial material.

[0122] In an alternative embodiment, the isolated bacteria and / or bacterial spores can be purified by any means known in the art, e.g., to eliminate contamination with undesirable bacterial types, host cells, and / or elements from the host microbial environment by repeatedly streaking single colonies on solid media until at least two repeated streaks from a series of single colonies show only single colony morphology. Purification can also be performed by repeated serial dilutions, e.g., 10 to 10 dilutions, to obtain single cells. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9To achieve a final dilution of 10 times or higher, multiple 10-fold serial dilutions can be performed. Any method known to those skilled in the art can be applied. The presence of only a single bacterial type can be confirmed by multiple methods, such as Gram staining, PCR, DNA sequencing, enzyme analysis, metabolic profiling / analysis, antigen analysis, and flow cytometry using appropriate identification reagents.

[0123] In an alternative embodiment, the purified population of vegetative bacteria incorporated into the therapeutic bacterial compositions provided herein or used to practice the methods provided herein are fermented in a growth medium. Suitable growth media include NUTRIENT BROTH(TM) (THERMO SCIENTIFIC(TM) OXOID(TM)), ANAEROBE BASAL BROTH(TM)(THERMO SCIENTIFIC(TM) OXOID(TM)), REINFORCED CLOSTRIDIAL MEDIUM(TM)(THERMO SCIENTIFIC(TM) OXOID(TM)), SCHAEDLER ANAEROBIC BROTH(TM)(THERMO SCIENTIFIC(TM) OXOID(TM)), MRS BROTH(TM)(MILLIPORE-SIGMA(TM)), VEGITONE ACTINOMYCES BROTH(TM)(MILLIPORE-SIGMA(TM)), VEGITONE INFUSION BROTH(TM)(MILLIPORE-SIGMA(TM)), VEGITONE CASEIN SOYA BROTH™ (Millipore-Sigma™), or one of the following media available from ANAEROBIC SYSTEMS™: BRAIN HEART INFUSION BROTH™ (BHI), Campylobacter-Thioglycollate Broth (CAMPY-THIO), Chopped Meat Broth (CM), Chopped Meat Carbohydrate Broth (CMC), CHOPPED MEAT GLUCOSE BROTH™ (CMG), Cycloserine Cefoxitin Mannitol Broth with Taurocholate Lysozyme Cysteine ​​(CCMB-TAL), Oral Treponeme Enrichment Broth (OTEB), MTGE-ANAEROBIC ENRICHMENT BROTH™ (MTGE), Thioglycollate Broth with Hemin, Vitamin K (THIO) without indicator, Thioglycollate without indicatorBroth and Hemin, Vit.K(THIO), Lactobacilli-MRS Broth(LMRS), Brucella Broth(BRU-BROTH), Peptone Yeast Extract Broth(PY), PY Glucose(PYG), PY Arabinose, PY Adonitol, PY Arginine, PY Amygdalin, PYG Bile, PY Cellobiose, PY DL-Threonine, PY Dulcitol, PY Erythritol, PY Esculin, PYG Formate / Fumarate for FA / GLCf, PY Fructose, PY Galactose, PYG Gelatin, PY Glycerol, Indole-Nitrate Broth, PY Inositol, PY Inulin, PY Lactate for FA / GLCf, PY Lactose, PY Maltose, PY Mannitol, PY Mannose, PY Melezitose, PY Melibiose, PY Pyruvic Acid, PY Raffinose, PY Rhamnose, PY Ribose, PY Salicin, PY Sorbitol, PY Starch, PY Sucrose, PY Trehalose, PY Xylan, PY Xylose, Reinforced Clostridial Broth(RCB), Yeast Casitone Fatty Acids Broth and Carbohydrates(YCFACBroth). In an alternative embodiment, the growth medium contains or is supplemented with reducing agents, such as L-cysteine, dithiothreitol, sodium thioglycolate, and sodium sulfide. In an alternative embodiment, fermentation is carried out in a stirred tank fermentation vessel with nitrogen sparging to maintain anaerobic conditions and is performed in either batch or fed-batch mode. pH is controlled by the addition of concentrated base, such as NH4OH or NaOH. In the fed-batch mode, the feed is the primary carbon source for growth of the microorganisms, such as glucose. In an alternative embodiment, the post-fermentation broth is harvested and / or the bacteria are isolated by ultrafiltration or centrifugation and lyophilized or freeze-dried prior to formulation.

[0124] In an alternative embodiment, the purified and isolated vegetative bacterial cells used in the therapeutic bacterial compositions provided herein or used to practice the methods provided herein are rendered dormant, and it should be noted that the bacterial spores are already dormant. Dormancy of vegetative bacterial cells can be achieved, for example, by incubating and maintaining the bacteria at a temperature below 4°C, freezing the bacteria, and / or lyophilizing them. Lyophilization can be achieved according to conventional bacterial freeze-drying procedures used by those skilled in the art, such as those published by the AMERICAN TYPE CULTURE COLLECTION™ (ATCC).

[0125] In alternative embodiments, the purified population of live dormant bacteria and / or bacterial spores has an undetectable level of pathogenic activity, e.g., the ability to cause infection and / or inflammation, toxicity, an autoimmune response, an undesirable metabolic response (e.g., diarrhea), or a neurological response.

[0126] In alternative embodiments, all of the live dormant bacteria or bacterial spore types present in the purified population are obtained from fecal material processed as described herein or otherwise known to those skilled in the art. In other embodiments, one or more of the live dormant bacteria or bacterial spore types present in the purified population are individually cultured and combined with one or more types obtained from the fecal material. In alternative embodiments, all of the live dormant bacteria or bacterial spore types present in the purified population are individually cultured. In yet other embodiments, one or all of the live dormant bacteria and / or bacterial spore types present in the purified population are non-naturally occurring or engineered. In still other embodiments, non-naturally occurring or engineered non-bacterial microorganisms are present with or without live dormant bacteria and / or bacterial spores.

[0127] In alternative embodiments, the bacterial compositions in the compositions provided herein or used to practice the methods provided herein comprise a combination of different bacteria, including, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bacterial types, or more than 20 bacterial types, or about 2-30 bacterial types.

[0128] In an alternative embodiment, the bacterial composition comprises at least about 10 2 , 10 3 ,10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or more (or about 10 2 ~10 15) microorganisms, such as live dormant bacteria and / or bacterial spores. In some embodiments, each bacterial type is equally represented in the total number of live dormant bacteria and / or bacterial spores. In other embodiments, at least one bacterial type is represented in a higher amount than other bacterial types found in the composition.

[0129] In alternative embodiments, the population of different bacterial types in the compositions provided herein or used to practice the methods provided herein can increase the microbial population found in the gastrointestinal (GI) tract of a subject (or individual in need thereof) by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, optionally up to 10,000% or more, or between about 5% and 2000% or more, or between about 1% and 10,000%, compared to the subject's microbiome gastrointestinal population before treatment, and optionally the individual in need thereof is an infant or newborn.

[0130] In alternative embodiments, the combination of microorganisms, e.g., combinations of bacterial cells and / or spores, used in the compositions provided herein or to practice the methods provided herein are mixed with pharmaceutically acceptable excipients, e.g., diluents, carriers, adjuvants, binders, fillers, salts, lubricants, glidants, disintegrants, coatings, colorants, etc. Examples of such excipients include acacia, alginates, alginic acid, aluminum acetate, benzyl alcohol, butylparaben, butylated hydroxytoluene, citric acid, calcium carbonate, candelilla wax, croscarmellose sodium, powdered sugar, colloidal silicon dioxide, cellulose, simple or anhydrous calcium phosphate, carnauba wax, etc.Wax), corn starch, carboxymethylcellulose calcium, calcium stearate, calcium EDTA disodium, copolyvidone, calcium hydrogen phosphate dihydrate, cetylpyridine chloride, cysteine ​​HCl, crosprovidone, di- or tribasic calcium phosphate, dibasic calcium phosphate, disodium hydrogen phosphate, dimethicone, sodium erythrosinate, ethylcellulose, gelatin, glyceryl monooleate, glycerin, glycine, glyceryl monostearate, glyceryl behenate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hypromellose, HPMC phthalate, inulin, iron oxide or ferric oxide, yellow iron oxide, red iron oxide or ferric oxide, hydrous, anhydrous, monohydrate or spray-dried lactose, magnesium stearate Ingredients: sodium, maltodextrin, microcrystalline cellulose, mannitol, methylcellulose, magnesium carbonate, mineral oil, methacrylic acid copolymer, magnesium oxide, methylparaben, providone or PVP, PEG, polysorbate 80, propylene glycol, polyethylene oxide, propyleneparaben, poloxamer 407 or 188, potassium bicarbonate, potassium sorbate, potato starch, phosphoric acid, polyoxyethylene 140 stearate, sodium starch glycolate, pregelatinized starch, sodium carmellose, sodium lauryl sulfate, starch, silicon dioxide, sodium benzoate, stearic acid, sucrose, sorbic acid, sodium carbonate, sodium saccharin, sodium alginate, silica gel, sorbitan monooleate monooleate), sodium stearyl fumarate, sodium chloride, sodium metabisulfite, sodium citrate dihydrate, sodium starch, sodium carboxymethylcellulose, succinic acid, sodium propionate, titanium dioxide, talc, triacetin, and triethyl citrate.

[0131] In alternative embodiments, the combinations of microorganisms, e.g., combinations of bacterial cells and / or spores, used in the compositions provided herein or to practice the methods provided herein are prepared as colonies or microflora-induced delivery systems, as described, for example, in Basit et al, J. Drug Targeting, 17:1, 64-71; Kotla, Int J Nanomedicine. 2016; 11: 1089-1095; Bansai et al, Polim Med. 2014 Apr-Jun;44(2):109-18; or Shah et al, Expert Opin Drug Deliv. 2011 Jun;8(6):779-96.

[0132] In an alternative embodiment, the combination of microorganisms, for example, bacterial cells and / or spores, used in the compositions or methods provided herein are encapsulated in at least one polymeric material, for example, a natural polymeric material, such that the core of the bacterial cells and / or spores is surrounded by a layer of polymeric material, for example, polysaccharide. Examples of suitable polymeric materials are those that have been proven to remain intact throughout the GI tract until they reach the small intestine or large intestine, where they are degraded by microbial enzymes. Exemplary natural polymeric materials include, but are not limited to, chitosan, inulin, guar gum, xanthan gum, amylose, alginate, dextran, pectin, kava, and albizia gum (Dafe et al. (2017) Int J Biol Macromol 97: 299-307; Kofla et al. (2016) Int J Nanomedicine 11: 1089-1095).

[0133] In alternative embodiments, the compositions provided herein are suitable for therapeutic administration to humans or other mammals in need thereof.In alternative embodiments, the compositions are produced by a process that includes, for example, (a) obtaining fecal material from a mammalian donor subject; (b) subjecting the fecal material to at least one purification treatment under conditions that produce a single bacterial type population of bacteria and / or bacterial spores, or a combination of bacterial types and / or bacterial spores; (c) optionally combining the purified population with another purified population obtained from the same or different fecal material, from culture conditions, or from a genetic stock center such as ATCC or DSMZ; (d) if the microorganisms, for example, bacterial cells, are not dormant, treating the purified population under conditions that cause vegetative bacterial cells to become dormant; and (e) placing the dormant bacteria and / or bacterial spores in a vehicle for administration.

[0134] In alternative embodiments, compositions, formulations, and pharmaceutical compositions comprising one or a mixture of microorganisms (e.g., bacteria), e.g., bacterial cells and / or spores, provided herein, or for practicing the methods provided herein, are formulated for oral, topical, aerosol, rectal, or gastric administration to a mammalian subject, e.g., a human subject, or an individual in need thereof, e.g., a human infant or newborn.

[0135] In alternative embodiments, the compositions, formulations and pharmaceutical compositions are formulated for oral administration in solid, semi-solid, gel or liquid form, for example, in the form of pills, tablets, capsules, lozenges, food, extracts or drinks. In alternative embodiments, the compositions, formulations and pharmaceutical compositions are formulated for administration to infants or newborns, for example, formulated with, mixed with or added to liquids or powders, including, for example, milk (e.g., human milk, cow's milk or soy protein, optionally fortified with vitamins, minerals and other nutrients), infant formula, soy-based formula, amino acid-based formula, hydrolyzed infant formula (made from cow's milk or soy protein that has been broken down into smaller proteins that are easier for infants to digest), supplemented (collected) human breast milk, etc.

[0136] In alternative embodiments, the compositions, formulations and pharmaceutical compositions are formulated with, mixed with or added to gel, liquid or powder or food, for example, food or gel that does not require much chewing, for example, any drink, juice, juice extract, yogurt, pudding, gelatin and ice cream.Examples of extracts include crude and processed pomegranate juice, strawberry, raspberry and blackberry.Examples of suitable drinks include cold drinks, for example, juice (pomegranate, raspberry, blackberry, blueberry, cranberry, acai, cloudberry, etc. and combinations thereof) and tea (green tea, black tea, etc.), and oaked wine.

[0137] In alternative embodiments, the formulations and pharmaceutical compositions further comprise, or the methods provided herein further comprise, administration of at least one prebiotic, metabolic precursor, drug, or nutrient; optionally, for example, antibiotics such as doxycycline, chlortetracycline, tetracycline hydrochloride, oxytetracycline, demeclocycline, methacycline, minocycline, penicillin, amoxicillin, erythromycin, vancomycin, clarithromycin, roxithromycin, azithromycin, spiramycin, or the like. Antibiotics include flucloxacin, oleandomycin, josamycin, kitasamycin, flurithromycin, nalidixic acid, oxolinic acid, norfloxacin, perfloxacin, amifloxacin, ofloxacin, ciprofloxacin, sparfloxacin, levofloxacin, rifabutin, rifampicin, rifapentine, sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfadoxine, sulfasalazine, sulfaphenazole, dapsone, sulfapyridine, linezolid, or any combination thereof. In alternative embodiments, an antibiotic or combination of antibiotics is administered before, during, and / or after administration of the formulations and pharmaceutical compositions provided herein.

[0138] Slow- or delayed-release formulations In alternative embodiments, exemplary compositions, formulations, or pharmaceutical preparations provided herein or used in the methods provided herein comprise, contain, or are coated with an enteric coating to protect the microorganisms, e.g., bacteria or mixtures of bacteria provided herein, in the formulations and pharmaceutical compositions provided herein, allowing them to pass through the stomach and small intestine (e.g., protect the combination of microorganisms administered such that a substantial majority of the microorganisms remain viable), although spores typically resist the stomach and small intestine.

[0139] In an alternative embodiment, the compositions and formulations provided herein, and the compositions and formulations used to practice the methods provided herein, are formulated with delayed-release compositions or formulations, coatings, or encapsulation. In an alternative embodiment, the compositions and formulations provided herein, and the compositions and formulations used to practice the methods provided herein, are designed or formulated for inoculation of live microorganisms, e.g., bacteria or spores, into the intestine, e.g., the intestinal tract and / or the distal small intestine and / or colon. In this embodiment, the live microorganisms, e.g., bacteria, pass through risk areas, e.g., gastric acid and pancreatic enzymes and bile, reach the intestinal tract substantially undamaged so as to remain viable, and inoculate the GI tract.

[0140] In alternative embodiments, the formulation or pharmaceutical preparation, or the combination of microorganisms contained therein, is liquid, or is frozen, lyophilized, or freeze-dried.In alternative embodiments, for example, for encapsulated formulations, or in powdered form, or in aerosol form, or spray form.In alternative embodiments, when the formulation or pharmaceutical preparation provided herein is in powdered form, lyophilized form, or freeze-dried form, the powdered form, lyophilized form, or freeze-dried form can be in a container such as a bottle, cartridge, packet, or sachet, and the powdered form, lyophilized form, or freeze-dried form can be moistened or reconstituted by adding a liquid, such as water, saline, juice, milk, formula (such as infant formula), etc., to the powdered form, lyophilized form, or freeze-dried form, for example, the powdered form, lyophilized form, or freeze-dried form can be added to a liquid. In an alternative embodiment, the powdered, lyophilized, or freeze-dried forms provided herein are in a bottle or container, a liquid is added to the bottle or container, and the mixture can be consumed by an individual in need thereof. In an alternative embodiment, the powdered, lyophilized, or freeze-dried forms provided herein are in a cartridge that can be part of a container or bottle, and the powdered, lyophilized, or freeze-dried forms can be mixed with a liquid, for example, as described in U.S. Patent No. 8,590,753. In alternative embodiments, the powdered, lyophilized, or freeze-dried forms provided herein may be contained in or added to a container or bottle, for example, as described in U.S. Pat. Nos. 10,315,815; 10,315,803; 10,281,317; 10,183,116; 9,809,374; 9,345,831; 9,173,999; and 7,874,420.

[0141] In an alternative embodiment, the compositions and formulations provided herein, and the compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow enteric release using cellulose acetate (CA) and polyethylene glycol (PEG), as described, for example, in Defang et al. (2005) Drug Develop. & Indust. Pharm. 31:677-685, which used cellulose acetate (CA) and polyethylene glycol (PEG) with sodium carbonate in a wet granulation manufacturing process.

[0142] In an alternative embodiment, the compositions and formulations provided herein, and the compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow enteric release using hydroxypropylmethylcellulose (HPMC), microcrystalline cellulose (MCC), and magnesium stearate, e.g., as described in Huang et al. (2004) European J. of Pharm. & Biopharm. 58: 607-614).

[0143] In an alternative embodiment, the compositions and formulations provided herein, and compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow enteric release using, for example, poly(meth)acrylates, such as methacrylic acid copolymer B, methyl methacrylate and / or methacrylic acid esters, polyvinylpyrrolidone (PVP) or PVP-K90, and EUDRAGIT® RL PO™, e.g., as described in Kuksal et al. (2006) AAPS Pharm. 7(1), article 1, E1 to E9.

[0144] In an alternative embodiment, the compositions and formulations provided herein, as well as compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow intestinal release, as described in U.S. Patent Application Publication No. 20100239667. In an alternative embodiment, the composition comprises a solid inner layer sandwiched between two outer layers. The solid inner layer can contain non-pathogenic bacteria and / or spores, and one or more disintegrating and / or bursting agents, or one or more effervescent agents or mixtures. Each outer layer can comprise a substantially water-soluble and / or crystalline polymer, or a mixture of substantially water-soluble and / or crystalline polymers, such as polyglycol, which can be tailored to achieve delivery of the living components to the intestinal tract.

[0145] In an alternative embodiment, the compositions and formulations provided herein, and compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow enteric release, as described in U.S. Patent Application Publication No. 20120183612, which describes a stable pharmaceutical formulation comprising an active agent in a non-swelling diffusion matrix. In an alternative embodiment, the compositions and formulations provided herein, and compositions and formulations used to practice the methods provided herein, are released in a sustained, consistent, and, if present, independent manner from a matrix, the matrix being determined for its substantial release characteristics by ethyl cellulose and at least one fatty alcohol to deliver bacteria distally.

[0146] In an alternative embodiment, the compositions and formulations provided herein, and compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow enteric release, as described in U.S. Pat. No. 6,284,274, which describes a bilayer tablet containing an active agent (e.g., an opiate analgesic), polyalkylene oxide, polyvinylpyrrolidone, and a lubricant in a first layer, and a second osmotic push layer containing polyethylene oxide or carboxymethylcellulose.

[0147] In an alternative embodiment, the compositions and formulations provided herein, and compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow enteric release, as described in U.S. Patent Application Publication No. 20030092724, which describes an extended release dosage form in which a non-opioid analgesic and an opioid analgesic are combined in an extended release layer and an immediate release layer, the extended release formulation comprising microcrystalline cellulose, EUDRAGIT RSPO™, CAB-O-SIL™, sodium lauryl sulfate, povidone, and magnesium stearate.

[0148] In an alternative embodiment, the compositions and formulations provided herein, as well as the compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow enteric release, as described in U.S. Patent Application Publication No. 20080299197, which describes a multilayer tablet for triple combination release of active agents in the GI tract. In an alternative embodiment, a multilayer tablet is used, which can include two outer drug-containing layers in a stacked arrangement on the opposite side of an oral dosage form that provides triple combination release of at least one active agent. In one embodiment, the dosage form is an osmotic device, or a gastroresistant coated core, or a matrix tablet, or a hard capsule. In these alternative embodiments, the outer layer may contain a biofilm dissolving agent and the inner layer may comprise viable / living bacteria, e.g., a formulation comprising one (e.g., as in a synbiotic, or a combination of one and a probiotic, e.g., a synbiotic combination, as shown in Table 8 or Table 32) or at least two different species or genera (or types) of non-pathogenic bacteria used to practice the methods provided herein.

[0149] In alternative embodiments, the compositions and formulations provided herein, and the compositions and formulations used to practice the methods provided herein, can be prepared using any of the methods described in, for example, U.S. Pat. No. 6,514,531 (disclosing coated tri-layer immediate / extended release tablets), U.S. Pat. No. 6,087,386 (disclosing tri-layer tablets), U.S. Pat. No. 5,213,807 (disclosing oral tri-layer tablets having a core containing an active agent and an intermediate coating comprising a material that is substantially impermeable / impermeable to the passage of a first active agent), and U.S. Pat. No. 6,926,907 (disclosing drug release tablets). Disclosed is a tri-layer tablet that separates a first active agent contained in a film coat from a core containing a controlled-release second active agent, formulated with an excipient that controls release, and the film coat can be an enteric coating configured to delay release of the active agent until the dosage form reaches an environment where the pH is greater than 4), formulated as a multi-layer tablet form, e.g., as described herein, where a first layer provides immediate release of a formulation or pharmaceutical preparation provided herein and a second layer provides controlled release of another (or the same) bacterium or drug, or another active agent.

[0150] In an alternative embodiment, the compositions and formulations provided herein, and compositions and formulations used to practice the methods provided herein, are formulated for delayed or slow enteric release, as described in U.S. Patent Application Publication No. 20120064133, which may include release-impeding matrix materials such as acrylic polymers, cellulose, waxes, fatty acids, shellac, zein, hydrogenated vegetable oils, hydrogenated castor oil, polyvinylpyrrolidone, vinyl acetate copolymers, vinyl alcohol copolymers, polyethylene oxide, acrylic and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate polymers, cyanoethyl methacrylate polymers, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymers, poly(methyl methacrylate), poly(methacrylic anhydride), methyl methacrylate polymers, polymethacrylate, poly(methyl methacrylate) copolymers, polyacrylamide, The present invention describes aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cross-linked sodium carboxymethylcellulose, cross-linked hydroxypropylcellulose, natural waxes, synthetic waxes, fatty alcohols, fatty acids, fatty acid esters, fatty acid glycerides, hydrogenated fats, hydrocarbon waxes, stearic acid, stearyl alcohol, beeswax, glycowax, castor wax, carnauba wax, polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, carboxymethyl starch, potassium methacrylate / divinylbenzene copolymer, cross-linked polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl alcohol copolymer, polyethylene glycol, non-cross-linked polyvinylpyrrolidone, polyvinyl acetate, polyvinyl acetate copolymer, or any combination thereof. In an alternative embodiment, spherical pellets are prepared using extrusion / spheronization techniques, many of which are well known in the pharmaceutical field. The pellets can contain one or more formulations or pharmaceutical preparations provided herein.

[0151] In alternative embodiments, the compositions and formulations provided herein, as well as those used to practice the methods provided herein, are formulated for delayed release, extended release, or slow enteric release, as described, for example, in U.S. Patent Application Publication No. 20110218216, which describes an extended-release pharmaceutical composition for oral administration that uses a hydrophilic polymer, a hydrophobic material, and a hydrophobic polymer, or a mixture thereof, along with a microenvironment pH adjuster. The hydrophobic polymer can be ethyl cellulose, cellulose acetate, cellulose propionate, cellulose butyrate, a methacrylic acid-acrylic acid copolymer, or a mixture thereof. The hydrophilic polymer can be polyvinylpyrrolidone, hydroxypropyl cellulose, methylcellulose, hydroxypropylmethylcellulose, polyethylene oxide, an acrylic acid copolymer, or a mixture thereof. The hydrophobic material can be hydrogenated vegetable oil, hydrogenated castor oil, carnauba wax, candelilla wax, beeswax, paraffin wax, stearic acid, glyceryl behenate, cetyl alcohol, cetostearyl alcohol, or a mixture thereof. The microenvironment pH adjuster can be an inorganic acid, an amino acid, an organic acid, or a mixture thereof. Alternatively, the microenvironment pH adjuster can be lauric acid, myristic acid, acetic acid, benzoic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, adipic acid, sebacic acid, fumaric acid, maleic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, sodium dihydrogen citrate, gluconic acid, salicylic acid, tosylic acid, cresylic acid, or malic acid, or a mixture thereof.

[0152] In an alternative embodiment, the therapeutic combinations or formulations, or medicaments or pharmaceutical preparations provided herein or used in the methods provided herein are formulated as delayed or slow enteric release compositions or formulations, and optionally the formulations include a gastroresistant coating designed to dissolve at pH 7 in the terminal ileum, e.g., the active ingredient is coated with an acrylic resin or equivalent, e.g., a poly(meth)acrylate, e.g., methacrylic acid copolymer B, NF, which dissolves at pH 7 or above, including, e.g., a multimatrix (MMX) formulation. In an alternative embodiment, the compositions and formulations provided herein and used to practice the methods provided herein are powders or aerosols that can be contained in a suitable carrier, such as a liquid, tablet, or suppository. In an alternative embodiment, the compositions and formulations provided herein and used to practice the methods provided herein are "reconstitution powders" used as liquids to be placed down the naso-duodenal tract for drinking, or as enemas for patients to take at home and self-administer. In alternative embodiments, the compositions and formulations provided herein, as well as the compositions and formulations used to practice the methods provided herein, are microencapsulated, tableted, and / or placed in capsules, particularly enteric-coated capsules. In alternative embodiments, the compositions provided herein are formulated to be effective in a given mammalian subject in a single dose or over multiple doses. In some embodiments, the substrates or prebiotics required by the bacterial strains in the formulations provided herein are administered prior to the administration of the microbial combinations provided herein, e.g., bacterial compositions. Such administration (e.g., of prebiotics) preloads the gastrointestinal tract with the substrates required by the bacterial strains in the composition, increasing the likelihood that the bacterial composition has sufficient resources to carry out the necessary metabolic reactions. In other embodiments, the compositions are administered simultaneously with the substrates required by the bacterial strains in the formulations provided herein.In yet other embodiments, the substrate or prebiotic is administered alone. In alternative embodiments, efficacy is measured by an increase in the population of that bacterial type in the subject's intestinal tract, or an increase in the population of that bacterial type originally found in the subject's intestinal tract before treatment.

[0153] In alternative embodiments, the compositions provided herein comprise, further comprise, or have added thereto at least one probiotic or prebiotic, optionally wherein the prebiotic comprises inulin, lactulose, artichoke extract, chicory root, oat, barley, various legumes, garlic, kale, beans or flakes or herbs, mammalian milk oligosaccharides, or mucin, and optionally wherein the probiotic comprises cultured or stool-extracted microorganisms or bacteria, or bacterial components, and optionally wherein the bacteria or bacterial components comprise or are derived from Bacteroidetes, Firmicutes, Proteobacteria, Verucomicrobia, Actinobacteria, Lactobacilli, Bifidobacteria, E. coli, Streptococcus faecalis, and equivalents.

[0154] In alternative embodiments, the compositions provided herein comprise, further comprise, or are added to at least one solidification agent, optionally comprising arrowroot starch or vegetable starch, wheat flour, powdered potato or potato starch, absorbent polymers, absorbable modifying polymers, and / or corn flour or corn starch; or saline solution, a vehicle, an antifoaming agent, a surfactant, a lubricant, an acid neutralizer, a marker, a cell marker, a drug, an antibiotic, a contrast agent, a dispersing agent, a buffer or buffering agent, a sweetener, a de-bittering agent, a flavoring agent, a pH stabilizer, an acid or further comprising or having added thereto at least one biofilm disrupting compound, optionally the biofilm disrupting compound being an enzyme, deoxyribonuclease (DNase), N-acetylcysteine, auranofin, alginate lyase, glycoside hydrolase dispersin B; quorum sensing inhibitors, ribonuclease III inhibitor peptides, Salvadora persica extract, competence stimulating peptides, patulin and penicillic acid; peptides - cathelicidin-derived peptides, small lytic peptides, PTP-7, nitric oxide, neo-emulsions;ozone, lytic bacteriophage, lactoferrin, xylitol hydrogel, synthetic iron chelator, statin (optionally lovastatin (optionally MEVACOR™), simvastatin (optionally ZOCOR™), atorvastatin (optionally LIPITOR™), pravastatin (optionally PRAVACHOL™), fluvastatin (optionally LESCOL™), or rosuvastatin (optionally CRESTOR™)), cranberry constituents, curcumin, silver nanoparticles, acetyl-11-keto-β-boswellic acid (AKBA), barley coffee constituents, probiotics, sinefungin, S-adenosylmethionine, S-adenosyl-homocysteine, Delisea furanones, N-sulfonylhomoserine lactone, or any combination thereof;

[0155] In alternative embodiments, the compositions provided herein comprise, further comprise, or have added to a flavoring or sweetening agent, aspartame, stevia, monk fruit, sucralose, saccharin, cyclamate, xylitol, vanilla, artificial vanilla or chocolate or strawberry flavor, artificial chocolate extract, or a mixture or combination thereof.

[0156] Manufactured Products and Kits Articles of manufacture, e.g., implants or medicaments, and kits containing components for practicing the methods provided herein are provided, e.g., including formulations including a microorganism provided herein, e.g., a freshly isolated microorganism, a cultured microorganism, or a genetically engineered microorganism, or a combination of one (e.g., as in a synbiotic, or a combination of one and a probiotic, e.g., a synbiotic combination, as shown in Table 8 or Table 32), or at least two different species or genera (or types) of non-pathogenic bacteria, each of which comprises (or is in the form of) a plurality of live non-pathogenic colony-forming bacteria, a plurality of non-pathogenic germinating bacterial spores, or a combination thereof, and optionally including instructions for practicing the methods provided herein.

[0157] Companion diagnostics and participant biomarkers Biomarkers are provided that indicate dysbiosis or eubiosis in adults at high risk for diseases, such as colorectal cancer. These biomarkers may be in the form of microbial species abundance in the intestine (or abundance in the colon), microbial gene expression or protein expression, or metabolite abundance in stool samples or bacterial samples obtained from the intestine. Alternatively, the biomarkers may be metabolite concentrations, cytokine profiles, or protein expression in the blood. These biomarkers are used to determine the level of dysbiosis in participants' intestines and to predict treatment methods to improve dysbiosis in order to reduce the risk associated with diseases, such as colorectal cancer.

[0158] Genetic modification of microbial therapeutics In alternative embodiments, the microorganisms, e.g., bacteria or mixes of bacteria, used in the compositions provided herein or used to practice the methods provided herein are genetically engineered (or genetically modified). In alternative embodiments, one, some (e.g., about 1% to 99%), or all of the combinations or mixes of microorganisms provided herein or used to practice the methods provided herein are genetically engineered.

[0159] In an alternative embodiment, a microorganism, e.g., a bacterium or mix of bacteria, used in the compositions provided herein or used to practice the methods provided herein is genetically engineered to metabolize or consume a prebiotic, e.g., a prebiotic listed in Table 3.

[0160] In alternative embodiments, the microorganisms, e.g., bacteria or mixtures of bacteria, used in the compositions provided herein or used to practice the methods provided herein are genetically engineered to increase their effectiveness, e.g., to increase the effectiveness of chemotherapy, radiation therapy, immune checkpoint inhibitors (e.g., checkpoint inhibitor therapy), chimeric antigen receptor (CAR) T-cell therapy (CAR-T), or other immunotherapy or cancer treatment.

[0161] In alternative embodiments, the microorganisms, e.g., bacteria or mixes of bacteria, used in the compositions provided herein or used to practice the methods provided herein are genetically engineered to substantially reduce, reduce, or eliminate their toxicity.

[0162] In an alternative embodiment, the microorganisms, e.g., bacteria or mix of bacteria, used in the compositions provided herein or used to practice the methods provided herein are genetically engineered to contain a death switch so that they can be inactivated after administration of an appropriate trigger or signal.

[0163] In an alternative embodiment, a microorganism, e.g., a bacterium or a mixture of bacteria, used in the compositions or used to practice the methods provided herein is genetically engineered to secrete an anti-inflammatory composition or have an anti-inflammatory effect.

[0164] In an alternative embodiment, a microorganism, e.g., a bacterium or a mixture of bacteria, used in the compositions or used to practice the methods provided herein is genetically engineered to secrete an anti-cancer or cytostatic substance.

[0165] Microorganisms, such as bacteria, used in the compositions provided herein or used to carry out the methods provided herein can be genetically engineered using any method known in the art, for example, as discussed in the Examples below. For example, one or more gene sequences and / or gene cassettes can be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from a plasmid is used to increase the expression of an inserted, e.g., heterologous nucleic acid, such as a gene or protein-coding sequence or an inhibitory nucleic acid, e.g., a nucleic acid encoding an antisense or siRNA. The inserted nucleic acid of interest can be inserted into the bacterial chromosome at one or more integration sites.

[0166] For example, in an alternative embodiment, the microorganism is genetically engineered to contain one or more gene sequences and / or gene cassettes for producing non-native anti-inflammatory and / or intestinal barrier function enhancer molecules. In an alternative embodiment, the anti-inflammatory and / or intestinal barrier function enhancer molecules include short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2, GLP-1, IL-10, IL-27, TGF-beta1, TGF-beta2, N-acylphosphatidylethanolamine (NAPES), elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, and kynurenine. The molecules may be primarily anti-inflammatory, e.g., IL-10, or primarily enhance intestinal barrier function, e.g., GLP-2. In an alternative embodiment, the microorganism is genetically engineered to contain one or more gene sequences and / or gene cassettes that are inhibitory to the activity of, or that substantially or completely inhibit the expression of, a bacterial virulence factor, toxin, or antibiotic resistance function.

[0167] Any of the above aspects and embodiments may be combined with any other aspect or embodiment disclosed herein in the Summary, Figures and / or Detailed Description sections.

[0168] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0169] Unless specifically stated otherwise or obvious from context, as used herein, the term "or" is understood to include and cover both "or" and "and."

[0170] Unless specifically stated or obvious from the context, the term "about" as used herein is understood to mean within the range of normal acceptable values ​​in the art, for example, within two standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0171] Unless specifically stated or obvious from the context, as used herein, the terms "substantially all," "substantially most of," "substantially all of," or "the majority of" encompass at least about 90%, 95%, 97%, 98%, 99%, or 99.5%, or more, of the referenced amount of a composition.

[0172] Each patent, patent application, publication, and document referenced herein is hereby incorporated by reference in its entirety. Citation of the above patents, patent applications, publications, and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or dates of these publications or documents. The incorporation by reference of these documents alone should not be construed as a claim or admission that any portion of the contents of any document is believed to be essential material for satisfying the statutory disclosure requirements of any country or region for patent applications. Nevertheless, we reserve the right, in appropriate cases, to rely on any such documents to provide material deemed essential to claimed subject matter by an examining authority or court.

[0173] Modifications may be made to the foregoing without departing from the basic aspects of the invention. While the present invention has been described in substantial detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and that these modifications and improvements are nonetheless within the scope and spirit of the invention. The invention illustratively described herein may suitably be practiced in the absence of any element not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. Therefore, the terms and expressions used are used as terms of description rather than of limitation, and equivalents of the features shown and described, or portions thereof, are not excluded, recognizing that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

[0174] The present invention will be further described in conjunction with the examples set forth herein; however, it should be understood that the present invention is not limited to such examples. [Example]

[0175] Unless otherwise stated in the examples, all recombinant DNA techniques are performed according to standard protocols, e.g., as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY, and Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for the polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0176] The following examples describe methods and compositions for practicing the embodiments provided herein, including methods for making and using compositions comprising non-pathogenic bacteria and non-pathogenic germinable bacterial spores used to practice the methods provided herein.

[0177] Example 1 Anaerobic culture conditions Preparation of anaerobic growth medium The exemplary bacterial strains described herein are obligate anaerobes that require anaerobic conditions for cultivation. Suitable growth media for culturing anaerobic bacteria include reducing agents, such as L-cysteine, sodium thioglycolate, and dithiothreitol, for the purpose of scavenging and removing oxygen. Suitable commercially available anaerobic growth media include, but are not limited to, ANAEROBE BASAL BROTH™ (OXOID / THERMO SCIENTIFIC™), REINFORCED CLOSTRIDIAL MEDIUM™ (OXOID / THERMO SCIENTIFIC™), WILKINS-CHALGREN ANAEROBE BROTH™ (OXOID / THERMO SCIENTIFIC™), SCHAEDLER ANAEROBE BROTH™ (OXOID / THERMO SCIENTIFIC™), and BRAIN HEART INFUSION BROTH™ (OXOID / THERMO SCIENTIFIC™). Animal-free media for anaerobic cultivation include, but are not limited to, VEGITONE ACTINOMYCES BROTH™ (MILLIPORE-SIGMA™), MRS BROTH™ (MILLIPORE-SIGMA™), VEGITONE INFUSION BROTH™ (MILLIPORE-SIGMA™), and VEGITONE CASEIN SOYA BROTH™ (MILLIPORE-SIGMA™).

[0178] One liter of anaerobic growth medium is prepared by combining the manufacturer's recommended amount of grams of dried growth medium powder with 800 ml of reagent-grade water (NERL™) along with 1 ml of 2.5 mg / ml resazurin (ACROS Organics™) in a 2-liter beaker and stirred on a heated stir plate until dissolved. The volume is adjusted to 1 liter by adding additional reagent-grade water, and the volume is then boiled with stirring until the red color stained by the resazurin becomes colorless, indicating the removal of oxygen from the solution. The volume is then removed from the stir plate and allowed to cool on the benchtop for 10 minutes before further manipulation.

[0179] From the 1 liter volume, 900 ml was transferred to a 1 liter anaerobic media bottle (CHEMGLASS LIFE SCIENCES™) and then placed back on the heated stir plate to remove any oxygen introduced during the transfer, as indicated by the color of the added resazurin. The anaerobic media bottle was then sealed with a butyl rubber stopper secured by a crimped aluminum collar and then placed in an anaerobic chamber (COY LAB TYPE A VINYL ANAEROBIC CHAMBER™, COY LABORATORY PRODUCTS™, Grass Lake, MI). Within the anaerobic chamber, the butyl rubber stopper was removed and the bottle was opened and allowed to equilibrate to anaerobic conditions while cooling to ambient temperature. The bottle was resealed with a new butyl rubber stopper, the aluminum collar was crimped, and removed from the chamber, then sterilized by autoclaving for 20 minutes, followed by slow draining.

[0180] Alternatively, the 1 liter volume can be aliquoted into smaller 50 ml volumes in 100 ml serum bottles (CHEMGLASS LIFE SCIENCES™, Vineland, New Jersey). The boiled 1 liter volume is transferred to a 1 liter screw-cap bottle, which is placed back on the heated stir plate to expel any oxygen introduced by the transfer. The bottle cap is then tightly screwed on, and the bottle is immediately placed in an anaerobic chamber, where the cap is loosened to allow the volume to equilibrate with the anaerobic atmosphere and allowed to cool for 1 hour. The volume is then transferred in 50 ml aliquots to 100 ml serum bottles using a serological pipette, and the liquid contents are then allowed to cool to ambient temperature. The bottle is sealed with a butyl rubber stopper, an aluminum collar is crimped, and removed from the chamber and sterilized by autoclaving for 20 minutes, followed by slow draining.

[0181] Alternatively, the 1 liter volume can be aliquoted into smaller 10 ml volumes in sealed Hungate tubes (CHEMGLASS LIFE SCIENCES™, Vineland, New Jersey) as follows: The boiled 1 liter volume is transferred to a 1 liter screw-cap bottle, which is placed back on the heated stir plate to expel any oxygen introduced by the transfer. The bottle cap is then tightly screwed on, and the bottle is immediately placed in an anaerobic chamber, where the cap is loosened to allow the volume to equilibrate with the anaerobic atmosphere and cool for 1 hour. The volume is then transferred into 10 ml aliquots and filled into racked Hungate tubes, which are then cooled to ambient temperature, after which each tube is tightly capped and sealed with a screw cap with a butyl rubber septum. The sealed Hungate tube aliquots are removed from the anaerobic chamber and sterilized by autoclaving for 20 minutes, followed by slow draining.

[0182] Alternatively, a 1 liter volume can be combined with 15 grams of agar (THERMO SCIENTIFIC™) to create solid media in culture plates as follows: The boiled 1 liter volume is poured into a 1 liter screw-cap bottle, then placed on a heated stir plate to remove any oxygen introduced by the transfer, as indicated by a colorless resazurin oxygen indicator. The bottle is loosely capped and then autoclaved for 20 minutes, followed by slow draining. Immediately after autoclaving, the bottle is capped and then placed in an anaerobic chamber. Once in the anaerobic chamber, the cap is loosened and the contents are allowed to cool for 30 minutes, then a 25 ml volume is poured into a culture plate and allowed to cool until solidified. The plate is then allowed to dry in the anaerobic chamber for 24 hours before use.

[0183] Low temperature storage of anaerobic microorganisms Individual microorganisms of interest are prepared for long-term cryogenic storage by inoculating a pure colony isolate grown on anaerobic solid medium into a prepared Hungate tube containing a liquid anaerobic growth medium previously determined to be optimal for the species. The inoculated Hungate tube is then incubated at 37°C until turbid evidence of exponential growth is observed. The Hungate culture is placed in an anaerobic chamber, and 1 ml is transferred by pipette into a 2 ml screw-cap cryotube containing 1 ml of anaerobic Biobank Buffer (filter-sterilized, nitrogen-gas-buffered phosphate-buffered saline (PBS) + 2% trehalose + 10% dimethyl sulfoxide) for long-term storage. The resulting 2 ml volume is thoroughly mixed by pipetting, tightly secured, and then placed in the gas phase of a liquid nitrogen Dewar flask or a -80°C freezer for long-term storage.

[0184] Microorganisms in fecal material can be cryogenically preserved for subsequent revival and new strain discovery as follows: Freshly obtained fecal material is placed in an anaerobic chamber, and 1 gram is weighed and mixed with a solution consisting of 5 ml of Anaerobe Basal Broth (ABB) and 5 ml of BIOBANK BUFFER™ in a 15 ml conical tube. The tube is tightly capped, and the fecal material is thoroughly suspended in the solution by vortexing for 20 minutes, followed by incubation upright on ice to allow large particles to settle. A 1 ml aliquot of the fecal suspension is then transferred by pipette to a 2 ml screw-cap cryotube, tightly fastened, and then placed in the gas phase of a liquid nitrogen Dewar flask or a -80°C freezer for long-term storage.

[0185] Example 2 Fecal material collection and processing Infant stool sample collection Donations of fecal material are obtained from infants aged 1 month to 3 years. If from the US, donor infants are representative of US statistics for mode of birth (C-section / vaginal) and feeding (breast-fed / mixed / formula-fed), as well as US racial and ethnic demographics. Donor infants are screened for antibiotic use prior to donation.

[0186] Donors receive a stool sample collection kit via mail to the contact address provided. Stool samples are collected by the subject at home. The stool sample collection kit consists of the following: gloves, instructions for stool collection, a welcome card, a freezer pack, a Styrofoam® container, a plastic scoop for stool collection, a DNA / RNA storage tube for immediate sample storage, a FEDEX™ shipping label, and a sticker to seal the kit before shipping. Subjects receive a freezer pack to keep the sample cool and are instructed to place it in their freezer overnight upon receipt of the sample collection kit. The stool sample collection kit also includes a plastic scoop so that the stool sample can be collected directly from the diaper. Subjects are instructed to use the scoops to collect the stool sample as soon as possible after the sample is generated using the first scoop and to collect any remaining material on the diaper using the second scoop provided with the DNA / RNA storage tube. Subjects are instructed to put on the gloves provided with the kit before scooping the stool sample. Subjects are instructed to seal the plastic container in a specimen bag and remove gloves. Subjects are then instructed to remove an ice pack from their home freezer and place it in a Styrofoam cooler box along with the bagged and sealed stool sample. Subjects are instructed to close the lid on the foam container, then close the box and seal it with a packing sticker. Subjects are instructed to schedule a FEDEX™ pickup at their home or take it to the nearest FEDEX™ location within 24 hours of stool collection. Under these conditions, stool has been demonstrated to remain chilled for up to 48 hours of delivery.

[0187] Upon receipt, the stool sample container is given a unique alphanumeric identifier that is subsequently used for sample tracking. The stool is emptied from the shipping box in a laboratory setting, and the temperature is assessed to ensure the sample has been properly stored. The sample is then homogenized, as described below, and divided into individual aliquots sufficient for all planned analyses before freezing and storage at -80°C. RNA storage aliquots are stored at -20°C upon arrival until further use. All aliquots also have an alphanumeric identifier corresponding to the target donor. Any remaining stool after aliquots have been obtained is discarded as biohazardous waste.

[0188] Preparation of infant fecal material samples for analysis Fecal material received from the donor can be processed using any method known in the art, for example, as described in USPN 10,493,111; 10,471,107; 10,286,012; 10,314,863; 9,623,056.

[0189] For example, the received fecal material in the container is placed on ice and then placed in an anaerobic chamber. The container is opened and the sample is diluted 1:1 with anaerobic PBS. The mixture is homogenized manually or, if the sample size is sufficient, with a blender cup to a smooth consistency.

[0190] The homogenized fecal material is then processed and aliquoted for cryopreservation for several different analyses as follows: 1) Live cryopreservation for fecal microbiome transfer (FMT) experiments in mice: Homogenized fecal material is combined with FMT buffer (phosphate-buffered saline + 1% L-cysteine ​​+ 2% trehalose + 30% glycerol). The tubes are then vortexed for 20 seconds and placed on ice. Using a pipette, 1 ml aliquots are transferred to 2 ml cryotubes, which are then tightly capped. The aliquoted samples are frozen and then stored at -80°C. 2) Bio-cryopreservation for microbial isolation and discovery: Homogenized fecal material is combined with Anaerobe Basal Broth and Biobank Buffer (phosphate-buffered saline + 2% trehalose + 10% dimethyl sulfoxide) in a conical tube, tightly capped, vortexed for 20 seconds, then placed upright on ice and allowed to settle for 10 minutes. Using a pipette, 1 ml aliquots are added to 2 ml cryotubes, which are then tightly capped. The aliquoted samples are frozen and then stored at -80°C. 3) For genomic, metabolomic, and immunophenotyping analyses: Aliquot the homogenized fecal material into 2 ml cryotubes at a volume of 1 ml. Freeze the aliquoted samples and store them at -80°C.

[0191] In addition to homogenized fecal samples, raw fecal samples are used to assess the pH of the samples.

[0192] Adult stool specimen collection Donation of fecal material is obtained from healthy volunteers and individuals who show disease symptoms.Donors can be cancer patients who are administered approved therapy or who participate in clinical trials that test various cancer treatment regimens.Donors can be healthy volunteers who do not show disease symptoms or who are at risk for disease based on family history or before diagnosis.

[0193] Donors receive a stool sample collection kit by mail to the contact address provided or by their doctor. Stool samples are collected by the subject at home or with necessary assistance if hospitalized. The stool sample collection kit consists of the following: gloves, instructions for stool collection, a welcome card, a freezer pack, a Styrofoam container, a plastic bucket and plastic commode to assist with stool collection, a FedEx shipping label, and a sticker to seal the kit before delivery. Subjects receive a freezer pack to cool the sample and are instructed to place it in their freezer overnight upon receipt of the sample collection kit. The stool sample collection kit also includes a plastic commode that can be safely and securely attached to a toilet seat, allowing the subject to defecate directly into the plastic container. Subjects are instructed to use the commode to capture the stool sample and then seal the sample container with the provided snap-cap lid. Subjects are instructed to put on the gloves provided with the kit before removing the sample container from the toilet. Subjects are instructed to seal the plastic container in a specimen bag and remove gloves. Subjects are then instructed to remove an ice pack from their home freezer and place it in a Styrofoam cooler box along with the bagged and sealed stool sample. Subjects are instructed to close the lid on the foam container, then close the box and seal it with a packing sticker. Subjects are instructed to schedule a FedEx pickup at their home or take it to the nearest FedEx location within 24 hours of stool collection. Under these conditions, stool has been demonstrated to remain chilled for up to 48 hours of delivery.

[0194] Upon receipt, the stool sample container is given a unique alphanumeric identifier that is subsequently used for sample tracking. The stool is emptied from the shipping box, homogenized, and divided into individual aliquots sufficient for all planned analyses in a laboratory setting, as described below, before freezing and storage at -80°C. All aliquots also bear an alphanumeric identifier corresponding to the target donor. Any remaining stool after aliquots are obtained is disposed of as biohazardous waste.

[0195] Preparation of adult fecal material samples for analysis Fecal material received from the donor can be processed using any method known in the art, for example, as described in USPN 10,493,111; 10,471,107; 10,286,012; 10,314,863; 9,623,056.

[0196] For example, the received fecal material in the container is placed on ice and then placed in an anaerobic chamber. The container is opened and approximately 40 g of stool is weighed into a tared specimen cup. 15 ml of sterile anaerobic PBS is then added, and the mixture is homogenized with a handheld homogenizer to achieve a smooth consistency.

[0197] The homogenized fecal material is then processed and aliquoted for cryopreservation for several different analyses as follows: 1) For genomic and transcriptomic analyses: Weigh the homogenized fecal material and then add an equal volume of RNALATER® (THERMO FISHER SCIENTIFIC™) solution based on weight. Cap the tube tightly, vortex for 20 seconds, and then place on ice. Use a pipette to transfer 1 ml aliquots to 2 ml Eppendorf tubes. Freeze the aliquoted samples on dry ice and then store at -80°C. 2) Live cryopreservation for fecal microbiome transfer (FMT) experiments in mice: Homogenized fecal material is combined with FMT buffer (phosphate-buffered saline + 1% L-cysteine ​​+ 2% trehalose + 30% glycerol). The tubes are then vortexed for 20 seconds and placed on ice. Using a pipette, 1 ml aliquots are transferred to 2 ml cryotubes, which are then tightly capped. The aliquoted samples are frozen on dry ice and then stored at -80°C. 3) Cryopreservation for Microbial Isolation and Discovery: Homogenized fecal material is combined with Anaerobe Basal Broth and Biobank Buffer (phosphate-buffered saline + 2% trehalose + 10% dimethyl sulfoxide) in a conical tube, tightly capped, vortexed for 20 seconds, then placed upright on ice and allowed to settle for 10 minutes. Using a pipette, 1 ml aliquots are added to 2 ml cryotubes, which are then tightly capped. The aliquoted samples are frozen on dry ice and then stored at -80°C. 4) For genomic, metabolomic, and immunophenotyping analyses: Aliquot the homogenized fecal material into 2 ml cryotubes at a volume of 1 ml. Freeze the aliquoted samples and store them at -80°C.

[0198] Example 3 Collecting and analyzing patient data from infant clinical trials MY BABY BIOME(TM) Clinical Research The MY BABY BIOME™ Clinical Study (NCT05472688) was designed to assess gut microbiome diversity among healthy infants in the United States. Samples were collected from over 400 infants aged 4 to 10 weeks (when immune development is crucial) and evaluated via metagenomics, metabolomics, and proteomics to determine key discriminatory biomarkers. To ensure an accurate understanding of the infant gut in the United States, participants were sampled from different birth (vaginal vs. C-incision) and feeding regimes (formula-fed, breast-fed, or mixed) with a population representing the racial, ethnic, and geographic diversity of the U.S. population.

[0199] Whole genome sequencing of infant fecal samples An aliquot of homogenized fecal material is thawed and centrifuged at 6000 g for 20 minutes to pellet the cells. After centrifugation, 0.8 ml of supernatant is carefully removed by pipette, leaving 0.1 ml of pellet and medium for gDNA processing. Total genomic DNA is extracted from the cell pellet using the MAGATTRACT POWERMICROBIOME™ DNA / RNA EP Kit (Qiagen). The genomic DNA is then prepared for whole genome sequencing analysis using the KAPA LIBRARY PREP™ Kit (Roche). Sequencing analysis is performed on an Illumina platform using paired-end 150 bp reads.

[0200] Sequencing data are first processed to remove low-quality reads and adapter contamination using TRIM GALORE™ (Babraham Bioinformatics, Cambridge, UK), a wrapper for CUTADAPT™, a tool for quality control of high-throughput sequencing reads.

[0201] Assembled genomes of microorganisms and archaea from the Genome Taxonomy Database (GTDB) (Parks et al. (2019) bioRxiv 771964, Meric et al. (2019) bioRxiv 712166) were used as references for classification using CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). CENTRIFUGE™ categorises sequencing reads from metagenomic fecal samples to reference sequences and uses expectation maximization to estimate the relative abundance of taxa present in the sample.

[0202] A second classification was performed using a custom gut bacteria-specific index and an alternative classification algorithm. The custom index was constructed in multiple steps. First, 1,085 gut and oral genera were identified using the Unified Human Gastrointestinal Genome (UHGG) (Almeida et al. (2021) Nature Biotechnology 39:105-114). Second, 132,128 bacterial genome assemblies for the identified gut and oral genera were batch downloaded from NCBI. The downloaded assemblies were then clustered and dereplicated using METAGENOMICS-INDEX CORRECTION™ software (https: / / github.com / rrwick / Metagenomics-Index-Correction) with a threshold of 0.01. NCBI taxonomic nomenclature was replaced with GTDB taxonomy to maintain a consistent interpretation with the primary classification. From the dereplicated NCBI assemblies, we constructed an index for use with the KRAKEN2™ (CCB, Johns Hopkins University) metagenomics classification package (Wood et al. (2019) Genome Biology 20:257). Using simulated and pseudocommunities, we validated this classification method and demonstrated increased accuracy in subspecies-level classification.

[0203] Whole-genome sequencing analysis of infant fecal samples Using GUNIFRAC™ (GUNIFRAC™, GITHUB™, San Francisco, CA) (Chen et al. (2012) Bioinformatics 28:2106-2113) to measure distances between samples and the Ward method of agglomerative clustering, metagenomics broadly grouped the samples into three clusters based on their microbial composition. The relationships between samples are shown by principal coordinate analysis (Figure 1), and compositional differences between clusters are illustrated using a bar graph (Figure 2). Specifically, one cluster (designated C1) is highly enriched in Actinobacteriota. Another cluster is dominated by Bacteroidota (C2), while the third (C3) is enriched in Firmicutes and Proteobacteria (Figures 2 and 3). C1 contains samples from both vaginally and C-section delivered infants, while C2 is almost exclusively vaginally delivered, and C3 is enriched in C-section delivered infants (Figures 4 and 5) (the chi-squared p-value for the association of delivery mode with the GUNIFRAC™ cluster is less than 0.0001). C1 is also exclusively depleted in formula-fed infants (Figure 6) (the chi-squared p-value for the association of feeding mode with the gUniFrac™ cluster is 0.05). These trends can also be seen in the phylogenetic tree, where the Ward method of agglomerative clustering of GUNIFRAC™ samples against sample similarity shows how samples cluster according to microbiome composition (Figures 7 and 8). Clusters C1, C2, and C3 form three separate branches of the phylogenetic tree.

[0204] The high abundance of Actinobacteriota in C1 is almost exclusively caused by the genus Bifidobacterium (Figure 9). Based on historical populations, metabolic output, and the presence of pathogens, it can be inferred that the Bifidobacterium-enriched cluster represents a eubiosis for infants, while the other clusters represent two unique dysbiosis.

[0205] The fold change difference and statistical significance (inverse p-value, Mann-Whitney U test) were calculated for taxon abundance in C1 relative to other clusters, and the results were displayed in a volcano plot (Figure 10). Each point represents a family, order, class, genus, or species. After eliminating taxa with low overall abundance, approximately 18 taxa are enriched in C1 with p-values ​​lower than 1E-5. Figures 11-13 and Table 1 show the abundances in each sample of the most significantly enriched species. Species enriched in C1 were Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, Collinsella sp900759335, and Limosilactobacillus pontis_A. In addition, nine taxa were enriched in C3 compared to C1, many of which are or contain potentially pathogenic species (Figure 14).

[0206] Table 1: List of taxa enriched or depleted among clusters or groups of clusters.

[0207] Similar results were obtained using the alternative KRAKEN2™, which uses a custom index classification method. Using the KRAKEN2™ classification results, we applied additional filters beyond statistical significance; using bootstrapping, we removed enrichments that were not robust, and we filtered out enrichments that were low in abundance compared to our estimated classification noise level. A summary of the cluster enrichments generated from this method is in Table 9.

[0208] Table 9: Summary list of taxa enriched among clusters or groups of clusters using KRAKEN2™ with a custom taxonomic index and filtering on statistical significance, robustness, and abundance.

[0209] Four of the Bifidobacterium species enriched in C1 were B. longum sub. longum (B. longum), B. longum sub. infantis (B. infantis), B. breve, and B. bifidum. These species have been reported to be important for a healthy infant gut, in part due to their ability to consume human milk oligosaccharides (HMOs) (Underwood, MA et al. (2015) Pediatr. Res. 77:229-235; Sakanaka, M. et al. (2020) Nutrients 12:1-21). Despite their importance, relatively few of our samples contained high levels of these organisms, while in many they were completely absent (Figure 15). The total abundance of these core species was even lower for infants born via C-section (Figure 16) and formula-fed infants (Figure 17). In general, the abundance of Bifidobacterium, and especially these four species, was high in most of the C1 samples (Figure 18).

[0210] [Table 1-1] [Table 1-2] [Table 1-3]

[0211] [Table 9-1] [Table 9-2]

[0212] Alternative groupings of the infant microbiome were performed using a Dirichlet multinomial mixture (DMM) clustering routine (Holms I. et al. (2012) PLOS One. 7(2):e30126). Dirichlet multinomial mixture analysis was performed using open-source software (https: / / microbiome.github.io / tutorials / DMM.html) and returned three clusters with significant overlap compared to the GUNIFRAC™ (gUniFrac) clusters (chi-squared p-value <0.0001). The relationships between GUNIFRAC™ clusters C1, C2, and C3 and DMM clusters DMM1, DMM2, and DMM3 are illustrated in a Sankey diagram (Figure 44). It can be seen that DMM3 is primarily composed of samples that are also members of C1, DMM1 is more closely related to C3, and DMM2 is primarily composed of a combination of C1 and C2.

[0213] GUNIFRAC™ clusters are groups of samples with species close to each other in the taxonomic tree, while Dirichlet multinomial mixtures group samples purely based on the joint taxon distribution, regardless of evolutionary history. An example of the fundamental difference between GUNIFRAC™ clusters and DMM clusters is shown in Figure 45, where the relative abundance of Bifidobacterium dentium is observed for both the DMM cluster and the GUNIFRAC™ cluster (C1, C2, and C3). GUNIFRAC™ groups samples with a high relative abundance of B. dentium with samples with a high relative abundance of other Bifidobacteria; i.e., C1. B. dentium is not typically associated with healthy infant gut, and in the DMM cluster, samples with high B. dentium are no longer in the healthy infant gut cluster (DMM3), but are instead located in the cluster considered to be dysbiotic gut (DMM1).

[0214] The Dirichlet multinomial mixture model had statistically significant associations with both mode of delivery (vaginal vs. incisional) and mode of feeding (breast-fed, mixed, or formula-fed); these associations are shown in Tables 37 and 38.

[0215] [Table 37]

[0216] [Table 38]

[0217] Figure 51 shows the relative abundance of Bifidobacterium consortia (combined B. infantis, B. breve, B. bifidum, and B. longum) isolated by both feeding and delivery mode. It can be seen that the median abundance of the consortia was highest for vaginally delivered, breast-fed infants, significantly superior to c-section infants who also received breast milk nutrition. This is likely a result of the ability of the consortia to consume HMOs. Notably, this trend is opposite for formula-fed infants. Clearly, other microorganisms typically introduced in vaginal births may be superior to the consortia in metabolizing the contents of typical formula.

[0218] We used the same methods used for the GUNIFRAC™ clusters described above to determine what species were enriched or depleted in the DMM clusters. An example of a volcano plot showing the taxa enriched in DMM3 versus the combination of DMM1 and DMM2 is shown in Figure 46. A summary of the enriched taxa for the three DMM clusters is provided in Table 39.

[0219] [Table 39]

[0220] Antibiotic resistance markers in metagenomic samples were detected using the NCBI NATIONAL DATABASE OF ANTIBIOTIC RESISTANT ORGANISMS™ (NDARO). The genomes used to construct the KRAKEN2™ database (described above) for classification were analyzed using prodigal (Hyatt, D. et. Al, BMC Bioinformatics 11, 119 (2010)) to identify open reading frames. These open reading frames were then BLAST searched against a set of genes in the NCBI Antimicrobial Gene Index to add functional annotations to the gene set. The metagenomics sequencing data for each infant fecal sample was then searched against an annotated gene list using CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The number of antimicrobial resistance (AMR) signatures detected for each sample was tabulated.

[0221] Boxplots showing the distribution of the number of AMR signatures for each sample separated by GUNIFRAC™ cluster are shown in Figure 47 and for the DMM cluster in Figure 48. It can be seen that clusters associated with healthy infant gut microbiomes C1 and DMM3 have a statistically significant reduced number of AMR signatures. Figure 49 shows the inverse correlation between Bifidobacterium abundance and the number of AMR signatures, and also shows the observed trends between feeding method and AMR signatures. Figure 50 shows statistically significant differences in the distribution of AMR signatures grouped by feeding method, with the breast-fed gut microbiome having the lowest median AMR count, followed by mixed, and finally formula-fed.

[0222] MY BABY BIOME™ Clinical Studies and Health Outcomes To link health outcomes to infant microbiome characteristics, we analyzed both publicly available data from the "three-country cohort" of the DIABIMMUNE™ study (Vatanen T. et al. (2016) Cell. 165:842-853) and updated health information from participants in the MY BABY BIOME™ study.

[0223] DIABIMMUNE(trademark) The DIABIMMUNE™ "Three Country Cohort" data followed children from birth to age 3 to better understand the prevalence of allergies and autoimmune diseases in industrialized societies. Fecal samples were obtained frequently for each participant from birth to age 3, and health status for each participant is provided covering the first three years of life.

[0224] The gut microbiome sequencing data from DIABIMMUNE™ is 16s rRNA (compared to MY BABY BIOME™ whole genome sequencing) and therefore cannot resolve all species- and strain-level features. We analyze data features at the genus level. Specifically, we examine the ability of Bifidobacterium abundance to predict IGE levels and allergy incidence obtained in two age cohorts: 104 samples obtained when participants were between birth and 110 days of age (most similar to the MY BABY BIOME™ cohort), and 189 samples obtained when participants were between 110 days and 1 year of age. We selected one sample for each participant in each age group, selecting the sample closest in age to the centroid age for the cohort.

[0225] From birth to 110 days, Table 33 provides the fold change and p-values ​​(Mann-Whitney U test) for Bifidobacterium abundance associated with 18 metadata traits. Mean_fc is the mean Bifidobacterium abundance for individuals with the true value for the metadata field divided by the mean Bifidobacterium abundance for those without that condition; i.e., a mean_fc of 0.62 for regular_formula means that Bifidobacterium tended to be higher in individuals who were not fed regular formula. The only trend (statistical significance below 0.1) observed in the birth to 110 day cohort was that infants receiving regular or hydrolyzed formula had lower Bifidobacterium abundance.

[0226] [Table 33]

[0227] From 110 days to 1 year of age, there is a more statistically significant association with Bifidobacterium abundance. Table 34 lists the observed associations between Bifidobacterium abundance and metadata fields. Here, there is a statistically significant association (Mann-Whitney U p-value less than 0.01) between low Bifidobacterium abundance and regular formula, hydrolyzed formula, any baby formula, and milk allergy (up to 3 years of age). There is also a trend (Mann-Whitney U p-value less than 0.1) for antibiotic and birch allergy at 1 year of age (up to 3 years of age), with peanut allergy and dust mite allergy (up to 3 years of age) just below the threshold for significant trend. Figure 37 graphs the fold change versus p-value for this cohort.

[0228] [Table 34]

[0229] A statistically significant trend was also observed between Bifidobacterium abundance from 110 days to 1 year and total IGE (a type 1 hypersensitivity marker) measurements by age 3 years (Spearman r=-0.185, p-value=0.013), implying that high Bifidobacterium is associated with low IGE. Figure 38 shows a scatter plot of Bifidobacterium abundance ranging from 110 days to 1 year and total IGE measurements at age 3 years.

[0230] Figure 39 shows that the gut microbiome over the 110 day to 1 year range reflects the country of origin, with Finland (representing an industrialized society) having the lowest Actinobacteria, Russia (representing Karelia, an agricultural society) having the highest Actinobacteria but very low Bacteroidota, and Estonia (transitioning from agriculture to industrialization) having a moderate amount of Actinobacteriota.

[0231] MY BABY BIOME™ Clinical Study Follow-Up Participants in the MY BABY BIOME™ study were surveyed at 6 months and 1 year of age with questions containing health outcome information related to allergies and other immune-related complications. 26 participants reported adverse health outcomes at 6 months (16 allergies, 2 eczema, 9 dermatitis, and 2 asthma); 46 participants reported adverse health outcomes at 1 year (35 allergies, 4 eczema, 8 dermatitis, and 3 asthma). These adverse outcomes were distributed across the sample population with little bias at 6 months relative to the cluster assignment of the original stool sample. Statistical trends were observed between clusters and 1-year outcomes.

[0232] Trends related to Bifidobacterium infantis, longum, breve, and bifidum abundance were searched for and associated with all four species combinations, but no statistically significant trends (Mann-Whitney U) were found for adverse outcomes for the combinations at 6 months.

[0233] Exploring only a subset of dermatitis and eczema outcomes reveals that these skin conditions were not found in the high Bifidobacterium region of the PCoA plot (Figure 40). Table 35 lists the abundance of Bifidobacterium infantis, longum, breve, and bifidum, as well as the fold change and p-value (Mann-Whitney U) for the combination of all four species. There is a large reduction in B. infantis and B. bifidum abundance between individuals with and without these skin conditions, but due to limited data, only B. bifidum has statistical significance. Abundances are plotted in Figure 41.

[0234] [Table 35]

[0235] The 6-month survey data also show a significant association between eczema and the C3 gUniFrac cluster (chi-squared p-val=0.049). Both participants with eczema at 6 months were in the C3 cluster. Both of these samples were also in DMM1, which has the highest overlap with C3, but the statistical significance is lower because there are more samples in DMM1 than in C3.

[0236] In the 1-year survey data, there is a trend toward fewer participants reporting eczema or dermatitis in DMM3 than would be expected for no association (chi-squared p-value=0.10). Table 36 shows the observed number of participants with either eczema or dermatitis at 1 year. A very similar trend was observed at 6 months, although with low statistical significance at that time point (chi-squared p-value at 6 months=0.2951 vs. 0.10 at 1 year).

[0237] [Table 36]

[0238] Gene function analysis of whole genome sequencing of infant fecal samples

[0239] Published genomes and novel isolates were probed for the presence of known genes involved in HMO utilization (Figure 19). These genes tend to group into five clusters, each involved in metabolizing a different class of HMO and the urease cluster (Sakanaka, M. et al. (2020) Nutrients 12:1-21). Only B. infantis isolates contain genes from all five HMO clusters plus the urease cluster, indicating they are the most versatile in HMO utilization. B. breve contains most of the genes in clusters H2, H4, and H5, while B. longum and B. bifidum contain only cluster H5, and B. scardoviii has most of the genes in the H4, H5, and urease clusters. Several other genomes contain various HMO utilization genes, but none have complete or near-complete clusters.

[0240] Next, metagenomics sequences from each sample were screened for known HMO utilization genes. Using DIAMOND, raw sequencing reads were mapped to a set of 56 genes belonging to six clusters: H1 (18 genes), H2 (4 genes), H3 (3 genes), H4 (12 genes), H5 (7 genes), and urease (12 genes). Similarly, the abundance of other gene functions of interest in the samples was determined. These included genes encoding for the production of acetate, lactate, butyrate, valerate, indole-3-lactate, indole-3-propionate, phenyllactate, phenylacetate, and bacteriocins.

[0241] Metabolomic analysis of infant samples Fecal PBS samples isolated from the infant study will be evaluated by liquid chromatography-mass spectrometry (LC-MS) / MS using a Sciex Exion UHPLC (ultra-high performance liquid chromatography) coupled to a SCIEX 5500+ TRIPLE QUADRUPOLE MASS SPECTROMETER™.A panel of 79 metabolites (2-methylbutyrate, 3-hydroxybenzoate, 3-hydroxyhippurate, 3-hydroxyphenylpropionate, 3-methylindole, 4-ethylphenol, 4-ethylphenylsulfate, 4-hydroxyphenylacetate, 4-hydroxyphenylacrylate, 4-hydroxyphenyllactate, 4-hydroxyphenylpropionate, acetate, agmatine, arginine, benzoate, betaine, butyrate, cadaverine, carnitine, chenodeoxycholate, cholate, choline, cinnamoylglycine, citrine, deoxycholate, enterodiol, enterolactone, glycochenodeoxycholate, glycocholate, hexanoate, hippurate, imidazolepropionate, indole, Indole-3-acetamide, indole-3-lactate, indole-3-propionate, indole acetate, indoleacetylglycine, indoleacrylate, indoleacrylglycine, indoxyl sulfate, inosine, isobutyrate, isoleucine, isovalerate, kynurenate, kynurenine, lactate, leucine, lithocholate, lysine, N-acetylserotonin, ornithine, p-cresol, p-cresol glucuronide, p-crethrin Resole sulfate, phenol, phenol glucuronide, phenol sulfate, phenylacetate, phenylacetylglutamine, phenylacetylglycine, phenylalanine, phenyllactate, phenylpropionate, phenylpropionylglycine, phenylpyruvate, propionate, putrescine, serotonin, thiamine, trimethylamine, tryptamine, tryptophan, tyramine, tyrosine, ursodeoxycholate, valerate, and valine). Absolute quantification for each sample is provided by a calibration curve and isotope-labeled internal standards. Values ​​are normalized to fecal dry weight.

[0242] Samples were analyzed in the context of birth mode and lactation mode, and results revealed lactation mode as a significant driver of metabolism (Figure 20). To eliminate the complexity of lactation mode when interpreting metabolomic results, additional samples were evaluated to establish the unique metabolome of our distinct clusters in the context of breastfeeding.

[0243] Protein and cytokine analysis of infant fecal samples Fecal PBS samples isolated from infant studies are evaluated for the presence of cytokines in the feces. This can be done in a number of ways, for example, by using the MESO QUICKPLEX SQ 120MM™ (Meso Scale Discovery) and MSD U-plex assays, or by using the LUMINEX™-based MILLIPLEX™ technology (Millipore). Panels of various sizes are used depending on the application, e.g., 71 different cytokines (6CKine, BCA-1, CTACK, EGF, ENA-78, eotaxin, eotaxin-2, eotaxin-3, FGF-2, Flt3L, fractalkine, G-CSF, GM-CSF, GROα, I-309, IFNα2, IFNγ, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49 ... A panel of cytokines (IL-17E / IL-25, IL-17F, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28, IL-33, IP-10, LIF, MCP-1, MCP-2, MCP-3, MCP-4, M-CSF, MDC, MIG, MIP-1α, MIP-1β, MIP-1δ, PDGF-AA, PDGF-AB / BB, RANTES, sCD40L, SCF, SDF-1α+β, TARC, TGFα, TNFα, TNFβ, TPO, TRAIL, TSLP, and VEGF-A) provides insight into broad cytokine expression in the intestine. In addition to cytokine analysis, other proteins, such as calprotectin, are evaluated as potential markers of inflammation.

[0244] For the MY BABY BIOME™ study, 44 infant samples were analyzed using MILLIPLEX™ technology to evaluate a panel of 71 cytokines listed above.

[0245] Network analysis of multi-omics data To identify relationships between high-dimensional microbiome, immune, and metabolomics data, we began by reducing the set of features using Pearson correlation. We then used the graphical lasso to estimate inverse covariance and analyzed the data using NETWORKX™ (Aric A. Hagberg, et al., "Exploring network structure, dynamics, and function using NETWORKX™"). TM ", in Proceedings of thNetwork representations of variables were constructed using the 7th Python in Science Conference (SciPy2008), Gaeel Varoquaux, et al. (Eds), (Pasadena, CA USA), pp. 11-15, Aug 2008. Starting with 50 metabolomics, shotgun metagenomics, and immune samples, the datasets were independently transformed using central log-ratio (CLR) before merging the three data types together. Pearson correlation was used to measure linear associations between pairs of variables in the dataset. P-values ​​were adjusted for multiple comparisons (Benjamini & Hochberg FDR), and only traits with adjusted p-values ​​<= 0.05 were retained. The graphical lasso method was used to estimate the inverse covariance matrix of variables in the reduced set of traits. The inverse covariance matrix provides information about the conditional independent relationships between variables and can be used to construct network representations. NETWORKX™, a Python library for analyzing graphs and networks (Los Alamos National Laboratory), was used to construct a network representation of variables based on the inverse covariance matrix estimated by the Lasso method. The network representation visualized the relationships between highly interconnected variables and identified network modules or clusters of variables. The Louvain community detection algorithm was applied in NETWORKX™ to identify network modules. These modules represent groups of variables that are highly interconnected and likely have similar biological functions or relationships. The modules were analyzed to identify important variables and understand the structure of the network.

[0246] Using the above approach, we found that a notable core Bifidobacterium consortium (B. infantis, B. breve, B. bifidum, and B. longum) clustered closely with most anti-inflammatory responses (immune and metabolites) (Figure 21). When we added all Bifidobacterium species present in our infant metagenomes, there was a subset of Bifidobacterium that clustered closely with the core Bifidobacterium consortium and anti-inflammatory responses, suggesting that they also possess anti-inflammatory behavior (Figure 22). We next explored network modules containing the Proteobacteria phylum, as it has been shown to be associated with inflammation and positively correlated with preterm infants. Our network analysis showed that Proteobacteria was indeed significantly associated with the pro-inflammatory cytokine MCP-1 (Figure 23).

[0247] The network analysis was repeated using the same approach but applied to the species composition of samples obtained from the new KRAKEN2™-based classifier described in Example 3 (Figure 42). Here, three Bifidobacterium species (B. infantis, B. breve, and B. longum) were found to have a positive association with the aromatic lactic acid derivatives indole-3-lactate and 4-phenyllactate. These molecules appear to be important for healthy immune system development in infants (Laursen M. et al. (2021) Nature Microbiology 6:1367-1382). B. longum was negatively associated with potentially harmful bacterial species, such as Klebsiella michiganensis, a known nosocomial pathogen (Simoni S. et al. (2022) Antimicrobial Chemotherapy), and metabolites, such as trimethylamine, which has been linked to various chronic health conditions (Jalandra R. et al. (2023) Frontiers in Immunology 13). In contrast to the initial results described above, B. bifidum now clusters somewhat apart from the other three species. Because the KRAKEN2™ classifier reduces problems with multiple mapping and false positives, this new analysis provides additional insight into species-level network connectivity.

[0248] Metatranscriptomic analysis A fecal PBS sample is isolated from the infant and, at the time of isolation, stored in an RNA storage buffer (such as ZYMO DNA / RNA SHIELD™ (ZYMO RESEARCH™)). RNA is extracted from the storage buffer using a kit such as ZYMOBIOMICS™ MAGBEAD RNA (Zymo Research). RNA is assessed for quality and quantity using fluorescent techniques, such as the QUBIT RNA HIGH SENSITIVITY ASSAY KIT™ (Invitrogen). Mammalian RNA is then recovered from the sample using a kit that employs poly-T hybridization, such as the DYNABEADS™ mRNA DIRECT™ purification kit. The RNA is processed by reverse transcription and amplification using a kit such as the TRUSEQ STRANDED MRNA KIT™ (Illumina), and then prepared and analyzed using the same pipeline described for whole genome sequencing of DNA samples. The remaining RNA is processed using a kit such as the RIBO-ZERO PLUS MICROBIOME RRNA DEPLETION KIT™ (Illumina) and sequenced and analyzed using the same pipeline described for whole genome sequencing of DNA samples.

[0249] The genomes of gut microbes identified in samples by metagenomics are used as a framework for metatranscriptomic analysis. Comparison of gene enrichment and transcript enrichment provides further insight into what is active in the gut environment.

[0250] Example 4 Data-driven approaches for the design of biotherapeutics Based on the analysis performed in Example 3, four core strains of Bifidobacterium were selected as keystone species for biotherapeutic drug design. Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum were all shown to be statistically significant species throughout the analysis performed in Example 3. Although additional species were also shown to be statistically significant (such as Bifidobacterium pseudocatenulatum), they did not clearly cluster with the core four Bifidobacteria based on gUnifrac cladistic analysis of the C1 sample (Figure 18, as outlined in Example 3). All four core Bifidobacteria have known HMO consumption capabilities, further demonstrating their relevance in the infant gut. Using the four keystone species, we developed a subset of biotherapeutic drug combinations that were then further refined with additional microorganisms (see, for example, Table 2 or Table 30). Most of these microorganisms were enriched in our own analyses (Table 1) or isolated as part of our bacterial isolation program (Example 5), demonstrating their relevance in the Bifidobacterium-dominated gut. A subset was identified as having complementary metabolism and was therefore included as well.

[0251] In some combinations, B. bifidum was excluded because it was not associated with the other three core species in the network analysis in Figure 24. Furthermore, B. bifidum harbors a gene capable of producing 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) from linoleic acid. Although beneficial in small amounts, elevated fecal concentrations of this metabolite in infants have been associated with atopy and asthma later in childhood (Levan, SR et al., Nat. Microbiol. 2019, 4(11): 1851-1861).

[0252] A set of prebiotics was selected to complement the bacteria identified as the live biotherapeutic core. This set of probiotics was selected based on the functionality (i.e., HMO utilization) and in vitro growth evaluation observed during the analysis performed in Example 3. Due to the selectivity of HMOs for supporting Bifidobacterium growth, they were selected as the core of different prebiotic combinations (Table 3). These prebiotic cores were then supplemented with prebiotics (resulting in synbiotics) that were expected to have a synergistic growth effect or that in vitro analysis showed to have a growth benefit.

[0253] In an alternative embodiment, the combinations of bacteria (or probiotics) and prebiotics shown in Table 8 below are provided. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11]

[0254] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0255] Example 5 Isolation and identification of pure microbial strains from fecal material Strain isolation

[0256] In alternative embodiments, the microorganisms used in the compositions, formulations and pharmaceutical combinations provided herein or used to practice the methods provided herein are isolated from fecal material and can be used in the form of pure microbial strains isolated from fecal material.

[0257] Individual bacterial strains can be isolated and cultured from fecal material for further study and assembly of probiotics and / or therapeutic biologics, i.e., for the production of the microbial combinations provided herein. Most live bacteria inhabiting fecal material tend to be obligate anaerobes; therefore, care must be taken to perform all culture and isolation procedures in an anaerobic chamber to prevent their exposure to oxygen, and to use various anaerobic growth media containing the reducing agent compounds described in Example 1. Growth media and plates favoring the growth of target bacteria can be used to improve the ability to find and isolate them as pure, live cultures. Specifically, Bifidobacterium selective agar can be used to isolate Bifidobacterium. Different anaerobic growth media can be used to allow the growth of different subsets of microorganisms, improving the overall ability to isolate and purify a comprehensive range of unique bacterial species from each individual fecal material sample.

[0258] To begin a microbial isolation and characterization campaign, one cryotube containing cryopreserved fecal material is removed from storage in a liquid nitrogen Dewar vessel, placed in an anaerobic chamber, and then gently thawed on ice. A total of 1 ml of the contents is added to 10 ml of Anaerobe Basal Broth (ABB) or another suitable anaerobic growth medium to establish a 1 / 10 dilution. Sequential 10-fold serial dilutions are then performed in ABB to establish 1 / 100, 1 / 1000, 1 / 100000, 1 / 100000, and 1 / 1000000 dilutions of fecal material. Four 0.1 ml volumes are removed from each of the 1 / 10000, 1 / 100000, and 1 / 1000000 dilutions and then added to and spread on an optimal solid anaerobic growth medium. Plates are incubated at 37°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days to allow for the growth of a wide variety of bacterial colonies. Typically, plates are evaluated after 1-2 days of growth. Plating is performed from several liquid dilutions of fecal material to ensure that there are multiple, but non-overlapping, colonies present for efficient colony picking.

[0259] Colonies are manually picked from plates using a sterile pipette tip. Colonies may also be picked by an automated colony picking machine placed in an anaerobic chamber. Colonies are picked in multiples of 96 to accommodate the subsequent 96-well-based genomic DNA isolation step and large-scale cryogenic storage. After visible colonies are revealed on the streak, single colonies are picked and then inoculated into individual wells of a 2 ml 96-well deep-well block, each well containing 1 ml of optimal liquid anaerobic growth medium. Once all wells of the deep-well block have been inoculated with different picked colonies, the deep-well block is covered with an adhesive gas-permeable seal and then incubated in an anaerobic chamber incubator at 37°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days to allow liquid growth from each isolated colony. Typically, 96-well plates are picked after 1–2 days of growth.

[0260] After turbid growth is observed in all wells, the gas-permeable seals are removed from the 96-well deep-well block, and viable stock presentation is performed by transferring 0.1 ml of culture from each well to the corresponding well of a second 96-well deep-well block, each well containing 0.4 ml of the same anaerobic growth medium + 0.5 ml of Biobank Buffer (phosphate-buffered saline + 2% trehalose + 10% dimethyl sulfoxide). The volume in each well is mixed thoroughly by pipetting up and down several times, and the deep-well block is then sealed with an impermeable foil seal rated for -80°C storage and stored in a -80°C freezer.

[0261] Sequencing and computational characterization of isolated fecal bacteria The remaining 0.9 ml of culture in the original 96-well deep-well plate is then used for whole-genome sequencing of the isolated strains as follows: the deep-well block is centrifuged at 6000 g for 20 minutes to pellet the cells. After centrifugation, 0.8 ml of supernatant is carefully removed by pipette, leaving 0.1 ml of pellet and medium for gDNA processing. Total genomic DNA is extracted from the cell pellet using a MAGATTRACT POWERMICROBIOME DNA / RNA EP KIT™ (Qiagen). The genomic DNA is then prepared for whole-genome sequencing analysis using a KAPA HYPERPLUS KIT™ (Roche). Sequencing analysis is performed on an Illumina platform using paired-end 150 bp reads.

[0262] Sequencing data are first processed to remove low-quality reads and adapter contamination using Trim Galore, a wrapper for cutadapt.

[0263] Assembled genomes of microorganisms and archaea from the Genome Taxonomy Database (GTDB) (Parks et al. (2019) bioRxiv 771964, Meric et al. (2019) bioRxiv 712166) were used as references for classification using CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). CENTRIFUGE™ categorises sequencing reads from metagenomic fecal samples to reference sequences and uses expectation maximization to estimate the relative abundance of taxa present in the sample.

[0264] Unique strains (Table 4) were isolated from the MY BABY BIOME™ study using the methodology described.

[0265] [Table 4-1] [Table 4-2]

[0266] In an alternative embodiment, a complete genome is created for a specific organism of interest using long-read sequencing. High-molecular-weight genomic DNA is prepared from the organism of interest using a commercially available kit, such as Genomic-tip (Qiagen). Library preparation for genomic DNA is performed using NATIVE BARCODING KIT 24 V14™ (Oxford Nanopore), and sequencing is performed using MINION™ (Oxford Nanopore). The reads are filtered and trimmed for quality, and assembly is performed using the assembler FLYE™ (Kolmogorov et al. (2019) Nature Biotechnology 37:540-546). The resulting assembly is completed using MEDAKA™ (Oxford Nanopore Technologies) using short reads to correct errors inherent in long-read sequencing. Genes are predicted in the completed genome using prodigal (Hyatt et al. (2010) BMC Bioinformatics 11:119), NCBI Prokaryotic Gene Annotation Pipeline (Tatusova et al. (2016) Nucleic Acids Res. 44(14):6614-24), or DIAMOND™ (Buchfink et al. (2021) Nature Methods 18:366-68).

[0267] In an alternative embodiment, strain-level differences were determined via pan-genome analysis, which uses complete genome sequences to compare entire sets of genes from strains within a clade. Here, pan-genome analysis was performed using 10 complete Bifidobacterium infantis genomes from NCBI and the assembled genome from a B. infantis isolate (PB-STR-093). 48% of the pan-genome, containing 1620 gene clusters, is a core genome conserved among strains, as shown by the continuous bars in Figure 24. The remainder are accessory genes present in some, but not all, strains. When the genomes themselves are clustered according to the number and identity of the gene clusters they share, they are separated into two groups, distinguished by shared blocks of gene clusters. A region of 13 genes unique to PB-STR-093 that is not found in other genomes is highlighted in medium gray shading in the SCG cluster band. Seven of these gene clusters are predicted to be involved in carbohydrate transport and metabolism (COG20_category), including four xylose transporters.

[0268] Strain definition for strains isolated from fecal material To determine a suitable definition of a unique strain, we downloaded publicly available genomes for B. infantis, B. breve, B. longum, and B. bifidum strains from the NCBI Genome and the Genome Taxonomy Database (GTDB) and compared these genomes with Persephone strains PB-STR-321, PB-STR-093, PB-STR-083, PB-STR-119, PB-STR-103, PB-STR-207, PB-STR-215, and PB-STR-220 (all isolated and subjected to long-read sequencing as described above). The accession IDs (GenBank or NCBI RefSeq assemblies) of the downloaded genomes are listed in Table 28. A functional phylogenetic tree was generated from the pan-genome analysis of each species using ANVI'O™ (or Anvi'o; Eren AM et al. (2020) Nature Microbio 6:3-6). These phylogenetic trees are shown in Figure 32 (B. infantis), Figure 33 (B. longum), Figure 34 (B. breve), and Figure 35 (B. bifidum). Similarity between genomes was calculated using ANIb (Goris et al. (2007) Int J Syst Evol Micr 57: 81-91) through the pyani.anib module (Pritchard et al. (2016) Anal. Methods, 8:12-24), which provides three useful metrics: average nucleotide identity (ANI), genome coverage, and the product of ANI and genome coverage. [Table 28-1] [Table 28-2]

[0269] PB-STR-220:B. longum Persephone strain PB-STR-220 is a member of the species B. longum. Comparative genomic analysis of PB-STR-220 was performed with the published B. longum genome listed in Table 28. The reference strain for B. longum is GCF_000196555.1. PB-STR-220 is distinguished from the reference strain by the following values: Accession: GCF_000196555.1, ani: 98.0%, Coverage: 74.2%; Product: 72.7%. The published genome most similar to PB-STR-220 is GCF_013393765.1 (as determined by the strain with the highest ANIb product). PB-STR-220 is distinguished from GCF_013393765.1 by the following values: Accession: GCF_013393765.1, ani:98.0, Coverage: 78.7%; Product: 77.1%. Table 10 provides a list of unique open reading frames (ORFs) from PB-STR-220. These ORFs were determined to be unique by BLAST search using ORFs from the above list of published B. longum genomes. If an ORF from PB-STR-220 does not have a corresponding ORF with any of the published genomes (having greater than 60% sequence identity), it is considered unique and is included in the table. If an ORF had less than 60% but greater than 20% sequence identity, the highest percent sequence identity (pident) is listed. If functional annotation was possible, it was included in the table. [Table 10-1] [Table 10-2] [Table 10-3] Table 10-4 Table 10-5 Table 10-6 Table 10-7 Table 10-8 Table 10-9 Table 10-10 Table 10-11 Table 10-12 Table 10-13 Table 10-14 Table 10-15 Table 10-16 Table 10-17 Table 10-18 Table 10-19 Table 10-20 [Table 10-21] [Table 10-22] [Table 10-23]

[0270] HMO utilization genes HMO utilization genes were detected in PB-STR-220 using a functional genomics pipeline built with CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The pipeline annotates the genome using functional annotations, including KEGG ORTHOLOGY database (KO) numbers. A list of HMO utilization genes and KO numbers associated with HMO utilization gene clusters was obtained from a published study (Henrick et al. (2021) Cell 184:P3884-3898). The Blon gene accession IDs associated with each observed KO value in PB-STR-220 are listed in Table 11, where the genes are grouped by HMO utilization gene cluster from Henrick et al.: [Table 11]

[0271] Bacteriocins Using the bacterial version of ANTISMASH™ (Blin et al. (2023) Nucleic Acids Research 51:W46-W50), the PB-STR-220 genome was searched for bacteriocins, peptides known to have antibacterial and immunological properties relevant to the infant gut environment (Benitez-chao D. et al. (2021) Frontiers in Microbiology 12), and the observed signatures are listed in Table 29.

[0272] [Table 29]

[0273] Antibiotic resistance genes The ORFs found in the genome of the PB-STR-220 strain were BLAST searched against the NCBI Antimicrobial Resistance Database, and no antimicrobial resistance genes were observed. Antimicrobial resistance genes are becoming increasingly common in infants (Shan Y. et al. (2019) Nature 574: 117-121) and pose potential health problems (Samarra A. et al. (2023) Gut Microbes 15: 2194797).

[0274] virulence factor The ORFs found in the genome for strain PB-STR-220 were BLAST searched against VFDB (Virulence Factor Database), and no virulence genes were observed.

[0275] PB-STR-207:B. longum Persephone strain PB-STR-207 is a member of the B. longum species. Comparative genomic analysis of PB-STR-207 was performed with the published B. longum genome found in Table 28. The type strain for B. longum is GCF_000196555.1. PB-STR-207 is distinguished from the type strain by the following values: Accession: GCF_000196555.1, ani: 98.7%, Coverage: 84.4%; Product: 83.3%. The published genome most similar to PB-STR-207 is GCF_000772485.1 (as determined by the strain with the highest ANIb product). PB-STR-207 is distinguished from GCF_000772485.1 by the following values: Accession: GCF_000772485.1, ani:98.8, Coverage: 89.0% Product: 88.0%. Table 12 provides a list of unique open reading frames (ORFs) from PB-STR-207. These ORFs were determined to be unique by BLAST search using ORFs from the above list of published B. longum genomes. If an ORF from PB-STR-207 does not have a corresponding ORF (with greater than 60% sequence identity) with any of the published genomes, it is considered unique and is included in the table. If an ORF had less than 60% but greater than 20% sequence identity, the highest sequence identity to the external strain is indicated as pident (percentage of identical matches). If functional annotation was possible, they were included in the table. [Table 12-1] [Table 12-2] [Table 12-3]

[0276] HMO utilization genes HMO utilization genes were detected in PB-STR-207 using a functional genomics pipeline built with CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The pipeline annotates the genome using functional annotations, including KEGG ORTHOLOGY™ database (KO) numbers. A list of HMO utilization genes and KO numbers associated with HMO utilization gene clusters was obtained from a published study (Henrick et al. (2021) Cell 184:P3884-3898). The Blon gene accession IDs associated with each observed KO value in PB-STR-207 are listed in Table 13, where genes are grouped by HMO utilization gene cluster from Henrick et al.: [Table 13]

[0277] Bacteriocins Using the bacterial version of ANTISMASH™ (Blin et al. (2023) Nucleic Acids Research 51:W46-W50), the PB-STR-207 genome was searched for bacteriocins, peptides known to have antibacterial and immunological properties relevant to the infant gut environment (Benitez-chao D. et al. (2021) Frontiers in Microbiology 12), but no bacteriocin signatures were observed.

[0278] Antibiotic resistance genes The ORFs found in the genome of strain PB-STR-207 were BLAST searched against the NCBI Antimicrobial Resistance Database, and no antimicrobial resistance genes were observed. Antimicrobial resistance genes are becoming increasingly common in infants (Shan Y. et al. (2019) Nature 574: 117-121) and pose potential health problems (Samarra A. et al. (2023) Gut Microbes 15: 2194797).

[0279] virulence factor The ORFs found in the genome for strain PB-STR-207 were BLAST searched against VFDB (Virulence Factor Database), and no virulence genes were observed.

[0280] PB-STR-215:B. longum Persephone strain PB-STR-215 is a member of the species B. longum. Comparative genomic analysis of PB-STR-215 was performed with the published B. longum genomes listed in Table 28. The type strain for B. longum is GCF_000196555.1. PB-STR-215 is distinguished from the type strain by the following: Accession: GCF_000196555.1, ani: 98.5%, Coverage: 78.4%; Product: 77.2%. The published genome most similar to PB-STR-215 is GCF_000219455.1 (as determined by the strain with the highest ANIb product). PB-STR-215 is distinguished from GCF_000219455.1 by the following values: Accession: GCF_000219455.1, ani:98.8, Coverage: 86.4%; Product: 85.3%. Table 14 provides a list of unique open reading frames (ORFs) from PB-STR-215. These ORFs were determined to be unique by BLAST search using ORFs from the above list of published B. longum genomes. If an ORF from PB-STR-215 does not have a corresponding ORF with any of the published genomes (with greater than 60% sequence identity), it is considered unique and is included in the table. If an ORF had less than 60% but greater than 20% sequence identity, the highest sequence identity to the external strain is indicated as pident. If functional annotation was possible, they were included in the table. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4]

[0281] HMO utilization genes HMO utilization genes were detected in PB-STR-215 using a functional genomics pipeline built using CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The pipeline annotates the genome using functional annotations, including KEGG ORTHOLOGY database (KO) numbers. A list of HMO utilization genes and KO numbers associated with HMO utilization gene clusters was obtained from a published study (Henrick et al. (2021) Cell 184:P3884-3898). The Blon gene accession IDs associated with each observed KO value in PB-STR-215 are listed in Table 15, where the genes are grouped by HMO utilization gene cluster from Henrick et al. [Table 15]

[0282] Bacteriocins Using the bacterial version of ANTISMASH™ (Blin et al. (2023) Nucleic Acids Research 51:W46-W50), the PB-STR-215 genome was searched for bacteriocins, peptides known to have antibacterial and immunological properties relevant to the infant gut environment (Benitez-chao D. et al. (2021) Frontiers in Microbiology 12), but no bacteriocin signatures were observed.

[0283] Antibiotic resistance genes The ORFs found in the genome of the PB-STR-215 strain were BLAST searched against the NCBI Antimicrobial Resistance Database. Antimicrobial resistance genes are becoming increasingly common in infants (Shan Y. et al. (2019) Nature 574: 117-121) and pose potential health problems (Samarra A. et al. (2023) Gut Microbes 15: 2194797). Two antimicrobial gene signatures were observed. Table 16 describes each of these signatures: [Table 16]

[0284] virulence factor The ORFs found in the genome for strain PB-STR-215 were BLAST searched against VFDB (Virulence Factor Database), and no virulence genes were observed.

[0285] PB-STR-093:B.infantis Persephone strain PB-STR-093 is a member of the B. infantis species. Comparative genomic analysis of PB-STR-093 was performed with the published B. infantis genomes listed in Table 28. The reference strain for B. infantis is GCF_000269965.1. PB-STR-093 is distinguished from the reference strain by the following values: Accession: GCF_000269965.1, ani: 97.8%, Coverage: 81.7%; Product: 79.9%. The published genome most similar to PB-STR-093 is GCF_001281305.1 (as determined by the strain with the highest ANIb product). PB-STR-093 is distinguished from GCF_001281305.1 by the following values: Accession: GCF_001281305.1, ani:100.0, Coverage: 99.5%, Product: 99.4%. Table 17 provides a list of unique open reading frames (ORFs) from PB-STR-093. These ORFs were determined to be unique by BLAST search using ORFs from the above list of published B. infantis genomes. If an ORF from PB-STR-093 does not have a corresponding ORF with any of the published genomes (with greater than 60% sequence identity), it is considered unique and is included in the table. If an ORF had less than 60% but greater than 20% sequence identity, the highest sequence identity to the external strain is indicated as pident. If functional annotation was possible, they were included in the table. [Table 17-1] [Table 17-2]

[0286] HMO utilization genes HMO utilization genes were detected in PB-STR-093 using a functional genomics pipeline built with CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The pipeline annotates the genome using functional annotations, including KEGG ORTHOLOGY database (KO) numbers. A list of HMO utilization genes and KO numbers associated with HMO utilization gene clusters was obtained from a published study (Henrick et al. (2021) Cell 184:P3884-3898). The Blon gene accession IDs associated with each observed KO value in PB-STR-093 are listed in Table 18, where genes are grouped by HMO utilization gene cluster from Henrick et al.: [Table 18]

[0287] Bacteriocins Using the bacterial version of ANTISMASH™ (Blin et al. (2023) Nucleic Acids Research 51:W46-W50), the PB-STR-093 genome was searched for bacteriocins, peptides known to have antibacterial and immunological properties relevant to the infant gut environment (Benitez-chao D. et al. (2021) Frontiers in Microbiology 12), and six signatures were observed. The observed signatures are listed in Table 29.

[0288] Antibiotic resistance genes The ORFs found in the genome of strain PB-STR-093 were BLAST searched against the NCBI Antimicrobial Resistance Database, and no antimicrobial resistance genes were observed. Antimicrobial resistance genes are becoming increasingly common in infants (Shan Y. et al. (2019) Nature 574: 117-121) and pose potential health problems (Samarra A. et al. (2023) Gut Microbes 15: 2194797).

[0289] virulence factor The ORFs found in the genome for strain PB-STR-093 were BLAST searched against VFDB (Virulence Factor Database), and no virulence genes were observed.

[0290] PB-STR-083:B. infantis Persephone strain PB-STR-083 is a member of the B. infantis species. Comparative genomic analysis of PB-STR-083 was performed with the published B. infantis genomes listed in Table 28. The reference strain for B. infantis is GCF_000269965.1. PB-STR-083 is distinguished from the reference strain by the following values: Accession: GCF_000269965.1, ani: 98.0%, Coverage: 81.1%; Product: 79.5%. The most similar published genome to PB-STR-083 is GCA_920939435.1 (as determined by the strain with the highest ANIb product). PB-STR-083 is distinguished from GCA_920939435.1 by the following values: Accession: GCA_920939435.1, ani:98.0, Coverage: 83.4%; Product: 81.8%. Table 19 provides a list of unique open reading frames (ORFs) from PB-STR-083. These ORFs were determined to be unique by BLAST search using ORFs from the above list of published B. infantis genomes. If an ORF from PB-STR-083 does not have a corresponding ORF (with greater than 60% sequence identity) with any of the published genomes, it is considered unique and is included in the table. If an ORF had less than 60% but greater than 20% sequence identity, the highest sequence identity to the external strain is indicated as pident. If functional annotation was possible, they were included in the table. [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4] [Table 19-5] [Table 19-6] [Table 19-7] [Table 19-8] [Table 19-9] [Table 19-10] [Table 19-11] [Table 19-12] [Table 19-13] [Table 19-14] [Table 19-15] [Table 19-16] [Table 19-17] [Table 19-18] [Table 19-19]

[0291] HMO utilization genes HMO utilization genes were detected in PB-STR-083 using a functional genomics pipeline built with CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The pipeline annotates the genome using functional annotations, including KEGG ORTHOLOGY database (KO) numbers. A list of HMO utilization genes and KO numbers associated with HMO utilization gene clusters was obtained from a published study (Henrick et al. (2021) Cell 184:P3884-3898). The Blon gene accession IDs associated with each observed KO value in PB-STR-083 are listed in Table 20, where genes are grouped by HMO utilization gene cluster from Henrick et al.: [Table 20]

[0292] Bacteriocins Using the bacterial version of ANTISMASH™ (Blin et al. (2023) Nucleic Acids Research 51:W46-W50), the PB-STR-083 genome was searched for bacteriocins, peptides known to have antibacterial and immunological properties relevant to the infant gut environment (Benitez-chao D. et al. (2021) Frontiers in Microbiology 12), and three signatures were observed. The observed signatures are listed in Table 29.

[0293] Antibiotic resistance genes The ORFs found in the genome of strain PB-STR-083 were BLAST searched against the NCBI Antimicrobial Resistance Database, and no antimicrobial resistance genes were observed. Antimicrobial resistance genes are becoming increasingly common in infants (Shan Y. et al. (2019) Nature 574: 117-121) and pose potential health problems (Samarra A. et al. (2023) Gut Microbes 15: 2194797).

[0294] virulence factor The ORFs found in the genome for strain PB-STR-083 were BLAST searched against VFDB (Virulence Factor Database), and no virulence genes were observed.

[0295] PB-STR-103:B. breve Persephone strain PB-STR-103 is a member of the B. breve species. Comparative genomic analysis of PB-STR-103 was performed with the published B. breve genome in Table 28. The reference strain for B. breve is GCF_001025175.1. PB-STR-103 is distinguished from the reference strain by the following values: Accession: GCF_001025175.1, ani: 98.4%, Coverage: 81.6%; Product: 80.3%. The published genome most similar to PB-STR-103 is GCF_002838305.1 (as determined by the strain with the highest ANIb product). PB-STR-103 is distinguished from GCF_002838305.1 by the following values: Accession: GCF_002838305.1, ani:98.6, Coverage: 87.6%; Product: 86.4%. Table 21 provides a list of unique open reading frames (ORFs) from PB-STR-103. These ORFs were determined to be unique by BLAST search using ORFs from the above list of published B. breve genomes. If an ORF from PB-STR-103 does not have a corresponding ORF with any of the published genomes (with greater than 60% sequence identity), it is considered unique and is included in the table. If an ORF had less than 60% but greater than 20% sequence identity, the highest sequence identity to the external strain is indicated as pident. If functional annotation was possible, they were included in the table. [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4] [Table 21-5]

[0296] HMO utilization genes HMO utilization genes were detected in PB-STR-103 using a functional genomics pipeline built with CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The pipeline annotates the genome using functional annotations, including KEGG ORTHOLOGY database (KO) numbers. A list of HMO utilization genes and KO numbers associated with HMO utilization gene clusters was obtained from a published study (Henrick et al. (2021) Cell 184:P3884-3898). The Blon gene accession IDs associated with each observed KO value in PB-STR-103 are listed in Table 22, where the genes are grouped by HMO utilization gene cluster from Henrick et al.: [Table 22]

[0297] Bacteriocins Using the bacterial version of ANTISMASH™ (Blin et al. (2023) Nucleic Acids Research 51:W46-W50), the PB-STR-103 genome was searched for bacteriocins, peptides known to have antibacterial and immunological properties relevant to the infant gut environment (Benitez-chao D. et al. (2021) Frontiers in Microbiology 12), but no bacteriocin signatures were observed.

[0298] Antibiotic resistance genes The ORFs found in the genome of the PB-STR-103 strain were BLAST searched against the NCBI Antimicrobial Resistance Database. Antimicrobial resistance genes are becoming increasingly common in infants (Shan Y. et al. (2019) Nature 574: 117-121) and pose potential health problems (Samarra A. et al. (2023) Gut Microbes 15: 2194797). Three antimicrobial gene signatures were observed. Table 23 describes each of these signatures: [Table 23]

[0299] virulence factor The ORFs found in the genome for strain PB-STR-103 were BLAST searched against VFDB (Virulence Factor Database), and no virulence genes were observed.

[0300] PB-STR-119:B. breve The Persephone strain PB-STR-119 is a member of the B. breve species. Comparative genomic analysis of PB-STR-119 was performed with the published B. breve genomes listed in Table 28. The reference strain for B. breve is GCF_001025175.1. PB-STR-119 is distinguished from the reference strain by the following values: Accession: GCF_001025175.1, ani: 98.6%, Coverage: 83.3%; Product: 82.2%. The published genome most similar to PB-STR-119 is GCF_002838565.1 (as determined by the strain with the highest ANIb product). PB-STR-119 is distinguished from GCF_002838565.1 by the following values: Accession: GCF_002838565.1, ani:100.0, Coverage: 98.1%, Product: 98.0%. Table 24 provides a list of unique open reading frames (ORFs) from PB-STR-119. These ORFs were determined to be unique by BLAST search using ORFs from the above list of published B. breve genomes. If an ORF from PB-STR-119 does not have a corresponding ORF with any of the published genomes (with greater than 60% sequence identity), it is considered unique and is included in the table. If an ORF had less than 60% but greater than 20% sequence identity, the highest sequence identity to the external strain is indicated as pident. If functional annotation was possible, they were included in the table. [Table 24-1] [Table 24-2] [Table 24-3]

[0301] HMO utilization genes

[0302] HMO utilization genes were detected in PB-STR-119 using a functional genomics pipeline built with CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The pipeline annotates the genome using functional annotations, including KEGG ORTHOLOGY database (KO) numbers. A list of HMO utilization genes and KO numbers associated with HMO utilization gene clusters was obtained from a published study (Henrick et al. (2021) Cell 184:P3884-3898). The Blon gene accession IDs associated with each observed KO value in PB-STR-119 are listed in Table 25, where the genes are grouped by HMO utilization gene cluster from Henrick et al.: [Table 25]

[0303] Bacteriocins

[0304] Using the bacterial version of ANTISMASH™ (Blin et al. (2023) Nucleic Acids Research 51:W46-W50), the PB-STR-119 genome was searched for bacteriocins, peptides known to have antibacterial and immunological properties relevant to the infant gut environment (Benitez-chao D. et al. (2021) Frontiers in Microbiology 12), but no bacteriocin signatures were observed.

[0305] Antibiotic resistance genes The ORFs found in the genome of the PB-STR-119 strain were BLAST searched against the NCBI Antimicrobial Resistance Database, and no antimicrobial resistance genes were observed. Antimicrobial resistance genes are becoming increasingly common in infants (Shan Y. et al. (2019) Nature 574: 117-121) and pose potential health problems (Samarra A. et al. (2023) Gut Microbes 15: 2194797).

[0306] virulence factor The ORFs found in the genome for strain PB-STR-119 were BLAST searched against VFDB (Virulence Factor Database), and no virulence genes were observed.

[0307] PB-STR-321:B. bifidum Persephone strain PB-STR-321 is a member of the B. bifidum species. Comparative genomic analysis of PB-STR-321 was performed with the published B. bifidum genomes listed in Table 28. The type strain for B. bifidum is GCF_001025135.1. PB-STR-321 is distinguished from the type strain by the following values: Accession: GCF_001025135.1, ani: 99.1%, Coverage: 94.1%; Product: 93.3%. GCF_001025135.1 is also the strain most similar to PB-STR-321 (as determined by the strain with the highest ANIb product).

[0308] Table 26 provides a list of unique open reading frames (ORFs) from PB-STR-321. These ORFs were determined to be unique by BLAST search using ORFs from the above list of published B. bifidum genomes. If an ORF from PB-STR-321 does not have a corresponding ORF with any of the published genomes (with greater than 60% sequence identity), it is considered unique and is included in the table. If an ORF had less than 60% but greater than 20% sequence identity, the highest sequence identity to the external strain is indicated as pident. If functional annotation was possible, they were included in the table. [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4] [Table 26-5] [Table 26-6] [Table 26-7] [Table 26-8] [Table 26-9]

[0309] HMO utilization genes HMO utilization genes were detected in PB-STR-321 using a functional genomics pipeline built with CENTRIFUGE™ (Kim et al. (2016) Genome Research 26:1721-1729). The pipeline annotates the genome using functional annotations, including KEGG ORTHOLOGY™ database (KO) numbers. A list of HMO utilization genes and KO numbers associated with HMO utilization gene clusters was obtained from a published study (Henrick et al. (2021) Cell 184:P3884-3898). The Blon gene accession IDs associated with each observed KO value in PB-STR-321 are listed in Table 27, where the genes are grouped by HMO utilization gene cluster from Henrick et al.: [Table 27]

[0310] Bacteriocins Using the bacterial version of ANTISMASH™ (Blin et al. (2023) Nucleic Acids Research 51:W46-W50), the PB-STR-321 genome was searched for bacteriocins, peptides known to have antibacterial and immunological properties relevant to the infant gut environment (Benitez-chao D. et al. (2021) Frontiers in Microbiology 12), but no bacteriocin signatures were observed.

[0311] Antibiotic resistance genes The ORFs found in the genome of strain PB-STR-321 were BLAST searched against the NCBI Antimicrobial Resistance Database, and no antimicrobial resistance genes were observed. Antimicrobial resistance genes are becoming increasingly common in infants (Shan Y. et al. (2019) Nature 574: 117-121) and pose potential health problems (Samarra A. et al. (2023) Gut Microbes 15: 2194797).

[0312] virulence factor The ORFs found in the genome for strain PB-STR-321 were BLAST searched against VFDB (Virulence Factor Database), and no virulence genes were observed.

[0313] Biotherapeutic Combinations Based on the strain definitions provided above, a set of new biotherapeutic combinations was generated with strain-level resolution (Table 30).

[0314] [Table 30-1] [Table 30-2] [Table 30-3] [Table 30-4] [Table 30-5] [Table 30-6] [Table 30-7] [Table 30-8]

[0315] Characterization of antibiotic resistance of strains isolated from fecal material

[0316] In an alternative embodiment, the complete genome sequence of each organism is screened using publicly available databases, such as DBETH55 (see, e.g., Chakraborty A, et al. (2012) Nucleic Acids Res. 40:615-620) and VFDB56 (Chen L, et al. (2005) Nucleic Acids Res. 33:325-328), to ensure that it does not contain genes encoding known virulence factors, toxins, or antibiotic resistance functions, or pathogenicity island gene clusters. Each organism is evaluated using standard antibiotic susceptibility profiling techniques, such as broth microdilution susceptibility panels, or plate-based methods, such as disk diffusion and antibiotic gradient assays (James H. Jorgensen and Mary Jane Ferraro 2009 Clinical Infectious Diseases 49:1749-1755). Such testing determines the minimum inhibitory concentration (MIC) of antibiotics against microbial growth. Antibiotics evaluated include, but are not limited to, amoxicillin, amoxicillin / clavulanic acid, carbapenems, methicillin, ampicillin, gentamicin, metronidazole, vancomycin, and neomycin. MIC determinations of novel microorganisms are compared to published values ​​for both susceptible and resistant related strains to assess susceptibility (CLSI Guideline M45: Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. Wayne, PA; 2015) to determine any possible associated increases in antibiotic resistance.

[0317] The PB-STR-093 strain of Bifidobacterium infantis was evaluated for antibiotic susceptibility and the results are presented in Table 5. [Table 5]

[0318] Example 6 Growth and characterization of isolated strains and strain consortia Experimental evaluation of metabolism

[0319] The growth of the isolated strains is evaluated to determine their ability to consume carbon sources and produce specific metabolites. The strains are grown in a minimal medium that allows control over the carbon source (e.g., 2 grams (2 ml, high quality) peptone water (peptone 10 g / L, sodium chloride 5 g / L), 2 grams yeast extract, 2 grams NaHCO3, 0.1 g NaCl, 0.04 g K2HPO4, 0.04 g KH2PO4, 0.01 g MgSO4.7H2O, 0.01 g CaCl2.6H2O, 2 ml Tween® 80, 10 g optimal carbohydrate, 10 μl vitamin K, 0.5 g cysteine, 0.5 g bile salts). Examples of carbon sources that were evaluated (independently or in combination) include, but are not limited to, glucose, lactose, galactose, fructose, xylose, galactooligosaccharides, fructooligosaccharides, xylooligosaccharides, lacto-N-tetraose, lacto-N-neotetraose, 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, and 6'-sialyllactose. Growth was monitored by OD 600 This can be done on individual samples using a cuvette or tube-based spectrometer (e.g., a CO 75000™ colorimeter) or on multiple samples in a plate-based format using a plate reader (e.g., a CYTATION 3™). In both cases, values ​​are normalized based on the signal from medium alone.

[0320] In addition to growth, metabolite production is assessed by spinning down the cell culture and isolating the supernatant for evaluation. The same panel used in Example 3 is used to assess metabolite production in the supernatant. In addition, human milk oligosaccharide consumption (lacto-N-tetraose, lacto-N-neotetraose, 2'-fucosyllactose, 3-fucosyllactose, and 3'-sialyllactose) is assessed where appropriate.

[0321] In addition to evaluating isolated strains, consortia of strains are also evaluated for both growth and metabolite production. The same techniques are used for growth and metabolic assessment, and if necessary, whole genome sequencing (see Example 3) is used to determine compositional information.

[0322] The combined growth and metabolic information is used to determine bacteria and consortia with key functional traits, including, but not limited to, human milk oligosaccharide consumption (lacto-N-tetraose, lacto-N-neotetraose, 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose), glycan degradation, short-chain fatty acid production, and tryptophan metabolism.

[0323] Application to libraries of isolated strains Selected bacteria were evaluated for their ability to consume different carbon sources to aid in the selection of the live biotherapeutic (probiotic) bacteria provided herein, as well as the bacterial combinations, mixes and consortia, and prebiotic combinations (see Table 6) used in the compositions and methods provided herein. [Table 6-1] [Table 6-2] [Table 6-3]

[0324] Example 7 Immunoreactivity of live biotherapeutic microorganisms Cytokine production and analysis in PBMCs for isolated strains

[0325] Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using a standard kit and stored at 1 × 10 per mL until use. 6 The cells are stored in liquid nitrogen. Prior to storage, PBMCs can be processed using flow sorting or antibody spin separation kits to select for certain purified lymphocyte subpopulations, such as T cells.

[0326] For use, PBMCs are thawed at 37°C and then transferred to growth medium consisting of RPMI-1640 (Lonza, Switzerland) supplemented with 10% heat-inactivated FCS, 0.1% penicillin-streptavidin, 1% L-glutamine, and 10 mg / mL DNase to prevent aggregation, or equivalent medium. Cells are centrifuged at 200 × g for 15 minutes, then counted using trypan blue and plated at 1 × 106 cells per well in 24-well plates (1 mL per well) (Kechaou et al. (2013) Applied and Environmental Microbiology 79:1491-1499; Martin et al. (2017) Frontiers in Microbiology 8:1226) or 250,000 cells per well in 96-well plates.

[0327] For evaluation of single bacteria, overnight bacterial cultures are inoculated with previously stocked isolated bacterial strains. The strains are grown for 10–20 hours at 37°C in Actinomyces veggie broth medium with added cellobiose (1 mg / mL), maltose (1 mg / mL), and cysteine ​​(0.5 mg / mL) in an anaerobic chamber filled with 85% nitrogen, 10% carbon dioxide, and 5% hydrogen (Martin et al., 2017). Other growth media, such as those outlined in Example 1, may be used instead. For evaluation of consortia rather than single strains (such as those identified in Table 2, Example 4 or Table 30, Example 5), overnight bacterial cultures for each included strain are inoculated with previously stocked isolated bacterial strains. The next day, the consortium bacteria are combined at the desired ratio (based on CFU, OD, or some other quantification method) and grown together.

[0328] At the end of anaerobic culture, the culture supernatant and bacterial cells alone are preserved for co-culture with PBMCs. The microbial culture supernatant is stored at -80°C immediately after centrifugation. The cells are preserved by washing with phosphate-buffered saline (PBS) and then storing in PBS with 15% glycerol. The bacteria are quantified using phase-contrast microscopy and stored at -80°C at a final concentration of 10 or 10 cells per mL (Haller et al. (2000) Infection and Immunity 68; Rossi et al. (2015) Scientific Reports 6:18507). The bacteria may also be pasteurized by treatment at 70°C for 30 minutes before storage (Plovier et al. (2017) Nature Medicine 23:107-113).

[0329] Prior to incubation with PBMCs, bacterial supernatants are thawed on ice and diluted 1:5 in PBMC growth medium. Microbial growth medium is used as a negative control. This supplemented PBMC growth medium is added 1:1 to each well with PBMCs, resulting in a final 10% dilution level of the microbial culture supernatant. Each combination of PBMCs and supernatants is performed in duplicate or triplicate. If bacteria are instead being evaluated, they are thawed on ice and then washed with PBMC growth medium at 4°C prior to co-culture. A bacterial suspension in PBMC growth medium is added 1:1 with 1 mL of PBMC culture in each well of the plate, resulting in 1 x 10 6 1 x 10 PBMCs and 1 x 10 5 or 1×10 6 A final 2 mL culture containing 10 (potentially pasteurized) bacteria is obtained. The PBMC and supernatant or purified bacterial co-culture is incubated at 37 °C in 10% carbon dioxide for a time ranging from 2 to 48 h.

[0330] After co-culture, the supernatant is collected and analyzed directly, or treated with protease inhibitors (Complete EDTA-Free Protease Inhibitor, Roche Applied Bioscience) to protect cytokines and stored directly at -80°C for cytokine profiling. Pelleted cells are treated with RNALATER™ (Thermo Fisher, USA) and stored for RNA sequencing. Cytokine analysis is performed on the stored co-culture supernatant using ELISA, LUMINEX™ system, Meso Scale Discovery system, or equivalent analytical method. Cytokines measured may include, but are not limited to, IL-10, IL-2, and IFN-gamma. RNA sequencing is performed on PBMCs stored in RNALATER™ after co-culture. Standard pseudoalignment is performed using Kallisto (Bray et al. (2016) Nature Biotechnology 34:525-527), and differential expression is analyzed using DESeq2 (Love et al. (2014) Genome Biology 15:550) to identify differential expression between different microorganisms and different PBMC donors. Statistical analysis is performed to identify microorganisms that exhibit desired immunomodulatory effects in vitro, including, but not limited to, induction of IFN-gamma production and reduced expression of genes associated with T cell exhaustion (PD1, CTLA4, VISTA, TIM3, TIGIT, LAG3).

[0331] In addition to the evaluation of the cell supernatants described, different matrices will also be evaluated for their immunostimulatory properties, for example, supernatants isolated from a simulated intestinal environment such as those described in Example 8.

[0332] In one example, simulated intestinal environments supplemented with Bifidobacterium infantis were evaluated for their ability to induce differential cytokine expression when compared to unsupplemented simulated intestinal environments. When performed in a C3 environment, a substantial shift was observed, with Bifidobacterium infantis supplementation greatly ameliorating the inflammation observed in the C3 intestinal environment alone (Figure 28).

[0333] Cytokine production in immature dendritic cells induced by live biotherapeutic (or probiotic) compositions Single bacterial strains, consortia of bacterial strains, and bacterial strains in a simulated gut environment are evaluated, alone and in combination with LPS, on cytokine production in immature dendritic cells. Monocyte populations are isolated from peripheral blood mononuclear cells (PBMCs). The monocytic cells are then differentiated into immature dendritic cells. Immature dendritic cells are plated at 200,000 cells / well and incubated for 10 min in RPMI medium with the addition of LPS at a final concentration of 100 ng / ml as needed. 7 The cells are then incubated with a live biotherapeutic composition at a final concentration of 100 ng / ml. Alternatively, the bacterial cells are centrifuged and the resulting supernatant is added to the dendritic cell preparation. A negative control involves incubating the cells with RPMI medium alone, and a positive control involves incubating the cells with LPS at a final concentration of 100 ng / ml. The cytokine content of the cells is then analyzed.

[0334] Example 8 Evaluation of live biotherapeutic (or probiotic) candidate strains in a simulated intestinal environment Experimental evaluation of strains in a simulated intestinal environment To understand the applicability of in vitro observations to the human intestine, strains are evaluated under simulated intestinal environments. These intestinal environments are generated using minimal media and inoculated with FMT aliquots (Example 2) to recreate an environment representative of the intestine. In these environments, the introduction of prebiotics (in the form of carbon sources, nitrogen sources, and other small molecules) and bacteria can be used to shift the community composition, metabolic output, and immunological impact of the intestinal environment. This allows for the rapid evaluation of combinations of prebiotics, probiotics, and synbiotics (prebiotics and probiotics) to confirm or reject observations determined in simpler systems such as single-strain microbial cultures. In subsequent embodiments, machine learning is used to further reduce the space that must be explored experimentally.

[0335] Application of simulated intestinal environment to isolated FMTs and strains The ability of carbon sources to shift the composition of the simulated gut environment was first examined by assessing the growth of C1 samples in the presence of human milk oligosaccharides or infant formula (Figure 25). In the presence of human milk oligosaccharides, the human gut environment maintained a C1 community structure, whereas in the presence of infant formula, the community structure shifted to that of a C3 community, demonstrating a significant effect of diet on the gut microbiome and the simulated gut environment.

[0336] The ability of bacterial addition (representing probiotic application) to shift the composition of the intestinal environment was evaluated by introducing Bifidobacterium infantis isolate PB-STR-093. Bifidobacterium infantis was able to shift the C3 composition toward a C1 composition, accounting for more than 50% of the sample after introduction (Figure 26). This was replicated across multiple FMTs, demonstrating the reproducibility of the method. In addition to the introduction of prebiotics and probiotics alone, the combination of Bifidobacterium infantis with lacto-N-tetraose (a human milk oligosaccharide) was evaluated, which resulted in increased growth and engraftment in the sample (Figure 26).

[0337] Notable among the compositional shifts was a significant reduction in potentially pathogenic bacterial strains (E. coli and S. vestibularis), particularly when growth was stimulated with lacto-N-tetraose compared to glucose (Figure 27). In addition to the compositional changes, the ability to shift metabolism was also demonstrated.

[0338] Based on the results of the MY BABY BIOME™ population-wide analysis, Bifidobacterium combinations were selected for their potential to improve infant health outcomes. Fifteen different combinations covering Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum were evaluated (shown in Table 31, where the number for each combination represents the ratio of microorganisms in the combination). In all cases, approximately the same number of bacteria was introduced into the system. In some combinations, multiple strains of the same species were evaluated, while in other combinations, the relative levels of these strains were varied. These 15 combinations were introduced into 20 unique gut environments (defined as combinations of FMT background and carbon source) covering a range of infant gut archetypes and evaluated for their ability to shift the composition of these backgrounds toward a C1 composition (Figures 29, 30, and 31). The compositions were also evaluated for their ability to reduce pathogenic species and their metabolic output. A sample of metabolic outputs showing the variation of the three metabolites across the different combinations is visualized in Figure 36. The results of this analysis demonstrated that certain combinations were superior in remodeling the microbiome in a simulated gut environment, suggesting that these combinations were worthy of further exploration for their effects on the infant gut microbiome.

[0339] [Table 31]

[0340] Example 9 Laboratory-scale fermentation and formulation of isolated anaerobic microorganisms

[0341] In alternative embodiments, the microorganisms used in the compositions provided herein or used to practice the methods provided herein include the use of isolated anaerobic microorganisms, e.g., anaerobic bacteria isolated from a donor, e.g., a fecal sample.

[0342] Laboratory-scale fermentations are carried out using a Sartorius BIOSTAT A™ bioreactor equipped with a 2 liter (L) vessel, using the growth medium described in Example 1. Still in the anaerobic chamber, 1 L of medium is transferred to a sterile feed bottle, which has two ports with tubing ends blocked by pinch clamps and covered with foil to maintain sterility.

[0343] The fermentation vessel is sterilized by autoclaving and then flushed with sequential purges of sterile, catalytically deoxygenated nitrogen gas. The two inlet ports are attached to tubing leading to connectors blocked with pinch clamps, and the sampling port is attached to tubing leading to a syringe. The vessel is also fitted with a thermowell containing a dissolved oxygen probe, a pH probe, and a temperature probe. Once anaerobic conditions are ensured, the medium is removed from the anaerobic chamber and connected to one of the inlet ports. The other feed bottle port is connected to a sterile nitrogen purge. The pinch clamp is removed, and the medium is transferred to the fermentation vessel by a peristaltic pump or by nitrogen pressure alone. Once the transfer is complete, both lines are resealed with pinch clamps, and the feed bottle is removed and returned to the anaerobic chamber.

[0344] The following genera (any one of which may be used to practice the compositions or methods provided herein): Agathobaculum (Tax ID: 2048137), Alistipes (Tax ID: 239759), Anaeromassilibacillus (Tax ID: 1924093), Anaerostipes (Tax ID: 207244), Asaccharobacter (Tax ID: 553372), Bactero ides(TaxID:816), Barnesiella(TaxID:397864), Bifidobacterium(TaxID:1678), Blautia(TaxID:572511), Butyrici coccus(TaxID:580596), Clostridium(TaxID:1485), Collinsella(TaxID:102106), Coprococcus(TaxID:33042), Dorea (TaxID:189330), Eubacterium(TaxID:1730), Faecalibacterium(TaxID:216851), Fusicatenibacter(TaxID:1407607 ), Gemmiger (TaxID:204475), Gordonibacter (TaxID:644652), Lachnoclostridium (TaxID:1506553), Methanobrevibac ter(TaxID:2172), Parabacteroides(TaxID:375288), Romboutsia(TaxID:1501226), Roseburia(TaxID:841), Ruminoco ccus (TaxID:1263), Erysipelotrichaceae (TaxID:128827), Coprobacillus (TaxID:100883), Erysipelotrichaceae sp.SNUG30099(TaxID:1982626), Erysipelatoclostridium(TaxID:1505663), Acetatifactor(TaxID:142 7378), Adlercreutzia (TaxID:447020), Agathobacter (TaxID:1766253), Anaerotruncus (TaxID:244127),Bariatricus(TaxID:1924081), Butyrivibrio(TaxID:830), Christensenellaceae(TaxID:990719), Clostridiales(TaxID:1868 02), Dialister(TaxID:39948), Drancourtella(TaxID:1903506), Eggerthella(TaxID:84111), Eisenbergiella(TaxID:1432051) , Enterocloster(TaxID:2719313), Enterococcus(TaxID:1350), Intestinibacter(TaxID:1505657), Lachnospira(TaxID:28050 ), Lachnospiraceae (TaxID:186803), Mediterraneibacter (TaxID:2316020), Negativibacillus (TaxID:1980693), Oscillibacte r(TaxID:459786), Phocaeicola(TaxID:909656), Pseudobutyrivibrio(TaxID:46205), Pseudoflavonifractor(TaxID:1017280) , Ruminococcaceae(TaxID:541000), Sellimonas(TaxID:1769710), Solobacterium(TaxID:123375), Terrisporobacter(TaxID:15 A 50 mL seed culture of one or more bacteria from the following groups is grown to mid-logarithmic phase in a sealed culture bottle using the same medium composition as above and transferred to a feed bottle in the anaerobic chamber. The transfer procedure is repeated, this time with the culture to inoculate the fermenter.

[0345] 5 M ammonium hydroxide is prepared in a separate feed bottle. One port is connected to sterile nitrogen and the bottle is purged for 5 minutes to remove all oxygen. The outlet tubing is then blocked with a pinch clamp and attached to the other inlet port in the fermentation vessel. The tubing is then passed through a peristaltic pump head and the pinch clamp is removed. Software built into the Biostat A™ unit is used to control the pump to maintain the pH at 7.0.

[0346] During culture growth, the temperature is maintained at 37°C using a temperature controller and a heating blanket on the vessel. The nitrogen purge is set at 0.5 L / min to maintain anaerobic conditions and positive pressure in the vessel, and the agitation is set at 500 rpm to keep the culture well mixed. Periodic samples are taken using a syringe attached to the sample port. For each sample, the optical density is measured at a wavelength of 600 nm using a spectrophotometer.

[0347] Example 10 Microbial Stability Testing In alternative embodiments, the microorganisms used in the compositions provided herein or used to practice the methods provided herein are selected from the family or genus (or class): Agathobaculum (Tax ID: 2048137), Alistipes (Tax ID: 239759), Anaeromassilibacillus (Tax ID: 1924093), Anaerostipes (Tax ID: 207244), Asaccharobacter r(TaxID:553372), Bacteroides(TaxID:816), Barnesiella(TaxID:397864), Bifidobacterium(TaxID:1678), Blautia(Tax ID:572511), Butyricicoccus(TaxID:580596), Clostridium(TaxID:1485), Collinsella(TaxID:102106), Coprococcus(Tax ID:33042), Dorea(TaxID:189330), Eubacterium(TaxID:1730), Faecalibacterium(TaxID:216851), Fusicatenibacter(Ta xID:1407607), Gemmiger(TaxID:204475), Gordonibacter(TaxID:644652), Lachnoclostridium(TaxID:1506553), Methanob revibacter(TaxID:2172), Parabacteroides(TaxID:375288), Romboutsia(TaxID:1501226), Roseburia(TaxID:841), Rumi nococcus (TaxID:1263), Erysipelotrichaceae (TaxID:128827), Coprobacillus (TaxID:100883), Erysipelotrichaceae sp.SNUG30099(TaxID:1982626), Erysipelatoclostridium(TaxID:1505663), Acetatifactor(TaxID:1427378), Adlercreutzia(TaxID:447020), Agat hobacter(TaxID:1766253), Anaerotruncus(TaxID:244127), Bariatricus(TaxID:1924081), Butyrivibrio(TaxID:830), Christensenellaceae(Ta xID:990719), Clostridiales(TaxID:186802), Dialister(TaxID:39948), Drancourtella(TaxID:1903506), Eggerthella(TaxID:84111), Eisenber giella (TaxID:1432051), Enterocloster (TaxID:2719313), Enterococcus (TaxID:1350), Intestinibacter (TaxID:1505657), Lachnospira (TaxID: 28050), Lachnospiraceae(TaxID:186803), Mediterraneibacter(TaxID:2316020), Negativibacillus(TaxID:1980693), Oscillibacter(TaxID:4 59786), Phocaeicola(TaxID:909656), Pseudobutyrivibrio(TaxID:46205), Pseudoflavonifractor(TaxID:1017280), Ruminococcaceae(TaxID:54 1000), Sellimonas (Tax ID: 1769710), Solobacterium (Tax ID: 123375), Terrisporobacter (Tax ID: 1505652), Tidjanibacter (Tax ID: 1929083), Veillonella (Tax ID: 29465), Lacticaseibacillus (Tax ID: 2759736), Limosilactobacillus (Tax ID: 2742598), or a combination thereof.

[0348] In alternative embodiments, any microorganism used in the compositions or methods provided herein, including, for example, those listed above, can be stored in a sealed container at, for example, 25° C. or 4° C., and the container can be placed in an atmosphere having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% relative humidity, or about 20% to 99% relative humidity. In alternative embodiments, after 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years, or 3 years, at least 50%, 60%, 70%, 80%, or 90% of the bacterial strain should remain, as measured by colony-forming units (CFUs) determined by standard protocols.

[0349] Example 11 Production of live biotherapeutics for evaluation of immunological effects in vivo In alternative embodiments, the microorganisms provided herein (including bacteria from all genera listed herein), e.g., the combinations of microorganisms provided herein, e.g., the exemplary combinations listed in Example 4 or Example 5, comprise anaerobic bacteria, e.g., anaerobic bacteria isolated from a fecal sample, cultured anaerobic bacteria, or a combination thereof.

[0350] Individual culture of anaerobic microorganisms for mouse studies Anaerobic microorganisms of interest are cultured in multiples of 1 liter volumes in anaerobic media bottles as follows: Microorganisms in cryogenic storage are plated on appropriate anaerobic solid media, allowed to root, and then cultured at 37°C to obtain isolated colonies. For each microorganism, a single colony is inoculated into a Hungate tube containing 10 ml of the appropriate anaerobic growth media and grown at 37°C until turbid, creating a starter culture. For each microorganism of interest, multiple 0.9 liter volumes of the appropriate liquid anaerobic media (as described in Example 1) in 1 L anaerobic bottles are inoculated with a needle and syringe, each containing 2 ml of starter culture. The number of 1 liter cultures for each microorganism depends on the final amount of live bacterial mass required for formulation into a live biotherapeutic for mouse studies. The inoculated bottles are placed upright on a platform shaker at 115 rpm at 37°C for 48 hours or until growth turbidity becomes apparent. Growth density is monitored by taking 1 ml samples over the course of the culture for optical density measurements at 600 nm. An optical density of 1.0 to 4.0 can be obtained after 48 hours, depending on the microorganism being cultured. Prior to large-scale cultivation, cell density is empirically determined for each microorganism by dilution plating and colony counting to determine colony-forming units (CFU) per ml at an optical density of 1.0.

[0351] Large-scale cultures were grown for 10 8 ~10 9After reaching a final viable density of CFU / ml, the culture bottles are then placed in an anaerobic chamber for viable cell mass harvest. Once in the chamber, the aluminum collars and butyl rubber stoppers are removed, and the 1 liter contents of each culture bottle are poured into two 500 ml centrifuge bottles equipped with screw caps with rubber gaskets. After decanting the growth medium, the centrifuge bottles are capped with airtight seals, removed from the anaerobic chamber, and centrifuged at 6000 g for 20 minutes at 4°C. The centrifuged bottles are then placed in the anaerobic chamber, uncapped, and the supernatant poured off and discarded. The remaining cell pellet is then combined with 250 ml of ice-cold vehicle buffer (phosphate-buffered saline + 1 g / L L-cysteine ​​+ 15% glycerol, filter-sterilized and made anaerobic by bubbling with filtered nitrogen). The cell pellets are carefully resuspended in vehicle buffer on ice, and the resuspended volumes of the two pellets are combined into one 500 ml bottle, recapped for an airtight seal, removed from the anaerobic chamber, and then centrifuged at 6000 g for 20 minutes at 4° C. After decanting the supernatant in the anaerobic chamber, the resulting cell pellets are then carefully resuspended once more in 250 ml of ice-cold vehicle buffer in the anaerobic chamber, removed from the anaerobic chamber, and then centrifuged at 6000 g for 20 minutes at 4° C. After removal of the supernatant in the anaerobic chamber, each pellet is resuspended in 100 ml of ice-cold vehicle buffer to establish a concentration 10 times the original culture cell density.

[0352] In the anaerobic chamber, the final resuspended cell pellet volumes for the anaerobic microorganisms of interest are combined in a sterile bottle and thoroughly mixed by gentle stirring on a stir plate on ice. The volumes are then dispensed into 25 ml aliquots in 50 ml conical tubes using a serological pipette, and a stream of sterile filtered argon gas is then introduced into each tube to displace the headspace and act as an oxygen barrier. Each tube is then tightly capped and the seal is wrapped with several layers of parafilm. The tubes are then placed upright in a rack, removed from the anaerobic chamber, and slowly frozen at -80°C. Smaller 5 ml aliquots are also made for each preparation and stored as described above. After 18 hours, 5 ml aliquots for each microbial strain of interest are removed and thawed upright in ice water within the anaerobic chamber. The thawed volume is mixed gently by inversion several times and then subjected to dilution plating on an appropriate solid anaerobic medium to determine the viable cell density (CFU / ml) after freezer storage.

[0353] Live Biotherapeutic Assembly for Mouse Studies A live biotherapeutic composition of the anaerobic microorganism of interest, including a combination of microorganisms provided herein, such as the exemplary combinations described in Example 4 or Example 5, is assembled in the appropriate volume for the planned mouse study. Sufficient aliquots for each microorganism of interest are removed from storage at -80°C and gently thawed in ice water in an anaerobic chamber. The thawed aliquots are combined in a sterile bottle, gently mixed, and then placed on ice. The volume of each microorganism added to the mix is ​​adjusted so that the determined viable cell density for each microorganism is equal, and the final total cell density can be adjusted by further adding ice-cold vehicle buffer. Once all the required volumes for each microorganism are added together in a larger sterile bottle, the volumes are gently mixed by stirring on a stir plate on ice.

[0354] The live biotherapeutic volume is then re-aliquoted into individual volumes each containing the planned daily dose of live bacteria in the prospective mouse study. The determined volume is dispensed into 15 ml conical tubes, up to 10 ml per aliquot. The volume in each tube is blanketed with a stream of sterile filtered argon to displace oxygen, followed by capping. The live biotherapeutic aliquot tubes are placed upright in a rack and slowly frozen at -80°C. After 48 hours, one aliquot from each microbial mix preparation is thawed and dilution plated to optimally obtain a 1.0 x 10 9 Verify the final total CFU / ml is higher than the CFU / ml.

[0355] Expanded production of stocks Strains PB-STR-093, PB-STR-083, PB-STR-119, and PB-STR-207 were produced at a larger scale, first at 1 liter and then at 7 liters, to generate sufficient material for further evaluation. All strains grew successfully in minimal medium and demonstrated maintained viability after freeze-drying. Examples of additives used in freeze-drying include, but are not limited to, acacia, alginate, alginic acid, aluminum acetate, benzyl alcohol, butylparaben, butylated hydroxytoluene, citric acid, calcium carbonate, candelilla wax, croscarmellose sodium, powdered sugar, colloidal silicon dioxide, cellulose, simple or anhydrous calcium phosphate, carnauba wax, corn starch, carboxymethylcellulose calcium, calcium stearate, calcium EDTA disodium, copolyvidone, calcium hydrogen phosphate dihydrate, cetylpyridine chloride, cysteine ​​HCl, crosprovidone, di- or tribasic calcium phosphate, dibasic calcium phosphate, disodium hydrogen phosphate, dimethicone, sodium erythrosinate, ethylcellulose, gelatin, glyceryl monooleate, glycerin, glycine, glyceryl monostearate, glyceryl behenate, hydroxypropyl cellulose, Ingredients: hydroxypropyl methylcellulose, hypromellose, HPMC phthalate, inulin, iron oxide or ferric oxide, yellow iron oxide, red iron oxide or ferric oxide, hydrous or anhydrous or monohydrate or spray-dried lactose, magnesium stearate, maltodextrin, microcrystalline cellulose, mannitol, methylcellulose, magnesium carbonate, mineral oil, methacrylic acid copolymer, magnesium oxide, methylparaben, providone or PVP, PEG, polysorbate 80, propylene glycol, polyethylene oxide, propylene paraben, poloxamer 407 or 188, potassium bicarbonate, potassium sorbate, potato starch, phosphoric acid, polyoxyethylene 140 stearate, sodium starch glycolate, pregelatinized starch, carmellose sodium, sodium lauryl sulfate, starch, silicon dioxide, sodium benzoate, stearic acid, sucrose, sorbic acid, sodium carbonate, sodium saccharinThe additives included sodium alginate, silica gel, sorbitan monooleate, sodium stearyl fumarate, sodium chloride, sodium metabisulfite, sodium citrate dihydrate, sodium starch, sodium carboxymethylcellulose, succinic acid, sodium propionate, titanium dioxide, talc, triacetin, and triethyl citrate. After freeze-drying, the isolated cells were evaluated for purity and quality, and viability was assessed as both colony-forming units (CFU) and active fluorescent units (AFU) (Figure 43). CFU and AFU were closely matched for each strain, and both were suggested as valid measures of viability.

[0356] Example 12 Demonstration of immunological effects in vivo Microorganisms in Mouse Studies The set of microorganisms to be administered is selected from those described in Example 4 or Example 5. Each microorganism is isolated from a healthy donor as described in Example 3. After assembly of the consortium as described in Example 11, PBS-CG is added to each live biotherapeutic to bring the total cell density of each live biotherapeutic to 1 x 10 7 / 0.2ml~1×10 12 The live biotherapeutic is aliquoted in single-use volumes into 15 ml conical tubes and stored at -20°C until needed.

[0357] animal Obtain BALB / c mice from SHANGHAI LINGCHANG BIOTECHNOLOGY CO., LTD™ (Shanghai, China), JACKSON LABORATORY™, or another mouse facility. Use 6- to 8-week-old female mice. To prepare mice for fecal microbiota transplantation, treat them daily with 200 µL of antibiotic solution via oral gavage for a period of 1 to 2 weeks. The antibiotic solution consisted of ampicillin (1 mg / mL) (Alfa Aesar J6380706), gentamicin (1 mg / mL) (Acros Organics AC455310050), metronidazole (1 mg / mL) (Acros Organics AC210440050), neomycin (1 mg / mL) (Alfa Aesar AAJ6149922), and vancomycin (0.5 mg / mL) (Alfa Aesar J6279006) administered via oral gavage. Animals were given a 24-hour rest period between antibiotic pretreatment and treatment phases to allow the antibiotics to pass through their systems, and cages were changed before inoculation with fecal microbiota transplant.

[0358] Fecal microbiota transplantation (FMT) Fecal microbiota transplantation (FMT) of human gut microbiomes into antibiotic-treated mice is a method for standardizing microbiome composition. To evaluate the impact of probiotic treatment on the immune system in vivo, mice are divided into two groups and treated with microbiomes representing a robust infant microbial composition (C1 from Example 3) or a deficient infant microbial composition (C3 as described in Example 3). This not only standardizes the mouse microbiome but also conditions them into two diverse immunological states (as shown in Example 5). After antibiotic pretreatment, colonization is performed by oral gavage with 200 μl of a suspension obtained by homogenizing fecal samples in PBS. Mouse fecal samples are collected one to two times during this period to allow for evaluation of the efficacy of FMT. A 5-7 day rest period is allowed after FMT before probiotic treatment.

[0359] Probiotic treatment Mice with each microbiome (C1 and C3) are randomized and divided into two groups: one control group and one treatment group. Mice are marked by ear tagging. Mice in the treatment group are treated with the microbial mix (10 per dose). 7 ~10 12 Mice are treated with oral gavage of 200 ul of colony forming units (CFU), and a control group of mice is treated with 200 ul of vehicle control. Treatment continues for 3 weeks or longer. Doses are administered at least twice a week and up to daily. Stool is collected at the time of inoculation and at least twice a week until the end of the study.

[0360] Peripheral blood extraction and processing Whole blood is obtained via cardiac puncture at the end of the experiment or via tail bleeding during the experiment and collected in EDTA tubes. Plasma is isolated from an aliquot of whole blood by centrifugation at 1500 × g for 10 minutes, and the supernatant is obtained. A second centrifugation is performed to remove any remaining blood cells. Peripheral blood mononuclear cells (PBMCs) are isolated from the blood using a standard kit and stored at 1 × 10 cells until use. 6 The PBMCs are stored in liquid nitrogen at 1000x the cell count / mL. Prior to storage, the PBMCs can be processed using flow sorting or antibody spin separation kits to select for certain purified lymphocyte subpopulations, such as T cells.

[0361] GI tract extraction and analysis After euthanasia at the end of the study, the intact digestive tract of each mouse, from the stomach to the rectum, was removed and placed in a 5 ml Eppendorf tube on ice before dissection. Forceps were sterilized by immersion in 100% ethanol and then used to remove a length of intestine and stretch it over a cellophane-covered work surface. Using ethanol-sterilized dissection scissors, 3 cm lengths of the jejunum closest to the stomach and the ilium closest to the cecum / large intestine were removed and then placed in 1.5 ml Eppendorf tubes with forceps and placed on ice. A 2 cm segment of the cecum / ascending colon was then removed, as were 2 cm segments of the transverse colon and descending colon, all placed in 1.5 ml Eppendorf tubes on ice. Dissection instruments were sterilized by immersion in 100% ethanol between removal of each intestinal segment. To each tube containing the dissected intestinal segments, 0.5 ml of ice-cold PBS buffer is added. A plastic pestle is used to apply pressure and massage the intestinal segments in each tube to expel ruminal matter, which is then removed with a pipette and placed into a new Eppendorf tube. The tubes containing the expelled ruminal matter from each intestinal segment are immediately placed on dry ice and then stored at -80°C for later analysis. The remaining intestinal tissue is then rinsed twice by adding and then removing 0.5 ml of ice-cold PBS. The rinsed intestinal segment tissue is then frozen on dry ice and then stored at -80°C for later analysis.

[0362] Analysis of dendritic cell subsets Cell suspensions from mouse spleens and lymph nodes are prepared by digestion with collagenase and Dnase for 60 minutes, then strained through a 70 mm mesh. Colonic and small intestinal lymphocytes are isolated as previously described (Viaud, S. et al. Science 80(342): 971-976 (2013)). Briefly, the cecum, colon, and small intestine are digested with shaking at 37°C in PBS containing 5 mM EDTA and 2 mM DTT. A plastic pestle is used to apply pressure and massage the intestinal segments in each tube to expel ruminal material, which is then removed by pipette and placed in a new Eppendorf tube. The tubes containing the expelled ruminal material from each intestinal segment are immediately placed on dry ice and then stored at -80°C for later analysis. The remaining intestinal tissue is then rinsed twice by adding and then removing 0.5 ml of ice-cold PBS. The rinsed intestinal fragment tissue is then frozen on dry ice in RNALATER™ (Thermo Fisher Scientific) and then stored at -80°C for later analysis.

[0363] After the initial digestion, colon and small intestine tissue fragments were digested in collagenase / DNase-containing RPMI medium for 30 minutes. The tissue fragments were further strained through a 70 mm mesh. For flow cytometry analysis, the cell suspension was stained with antibodies against the following surface markers: CD11c (N418), CD11b (M1 / 70), Ly6c (HK1.4), MHC class II (M5 / 114.15.2), CD24 (M1 / 69), CD64 (X54-5 / 7.1), CD317 (ebio927), CD45 (30-F11), F4 / 80 (C1:A3-1), and CD8α (53-6.7). DAPI was used to exclude dead cells. Antibodies were purchased from EBIOSCIENCES, BD BIOSCIENCES™, or BIOLEGEND™, respectively. Cell populations are gated as follows: small intestine (mobile fraction): CD103+ DC (CD45+CD11c+MHC-II+CD103+CD24+), CD11b+CD103+ (CD45+CD11c+MHC-II+CD103+CD11b+CD24+), CD11b+ (CD45+CD11c+MHC-II+CD11b+CD24+), inflammatory DC(CD45+CD11c+MHC-II+CD11b+CD64+Ly6c+), large intestine:CD103+DC(CD45+CD11c+MHC-II+CD103+CD24+) , CD11b+ (CD45+CD11c+MHC-II+CD11b+CD24+), inflammatory DC (CD45+CD11c+MHC-II+CD11b+CD64+Ly6c+).

[0364] Whole genome sequencing Fecal gDNA is extracted for whole genome sequencing (WGS). The experimental method for DNA extraction and library preparation is performed using a protocol modeled after the Human Microbiome Project (Lloyd-Price et al. (2017) Nature 550(7674):61-66) and validated using samples from healthy volunteers. Sequencing is performed by an external service provider using HISEQ-X® (Illumina) with 2 x 150 bp paired-end reads, providing approximately 4 million reads per sample. Analysis software, such as Centrifuge (Kim, D., et al., Centrifuge: rapid and sensitive classification of metagenomic sequences. Genome Res, 2016. 26(12): p. 1721-1729), is used to align sequence reads to the reference genome and obtain species and strain-level identification.

[0365] Metabolomics Metabolites are extracted from fecal material or plasma using methanol under vigorous shaking for 2 minutes (GENOGRINDER 2000™ (Glen Mills)) to precipitate proteins and dissociate small molecules bound to proteins or trapped in the precipitated protein matrix, followed by centrifugation to recover chemically diverse metabolites. The resulting extracts are evaluated by targeted metabolomics as described in Example 4 or by non-targeted metabolomics. For targeted metabolomics, samples are placed on a TURBOVAP® (Zymark) to remove organic solvents before evaluation. Compounds are identified by comparison with known standards using relevant calibration curves. Absolute quantification is achieved by the use of isotopically labeled internal standards. For untargeted metabolomics, samples are loaded onto a TURBOVAP® (Zymark) column to remove organic solvents and then evaluated by one of the following methods: reversed-phase (RP) / UPLC-MS / MS using electrospray ionization (ESI) in positive ion mode, RP / UPLC-MS / MS using ESI in negative ion mode, HILIC / UPLC-MS / MS using ESI in negative ion mode, or HILIC / UPLC-MS / MS using ESI in positive ion mode. Compounds are identified by comparison with library entries of purified standards containing retention time / index (RI), mass-to-charge ratio (m / z), and chromatographic data (including MS / MS spectral data) for all molecules present in the library. Furthermore, biochemical identification is based on three criteria: retention index within a proposed, specified, narrow RI window, accurate mass matching to the library + / - 10 ppm, and MS / MS forward and reverse scores. MS / MS scores are based on comparison of ions present in the experimental spectrum with those present in the library entry spectra. Although there may be similarities between these molecules based on one of these factors, the use of all three data points can be utilized to distinguish and identify the biochemicals. Peaks are quantified as area under the curve detector ion counts.

[0366] Immunophenotyping assays Whole blood immune profiling is used to assess T cell activation in response to microbial treatment. In some experiments, immunophenotyping is also performed on tissue obtained from the GI tract. For flow cytometry analysis, 1 mL of RBC lysis buffer is added to 0.1 mL of whole blood or homogenized tissue and allowed to incubate at room temperature for 10 minutes. Lysis is quenched by adding 10 mL of cold DPBS. The sample is centrifuged at 1500 rpm for 5 minutes at 4°C. The pellet is aspirated and resuspended in another 10 mL of cold DPBS. The sample is centrifuged at 1500 rpm for 5 minutes at 4°C. The sample is resuspended in 500 μL of FACS buffer and transferred to a 96-well plate. Samples are stained with Fixable Viability ef780™ (eBioscience), CD45-Pecy7 (BioLegend), CD3-BV605™ (BioLegend), CD8-AF700™ (BioLegend), and CD4-AF488™ (BioLegend). Stained samples are run on a BD LSRFortessa™ flow cytometer, and analysis is performed using FLOWJO™ (Tree Star).

[0367] Alternatively, CyTOF® is applied to characterize the immune profile of PBMCs. This work, performed by the Bioanalytical and Single-Cell Facility at the University of Texas, San Antonio, involves a comprehensive panel of 29 different immune markers, allowing for deep interrogation of cell phenotype and function. To complement these results, RNA sequencing is applied to whole populations of PBMCs, sorted populations, and even single cells. Single-cell RNA sequencing is applied using methods developed by 10X GENOMICS™. Finally, cytokine levels are determined using assays such as the HUMAN CYTOKINE 30-PLEX LUMINEX™ assay.

[0368] Example 13 Observational and Interventional Clinical Studies of Colorectal Cancer Risk Stool and blood samples are collected from cancer patients and healthy individuals classified as high or low risk for colorectal cancer (CRC) based on family history, previous CRC, or previous colonoscopy findings. Specifically, high-risk subjects are those who meet one of the following criteria: 1. Family history of CRC (one or more first-degree relatives) OR 2. One or more of the following findings during a previous colonoscopy: Adenomas larger than 10 mm (>10 mm) Adenoma with ductal / villous histology Adenoma with high-grade dysplasia CRC Three or more non-advanced adenomas, regardless of size, histology, or dysplasia, found during a single screening or surveillance visit

[0369] Subjects who meet the entry criteria will provide a baseline stool sample and up to four samples over a two-year period. Stool sample collection will be performed as described in Example 2. Blood samples will also be collected from some participants. Electronic health records will be obtained, and patient lifestyle questionnaires will be administered at various times over an additional three to six years. In some cases, additional samples will be collected in conjunction with significant clinical events, such as another standard-of-care colonoscopy or CRC recurrence with or without surgical intervention.

[0370] A subset of high-risk subjects will receive an intervention to determine the effect of nutritional counseling or probiotic supplementation on microbiome composition and function. These subjects will be assigned to cohorts as follows:

[0371] Cohort 1 was the no-intervention control group.

[0372] Cohort 2 participants will receive six telehealth visits with a dietitian to provide dietary guidance. The first visit will be within three weeks of providing a stool sample, followed by monthly visits. The dietitian will work with participants to design an optimized meal plan to improve microbiome health and meet their overall health goals.

[0373] Participants in Cohort 3 will be given a probiotic supplement daily for the duration of the study, starting within 3 weeks of their first stool sample.

[0374] Participants in Cohort 4 will receive both daily probiotics and dietary counseling.

[0375] Stool samples will be collected at 3 and 6 months post-intervention. Metagenomics, metabolomics, and cytokine analysis will be performed as described in Example 3.

[0376] Example 14 Exemplary Methods for Collection and Analysis of Stool from Mothers and Infants In a clinical study focused on maternal health, stool samples are collected from pregnant women during the third trimester of pregnancy. Samples are then collected from infants 4-10 weeks after birth. Mothers provide demographic, dietary, and lifestyle information, and delivery and breastfeeding methods are documented. Antibiotic or probiotic use by either the mother or infant is also captured. Periodic surveillance is conducted for up to seven years to capture health information as the babies grow. Subsequent stool samples are also collected from some of the babies. Some mothers provide breast milk samples. Samples are processed as described in Example 2. Metagenomics, metabolomics, and proteomics are performed as described in Example 3 to assess multigenerational transitions of gut microbes.

[0377] Example 15 Exemplary Methods of Treating Infants with Live Biotherapeutics (or Probiotics) for the Treatment and Prevention of Dysbiosis that Can Lead to Disease This example describes the administration of a live biotherapeutic (or probiotic) provided herein, including, in alternative embodiments, one bacterium and a probiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively, a bacterial combination or mix (or consortium) provided herein, e.g., those shown in Table 2, Example 4, or Table 30, Example 5, and / or a prebiotic provided herein, including a prebiotic combination provided herein, e.g., those shown in Table 3, Example 4, to an infant or newborn in need thereof.

[0378] Infant intestines are dysbiotic or at risk of developing dysbiosis. Infant dysbiosis can be defined as, but not limited to, infants with the bacterial composition described in Example 3, infants with a predominant HMO consumer that is not Bifidobacterium, infants whose intestinal metabolism and immunological status are substantially different from those of intestines dominated by Bifidobacterium, infants in which predominant Bifidobacterium is rarely found in the dataset described in Example 3, and infants with a microbiome composition associated with later-life diseases such as asthma, allergies, obesity, and diabetes. To address dysbiosis, infants are administered a live biotherapeutic composition, i.e., a formulation consisting of some combination of the microorganisms outlined in Example 4 or Example 5, either alone or in combination with prebiotics or supplements outlined in Example 4.

[0379] In alternative embodiments, each or one of the microorganisms used in the bacterial combination is either (at least initially) isolated from a healthy donor(s) as described in Example 5, or is a genetically modified or cultured derivative as described in Example 21.

[0380] In an alternative embodiment, the patient receives5 ~10 15 The composition, formulation, or pharmaceutical preparation (e.g., probiotic) is administered at a dose of bacteria once, twice, three times, or more frequently per day. The administration can be in a lyophilized form, i.e., a freeze-dried powder, given orally to the infant directly, through a dropper, or through a bottle.

[0381] In another embodiment, the infant may be medicated with the composition, formulation or pharmaceutical preparation (eg, probiotics) before, during, and / or immediately after feeding.

[0382] In another embodiment, dosing of the composition, formulation or pharmaceutical preparation (eg, probiotic) continues for one month, six months, one year, or longer.

[0383] In an alternative embodiment, the composition of the infant's gut microbiome, as well as the metabolic and immunological status of the infant's gut, are used as measures of treatment success.

[0384] A non-intensive, placebo-controlled study of 20 infants was used to evaluate the probiotic combinations outlined in Example 4 or Example 5. Stool samples were collected at multiple time points (as outlined in Example 2) and evaluated for stability of Bifidobacterium populations, the effect of Bifidobacterium on gut metabolism, and reduction of pathogenic or undesirable microbial content.

[0385] Example 16 Methods for treating infants after disruption of the microbiome due to cesarean delivery or antibiotic use This example describes the administration of a live biotherapeutic (or probiotic) provided herein, including, in alternative embodiments, one bacterium and a probiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively, a bacterial combination or mix (or consortium) provided herein, e.g., those shown in Table 2, Example 4, or Table 30, Example 5, and / or a prebiotic provided herein, including a prebiotic combination provided herein, e.g., those shown in Table 3, Example 4, to an infant or newborn in need thereof.

[0386] Infants undergo events such as cesarean section or antibiotic use that can result in disruption of the microbiome. Microbiome disruption in infants can be defined as, but is not limited to, a reduction in Bifidobacterium, altered gut metabolism and immunological status, an increase in pathogens or undesirable microorganisms, or altered microbiome composition, such as those associated with diseases later in life, such as asthma, allergies, obesity, and diabetes. To address this disruption, infants are administered a live biotherapeutic composition, i.e., a formulation consisting of some combination of the microorganisms outlined in Example 4 or Example 5, either alone or in combination with prebiotics or supplements outlined in Example 4.

[0387] In alternative embodiments, each or one of the microorganisms used in the bacterial combination is either (at least initially) isolated from a healthy donor(s) as described in Example 5, or is a genetically modified or cultured derivative as described in Example 21.

[0388] In an alternative embodiment, the patient receives 5 ~10 15The composition, formulation, or pharmaceutical preparation (e.g., probiotic) is administered at a dose of bacteria once, twice, three times, or more frequently per day. The administration can be in a lyophilized form, i.e., a freeze-dried powder, given orally to the infant directly, through a dropper, or through a bottle. In another embodiment, the infant may be medicated with the composition, formulation or pharmaceutical preparation (eg, probiotics) before, during, and / or immediately after feeding. In another embodiment, dosing of the composition, formulation or pharmaceutical preparation (eg, probiotic) continues for one month, six months, one year, or longer. In an alternative embodiment, the composition of the infant's gut microbiome, as well as the metabolic and immunological status of the infant's gut, are used as measures of treatment success.

[0389] A non-intensive, placebo-controlled study of 20 infants was used to evaluate the probiotic combinations outlined in Example 4 or Example 5. Stool samples were collected at multiple time points (as outlined in Example 2) and evaluated for stability of Bifidobacterium populations, the effect of Bifidobacterium on gut metabolism, and reduction of pathogenic or undesirable microbial content.

[0390] Example 16 Methods of treating infants suffering from immune-related disorders This example describes the administration of a live biotherapeutic (or probiotic) provided herein, including, in alternative embodiments, one bacterium and a probiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively, a bacterial combination or mix (or consortium) provided herein, e.g., those shown in Table 2, Example 4, or Table 30, Example 5, and / or a prebiotic provided herein, including a prebiotic combination provided herein, e.g., those shown in Table 3, Example 4, to an infant or newborn in need thereof.

[0391] The infant is beginning to exhibit symptoms of an immune-related disorder, such as the development of allergies, dermatitis, asthma, or obesity. The infant is administered a live biotherapeutic composition, i.e., a formulation consisting of some combination of the microorganisms outlined in Example 4 or Example 5, either alone or in combination with a prebiotic or supplement outlined in Example 4, to address the immune disorder.

[0392] In alternative embodiments, each or one of the microorganisms used in the bacterial combination is either (at least initially) isolated from a healthy donor(s) as described in Example 5, or is a genetically modified or cultured derivative as described in Example 21.

[0393] In an alternative embodiment, the patient receives 5 ~10 15 The composition, formulation, or pharmaceutical preparation (e.g., probiotic) is administered at a dose of bacteria once, twice, three times, or more frequently per day. The administration can be in a lyophilized form, i.e., a freeze-dried powder, given orally to the infant directly, through a dropper, or through a bottle. In another embodiment, the infant may be medicated with the composition, formulation or pharmaceutical preparation (eg, probiotics) before, during, and / or immediately after feeding. In another embodiment, dosing of the composition, formulation or pharmaceutical preparation (eg, probiotic) continues for one month, six months, one year, or longer. In an alternative embodiment, the composition of the infant's gut microbiome, as well as the metabolic and immunological status of the infant's gut, are used as measures of treatment success. In an alternative embodiment, remission of the immune disorder or inhibition of its progression is used as a measure of successful treatment.

[0394] Example 16 Methods of treating infants based on fecal biomarkers This example describes the successful therapeutic administration to an infant or newborn in need thereof of a live biotherapeutic (or probiotic) provided herein, which in alternative embodiments includes one bacterium and a probiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively includes a combination or mix or consortium of bacteria provided herein, e.g., those shown in Table 1 or Table 4, or a live biotherapeutic (also called probiotic) composition or combination of bacteria shown in Table 2 or Table 30, and / or at least one prebiotic provided herein, including one or a combination of prebiotics provided herein, e.g., those shown in Table 3; wherein in alternative embodiments, the administration treats or ameliorate dysbiosis in the infant or newborn, thereby increasing the infant's ability to develop or resist disease or infection, as needed.

[0395] In an alternative embodiment, the compositions, formulations, and pharmaceutical compositions provided herein, as well as the methods provided herein, are used to treat infants with dysbiosis. Events that predispose infants to dysbiosis include, but are not limited to, preterm birth, extended stay in a neonatal intensive care unit, antibiotic treatment, maternal antibiotic treatment before birth, cesarean section delivery, formula feeding, and known dysbiosis in the mother. To verify dysbiosis, infant stool is sampled as described in Example 2 and evaluated using methods equivalent to those described in Example 3. Upon indication of dysbiosis based on metabolic, immunological, and microbial biomarkers in infant stool as defined in Example 3, the infant is administered a live biotherapeutic composition, i.e., a formulation consisting of some combination of the microorganisms outlined in Example 4 or Example 5, either alone or in combination with prebiotics or supplements as outlined in Example 4, to address the dysbiosis.

[0396] In alternative embodiments, each or one of the microorganisms used in the bacterial combination is (at least initially) isolated from a healthy donor(s) as described in Example 5, or is a genetically modified derivative as described in Example 21, or is a cultured derivative of either.

[0397] In an alternative embodiment, the patient receives 5 ~10 15 The synbiotic is administered in a dose of bacteria once, twice, three times, or more frequently per day. The medication can be in a lyophilized form, i.e., a freeze-dried powder, given orally to the infant directly, through a dropper, or through a bottle.

[0398] In another embodiment, the infant may be medicated with probiotics before, during, and / or immediately after breastfeeding.

[0399] In another embodiment, the probiotic dosage continues for one month, six months, one year, or longer. In an alternative embodiment, the composition of the infant's gut microbiome, as well as the metabolic and immunological status of the infant's gut, are used as measures of treatment success.

[0400] Example 17 Exemplary Methods of Treating Children with Live Biotherapeutics (or Probiotics) for the Treatment and Prevention of Dysbiosis, Which May Lead to Disease or Reduce Treatment Effectiveness This example describes the successful therapeutic administration of a live biotherapeutic (or probiotic) provided herein, including, in alternative embodiments, one bacterium and a probiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively, a bacterial combination or mix or consortium provided herein, e.g., those shown in Table 2, Example 4, or Table 30, Example 5, and / or the administration of a prebiotic provided herein, including a prebiotic combination provided herein, e.g., those shown in Table 3, Example 4, to a child in need thereof.

[0401] In alternative embodiments, the compositions, formulations, and pharmaceutical compositions provided herein, and methods provided herein, are used to treat dysbiosis in children, which can be defined, but not limited to, as a microbial population associated with high levels of pathogenic bacteria, high levels of antibiotic resistance, a skewed metabolic balance that differs from that of a healthy population, a skewed immunological state that differs from that of a healthy population, a loss of metabolic function associated with a healthy population, an increase in bacteria associated with a particular disease state, or insufficient therapeutic efficacy.

[0402] In alternative embodiments, the compositions, formulations, and pharmaceutical compositions provided herein, and methods provided herein, are administered to treat or ameliorate a disease state associated with dysbiosis, including, but not limited to, cancer, diabetes, obesity, allergies, dermatitis, asthma, gout, Alzheimer's disease, and Parkinson's disease.

[0403] In an alternative embodiment, a child is administered a live biotherapeutic composition (or probiotic), e.g., a pharmaceutical composition or formulation, comprising or consisting of one bacterium and a prebiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively, comprising one or a combination mix or consortium of the microorganisms outlined in Tables 1, 2, 4, and 30, alone or in combination with a prebiotic or supplement (e.g., those outlined in Table 3), to address dysbiosis and to reduce the risk of disease or correct a lack of therapeutic efficacy.

[0404] In alternative embodiments, each or one of the microorganisms used in the bacterial combination is (at least initially) isolated from a healthy donor(s) as described in Example 5, or is a genetically modified derivative as described in Example 21, or is a cultured derivative of either.

[0405] In an alternative embodiment, the patient receives about 10 5 ~10 15 at a dose of bacteria, or about 10 10 , 10 11 Or 10 12 A live biotherapeutic is administered at a dose of bacteria, which may be, for example, in lyophilized form or formulated in an enteric-coated capsule. In alternative embodiments, the patient takes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more live biotherapeutic capsules (e.g., by mouth or suppository) once, twice, or three times or more per day, and the patient can resume a normal diet after about 1, 2, 4, 8, 12, or 24 hours, or longer. In another embodiment, the patient may take the live biotherapeutic capsule by mouth before, during, and / or immediately after a meal.

[0406] In another embodiment, the patient is given a course of antibiotics prior to treatment, for example, 1 to 7 days, or about 1 to 2 weeks, before the first dose of live biotherapeutic (e.g., as a capsule), or 3 weeks, or 4 weeks, or up to 6 months before the first dose of live biotherapeutic.

[0407] In another embodiment, dosing of the live biotherapeutic (or probiotic) capsule is continued for about 1 month, 6 months, 1 year, or longer, or for about 1 week to 2 years after the end of treatment or therapy. In an alternative embodiment, the composition of the child's gut microbiome, as well as the metabolic and immunological state of the child's gut, are used as measures of treatment success. In an alternative embodiment, restored efficacy of a therapeutic agent (such as a drug, eg, a cancer therapeutic agent) is used as a measure of treatment success.

[0408] Example 17 Exemplary Methods of Treating Adults with Live Biotherapeutics (or Probiotics) for the Treatment and Prevention of Dysbiosis, Which May Lead to Disease or Reduce Therapeutic Effectiveness This example describes the successful therapeutic administration of a live biotherapeutic (or probiotic) provided herein, including, in alternative embodiments, one bacterium and a probiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively, a combination or mix or consortium of bacteria provided herein, e.g., those shown in Table 2, Example 4, or Table 30, Example 5, and / or the administration of a prebiotic provided herein, including a combination of prebiotics provided herein, e.g., those shown in Table 3, Example 4, to an individual in need thereof.

[0409] In alternative embodiments, the compositions, formulations, and pharmaceutical compositions provided herein, and methods provided herein, are used to treat adults with dysbiosis, e.g., to treat adult gut that is dysbiotic or at risk of becoming dysbiotic, which may include high levels of pathogenic bacteria, high levels of antibiotic resistance, a skewed metabolic balance that differs from that of a healthy population, and a skewed immunological state that differs from that of a healthy population, a loss of metabolic function associated with a healthy population, an increase in bacteria associated with a particular disease state, or a microbial population associated with insufficient therapeutic efficacy.

[0410] In alternative embodiments, the compositions, formulations, and pharmaceutical compositions provided herein, and methods provided herein, are administered to treat or ameliorate disease conditions associated with dysbiosis, including, but not limited to, cancer, diabetes, obesity, allergies, asthma, dermatitis, gout, Alzheimer's disease, and Parkinson's disease.

[0411] In an alternative embodiment, an adult is administered a live biotherapeutic composition (or probiotic), e.g., a pharmaceutical composition or formulation, alone or in combination with a prebiotic or supplement (e.g., those outlined in Table 3), comprising or consisting of one bacterium and a prebiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively, comprising one or a combination mix or consortium of the microorganisms outlined in Tables 1, 2, 4, and 30, to address dysbiosis and reduce the risk of disease or correct a lack of therapeutic efficacy.

[0412] In alternative embodiments, each or one of the microorganisms used in the bacterial combination is (at least initially) isolated from a healthy donor(s) as described in Example 5, or is a genetically modified derivative as described in Example 21, or is a cultured derivative of either.

[0413] In an alternative embodiment, the patient receives about 10 5 ~10 15 at a dose of bacteria, or about 10 10 , 10 11 Or 10 12 A live biotherapeutic is administered at a dose of bacteria, which may be, for example, in lyophilized form or formulated in an enteric-coated capsule. In alternative embodiments, the patient takes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more live biotherapeutic capsules (e.g., by mouth or suppository) once, twice, or three times or more per day, and the patient can resume a normal diet after about 1, 2, 4, 8, 12, or 24 hours, or longer.

[0414] In another embodiment, the patient may take the live biotherapeutic capsule by mouth before, during, and / or immediately after a meal.

[0415] In another embodiment, the patient is given a course of antibiotics prior to treatment, for example, 1 to 7 days, or about 1 to 2 weeks, before the first dose of live biotherapeutic (e.g., as a capsule), or 3 weeks, or 4 weeks, or up to 6 months before the first dose of live biotherapeutic.

[0416] In another embodiment, dosing of the live biotherapeutic (or probiotic) capsule is continued for about 1 month, 6 months, 1 year, or longer, or for about 1 week to 2 years after the end of treatment or therapy.

[0417] In an alternative embodiment, the composition of the adult gut microbiome, as well as the metabolic and immunological state of the adult gut, are used as measures of treatment success.

[0418] In an alternative embodiment, restored efficacy of a therapeutic agent (such as a drug, eg, a cancer therapeutic agent) is used as a measure of treatment success.

[0419] Example 18 Exemplary methods of treating disease based on stool biomarkers This example describes the successful therapeutic administration of a live biotherapeutic (or probiotic) provided herein, including, in alternative embodiments, one bacterium and a probiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively, a combination, mix, or consortium of bacteria provided herein, e.g., those shown in Table 1, Table 2, Table 4, Table 30, and / or the administration of a prebiotic provided herein, including a combination of prebiotics provided herein, e.g., those shown in Table 3, to an individual in need thereof.

[0420] In alternative embodiments, the compositions, formulations, and pharmaceutical compositions provided herein, and methods provided herein, are administered to treat or ameliorate an adult having a microbiome-mediated condition or disease, including, but not limited to, cancer, diabetes, obesity, allergies, asthma, gout, Alzheimer's disease, and Parkinson's disease.

[0421] Patient stool is collected and analyzed using the method described in Example 3. In one embodiment, whole genome sequencing is performed to assess the presence of microorganisms characteristic of healthy or diseased individuals. Based on the abundance profiles of healthy and diseased individuals, a classifier is developed to predict whether any given microbiome composition represents a healthy or diseased patient. This may be based on the amount of one or more specific organisms present, or other criteria that combine aspects of the whole genome sequence data. This classifier is applied to the patient's microbiome composition to predict whether the patient will require a biotherapeutic intervention.

[0422] In another embodiment, metabolomics is performed on stool or plasma, and a classifier is developed based on the concentration of one or more metabolites in all patient data collected to date and the composition of their microbiomes. This classifier is applied to the patient's data to predict whether the patient will require a biotherapeutic intervention.

[0423] In another embodiment, immunological analysis is performed on stool or plasma, and a classifier is developed based on the concentration of one or more immunological markers in all patient data collected to date and the composition of their microbiome. This classifier is applied to the patient's data to predict whether the patient will require a live biotherapeutic intervention. If the patient is classified as requiring intervention, a live biotherapeutic is administered to shift the microbiome away from the dysbiosis associated with the disease state.

[0424] In alternative embodiments, each or one of the microorganisms used in the bacterial combination is (at least initially) isolated from a healthy donor(s) as described in Example 5, or is a genetically modified derivative as described in Example 21, or is a cultured derivative of either.

[0425] In an alternative embodiment, the patient receives about 10 5 ~10 15 at a dose of bacteria, or about 10 10 , 10 11 Or 10 12A live biotherapeutic is administered at a dose of bacteria, which may be, for example, in lyophilized form or formulated in an enteric-coated capsule. In alternative embodiments, the patient takes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more live biotherapeutic capsules (e.g., by mouth or suppository) once, twice, or three times or more per day, and the patient can resume a normal diet after about 1, 2, 4, 8, 12, or 24 hours, or longer.

[0426] In another embodiment, the patient may take the live biotherapeutic capsule by mouth before, during, and / or immediately after a meal.

[0427] In another embodiment, the patient is given a course of antibiotics prior to treatment, for example, 1 to 7 days, or about 1 to 2 weeks, before the first dose of live biotherapeutic (e.g., as a capsule), or 3 weeks, or 4 weeks, or up to 6 months before the first dose of live biotherapeutic.

[0428] In another embodiment, dosing of the live biotherapeutic capsule is continued for one month, six months, one year, or longer, or for about one week to two years after treatment has ended.

[0429] In an alternative embodiment, the composition of the adult gut microbiome, as well as the metabolic and immunological state of the adult gut, are used as measures of treatment success.

[0430] Example 19 Exemplary Methods of Treating Pregnant Women with Live Biotherapeutics (or Probiotics) for the Treatment and Prevention of Dysbiosis, Which Can Lead to Disease in the Mother or Infant This example describes the administration to an individual in need thereof of a live biotherapeutic (or probiotic) provided herein, which in alternative embodiments includes one bacterium and a probiotic (or synbiotic, e.g., as shown in Table 8 or Table 32 below), or alternatively includes at least one of the bacteria provided herein, or a combination, mix, or consortium, e.g., those shown in Table 1, Table 4, Table 2, or Table 30, and / or a prebiotic provided herein, including a combination of prebiotics provided herein, e.g., those shown in Table 3.

[0431] In alternative embodiments, the compositions, formulations, and pharmaceutical compositions provided herein, as well as the methods provided herein, are administered to treat or improve the intestine of a pregnant woman or an individual expecting pregnancy when the intestine is dysbiotic or at risk of developing dysbiosis. For example, the dysbiosis being treated can be the presence of pathogenic bacteria, e.g., high levels of pathogenic bacteria, high levels of antibiotic resistance, a distorted metabolic balance that differs from that of a healthy population, and a distorted immunological state that differs from that of a healthy population, a loss of metabolic function associated with a healthy population, or an increase in bacteria associated with adverse events for the mother and her child. Dysbiosis can be caused by any number of lifestyle factors, including, but not limited to, the mother's mode of delivery, the mother's antibiotic use, the mother's diet, and the mother's GI condition.

[0432] In an alternative embodiment, pregnant women are administered a live biotherapeutic composition, i.e., a formulation consisting of some combination, mix, or consortium of microorganisms outlined in Table 4, or listed in Tables 1, 2, or 30, alone or in combination with a prebiotic or supplement outlined in Table 3, to address dysbiosis with the goal of preventing disease in the mother or infant.

[0433] In alternative embodiments, each or one of the microorganisms used in the bacterial combination, mix or consortium is (at least initially) isolated from a healthy donor(s) as described in Example 5, or is a genetically modified derivative as described in Example 21, or is a cultured derivative of either.

[0434] In an alternative embodiment, a patient (e.g., a pregnant woman) receives about 10 doses in total or per dose. 5 ~10 15 ...

Claims

1. - a method for controlling, ameliorating, reducing or preventing the symptoms or mortality of a dysbiosis or infection in an individual in need thereof, comprising: Optionally, the infection is a bacterial infection or a viral infection; optionally, the dysbiosis causes or exacerbates failure to thrive (FTT) in the individual, optionally the dysbiosis is in an infant, a child, a pregnant woman or a mother (maternal dysbiosis), optionally the infant is 0-36 months of age; Optionally, the dysbiosis may be in the presence of pathogenic bacteria, optionally having high levels of pathogenic bacteria, or the dysbiosis may be caused by high levels of antibiotic resistance, or a skewed metabolic balance that differs from that of a healthy population, or a skewed immunological state that differs from that of a healthy population, or a loss of metabolic function associated with a healthy population, or an increase in bacteria associated with adverse events to the mother and her child. - a method for modulating the microbiome of an individual, comprising: Optionally, the individual is a human, optionally wherein the human is a human child or human infant, optionally wherein the infant is 0-36 months of age; optionally, the microbiome of said individual is modulated to positively affect the growth, development or health of said individual (or to increase the individual's ability to develop), or to enhance the effectiveness of a treatment in an individual in need thereof, optionally wherein said treatment is a drug treatment, or a treatment for cancer; - a method for treating, ameliorating, reducing the symptoms or severity of, or preventing a disease or condition caused by dysbiosis in an individual in need thereof, comprising: Optionally, the individual is a human, optionally wherein the human is a human child or human infant, optionally wherein the infant is 0-36 months of age; Optionally, the disease or condition is failure to thrive (FTT); - optionally a method wherein the dysbiosis to be treated or the condition to be treated or ameliorated comprises dysbiosis caused or exacerbated by preterm birth, prolonged stay in a neonatal intensive care unit, drug or antibiotic treatment, drug or antibiotic treatment of the mother before birth, birth by Caesarean section, formula feeding, and a known dysbiosis of the mother, - for treating, ameliorating, reducing the symptoms or severity of, or preventing allergic reactions (optionally food allergies), dermatitis (optionally atopic dermatitis), atopic eczema, allergic rhinitis (hay fever), gastroesophageal reflux disease (GERD), sinusitis, obstructive sleep apnea, celiac disease, irritable bowel syndrome (IBS), Crohn's disease, rheumatoid arthritis, Sjogren's syndrome and / or asthma, - a method for treating, ameliorating, reducing the symptoms or severity of, or preventing obesity or metabolic syndrome, non-alcoholic fatty liver (NAFL), or metabolic associated fatty liver disease (MASLD); - a method for treating, ameliorating, reducing the symptoms or severity of, or preventing diabetes (optionally gestational diabetes, type 1 diabetes (T1D) or juvenile diabetes, or type 2 diabetes (T2D) or adult-onset diabetes), acute or chronic hyperglycemia; and / or The method comprises: (a) (i) at least two different species or genera (or types) of non-pathogenic bacteria (also called probiotics) and / or non-pathogenic bacterial spores; or (ii) at least one live non-pathogenic bacteria and / or non-pathogenic bacterial spores and at least one probiotic (also called a synbiotic, or a combination of a probiotic and a prebiotic); administering or having administered to an individual in need thereof a composition or formulation comprising each of said non-pathogenic bacteria comprises (or is in the form of) a plurality of non-pathogenic live colony-forming bacteria, a plurality of non-pathogenic germinating bacterial spores, or a combination or mix thereof; step; or (b)(i)(1) at least two different species or genera (or types) of non-pathogenic bacteria, each of said non-pathogenic bacteria comprising (or being in the form of) a plurality of live non-pathogenic colony-forming bacteria, a plurality of non-pathogenic germinating bacterial spores, or a combination thereof; or (2) at least one live non-pathogenic bacteria and / or non-pathogenic bacterial spores and at least one probiotic (also called a synbiotic, or a combination of a probiotic and a prebiotic); providing a composition or formulation comprising: Optionally, the at least two different species or genera (or types) of non-pathogenic bacteria of (b)(i)(1), or at least one live non-pathogenic bacterium and / or non-pathogenic bacterial spore of (b)(i)(2), have been genetically engineered to contain or express a new or heterologous trait or phenotype; and (ii) administering or maintaining the composition or formulation administered to an individual in need thereof. Including; Optionally, the composition or formulation comprises one or any combination or mix (or consortium) of one (optionally a synbiotic, or a combination of one species and a probiotic, optionally as in a synbiotic combination shown in Table 8 or Table 32) or at least two different species or genera of live non-pathogenic bacteria (or spores, if said bacteria form spores) set forth in Table 1 or Table 4, or live biotherapeutic (also called probiotic) compositions or combinations of bacteria shown in Table 2 or Table 30, or wherein said at least one live non-pathogenic bacteria and / or non-pathogenic bacterial spores and at least one probiotic (or synbiotic) comprises a combination as shown in Table 8 or Table 32, Optionally, at least one of the bacteria in the synbiotic provided herein, or the combination, mix (or consortium) provided herein, is a Bifidobacterium or Bacillus species, optionally Bifidobacterium infantis species; Optionally, the different species or genera (or types) of live non-pathogenic bacteria are present in approximately equal amounts, or each of the different species or genera (or types) of live non-pathogenic bacteria or non-pathogenic germinable bacterial spores represents at least about 1%, 5%, 10%, 20%, 30%, 40%, or 50% or more, or about 1% to 75%, or about 0.5 to 99% of the total amount of live non-pathogenic bacteria and non-pathogenic germinable bacterial spores in the formulation; Optionally, only live non-pathogenic bacteria or substantially only live non-pathogenic bacteria are present in the formulation, or only non-pathogenic germinable bacterial spores or substantially only non-pathogenic germinable bacterial spores are present in the formulation, or approximately equal amounts of live non-pathogenic bacteria and non-pathogenic germinable bacterial spores are present in the formulation. method.

2. and further comprising administering or having administered one or any one of the following: a treatment, prebiotic, synbiotic (or a combination of prebiotics and probiotics shown in Table 8 or Table 32, optionally a synbiotic), metabolite or drug, optionally an antiviral or antibacterial treatment or drug; an immune checkpoint inhibitor; a chimeric antigen receptor (CAR) T-cell therapy (CAR-T), or an immunotherapy (optionally an immune-enhancing therapy); or a combination thereof, Optionally, the method comprises administering an antibacterial, optionally an antiviral, antibacterial, antifungal or antimalarial agent, and optionally the antibacterial (optionally the antiviral) is selected from the group consisting of lopinavir; ritonavir; oseltamivir (optionally TAMIFLU™); lopinavir combined (formulated) with ritonavir, or KALETRA™; chloroquine phosphate (optionally RESOCHIN™), chloroquine diphosphate, hydroxychloroquine (optionally PLAQUENIL™) or oral chloroquine (optionally ARALEN™); remdesivir (optionally GS-5734™, Gilead Sciences); nevirapine, efavirenz, emtricitabine, tenofovir (or a combination of efavirenz, emtricitabine, and tenofovir, or ATRIPLA™); amprenavir (optionally AGENERASE™); nelfinavir (optionally VIRACEPT™); a drug of the thiazolide class, optionally nitazoxanide (or ALINIA™, NIZONIDE™) or tizoxanide (or 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide); plitidepsin (also known as dehydrodidemnin B), or APLIDIN™ (PharmaMar, S.A.); an inhibitor of S-phase kinase-associated protein 2 (SKP2), or dioscin, or niclosamide , or NICLOCIDE™, FENASAL™, or PHENASAL™; ribavirin; interferons, such as interferon alpha, interferon beta, type I interferon, type II interferon and / or type III interferon, or a combination of ribavirin and interferon beta, or a combination of lopinavir and ritonavir and interferon-beta-1b; abacavir, Actemra, acyclovir, optionally (ACICLOVIR™), adefovir, amantadine, Ampligen, amprenavir (optionally AGENERASE™), aprepitant, atazanavir, baravir, baloxavir marboxil (XOFLUZA™), bepotastine, bevirima,Biktegravir, Biktarvy, Brilacidin, Cidofovir, Caspofungin, Lamivudine and Zidovudine (Optionally COMBVIR™), Cobicistat, Colistin, Cocaine, Danoprevir or Danoprevir and Ritonavir (Optionally GANOVO™), Darunavir (or Darunavir and Cobicistat, Optionally PREZCOBIX™), Delavirdine, Descovy, Didanosine, Docosanol, Dolutegravir, Ecolievel, Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide ... Intecavir, epirubicin, epoprostenol, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ibacitabine, icatibant, idoxuridine, ifenprodil, imiquimod, Immunovir, indinavir, inosine, lamivudine, lopinavir, loviride, ledipasvir, leronlimab, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide, norvir, nucleoside analogs (optionally brincidofovir, didanosine, favipiravir (also known as T-705), Avigan, or favipiravir, Toyama Chemical, Fujifilm, Japan), vidarabine, galidesivir (optionally BCX4430 by Biocryst, IMMUCILLIN-A™), remdesivir (optionally GS-5734™, Gilead Sciences), cytarabine, gemcitabine, emtricitabine, zalcitabine, stavudine, telbivudine, zidovudine, idoxuridine and / or trifluridine, or any combination thereof), oseltamivir (or TAMIFLU™), peginterferon alfa-2a, penciclovir, peramivir (optionally RAPIVAB™), perphenazine, pleconaril, plurifloxacin, podophyllotoxin, pyramidine, raltegravir, rifampicin, ribavirin, rilpivirine, rimantadine, ritonavir, saquinavir, sofosbuvir, telaprevir, tegobuv, tenofovir alafenamide,tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir (optionally VALTREX™), valganciclovir, valrubicin, vapreotide, vicriviroc, vidarabine, viramidine, velpatasvir, vibecon, zalcitabine, zanamivir (optionally RELENZA™) or zidovudine; a serine protease inhibitor, optionally camostat; an anti-PD-1 checkpoint inhibitor, optionally camrelizumab; a compound that binds complement factor C5, a compound or antibody capable of blocking membrane attack complex formation, optionally eculizumab; a cathepsin inhibitor, optionally a cathepsin K, B, or L inhibitor, optionally relacatib; thalidomide, or thalidomide and a glucocorticoid (optionally a low-dose glucocorticoid), or and thalidomide and celecoxib; an antibacterial antibiotic, or optionally azithromycin (optionally ZITHROMAX™, or AZITHROCIN™), clarithromycin (optionally BIAXIN™), (TM)), erythromycin (optionally ERYTHROCIN™), or fidaxomicin (optionally DIFICID™ or DIFICLIR™), troleandomycin (optionally TEKMISIN™), tylosin (optionally TYLOCINE™ or TYLAN™), solithromycin (optionally SOLITHERA™), oleandomycin (or SIGMAMYCINE™), midecamycin, roxithromycin, kitasamycin, or thurimonosomycin. macrolide drugs including mycobacterium globulin, josamycin, carbomycin or magnamycin, and / or spiramycin; opaganib or YELIVA™; anti-interleukin-6 antibodies (e.g., tocilizumab or tocilizumab and favipiravir, optionally ACTEMRA™); sarilumab (optionally KEVZARA™); umifenovir (optionally ARBIDOL™); colchicine, or COLCRYS™, MITIGARE™; drugs of the corticosteroid class, for examplebudesonide (or RHINOCORT™ or PULMICORT™), prednisolone (or ORAPRED™), methyl-prednisolone, prednisone (or DELTASONE™ or ORASONE™), or hydrocortisone (or CORTEF™); antiandrogens, or bicalutamide; hydrocortisone or cortisol (or CORTEF™, SOLUCORTEF™), or hydrocortisone sodium succinate or hydrocortisone acetate, or dexamethasome (or DEXTENZA™, OZURDEX™, NEOFORDEX™); famotidine, or PEPCID™; drugs of the antihistamine class, such as azelastine, or ASTELIN™, OPTIVAR™, ALLERGODIL™, brompheniramine, fexofenadine, or ALLEGRA™, pheniramine or AVIL™, or chlorpheniramine; dendrimers, or astodrimer sodium (Starpharma, Melbourne, Australia); drugs of the selective serotonin reuptake inhibitor (SSRI) class, optionally fluvoxamine, or LUVOX™, FAVERIN™, FLUVOXIN™; nicotinic antagonists, dopamine agonists, or non-competitive N-methyl-d-aspartic acid or N-methyl-d-asparagine an NMDA antagonist; an immunosuppressant, or tocilizumab or atlizumab, or ACTEMRA™, or ROACTEMRA™, or a calcineurin inhibitor (CNI), or cyclosporin or cyclosporine or cyclosporin; or any two, three or more thereof, or combinations thereof; Optionally, the antiviral treatment or drug, the immune checkpoint inhibitor, the chimeric antigen receptor (CAR) T-cell therapy (CAR-T) or the immunotherapy, or the combination thereof, is administered before, during (concurrently with), and / or after administration of the formulation. The method of claim 1.

3. (a) the composition or formulation comprises an inner core surrounded by an outer layer of a polymeric material that encapsulates the inner core, wherein the non-pathogenic bacteria or the non-pathogenic germinable bacterial spores are substantially within the inner core, and optionally the polymeric material comprises a natural polymeric material; (b) the composition or formulation is formulated or manufactured as a nanosuspension delivery system; as or in an encapsulated formulation; or as a multi-layered crystalline helical structure without an internal aqueous space; the composition or formulation is formulated or manufactured as a delayed or slow enteric release composition or formulation, optionally the formulation comprises a gastro-resistant coating designed to dissolve at pH 7 in the terminal ileum, optionally the active ingredient is coated with an acrylic resin or equivalent, optionally a poly(meth)acrylate, optionally methacrylic acid copolymer B, NF, optionally EUDRAGIT S™ (Evonik Industries AG, Essen, Germany), which dissolves at a pH of 7 or higher, optionally comprises a multi-matrix (MMX) formulation, and optionally is manufactured with an enteric coating to avoid stomach acid and duodenal bile; The method according to claim 1 or claim 2.

4. the plurality of live non-pathogenic colony-forming bacteria are substantially dormant live colony-forming bacteria, or the plurality of live non-pathogenic colony-forming bacteria or the plurality of non-pathogenic germinable bacterial spores are freeze-dried; Optionally, the dormant, live, colony-forming bacteria comprise live, vegetative bacterial cells that have been rendered dormant by lyophilization or freeze-drying. A method according to any of the preceding claims or a method according to claims 1 to 3.

5. The formulation comprises at least about 1 x 10 ４ Colony forming units (CFU), or approximately 1 x 10 １ ~1 x 10 １３ CFU, 1 x 10 ２ ~1 x 10 １０ CFU, 1 x 10 ２ ~1 x 10 ８ CFU, 1 x 10 ３ ~1 x 10 ７ CFU, or 1 x 10 ４ ~1 x 10 ６ 5. A method according to any preceding claim or a method according to claims 1 to 4, comprising CFUs of live non-pathogenic bacteria and / or non-pathogenic germinating bacterial spores.

6. The preparation belongs to the family or genus (or class): Agathobaculum (TaxID: 2048137), Alistipes (TaxID: 239759), Anaeromassilibacillus (TaxID: 1924093), Anaerostipes (TaxID: 207244), Asaccharobacter (TaxID: 553372), Bacteroides (TaxID: 816), Barnesiella (TaxID: 397864), Bifidobacterium (TaxID: 1678), Blautia (TaxID: 572511), Butyricicoccus (TaxID: 580596), Clostridium (TaxID: 1485), Collinsella (TaxID: 102106), Coprococcus (TaxID: 33042), Dorea (TaxID: 189330), Eubacterium (TaxID: 1730), Faecalibacterium (TaxID: 216851), Fusicatennibacter (TaxID: 1407607), Gemmiger (TaxID: 204475), Gordonibacter (TaxID: 644652), Lachnoclostridium (TaxID: 1506553), Methanobrevibacter (TaxID: 2172), Parabacteroides (TaxID: 375288), Romboutsia (TaxID: 1501226), Roseburia (TaxID: 841), Ruminococcus (TaxID: 1263), Erysipelotrichaceae (TaxID: 128827), Coprobacillus (TaxID: 100883), ErysipelatoclostridiumSNUG30099 (TaxID: 1982626), Erysipelatoclostridium (TaxID: 1505663), or combinations thereof (optionally a synbiotic, or a combination of one species and a probiotic, optionally as in the synbiotic combinations shown in Table 8 or Table 32), or (or any one, some, or all) non-pathogenic bacteria or spores of:

7. 7. The method of any preceding claim, or the method of claims 1-6, wherein the formulation comprises water, sterile water, saline, sterile saline, a pharmaceutically acceptable preservative, carrier, buffer, diluent, adjuvant or combinations thereof.

8. 8. The method of any preceding claim, or the method of claims 1 to 7, wherein the composition or formulation is administered orally or rectally, or is formulated and / or administered as a freeze-dried composition, liposome, liquid, food, gel, supplement, gummy, candy, ice, lozenge, tablet, pill or capsule, or suppository or enema, or the formulation is administered as or in a form for aerosol, topical, sublingual, oral, rectal or colonic administration.

9. 9. A method according to any preceding claim, or a method according to claims 1 to 8, wherein the composition or formulation comprises or is mixed with milk (optionally human milk, cow's milk or soy protein, optionally fortified with vitamins, minerals and other nutrients), infant formula, soy-based formula, amino acid-based formula, hydrolysed infant formula (optionally made from cow's milk or soy protein that has been broken down into smaller proteins that are easier for infants to digest), or supplemented (collected) human breast milk.

10. 10. The method of any preceding claim, or the method of claims 1-9, wherein the composition or formulation is administered to the individual in need thereof in one, two, three or four or more doses, wherein the one, two, three or four or more doses are administered daily (optionally once daily, bid or tid), every other day, every third day, or about once a week, and wherein, optionally, the two, three or four or more doses are administered at least one week apart (or the doses are separated by about one week).

11. the composition or formulation further comprises a prebiotic, a synbiotic (or a combination of prebiotics and probiotics shown in Table 8 or Table 32, optionally a synbiotic), a nutrient, a metabolite, or a drug, and optionally the drug comprises an antibiotic; or the method further comprises administration of a prebiotic, a synbiotic (or a combination of prebiotics and probiotics shown in Table 8 or Table 32, optionally a synbiotic), a nutrient, a metabolite, or a drug; 2. The method of any preceding claim or the method of claims 1-1, wherein optionally at least one dose of the prebiotic, synbiotic, nutrient, metabolite or drug is administered before the first administration of the bacterial formulation, mix or consortium, and optionally at least one dose of the drug (or antibiotic), prebiotic, synbiotic, nutrient or metabolite is administered one or two days or more before the first administration of the formulation.

12. 12. The method of any of the preceding claims, or the method of claims 1 to 11, wherein the drug (or a combination of prebiotics and probiotics, optionally synbiotics, as shown in Table 8 or Table 32), prebiotic, said synbiotic, metabolite, metabolic precursor, or nutrient is administered by aerosol, spray, intravenous (IV) injection, intramuscular (IM) injection, intratumoral injection, or subcutaneous injection; or is administered orally or by suppository.

13. (a) a combination, mix or consortium of microorganisms shown in Table 1 or Table 4, or a live biotherapeutic composition or combination of bacteria shown in Table 2 or Table 30; (b) a combination, mix or consortium of microorganisms as used in any of the preceding claims or as used in a method according to any of claims 1 to 12; and / or (c) at least two different species or genera (or types) of non-pathogenic bacteria, each of said non-pathogenic bacteria comprising (or being in the form of) a plurality of live non-pathogenic colony-forming bacteria, a plurality of non-pathogenic germinating non-pathogenic bacterial spores, or a combination thereof; wherein said formulation comprises at least one (or any one, some or all) of the non-pathogenic bacteria or spores of said family or genus (or class), Composition or formulation or pharmaceutical composition.

14. the composition or formulation or pharmaceutical composition comprises at least one (optionally a synbiotic, or a combination of one species and a probiotic, optionally as in the synbiotic combinations shown in Table 8 or Table 32) or mix or consortium of bacteria having at least two different species or genera (or types) of non-pathogenic bacteria, each of said non-pathogenic bacteria comprising (or being in the form of) a plurality of live non-pathogenic colony-forming bacteria, a plurality of non-pathogenic germinating non-pathogenic bacterial spores, or a combination thereof, and the formulation , Family or Genus (or Class): Agathobaculum (TaxID: 2048137), Alistipes (TaxID: 239 759), Anaeromassilibacillus (TaxID: 1924093), Anaerostipes (TaxID: 20 7244), Asaccharobacter (TaxID: 553372), Bacteroides (TaxID: 816), Bar nesiella (TaxID: 397864), Bifidobacterium (TaxID: 1678), Blautia (TaxI D:572511), Butyricicoccus (TaxID:580596), Clostridium (TaxID:1485) , Collinsella (TaxID: 102106), Coprococcus (TaxID: 33042), Dorea (TaxID : 189330), Eubacterium (TaxID: 1730), Faecalibacterium (TaxID: 216851) ), Fusicatenibacter (TaxID: 1407607), Gemmiger (TaxID: 204475), Gordon ibacter (TaxID: 644652), Lachnoclostridium (TaxID: 1506553), Methano brevibacter (TaxID: 2172), Parabacteroides (TaxID: 375288), Romboutsi a (TaxID: 1501226), Roseburia (TaxID: 841), Ruminococcus (TaxID: 1263) , Erysipelotrichaceae (TaxID: 128827), Coprobacillus (TaxID: 100883),14. The composition or formulation or pharmaceutical composition of claim 13, comprising at least one (or any one, some, or all) of the non-pathogenic bacteria or spores of Erysipelatoclostridium sp. SNUG30099 (Tax ID: 1982626), Erysipelatoclostridium (Tax ID: 1505663), or a combination thereof.

15. (a) the composition or formulation or pharmaceutical composition comprises an inner core surrounded by an outer layer of a polymeric material that encapsulates the inner core, wherein the non-pathogenic bacteria or non-pathogenic germinable bacterial spores are substantially within the inner core, and optionally the polymeric material comprises a natural polymeric material; (b) the composition or formulation is formulated or manufactured as a nanosuspension delivery system; as or in an encapsulated formulation; or as a multi-layered crystalline helical structure without an internal aqueous space; (c) the composition or preparation is formulated or manufactured as a delayed or slow enteric release composition or preparation, optionally the preparation comprises a gastro-resistant coating designed to dissolve at a pH of 7 in the terminal ileum, optionally the active ingredient is coated with an acrylic resin or equivalent, optionally a poly(meth)acrylate, optionally methacrylic acid copolymer B, NF, optionally EUDRAGIT S™ (Evonik Industries AG, Essen, Germany), which dissolves at a pH of 7 or higher, optionally comprises a multi-matrix (MMX) formulation, and optionally is manufactured with an enteric coating to avoid stomach acid and duodenal bile; 15. A composition or formulation or pharmaceutical composition according to claim 13 or claim 14.

16. 16. The composition or formulation or pharmaceutical composition of any of claims 13 to 15, wherein the composition or formulation comprises water, sterile water, saline, sterile saline, a pharmaceutically acceptable preservative, carrier, buffer, diluent, adjuvant or combinations thereof.

17. 17. The composition or formulation or pharmaceutical composition of any of claims 13 to 16, wherein the composition or formulation is formulated for oral or rectal administration, or is formulated for administration as a freeze-dried composition, liposome, liquid, food, gel, supplement, gummy, candy, ice, lozenge, tablet, pill or capsule, or suppository or enema, or the formulation is formulated for or in a form for administration by aerosol, topical, sublingual, oral, rectal or colonic administration.

18. 18. A composition or formulation or pharmaceutical composition according to any of claims 13 to 17, wherein the composition or formulation comprises or is mixed with milk (optionally human milk, cow's milk or soy protein, optionally fortified with vitamins, minerals and other nutrients), infant formula, soy-based formula, amino acid-based formula, hydrolysed infant formula (optionally made from cow's milk or soy protein that has been broken down into smaller proteins that are easier for infants to digest), or supplemented (collected) human breast milk.

19. 19. The composition or formulation or pharmaceutical composition of any of claims 13 to 18, wherein the composition or formulation is formulated for administration to an individual in need thereof in 1, 2, 3 or 4 or more doses, wherein the 1, 2, 3 or 4 or more doses are formulated for administration daily (optionally once daily, bid or tid), every other day, every third day, or about once a week, and wherein, optionally, the 2, 3 or 4 or more doses are formulated for administration at least one week apart (or the doses are separated by only about one week).

20. 20. The composition or formulation or pharmaceutical composition of any of claims 13 to 19, wherein the composition or formulation further comprises a prebiotic, nutrient, metabolite or drug, optionally wherein the drug comprises an antibiotic, optionally wherein the drug comprises an antibiotic, optionally wherein at least one dose of the prebiotic, nutrient, metabolite or drug is administered prior to the first administration of the bacterial formulation, mix or consortium, and optionally wherein at least one dose of the antibiotic is administered one or two days or more before the first administration of the formulation.

21. 21. The composition or formulation or pharmaceutical composition of any of claims 13 to 20, wherein the drug, prebiotic, metabolite, metabolic precursor, or nutrient is formulated for administration by aerosol, spray, intravenous (IV), intramuscular (IM), intratumoral, or subcutaneous injection; or is administered orally or by suppository.

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