Long-term stabilization, formulation, and tableting of live microbial cells

Abstract

Provided herein are compositions comprising Escherichia coli (E. coli), Saccharomyces boulardii (S. boulardii), Lactobacillus plantarum (L. plantarum), and / or Ensifer meliloti (E. meliloti) and a first stabilizing excipient. Also provided herein are methods for delivering E. coli, S. boulardii, L. plantarum, and / or E. meliloti to a subject in need thereof, methods for inducing bacterial growth in a subject or in a cell, tissue, or biological sample, and methods for inhibiting enteric pathogens in a subject or in a cell, tissue, or biological sample. Further provided herein are methods for treating dysbiosis in a subject in need thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 428,708, filed November 29, 2022, entitled "LONG-TERM STABILIZATION, FORMULATION AND TABLETING OF LIVE MICROBIAL CELLS," the entire contents of which are incorporated herein by reference.

[0002] government support This invention was made with government support under NNX16A069A awarded by the National Aeronautics and Space Administration and under FA8650-21-2-7120 awarded by the Air Force Office of Scientific Research. The government has certain rights in this invention. Summary of the Invention

[0003] overview In one aspect, provided herein is a composition comprising Escherichia coli (E. coli) and a first stabilizing excipient selected from an enzymatic digest of soybean (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, potassium gluconate, melibiose, sucrose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), L-rhamnose monohydrate, (+)-L-sodium ascorbate, an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), trehalose dihydrate, D-(+)-galactose, the water-soluble portion of malted barley (e.g., Bacto malt extract), D-(+)-melezitose monohydrate, β-lactose, D(-)-fructose, glucose, 1-kestose, or a combination thereof.

[0004] In another aspect, provided herein is a composition comprising Saccharomyces boulardii (S. boulardii) and a first stabilizing excipient selected from L-glutamic acid monosodium salt monohydrate, skim milk powder, D-(+)-turanose, the water-soluble portion of malted barley (e.g., Bacto malt extract), maltitol, melibiose, lactulose, D-(+)-raffinose pentahydrate, palatinose hydrate, sucrose, animal-origin pancreatic digest of casein (e.g., Bacto tryptone), glucose, enzymatic digest of soybean (e.g., Bacto soytone), potassium gluconate, polydextrose, sodium gluconate, or a combination thereof.

[0005] In another aspect, provided herein is a composition comprising Lactobacillus plantarum (L. plantarum) and a first stabilizing excipient selected from a pancreatic digest of gelatin (e.g., Gelysate peptone), the water-soluble portion of malted barley (e.g., Bacto malt extract), an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), sucrose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), D-sorbitol, polydextrose, trehalose dihydrate, L-glutamic acid monosodium salt monohydrate, β-lactose, maltodextrin, L-rhamnose monohydrate, 1-kestose, an animal-origin enzymatic digest of bovine and porcine animal proteins (e.g., Bacto peptone), chondroitin sulfate A, or a combination thereof.

[0006] In another aspect, provided herein is a composition comprising Ensifer meliloti (E. meliloti) and a first stabilizing excipient selected from an enzymatic digest of soybean (e.g., Bacto soytone), skim milk powder, polydextrose, the water-soluble portion of malted barley (e.g., Bacto malt extract), L-glutamic acid monosodium salt monohydrate, maltodextrin, palatinose hydrate, D-(+)-melezitose monohydrate, trehalose dihydrate, 1-kestose, maltose monohydrate, α-lactose monohydrate, sucrose, D-cellobiose, D-(+)-raffinose pentahydrate, melibiose, or a combination thereof.

[0007] Details of certain embodiments of the present disclosure are set forth in the Detailed Description of Certain Embodiments, as set forth below. Other features, objects, and advantages of the present disclosure will become apparent from the definitions, examples, and claims. [Brief explanation of the drawings]

[0008] Brief description of the drawings [Figure 1]Survey of commercially available dried formulations of microbial probiotics. The viability of a wide range of representative commercial probiotic products was quantified (Table 1). Viable cell counts were defined as the number of colony-forming units (CFUs) assessed through dilution plating on appropriate solid media and quantified via programmatic image analysis (see Example 8). Percent viability relative to the expected cells was calculated by dividing the CFU count by the CFU count printed on the product label. Percent viability relative to the total cells was calculated by dividing the CFU count by the total countable cell number determined via automated fluorescence cytometry. N = 4 or 5 independent dosage forms. Geometric means and geometric 95% confidence intervals are plotted in black over individual replicates. The broad phylogenetic classification of the constituent microorganisms is indicated by color, and specific genera are italicized (see Figures 5A-B for detailed organization). neg. = negative; pos. = positive; Bifido. = Bifidobacterium; Lacto. = Lactobacillus; caps. = capsules.

[0009] [Figure 2]Development and validation of a high-throughput pipeline for dried stabilized microbial materials. Figure 2A. A high-throughput pipeline (batch mixing, lyophilization, plating, and quantification) was developed to assess the ability of a library of generally regarded as safe (GRAS) materials to stabilize microorganisms (see Example 8). The illustration was generated using BioRender.com. Figure 2B. Representative dried formulations generated in high-throughput and corresponding colony count images. Scale bar = 1 cm. Figure 2C. Results of the top 33% of materials showing increased viability after drying and 24-hour storage at room temperature across four organisms and two concentrations, sorted in descending order of their ability to stabilize E. coli Nissle 1917 at a 5X concentration. Viability scores are composite colony counts normalized to maximum observable growth, defined as 1 (see Example 8). See Table 2 for material identities and concentrations corresponding to the noted material indexes and Figure 8 for complete results. Figure 2D. Correlation analysis of top-performing materials for each organism. Figure 2E. Validation of the top materials defined in Figure 2C through a more precise assay (lyophilization in vials and individual CFU counts on plates) and their ability to preserve viability at room temperature for 1 and 30 days. Material indexes marked with an asterisk are concentrations at 1X, otherwise at 5X. The horizontal dashed line represents the limit of detection. Where indicated by a vertical bar, water samples were assayed at higher concentrations to achieve a lower limit of detection. N = 4-8 independent dried vials. Means and SEM are plotted over individual replicates.

[0010] [Figure 3]Application of the pipeline to make E. coli Nissle 1917 a synthetic extremophile. Figure 3A. A library of materials was assayed individually (X-axis) and in combination with melibiose (Y-axis) for their ability to stabilize E. coli Nissle 1917 at room temperature and 50°C for 24 hours. Black circles along both dimensions indicate the mean viability score (see Example 8), and error bars represent SEM. N=3. Colored shapes indicate combinations of melibiose and materials selected for further characterization. The horizontal dashed line is a reference showing the viability score of melibiose in combination with vehicle (water, blue circles). The diagonal dashed line is identity. All concentrations were 1X as defined in Table 2. FOS: fructooligosaccharides (fructooligosacharides). Figure 3B. The relative concentrations of each of the components in selected two-ingredient combinations (melibiose + {yeast extract or caffeine}) were varied, and the resulting effect on viability was measured after 23 days of storage at 37°C. All concentrations are relative to 1X, as defined in Table 2. The letters "D" and "E" indicate formulations selected for further characterization. Figure 3C. Head-to-head stability comparison of formulation "D" (as noted in Figure 3B), the parent formulation (melibiose), and the commercial E. coli Nissle 1917 product Mutaflor capsules after 1 month of storage at room temperature. Means and SEM are plotted over individual replicates. N=3-5. ****, P<0.0001. Figure 3D. Ultra-long-term stability profile at 37°C compared to maltodextrin (the stabilizer used in Mutaflor). Means and SEM are plotted for each time point. N=3. The lower dashed line represents the limit of detection. Figure 3E. Transmission electron micrographs of E. coli Nissle 1917 cells after drying in Formulation D or maltodextrin and optional exposure to elevated temperatures. Black arrowheads indicate detached inner membranes.

[0011] [Figure 4]Synthetic extremophiles withstand rigorous processing to create microbial therapeutics for human, agricultural, and space applications. Figure 4A. Microbial tableting and coating allows for easy dosing, delivery, and release control for access to a wide range of applications. Figure 4B. Representative photographs and scanning electron micrographs of synthetic extremophiles processed through industrially relevant processes of milling, wet granulation, tableting, and spray coating. Figure 4C. Through-process stability of E. coli Nissle 1917 synthetic extremophile (Formulation D) relative to the parent powder. Maltodextrin is a stabilizer used in the commercial product Mutaflor. The horizontal dotted line indicates the limit of detection. Means and SEMs are plotted over individual replicates. N=3. Figure 4D. Tableting allows for tailoring of release kinetics through the inclusion of a matrix-forming agent (hydroxypropyl methylcellulose, HPMC). Synthetic extremophile E. coli Nissle 1917 cells harboring the biosynthetic pathway (luxCDABE plasmid) were monitored for luminescence emitted over time. Lines connect means. Individual replicates are plotted. N=2. Figure 4E. High viability in the dry state allows for greater resistance to ionizing radiation. Synthetic extremophile E. coli Nissle 1917 cells or liquid suspensions of the same cells in PBS were exposed to the indicated doses of ionizing radiation. Lines connect means. Individual replicates are plotted. N=4. Two-way ANOVA: ****, P<0.0001. Figure 4F. Both fresh E. coli Nissle 1917 and synthetic extremophile (formulation D) inhibit the enteric pathogen S. flexneri. A commercial competitor (maltodextrin) performed comparable to a laboratory E. coli strain (DH5a). Means and SEM are plotted over individual replicates. N=3. One-way ANOVA: ***, P<0.001; **, P<0.01. Figure 4G. E. meliloti is a nitrogen-fixing bacterium that provides nitrogen to plants in symbiotic root nodules (inset). The synthetic extremophile E. meliloti was exposed to 50°C, rehydrated, and tested for functionality in a plant root nodulation assay with M. truncatula.At 12 days after root inoculation, nodulated seedlings (arrows) could be quantified. Figure 4H. Quantification of the nodulation assay (Figure 4F) compared to material viability scores. Means and SEMs are plotted over individual replicates. N = 3–5 and 8 for the control (left of dashed line).

[0012] [Figure 5] Raw CFU / g values ​​from the probiotic survey. Figure 5A. Raw data used to calculate the percent viability graphed in Figure 1. Means and standard errors of the mean (SEM) are plotted over individual replicates. Figure 5B. Phylogenetic composition of the commercial products assessed. Numbers in boxes indicate the number of individual strains in each clade. Full product details are summarized in Table 1.

[0013] [Figure 6] Commercial probiotic products stress tested at 50°C for 24 hours. Figure 6A. Each product was stored at 4°C, 23°C, or 50°C for 24 hours, and the resulting viability was assessed (see Example 8). Only Mutaflor and VSL#3 were stored at 4°C, as recommended by the manufacturer. Figure 6B. Analysis of the data in Figure 6A. The overall viability value at 50°C was divided by the average viability at the manufacturer's recommended storage temperature (4°C for Mutaflor and VSL#3; 23°C for the remaining products). For both panels, the mean and standard error of the mean (SEM) are plotted overlaid on the individual replicates.

[0014] [Figure 7]High-throughput pipeline for assessing viability scores. Figure 7A. Dried bacterial samples are reconstituted, diluted, and batch-plated onto 1-well plates and incubated. The resulting array of spots is imaged, and the image of each spot ("raw") is programmatically segmented ("masked") into regions of interest (i.e., regions containing bacterial colonies). The segmented regions are further divided into countable "particles" using a watershed algorithm in Fiji. Figure 7B. Additional image parameters from the segmented regions, such as size relative to the control lawn size (spot is a percentage) and mean signal intensity relative to the area outside the segmented region (intensity relative to background), can be used to automatically classify spots into "lawn," "blank," and "countable" spots. Particle counts for blank spots are set to zero. Particle counts for countable spots are interpreted as colony numbers. Figure 7C. The particle count value for the flora is set to an organism-specific value determined by extrapolating a best-fit line of all countable spots (i.e., spots below the gray area) to the particle count value corresponding to a spot area percent of 120%.

[0015] [Figure 8] Detailed results for the material stabilizer library. Extended data from Figure 2C plotted using a continuous color scale. Gray denotes no observed viability. Vertical black boxes indicate positive controls (#18: ATCC Reagent 20 and #35: Trehalose), and vertical red boxes indicate negative controls (#224: Sodium Hydroxide and #238: Sodium Metabisulfite). The viability score is a composite score of colony counts across three plating dilutions (see Example 8). "Rel.mat.conc." - Relative material concentration as defined for 5X and 1X in Table 2.

[0016] [Figure 9]Compound classification over-represented in the hit material. All compounds were classified by chemical structure. The top performing compounds are the same as those included in the correlation analysis (see Figure 2D, Example 8). Extract refers to cell or plant extracts, such as yeast extract or malt extract. Peptone refers to both protein digests (tryptone) or other protein-based materials (catalase). Mixtures of individually prepared components were omitted from this analysis.

[0017] [Figure 10] Validation of two-material formulations at 37°C. Selected materials from the two-material library were validated on a larger scale (see Example 8) to determine precise viability retention when stored at 37°C for the specified time periods. "Powder only" refers to freeze-dried, milled powder stored with desiccant in nitrogen-flushed bags. "Powder + excipients" refers to milled microbial powder mixed with excipients (binders, fillers, lubricants) and stored as described in Example 8. For both panels, the mean and standard error of the mean (SEM) are plotted over individual replicates.

[0018] [Figure 11] Individual viability signatures at different storage temperatures for top material formulations. Extended data from Figure 2D. Lyophilized microbial powder was stored after grinding and mixing with excipients (see Example 8). Storage was in glass vials with desiccant in the dark at the specified temperatures. Lines connect geometric means and error bars indicate 95% confidence intervals. N=3.

[0019] [Figure 12]Preculture medium influences ultimate survival through lyophilization. E. coli Nissle 1917 was grown in various preculture media, washed in PBS, mixed with trehalose, lyophilized, rehydrated, inoculated into fresh LB, and then mixed with Presto Blue to assess viability. Values ​​represent the fold increase in Presto Blue signal relative to control medium (LB). Each medium had three components: peptone (P#) at 10 g / L, extract at 5 g / L (E#), and salt (NaCl) concentration (S#): P1: tryptone, P2: soytone, P3: gelysate peptone, P4: bacto peptone, E1: beef extract, E2: yeast extract, E3: malt extract, E4: beef and yeast extract, S0: 86 mM NaCl, S1: 250 mM NaCl, S2: 600 mM NaCl. Control LB wells are P1:E2:S0.

[0020] [Figure 13] The time of bacterial harvest affects ultimate survival through lyophilization. Liquid flask cultures of E. coli Nissle 1917 were harvested at the indicated times after inoculation. Culture viability at harvest was assessed by plating, and percent viability was calculated by dividing by the estimated CFU / mL estimated from optical density measurements (1 OD600 approximately 1E9 CFU / mL). Each sample of harvested cells was lyophilized, and the resulting viability was assessed after 24 hours of storage at 23°C (see Example 8). Lines connect means, and error bars indicate the standard error of the mean. N=2.

[0021] [Figure 14]Bacterial loading can be increased in powders and tablets. E. coli Nissle 1917 was lyophilized with Formulation D at three different concentrations of the initial cell suspension. The resulting powder was milled and mixed with excipients (see Example 8) and either stored as mixed (right) or compressed into tablets and then stored (left). All samples were stored at 37°C in nitrogen-flushed bags with desiccant for the indicated times. The mean and standard error of the mean (SEM) are plotted over each replicate.

[0022] [Figure 15] The combination of pre-culture timing and increased bacterial loading results in a viability of greater than 10 CFU per gram. Either the commercial product Mutaflor or the synthetic extremophile E. coli Nissle 1917 (Melibiose 1X) was evaluated for their initial viability and for viability after long-term storage of 32 days at 23°C. "100X" refers to a 100-fold increase in bacterial concentration in the bacterial suspension mixed with the stabilizer. "Ideal time" refers to harvesting the bacterial flask culture at the optimal time of 9 hours, as indicated by the data in Figure 14. Geometric means and 95% confidence intervals are plotted over individual replicates.

[0023] [Figure 16] Tableting pressure controls bacterial stability at 37°C. E. coli Nissle 1917 was lyophilized with Formulation D at 10X cell concentration of the initial cell suspension. The resulting material was ground, mixed with excipients, and compressed into tablets at the indicated pressures (see Example 8). The mean and standard error of the mean (SEM) are plotted over each replicate.

[0024] [Figure 17]The synthetic extremophile E. coli Nissle 1917 extremophile survives for 1 hour in SGF. Extended data from Figure 4C. The synthetic extremophile E. coli Nissle 1917 (Formulation D) or a commercial comparator (lyophilized with 5% maltodextrin) was ground, compressed into tablets, and coated with Eudragit S100 (see Example 8). The coated tablets were then immersed in simulated gastric fluid (SGF) for 1 hour at 37°C to mimic digestion and passage through the stomach. The viability of each sample was assessed by plating. Viability is normalized to that of the ground powder. The coated tablets were cut in half immediately before reconstitution to expose the contents. Geometric means and 95% confidence intervals are plotted over individual replicates. The horizontal dotted line indicates the limit of detection.

[0025] [Figure 18] E. meliloti stabilizers for 24 hours at 50°C. Extended data from Figure 8. E. meliloti was mixed with the material library at 1X concentration, lyophilized, and exposed to 50°C for 24 hours. Viability scores were characterized as in Figure 8, and the data are ordered by comparative viability scores when stored at 23°C.

[0026] [Figure 19]Ultrastructural characterization of E. coli Nissle 1917 cell envelopes after drying and rehydration. Figure 19A. Representative transmission electron micrographs of dried and rehydrated bacterial cells. Cells were dried in either Formulation D or a commercial comparator (maltodextrin) and optionally exposed to 50°C (see Example 8). Figure 19B. Enlargement of the area indicated by the dashed rectangle in Figure 19A. An additional dashed rectangle indicates the area encompassed in Figure 3E. Figure 19C. Objects in three image planes for each of the four conditions were classified into the three categories indicated and quantified (see Example 8). N=3 image planes. Error bars represent the standard error of the mean (SEM). Insets show multiple representative objects in each category. The field of view in the inset is 1 um x 1 um. Figure 19D. Analysis of the data in Figure 19C. The number of cells with detached inner membranes was divided by the total number of cells (excluding cell debris). Ordinary one-way ANOVA: ****, P<0.0001.

[0027] [Figure 20] Inhibition of the enteric pathogen Shigella flexneri by E. coli Nissle 1917 is not affected by the stabilizer materials. Figure 20A. S. flexneri was cultured with fresh E. coli Nissle 1917 cells or either with the vehicle control (water), the material used to make Formulation D ("D material"), or mixed with maltodextrin. Shigella inhibition was quantified by qPCR (see Example 8). N=3. Ordinary one-way ANOVA: ns, P>0.05. Figure 20B. S. flexneri was cultured with the vehicle control (water) or co-cultured with fresh E. coli Nissle 1917 cells, either alone or mixed with the material used to make Formulation D ("D material") or maltodextrin. Shigella inhibition was quantified by qPCR (see Example 8). N=3. Ordinary one-way ANOVA: ns, P > 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition The terms "composition" and "formulation" are used interchangeably.

[0029] A "subject" to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, or adolescent), or an adult subject (e.g., a young adult, middle-aged adult, or elderly adult)), or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially suitable mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog), or a bird (e.g., a commercially relevant bird such as a chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be male or female at any stage of development. The non-human animal may be a transgenic or genetically engineered animal. The term "patient" refers to a human subject in need of treatment for a disease. The subject may also be a plant. In certain embodiments, the plant is a land plant. In certain embodiments, the plant is a non-vascular land plant. In certain embodiments, the plant is a vascular land plant. In certain embodiments, the plant is a seed plant. In certain embodiments, the plant is a cultivated plant. In certain embodiments, the plant is a dicotyledonous plant. In certain embodiments, the plant is a monocotyledonous plant. In certain embodiments, the plant is a flowering plant. In some embodiments, the plant is a cereal plant, for example, maize, corn, wheat, rice, oat, barley, rye, or millet. In some embodiments, the plant is a legume, for example, a bean plant, for example, a soybean plant. In some embodiments, the plant is a tree or shrub.

[0030] The term "biological sample" refers to any sample that includes a tissue sample (such as a tissue section and a needle biopsy of tissue); a cell sample (e.g., a cytological smear (such as a Pap or blood smear) or a sample of cells obtained by microdissection); a sample of a whole organism (such as a yeast or bacterial sample); or a cell fraction, fragment, or organelle (e.g., obtained by lysing cells and centrifuging or otherwise separating their components). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical or needle biopsy), nipple aspirate, milk, vaginal fluid, saliva, a swab (e.g., a buccal swab), or any material containing biomolecules derived from a first biological sample.

[0031] The term "target tissue" refers to any biological tissue (including cell groups, body parts, or organs) or part thereof of a subject, including blood vessels and / or lymphatic vessels, to which the compounds, particles, and / or compositions of the present invention are delivered. The target tissue may be an abnormal or unhealthy tissue that may need to be treated. The target tissue may also be a normal or healthy tissue that is at a higher than normal risk of becoming abnormal or unhealthy, which may need to be prevented. In certain embodiments, the target tissue is the stomach and / or intestine. A "non-target tissue" is any biological tissue (including cell groups, body parts, or organs) of a subject, including blood vessels and / or lymphatic vessels, that is not a target tissue.

[0032] The terms "administer," "administering," or "administration" refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein or a composition thereof into or onto a subject.

[0033] The terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of, a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of a disease have developed or been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or exposure to a pathogen). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence.

[0034] The terms "condition," "disease," and "disorder" are used interchangeably.

[0035] The "effective amount" of the compound described herein refers to an amount sufficient to induce a desired biological response.The effective amount of the compound described herein may vary depending on factors such as the desired biological endpoint, side effects, the severity of the disease or disorder, the characteristics, pharmacokinetics, and pharmacodynamics of the specific compound, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age, and health or general condition of the subject.In certain embodiments, the effective amount is a therapeutically effective amount.In certain embodiments, the effective amount is a preventive treatment.In certain embodiments, the effective amount is the amount in a single dose of the compound described herein.In certain embodiments, the effective amount is the combined amount in multiple doses of the compound described herein.In certain embodiments, the desired dosage is delivered three times a day, twice a day, once a day, every other day, every other day, every week, every other week, every three weeks, or every four weeks. In certain embodiments, the desired dose is delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations).

[0036] In certain embodiments, an effective amount of a compound for administration to a 70 kg adult one or more times per day comprises from about 0.0001 mg to about 3000 mg, from about 0.0001 mg to about 2000 mg, from about 0.0001 mg to about 1000 mg, from about 0.001 mg to about 1000 mg, from about 0.01 mg to about 1000 mg, from about 0.1 mg to about 1000 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 100 mg, from about 10 mg to about 1000 mg, or from about 100 mg to about 1000 mg of the compound per unit dosage form.

[0037] In certain embodiments, the compounds of the invention may be administered orally or parenterally, one or more times daily, at a dosage level sufficient to deliver from about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 40 mg / kg, preferably from about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, and more preferably from about 1 mg / kg to about 25 mg / kg of body weight per day to achieve the desired therapeutic effect.

[0038] It will be understood that the dosage ranges as set forth herein provide guidance for administration of the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent can be determined by a medical professional or person skilled in the art and may be less than or the same as the amount administered to an adult.

[0039] A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in treating a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a composition refers to an amount of a therapeutic agent that, alone or in combination with other treatments, provides a therapeutic benefit in treating a condition. The term "therapeutically effective amount" can encompass an amount that improves overall treatment, reduces or avoids symptoms, signs, or causes of a condition, and / or enhances the therapeutic effectiveness of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient to treat dysbiosis.

[0040] A "prophylactically effective amount" of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with a condition, or to prevent its recurrence. A prophylactically effective amount of a compound refers to the amount of a therapeutic agent that, alone or in combination with other agents, provides a prophylactic benefit in preventing a condition. The term "prophylactically effective amount" can encompass an amount that improves overall prevention or enhances the prophylactic effectiveness of another prophylactic agent. In certain embodiments, a prophylactically effective amount is an amount sufficient to prevent dysbiosis.

[0041] The terms "prevent," "preventing," or "prevention" refer to the prophylactic treatment of a subject who is either currently or previously disease-free but is at risk of developing the disease, or who previously had the disease and is currently disease-free but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than the average healthy member of the population.

[0042] In certain aspects, the subject is a human. The subject may be of either gender and at any stage of development. In certain aspects, the subject described herein is a human. In certain aspects, the subject is a non-human animal. In certain aspects, the subject is a mammal. In certain aspects, the subject is a non-human mammal. In certain aspects, the subject is a domestic animal such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain aspects, the subject is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain aspects, the subject is a zoo animal. In another aspect, the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain aspects, the animal is a genetically engineered animal. In certain aspects, the animal is a transgenic animal (e.g., transgenic mouse and transgenic pig). In certain embodiments, the subject is a fish or reptile.

[0043] In certain embodiments, the cell is present in vitro. In certain embodiments, the cell is present in vivo.

[0044] The pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacy.In general, such preparation methods include bringing the compounds described herein (i.e., "active ingredient") into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into a desired single or multiple dosage unit.

[0045] Pharmaceutical compositions may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. A "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0046] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in the pharmaceutical compositions described herein will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition will be administered. The compositions may contain from 0.1% to 100% (w / w) of the active ingredient.

[0047] Pharmaceutically acceptable excipients used in the preparation of the provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and perfumes may also be present in the compositions.

[0048] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0049] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0050] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters ( Examples include polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., Examples of suitable oleic acid esters include polyoxyethylene monostearate (e.g., Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate,Examples include sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

[0051] Exemplary binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, psyllium husk mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabinogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0052] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0053] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0054] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0055] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0056] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0057] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0058] Other preservatives include tocopherol, tocopheryl acetate, deferoxamine mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone®, Kathon®, and Euxyl®.

[0059] Exemplary buffering agents include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium phosphate, monosodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0060] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0061] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, lily of the valley, and macadamia. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0062] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil, groundnut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may contain adjuvants such as wetting agents, emulsifiers, and suspending agents, sweeteners, flavoring agents, and perfumes. In certain embodiments for parenteral administration, the conjugates described herein are mixed with a solubilizing agent such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0063] Injectable preparations, for example, sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils have traditionally been used as solvents or suspending media. For this purpose, any non-irritating fixed oil, including synthetic monoglycerides or diglycerides, can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0064] Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0065] In order to prolong the effect of drugs, it is often desirable to delay the absorption of drugs from subcutaneous or intramuscular injection.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility.In this case, the absorption rate of the drug depends on its dissolution rate, and the dissolution rate can also depend on crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0066] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active ingredient.

[0067] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicic acids, and sodium carbonate; (e) solution retarders, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) humectants, such as, for example, cetyl alcohol and glycerol monostearate; (h) absorbents, such as kaolin and bentonite clay; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.

[0068] Solid compositions of a similar type can be used as fillers in hard and soft gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols, etc. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmacological arts. They may optionally contain opacifying agents and can be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of encapsulating compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can be used as fillers in hard and soft gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols, etc.

[0069] The active ingredient may be in microencapsulated form with one or more excipients as described above.Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical industry.In such solid dosage forms, the active ingredient may be mixed with at least one inert diluent, such as sucrose, lactose, or starch.Such dosage forms may contain additional substances other than inert diluents, as is customary, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose.In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents.They may optionally contain opacifying agents, and may be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner.Examples of encapsulating agents that can be used include polymeric substances and waxes.

[0070] The dosage form for topical and / or transdermal administration of the compounds described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches.Generally, the active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier or excipient, and / or any preservatives and / or buffers that may be required.In addition, the present disclosure contemplates the use of transdermal patches, which often have the additional advantage of providing controlled delivery of the active ingredient to the body.Such dosage forms can be prepared, for example, by dissolving and / or dispensing the active ingredient in a suitable medium.Alternatively or additionally, the rate can be controlled by providing a rate-controlling membrane and / or dispersing the active ingredient in a polymer matrix and / or gel.

[0071] The device suitable for use in delivering the intradermal pharmaceutical composition described herein includes a short needle device.The intradermal composition can be administered by a device that limits the effective penetration length of the needle into the skin.Alternatively, or in addition, a conventional syringe can be used in the classical Mantoux method of intradermal administration.Jet injection devices are suitable, which deliver liquid formulations to the dermis via a liquid jet injector and / or via a needle that punctures the stratum corneum and generates a jet that reaches the dermis.Ballistic powder / particle delivery devices are suitable, which use compressed gas to accelerate powder-formed compounds through the outer layer of the skin to the dermis.

[0072] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid preparations, such as liniments, lotions, oil-in-water and / or water-in-oil emulsions, and / or solutions and / or suspensions, including creams, ointments, and / or pastes. Topically administrable formulations may contain, for example, about 1% to about 10% (w / w) of the active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further include one or more of the additional ingredients described herein.

[0073] The pharmaceutical compositions described herein may be prepared, packaged, and / or sold as formulations suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles comprising the active ingredient and having diameters ranging from about 0.5 to about 7 nanometers or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir into which a stream of propellant can be directed to disperse the powder, and / or using a self-propelling solvent / powder dispensing container, such as a device containing the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles in which at least 98% of the particles (by weight) have a diameter greater than 0.5 nanometers and at least 95% of the particles (by number) have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles (by weight) have a diameter greater than 1 nanometer and at least 90% of the particles (by number) have a diameter less than 6 nanometers. Dry powder compositions may also include a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0074] Low boiling point propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition, and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional ingredients such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size on the same order as the particles containing the active ingredient).

[0075] Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions, optionally sterile, containing the active ingredient, and may be conveniently administered using an inhalation administration device and / or an atomization device. Such formulations may further contain one or more additional ingredients, including, but not limited to, flavoring agents such as saccharin sodium, volatile oils, buffers, surfactants, and / or preservatives such as methylhydroxybenzoate. The droplets provided by this administration route may have an average diameter ranging from about 0.1 to about 200 nanometers.

[0076] The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions described herein. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 to 500 micrometers. Such formulations are administered by high-velocity inhalation through the nasal passages from a container of the powder held close to the nostrils.

[0077] Formulations for nasal administration may contain, for example, as little as about 0.1% (w / w) to as much as 100% (w / w) of the active ingredient, and may include one or more of the additional ingredients described herein. The pharmaceutical compositions described herein may be prepared, packaged, and / or sold as formulations for buccal administration. Such formulations may be in the form of, for example, tablets and / or lozenges made using conventional methods and may contain, for example, 0.1-20% (w / w) of the active ingredient, the remainder comprising an orally soluble and / or degradable composition, and, optionally, one or more of the additional ingredients described herein. Alternatively, formulations for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range of about 0.1 to about 200 nanometers and may further comprise one or more of the additional ingredients described herein.

[0078] The pharmaceutical compositions described herein may be prepared, packaged, and / or sold as formulations for ocular administration. Such formulations may be in the form of, for example, eye drops comprising a 0.1 to 1.0% (w / w) solution and / or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and / or one or more other additional ingredients described herein. Other useful ophthalmically administrable formulations include formulations comprising the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are also contemplated within the scope of this disclosure.

[0079] Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and veterinary pharmacologists of ordinary skill in the art can design and / or perform such modifications using routine experimentation.

[0080] The compound provided herein is usually formulated in unit dosage form for ease of administration and dosage uniformity.However, it is understood that the total daily use amount of the compositions described herein is determined by physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any specific subject or organism will depend on various factors, including the disease and disorder severity being treated; the activity of the specific active ingredient used; the specific composition used; the age, weight, general health condition, sex and diet of subject; the administration time, administration route and excretion rate of the specific active ingredient used; treatment period; the drug used in combination with or simultaneously with the specific active ingredient used; and similar factors well known in the medical field.

[0081] The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intracerebroventricular, transdermal, intradermal, rectal, vaginal, intraperitoneal, topical (by powder, ointment, cream, and / or drops), mucosal, nasal, buccal, sublingual; intratracheal instillation, intrabronchial instillation, and / or inhalation; and / or oral spray, nasal spray, and / or aerosol.Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), local administration via blood and / or lymphatic supply, and / or direct administration to the affected area.Generally, the most suitable administration route depends on various factors, including the nature of the agent (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration).In certain embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the eye of a subject.

[0082] The exact amount of compound required to achieve an effective dose will vary from subject to subject, depending, for example, on the subject's species, age, and general condition, the severity of side effects or disorders, the identity of the specific compound, the mode of administration, etc. An effective amount may be contained in a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two of the multiple doses contain different or substantially the same amounts of the compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency with which the multiple doses are administered to a subject or applied to a tissue or cell is three doses per day, two doses per day, one dose per day, one dose every other day, one dose every two days, one dose per week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to tissue or cells is 1 dose per day.In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to tissue or cells is 2 doses per day.In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to tissue or cells is 3 doses per day.In certain embodiments, when administering multiple doses to a subject or applying multiple doses to tissue or cells, the period between the first dose and the last dose of the multiple doses is 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years, 15 years, 20 years, or the lifespan of the subject, tissue, or cell.In certain embodiments, the period between the first dose and the last dose of the multiple doses is 3 months, 6 months, or 1 year. In certain embodiments, the period between the first and last dose of the multiple doses is the lifespan of the subject, tissue, or cell.In certain embodiments, the doses described herein (e.g., either a single dose or multiple doses) independently include 0.1 μg to 1 μg, 0.001 mg to 0.01 mg, 0.01 mg to 0.1 mg, 0.1 mg to 1 mg, 1 mg to 3 mg, 3 mg to 10 mg, 10 mg to 30 mg, 30 mg to 100 mg, 100 mg to 300 mg, 300 mg to 1,000 mg, or 1 g to 10 g (inclusive) of a compound described herein. In certain embodiments, the doses described herein independently include 1 mg to 3 mg (inclusive) of a compound described herein. In certain embodiments, the doses described herein independently include 3 mg to 10 mg (inclusive) of a compound described herein. In certain embodiments, the doses described herein independently include 10 mg to 30 mg (inclusive) of a compound described herein. In certain embodiments, the doses described herein independently encompass between 30 mg and 100 mg, inclusive, of a compound described herein.

[0083] The dosage ranges described herein provide guidance for administering the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent can be determined by a medical professional or person skilled in the art and can be less than or the same as the amount administered to an adult.

[0084] The compounds or compositions described herein can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and / or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., efficacy and / or effectiveness) in treating a disease in a subject in need of such treatment, preventing a disease in a subject in need of such treatment, or reducing the risk of developing a disease in a subject in need of such treatment), improve bioavailability, improve safety, reduce drug resistance, reduce and / or alter metabolism, inhibit excretion, and / or alter distribution in a subject or cell. It will also be understood that the treatments used may achieve a desired effect for the same disorder and / or may achieve different effects. In certain embodiments, the pharmaceutical compositions described herein that include a compound described herein and an additional pharmaceutical agent exhibit a synergistic effect that is absent in pharmaceutical compositions that include one, but not both, of the compound and the additional pharmaceutical agent. In some embodiments, the additional pharmaceutical agent achieves a desired effect for the same disorder. In some embodiments, the additional pharmaceutical agent achieves a different effect.

[0085] The compound or pharmaceutical composition may be administered simultaneously with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful, for example, as a combination therapy. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration, as defined in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and small organic molecules such as cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for the treatment and / or prevention of a disease (e.g., a proliferative disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disorder, or a metabolic disorder). Each additional pharmaceutical agent may be administered at a dose and / or time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and / or with the compounds or compositions described herein in a single dose or in a single composition, or may be administered separately in different doses or in different compositions. The particular combination used in the regimen will take into account the compatibility of the compounds described herein with the additional pharmaceutical agent(s) and / or the desired therapeutic and / or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) to be combined will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than the levels at which they are utilized individually.

[0086] Additional pharmaceutical agents include, but are not limited to, antiproliferative agents, anticancer agents, antiangiogenic agents, steroidal or nonsteroidal anti-inflammatory agents, immunosuppressants, antibacterial agents, antiviral agents, cardiovascular agents, cholesterol-lowering agents, antidiabetic agents, antiallergic agents, contraceptives, analgesics, anesthetics, anticoagulants, enzyme inhibitors, steroids, steroidal or antihistamines, antigens, vaccines, antibodies, decongestants, sedatives, opioids, analgesics, antipyretics, hormones, and prostaglandins. In certain embodiments, the additional pharmaceutical agent is an antiproliferative agent. In certain embodiments, the additional pharmaceutical agent is an anticancer agent. In certain embodiments, the additional pharmaceutical agent is an antiviral agent. In certain embodiments, the additional pharmaceutical agent is a protein kinase binder or inhibitor. In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic agents (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine kinase inhibitors), protein stability modulators (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acid, and other agents that promote differentiation. In certain embodiments, the compounds or pharmaceutical compositions described herein may be administered in combination with anticancer therapies, including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy. Additional pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved by the U.S. Food and Drug Administration as defined in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.

[0087] The present disclosure also encompasses kits (for example, pharmaceutical packs).The provided kits may include the pharmaceutical compositions or compounds described herein and containers (for example, vials, ampoules, bottles, syringes, and / or dispenser packages or other suitable containers).In some embodiments, the provided kits may optionally further include a second container containing pharmaceutical excipients for diluting or suspending the pharmaceutical compositions or compounds described herein.In some embodiments, the pharmaceutical compositions or compounds described herein provided in the first container and the second container are combined to form a single unit dosage form.

[0088] Thus, in one aspect, a kit is provided, comprising a first container containing the compound or pharmaceutical composition described herein. In certain embodiments, the kit is useful for treating a subject in need of treatment for a disease (e.g., a proliferative disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disorder, or a metabolic disorder). In certain embodiments, the kit is useful for preventing a subject in need of prevention for a disease (e.g., a proliferative disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disorder, or a metabolic disorder). In certain embodiments, the kit is useful for reducing the risk of developing a disease (e.g., a proliferative disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disorder, or a metabolic disorder) in a subject in need of such prevention. In certain embodiments, the kit is useful for inhibiting the activity (e.g., abnormal activity, such as increased activity) of protein kinase in a subject or cell.

[0089] In certain embodiments, the kits described herein further include instructions for using the kit. The kits described herein may also include information as required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescribing information. In certain embodiments, the kits and instructions provide for the treatment of a subject in need of treatment for a disease (e.g., a proliferative disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disorder, or a metabolic disorder). In certain embodiments, the kits and instructions provide for the prevention of a subject in need of prevention for a disease (e.g., a proliferative disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disorder, or a metabolic disorder). In certain embodiments, the kits and instructions provide for the reduction of a subject's risk of developing a disease (e.g., a proliferative disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disorder, or a metabolic disorder) in a subject in need of such prevention. In certain embodiments, the kits and instructions provide for the inhibition of protein kinase activity (e.g., abnormal activity, such as increased activity) in a subject or cell. The kits described herein may also include one or more additional pharmaceutical agents described herein as separate compositions.

[0090] The term "polymer" refers to a compound comprising 11 or more covalently joined repeating units. In certain embodiments, the polymer is naturally occurring. In certain embodiments, the polymer is synthetic (i.e., not naturally occurring).

[0091] The term "about X", where X is a number or percentage, refers to a number or percentage that is between 99.5% and 100.5%, between 99% and 101%, between 98% and 102%, between 97% and 103%, between 96% and 104%, between 95% and 105%, between 92% and 108%, or between 90% and 110% (inclusive) of X.

[0092] The term "particle" refers to a small object, fragment, or piece of material, which may be a single element, an inorganic material, an organic material, or a mixture thereof. Examples of particles include polymer particles, single emulsion particles, double emulsion particles, coacervates, liposomes, microparticles, nanoparticles, macroparticles, pellets, crystals, aggregates, complexes, micronized, crushed, or otherwise disrupted matrices, and cross-linked protein or polysaccharide particles, each of which has a characteristic average dimension of less than about 1 mm and at least 1 nm, where the characteristic dimension or "critical dimension" of a particle is the smallest cross-sectional dimension of the particle. A particle may be composed of a single material or multiple materials. In certain embodiments, the particle is not a virus particle. In other embodiments, the particle is not a liposome. In certain embodiments, the particle is not a micelle. In certain embodiments, all of the particles are substantially solid. In certain embodiments, the particle is a nanoparticle. In certain embodiments, the particle is a microparticle.

[0093] Detailed Description of Specific Embodiments The aspects described herein are not limited to specific embodiments, systems, compositions, methods, or configurations, which, as such, can, of course, vary. The terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, unless specifically defined herein.

[0094] composition Escherichia coli In one aspect, provided herein is a composition comprising Escherichia coli (E. coli) and a first stabilizing excipient selected from an enzymatic digest of soybean (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, potassium gluconate, melibiose, sucrose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), L-rhamnose monohydrate, (+)-L-sodium ascorbate, an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), trehalose dihydrate, D-(+)-galactose, the water-soluble portion of malted barley (e.g., Bacto malt extract), D-(+)-melezitose monohydrate, β-lactose, D(-)-fructose, glucose, 1-kestose, or a combination thereof.

[0095] In certain embodiments, the first stabilizing excipient is selected from an enzymatic digest of soybeans (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, melibiose, sodium (+)-L-ascorbate, trehalose dihydrate, β-lactose, glucose, potassium gluconate, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from an enzymatic digest of soybeans (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, melibiose, sodium (+)-L-ascorbate, trehalose dihydrate, β-lactose, glucose, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from an enzymatic digest of soybean (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, melibiose, sodium (+)-L-ascorbate, trehalose dihydrate, β-lactose, glucose, or a combination thereof. In certain embodiments, the first stabilizing excipient is melibiose.

[0096] In certain embodiments, the composition comprises a second stabilizing excipient selected from short-chain inulin, a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract), an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), porcine mucin type III, 1,4-benzenedimethanol, caffeine, an enzymatic digest of soybean (e.g., Bacto soytone), sucralose, dioctyl sulfosuccinate, 2-methyl-1-propanol, propyl gallate, β-glycerophosphate disodium salt hydrate, DL-β-(2-thienyl)serine, melibiose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), sodium citrate dihydrate, N-phenylthiourea, 4-guanidinobutyric acid, calcium D-gluconate, or a combination thereof. In certain embodiments, the second stabilizing excipient is selected from short-chain inulin, a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract), caffeine, or a combination thereof. In certain embodiments, the second stabilizing excipient is selected from a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract), caffeine, or a combination thereof.

[0097] In certain embodiments, the first stabilizing excipient is melibiose, and the second stabilizing excipient is a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract) or caffeine. In certain embodiments, the first stabilizing excipient is present at a concentration of about 7% to about 50% (w / w). In certain embodiments, the first stabilizing excipient is melibiose, present at a concentration of about 0.3% to about 9% (w / w). In certain embodiments, the second stabilizing excipient is present at a concentration of about 0.01% to about 7% (w / w).

[0098] In certain embodiments, the second stabilizing excipient is a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract) present at a concentration of about 0.04% to about 7% (w / w). In certain embodiments, the first stabilizing excipient is melibiose present at a concentration of about 1% (w / w) and the second stabilizing excipient is a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract) present at a concentration of about 0.04% (w / w) or about 5% (w / w).

[0099] In certain embodiments, the second stabilizing excipient is caffeine present at a concentration of about 0.01% to about 0.4% (w / w). In certain embodiments, the first stabilizing excipient is melibiose present at a concentration of about 1% (w / w) and the second stabilizing excipient is caffeine present at a concentration of about 0.01% (w / w).

[0100] In a particular embodiment, the E. coli is E. coli Nissle 1917.

[0101] Saccharomyces boulardii In another aspect, provided herein is a composition comprising Saccharomyces boulardii (S. boulardii) and a first stabilizing excipient selected from L-glutamic acid monosodium salt monohydrate, skim milk powder, D-(+)-turanose, the water-soluble portion of malted barley (e.g., Bacto malt extract), maltitol, melibiose, lactulose, D-(+)-raffinose pentahydrate, palatinose hydrate, sucrose, animal-origin pancreatic digest of casein (e.g., Bacto tryptone), glucose, enzymatic digest of soybean (e.g., Bacto soytone), potassium gluconate, polydextrose, sodium gluconate, or a combination thereof.

[0102] In certain embodiments, the first stabilizing excipient is selected from L-glutamic acid monosodium salt monohydrate, skim milk powder, D-(+)-turanose, the water-soluble portion of malted barley (e.g., Bacto malt extract), maltitol, melibiose, D-(+)-raffinose pentahydrate, palatinose hydrate, sucrose, animal-origin pancreatic digest of casein (e.g., Bacto tryptone), glucose, potassium gluconate, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from the water-soluble portion of malted barley (e.g., Bacto malt extract), D-(+)-turanose, D-(+)-raffinose pentahydrate, L-glutamic acid monosodium salt monohydrate, maltitol, palatinose hydrate, skim milk powder, melibiose, sucrose, glucose, animal-derived pancreatic digest of casein (e.g., Bacto tryptone), or combinations thereof.

[0103] Lactobacillus plantarum In another aspect, provided herein is a composition comprising Lactobacillus plantarum (L. plantarum) and a first stabilizing excipient selected from a pancreatic digest of gelatin (e.g., Gelysate peptone), the water-soluble portion of malted barley (e.g., Bacto malt extract), an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), sucrose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), D-sorbitol, polydextrose, trehalose dihydrate, L-glutamic acid monosodium salt monohydrate, β-lactose, maltodextrin, L-rhamnose monohydrate, 1-kestose, an animal-origin enzymatic digest of bovine and porcine animal proteins (e.g., Bacto peptone), chondroitin sulfate A, or a combination thereof.

[0104] In certain embodiments, the first stabilizing excipient is selected from the water-soluble portion of malted barley (e.g., Bacto malt extract), a pancreatic digest of gelatin (e.g., Gelysate peptone), an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), sucrose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), D-sorbitol, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from the animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), sucrose, a pancreatic digest of gelatin (e.g., Gelysate peptone), D-sorbitol, an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), or a combination thereof.

[0105] Ensifer meliloti In another aspect, provided herein is a composition comprising Ensifer meliloti (E. meliloti) and a first stabilizing excipient selected from an enzymatic digest of soybean (e.g., Bacto soytone), skim milk powder, polydextrose, the water-soluble portion of malted barley (e.g., Bacto malt extract), L-glutamic acid monosodium salt monohydrate, maltodextrin, palatinose hydrate, D-(+)-melezitose monohydrate, trehalose dihydrate, 1-kestose, maltose monohydrate, α-lactose monohydrate, sucrose, D-cellobiose, D-(+)-raffinose pentahydrate, melibiose, or a combination thereof.

[0106] In certain embodiments, the first stabilizing excipient is selected from an enzymatic digest of soybeans (e.g., Bacto soytone), skim milk powder, polydextrose, the water-soluble portion of malted barley (e.g., Bacto malt extract), L-glutamic acid monosodium salt monohydrate, maltodextrin, palatinose hydrate, D-(+)-melezitose monohydrate, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from an enzymatic digest of soybeans (e.g., Bacto soytone), skim milk powder, polydextrose, the water-soluble portion of malted barley (e.g., Bacto malt extract), L-glutamic acid monosodium salt monohydrate, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from an enzymatic digest of soybeans (e.g., Bacto soytone), skim milk powder, L-glutamic acid monosodium salt monohydrate, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from trehalose dihydrate, 1-kestose, palatinose hydrate, maltose monohydrate, α-lactose monohydrate, an enzymatic digest of soybean (e.g., Bacto soytone), D-(+)-melezitose monohydrate, sucrose, D-cellobiose, D-(+)-raffinose pentahydrate, melibiose, polydextrose, maltodextrin, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from trehalose dihydrate, 1-kestose, palatinose hydrate, maltose monohydrate, α-lactose monohydrate, enzymatic digest of soybean (e.g., Bacto soytone), D-(+)-melezitose monohydrate, sucrose, D-cellobiose, D-(+)-raffinose pentahydrate, melibiose, or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from sucrose, maltose monohydrate, polydextrose, maltodextrin, enzymatic digest of soybean (e.g., Bacto soytone), or a combination thereof. In certain embodiments, the first stabilizing excipient is selected from sucrose, maltose monohydrate, polydextrose, or a combination thereof.In certain embodiments, E. meliloti is equivalent to Sinorhizobium meliloti (S. meliloti).

[0107] Additional Aspects In certain embodiments, the composition is any of the compositions disclosed herein.

[0108] In certain embodiments, the composition has a cell proliferation rate of at least about 1 x 10 after 30 days at 23°C. 6 , at least about 1 x 10 7 , at least about 1 x 10 8 , at least about 1 x 10 9 , or at least about 1 × 10 10 The colony forming units (CFU) / gram are shown.

[0109] In certain embodiments, the composition is stable at about 23°C, about 37°C, or about 50°C for at least one day. In certain embodiments, the composition is stable at about 23°C, about 37°C, or about 50°C for at least one month. In certain embodiments, the composition is stable at less than about 5% relative humidity for at least one day. In certain embodiments, the composition is stable at less than about 5% relative humidity for at least one month. In certain embodiments, the composition is stable at about 23°C, about 37°C, or about 50°C and less than about 5% relative humidity for at least one day. In certain embodiments, the composition is stable at about 23°C, about 37°C, or about 50°C and less than about 5% relative humidity for at least one month. In certain embodiments, the composition maintains at least about 10% viability at about 23°C, about 37°C, or about 50°C for at least about 6.5 months.

[0110] In certain embodiments, the composition is stable upon exposure to organic solvents, hi certain embodiments, the organic solvent is acetone or isopropanol.

[0111] In certain embodiments, the composition maintains at least about 5%, at least about 10%, or at least about 15% viability upon comminution, hi certain embodiments, comminution comprises exposure to shear forces.

[0112] In certain embodiments, the composition maintains at least about 5%, at least about 10%, or at least about 15% viability upon tableting. In certain embodiments, tableting involves application of a force of about 3.4 kN per tablet. In certain embodiments, tableting involves application of a force of up to about 6 kN per tablet. In certain embodiments, tableting involves exposure to increased pressure. In certain embodiments, the increased pressure is about 173 uPa per tablet. In certain embodiments, the increased pressure is up to about 650 uPa per tablet.

[0113] In certain embodiments, the composition maintains at least about 20% or at least about 28% viability during wet granulation. In certain embodiments, the wet granulation comprises exposure to an organic solvent and baking. In certain embodiments, the organic solvent is isopropanol. In certain embodiments, the baking comprises exposure to a temperature of at least about 45°C.

[0114] In certain embodiments, the composition maintains at least about 0.2%, at least about 1%, or at least about 3% viability upon spray coating. In certain embodiments, the spray coating comprises exposure to an organic solvent and a polymer, and baking. In certain embodiments, the organic solvent is acetone and / or isopropanol. In certain embodiments, the polymer is an enteric polymer. In certain embodiments, the enteric polymer is Eudragit L100-55, Eudragit L100, Eudragit S100, or a combination thereof. In certain embodiments, the baking comprises exposure to a temperature of at least about 40°C.

[0115] In certain embodiments, the composition is stable upon exposure to acid. In certain embodiments, the composition is stable after exposure to a solution having a pH of about 1.2 for at least about 1 hour at about 37° C. In certain embodiments, the composition maintains at least about 90% or at least about 95% viability after exposure to a solution having a pH of about 1.2 for at least about 1 hour at about 37° C.

[0116] In certain embodiments, the composition is stable upon exposure to ionizing radiation, hi certain embodiments, the ionizing radiation is at least about 0.01 kGy, at least about 0.1 kGy, or at least about 1 kGy.

[0117] In certain embodiments, the composition is stable upon exposure to ionizing radiation in space. In certain embodiments, the composition is stable upon exposure to ionizing radiation in space between Earth and Mars. In certain embodiments, the composition is stable upon exposure to ionizing radiation on the Earth's surface. In certain embodiments, the composition is stable upon exposure to ionizing radiation on the surface of Mars. In certain embodiments, the composition is stable upon exposure to ionizing radiation in space between Earth and the International Space Station (ISS). In certain embodiments, the composition is stable upon exposure to ionizing radiation outside the ISS.

[0118] In certain embodiments, the composition is capable of inducing bacterial growth in the plant. In certain embodiments, inducing bacterial growth comprises inducing nodulation in the plant.

[0119] In certain embodiments, the composition is capable of inhibiting an enteric pathogen, hi certain embodiments, the enteric pathogen is Shigella flexneri.

[0120] In certain embodiments, the composition comprises a solid dosage form. In certain embodiments, the solid dosage form is a tablet. In certain embodiments, the solid dosage form comprises an immediate-release layer. In certain embodiments, the solid dosage form comprises a sustained-release layer. In certain embodiments, the sustained-release layer comprises a matrix-forming polymer. In certain embodiments, the matrix-forming polymer is hydroxypropylmethylcellulose (HPMC).

[0121] In certain embodiments, the composition further comprises an additional microorganism.

[0122] In certain embodiments, E. coli, S. boulardii, L. plantarum, or E. meliloti is pre-cultured in a medium containing peptone, extract, and salt concentration. In certain embodiments, the peptone is an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), an enzymatic digest of soybean (e.g., Bacto soytone), a pancreatic digest of gelatin (e.g., gelysate peptone), an animal-origin enzymatic digest of bovine and porcine animal proteins (e.g., Bacto peptone), or a combination thereof. In certain embodiments, the extract is an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract), the water-soluble portion of malted barley (e.g., Bacto malt extract), or a combination thereof. In certain embodiments, the salt concentration is from about 50 mM to about 800 mM NaCl.

[0123] Methods of Microbial Delivery, Induction of Bacterial Growth, Inhibition of Enteric Pathogens, and Treatment - Patent application Provided herein are methods of microbial delivery, bacterial growth induction, enteric pathogen inhibition, and treatment, which comprise administering a "composition" or "provided composition," wherein the composition or provided composition is any of the compositions disclosed herein.

[0124] In another aspect, provided herein are methods for delivering Escherichia coli (E. coli) to a subject in need thereof, comprising administering to the subject a provided composition. In certain embodiments, a method for delivering E. coli comprises administering to a subject a composition comprising E. coli and a first stabilizing excipient selected from an enzymatic digest of soybean (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, potassium gluconate, melibiose, sucrose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), L-rhamnose monohydrate, (+)-L-sodium ascorbate, an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), trehalose dihydrate, D-(+)-galactose, the water-soluble portion of malted barley (e.g., Bacto malt extract), D-(+)-melezitose monohydrate, β-lactose, D(-)-fructose, glucose, 1-kestose, or a combination thereof.

[0125] In another aspect, provided herein is a method for delivering Saccharomyces boulardii (S. boulardii) to a subject in need thereof, the method comprising administering to the subject a provided composition. In certain embodiments, a method for delivering S. boulardii comprises administering to a subject a composition comprising S. boulardii and a first stabilizing excipient selected from L-glutamic acid monosodium salt monohydrate, skim milk powder, D-(+)-turanose, the water-soluble portion of malted barley (e.g., Bacto malt extract), maltitol, melibiose, lactulose, D-(+)-raffinose pentahydrate, palatinose hydrate, sucrose, animal-origin pancreatic digest of casein (e.g., Bacto tryptone), glucose, enzymatic digest of soybean (e.g., Bacto soytone), potassium gluconate, polydextrose, sodium gluconate, or a combination thereof.

[0126] In another aspect, provided herein is a method for delivering Lactobacillus plantarum (L. plantarum) to a subject in need thereof, the method comprising administering to the subject a provided composition. In certain embodiments, a method for delivering L. plantarum includes administering to a subject a composition comprising L. plantarum and a first stabilizing excipient selected from a pancreatic digest of gelatin (e.g., Gelysate peptone), the water-soluble portion of malted barley (e.g., Bacto malt extract), an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), sucrose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), D-sorbitol, polydextrose, trehalose dihydrate, L-glutamic acid monosodium salt monohydrate, β-lactose, maltodextrin, L-rhamnose monohydrate, 1-kestose, an animal-origin enzymatic digest of bovine and porcine animal proteins (e.g., Bacto peptone), chondroitin sulfate A, or a combination thereof.

[0127] In another aspect, provided herein is a method for delivering Ensifer meliloti (E. meliloti) to a subject in need thereof, the method comprising administering to the subject a provided composition. In certain embodiments, a method for delivering E. meliloti comprises administering to a subject a composition comprising E. meliloti and a first stabilizing excipient selected from an enzymatic digest of soybean (e.g., Bacto soytone), skim milk powder, polydextrose, the water-soluble portion of malted barley (e.g., Bacto malt extract), L-glutamic acid monosodium salt monohydrate, maltodextrin, palatinose hydrate, D-(+)-melezitose monohydrate, trehalose dihydrate, 1-kestose, maltose monohydrate, α-lactose monohydrate, sucrose, D-cellobiose, D-(+)-raffinose pentahydrate, melibiose, or a combination thereof.

[0128] In another aspect, the present invention provides a method for inducing bacterial growth in a subject or in a cell, tissue, or biological sample, comprising administering to the subject or contacting with the provided composition the cell, tissue, or biological sample.In certain embodiments, the cell, tissue, or biological sample is a plant.In certain embodiments, the method comprises inducing nodule formation in the plant.

[0129] In another aspect, provided herein is a method for inhibiting an enteric pathogen in a subject or in a cell, tissue, or biological sample, comprising administering to the subject or contacting with the cell, tissue, or biological sample a provided composition. In certain embodiments, the enteric pathogen is Shigella flexneri.

[0130] In another aspect, provided herein are methods of treating dysbiosis in a subject in need thereof, comprising administering to the subject a provided composition. In certain embodiments, the subject in need thereof has a gastrointestinal disorder.

[0131] example Example 1 Microorganisms are central to human technological advances, from the production of food (e.g., bread and baked goods) to the production of biopharmaceuticals (e.g., synthetic insulin). 1 Microbial cells remain important in a wide range of fields, from the production of pharmaceuticals to the manufacture of pharmaceuticals. However, by and large, microbial cells are kept alive only during the manufacturing process and are destroyed, inactivated, or removed from the final product. However, through the advent of culture-independent sequencing techniques and synthetic biology, the fields of medicine, agriculture, and space health are now exploring the potential of microbial cells to treat disease. 2 to increase crop yields 3 for and on-demand bioproduction 4 We are focusing on the development of live microorganisms as end products for

[0132] Critical to these new microbial technologies is the maintenance of high cell viability throughout the product's life cycle. To achieve this, researchers and commercial companies are developing methods for microbial cryopreservation. 5 or organisms with natural extremophilic properties (e.g., dehydration resistance, acid tolerance) 6 However, the need for carefully selected organisms or an expensive and inflexible cold chain severely limits the use cases of live microbial products.

[0133] An alternative solution would be to develop shelf-stable dried microbial materials that are easy to package, transport, and use. Although freeze-drying preservation of microorganisms has been widely studied, these previous studies have focused primarily on the storage of microorganisms in culture collections, which require only an "acceptable" level of viability (i.e., enough live cells for recovery through culture amplification). 10Furthermore, although these studies generally define sugars and peptides with high glass transition temperatures as good stabilizers, they also show that specific viability results vary widely depending on the microorganism in question. 10 .

[0134] Therefore, applications of this freeze-drying preservation knowledge were explored with the goal of generating the high viability that would be required for functional microbial products. To understand the current state of microbial stabilization, available commercial examples of dried microbial products (i.e., probiotics) were evaluated. Low viability was generally found, with particularly poor results for Gram-negative bacteria (e.g., E. coli). To fill this gap, materials-based synthetic extremophiles were developed that can be stored at ambient temperature without refrigeration. These stabilized microorganisms can withstand extreme conditions (e.g., heat, pressure, and solvents) encountered in pharmaceutical manufacturing pipelines. This stabilization approach preserves the functional competence of the microorganisms in both bioluminescence and plant root nodulation assays.

[0135] Example 2: Survey of commercial probiotic products One striking example of a dried microbial product is the currently commercially available probiotics marketed for human use. These products represent the current state-of-the-art in microbial stabilization for commercial applications. However, when viable cell counts (colony-forming units, CFU) were examined across a variety of pre-prepared probiotics (Table 1), only 7 of 13 products were found to contain viable cell counts at or above the label-predicted amount (Figure 1, Figures 5A-5B), with a (geometric) mean viability of only approximately 21% of the predicted viability. Nevertheless, when total cell counts (both dead and live cells) were assessed microscopically, all products contained total cell numbers exceeding the predicted cell count (Figure 5A), suggesting a loss of viability during and after manufacturing. When the viable cell counts were compared to this microscopically determined total cell count, only 1 of 13 products had a viability greater than 40%, and 6 of 13 products had a viability less than 2%, for an overall mean viability of only 1.9% of total cells (Figure 1).

[0136] In addition to this overall low viability, a lack of diversity in terms of microbial species identity was also found (Figure 5B). The majority of products (9 of 13) used microorganisms from only two groups (Lactobacillus and Bifidobacterium), which encompassed all products with higher viability (Figure 5B). There was only one commercially available representative of a large clade of Gram-negative bacteria (Mutaflor, Escherichia coli Nissle 1917), which had the lowest viability (0.05% of potential cells and 0.01% of total cells). To support this finding, one additional commercial Gram-negative product (Nitragin Gold, Ensifer meliloti) marketed for plant use was tested and also found to have low viability (10% of potential cells and 1% of total cells) (Figure 1).

[0137] To further assess their inherent extremophilic qualities, these products were stress-tested by exposure to temperatures of 50°C for 24 hours. Nearly all products were found to maintain acceptable viability of >10% relative to non-stressed samples (Figures 6A-6B). However, the E. coli Nissle 1917 product Mutaflor maintained only 0.002% viability (Figures 6A-6B). Overall, the above studies point to an industry focused on a small number of easily stabilized Gram-positive microorganisms and poor options for Gram-negative organisms such as E. coli Nissle 1917. In contrast, the fields of synthetic biology and microbial therapeutics research in academia are focused on Gram-negative bacteria, with many of the most promising examples (e.g., cancer treatments) specifically using E. coli Nissle 1917. 2、7、8 .

[0138] Example 3: A high-throughput pipeline for generating synthetic extremophiles To overcome the discrepancy between the commercial and academic fields, a high-throughput pipeline for generating synthetic extremophiles was developed (Figure 2A, Example 8). Synthetic extremophiles were defined as microbial preparations with an enhanced ability to survive insults such as desiccation, heat, and exposure to pressure, acid, organic solvents, and ionizing radiation. Similar to natural extremophiles (e.g., spore-forming organisms, tardigrades), such resilience would enable synthetic extremophiles to survive various manufacturing processes and long-term storage demands in the pharmaceutical, agricultural, and space health fields.

[0139] The synthetic extremophiles that survive in the dry state are then applied to a dry powder using material formulation techniques (e.g., coating). 9It was hypothesized that the 260 materials could be enhanced to survive additional environmental hazards. Therefore, a library composed primarily of materials generally regarded as safe (GRAS) by the Food and Drug Administration (FDA) was screened for their ability to protect microorganisms through lyophilization and survive at room temperature for 24 hours (Table 2). This material library was applied to four technologically important and genetically traceable microorganisms (Escherichia coli Nissle 1917, Ensifer meliloti, Saccharomyces boulardii, and Lactobacillus plantarum) spanning a wide range of phylogenetic backgrounds. Each microorganism was mixed with each of the 260 materials at two different concentrations to yield 2,080 microorganism-material formulations.

[0140] The residual viability of each formulation was measured via colony counts assessed in a high-throughput fashion, resulting in a normalized viability score (Figure 2B, Figures 7A-7C, and Example 8). The included negative controls (i.e., the antimicrobial compounds sodium metabisulfite and sodium hydroxide) consistently yielded a viability score of 0, while the positive controls (i.e., the known lyoprotectants trehalose and ATCC Reagent 20) consistently yielded measurable viability (Figure 2C and Figure 8, black boxes). In addition, sugars and peptones were over-represented in the top-performing formulations (Figure 9). Surprisingly, neither the positive control nor any single material was a top performer across the various organisms. Instead, these results indicate ideal species-specific formulations with little overlap in best-performing materials even between two Gram-negative representatives (i.e., E. coli Nissle 1917 and E. meliloti) (Figure 2D).

[0141] The top-performing materials for each of the four microorganisms were then further stratified by assessing colony counts after long-term storage of 1 month at room temperature and <5% relative humidity (Figure 2E). These conditions were chosen to differentiate microorganism-material combinations by their heat tolerance. This contrasts with current microbial freeze-drying practices, which still require refrigeration to retain acceptable viability in the dry state. 10 This stratification also provided a fingerprint of the intrinsic extremophilic qualities of the selected organisms. For example, these results indicate that L. plantarum already possessed good heat tolerance, as nearly all top-performing materials were indistinguishable after storage (Figure 2E). Conversely, the two Gram-negative bacteria showed a sharp decline, with only a few materials retaining their initial viability over time. These results further support the probiotic viability studies described herein and suggest that the lactic acid bacteria that dominate the market may be inherently better tolerated and more susceptible to stabilization by a wide range of materials.

[0142] Example 4: Optimization of E. coli Nissle 1917 synthetic extremophile Due to its importance in the fields of microbial therapeutics and synthetic biology, the extremophilic qualities of E. coli Nissle 1917 were further enhanced. This screening showed that previously established mixtures, such as the positive control ATCC Reagent 18, tended to outperform the individual components that made up the mixture (i.e., tryptic soy broth, sucrose, and albumin for ATCC Reagent 18). Therefore, two-material combinations of library members and the best-performing material (melibiose) were next compared to single-material formulations, and residual viability after 24 hours was assessed both at room temperature and at 50°C (Figure 3A). Top hits were defined as material combinations with viability scores above the control (melibiose + vehicle). Library members that were themselves mixtures (e.g., LB broth) or of animal origin (e.g., mucin) were excluded to facilitate future manufacturing. The stability of selected two-ingredient formulations (melibiose combined with fructooligosaccharides, caffeine, or yeast extract) was analyzed in larger formats over extended storage times at 37°C, and mixtures with caffeine or yeast extract were found to be robust (Figure 10). The relative concentrations of melibiose, caffeine, and yeast extract were titrated to find optimal formulations D and E (Figure 2B).

[0143] When stored at room temperature for 1 month, both melibiose and Formulation D outperformed the commercial product Mutaflor (E. coli Nissle 1917) by more than 3.5 orders of magnitude (Figure 2C). To confirm these results and eliminate any manufacturing bias (i.e., freshly made vs. ready-made), E. coli Nissle 1917 was formulated in maltodextrin (the stabilizer used in Mutaflor) using the disclosed freeze-drying process. This "commercially-as-prepared" dried E. coli Nissle 1917 was compared to the top-performing formulation. At 37°C, the maltodextrin-stabilized material was found to have lost all measurable viability (>4 orders of magnitude) within 11 days (Figure 2D). In contrast, Formulation D maintained >10% viability even after 6.5 months at 37°C (Figure 2D and Figure 11). This was >2 orders of magnitude better than the non-optimized melibiose formulation. In addition to preserving relative viability, absolute CFU counts can be optimized by varying the growth medium (Figure 12), time of harvest (Figure 13), and percent bacterial loading (Figure 14), thereby achieving >10 CFU counts per gram. 10 It was also shown that CFU viable synthetic extremophiles could be produced (Figure 15).

[0144] To understand the mechanism of protection, transmission electron microscopy (TEM) was used to compare the ultrastructure of E. coli Nissle 1917 cells dried in either the high-performing formulation D or a low-performing commercial formulation (maltodextrin). Formulation D samples had a significantly lower fraction of cells with defective cell envelopes (i.e., periplasmic spaces filled with electron-dense material and the inner membrane separated from the outer membrane) (Figure 3E and Figures 19A-19D). This difference between formulation D and maltodextrin was even greater for samples exposed to 50°C (Figure 19D). Protection against this membrane defect could occur through several mechanisms, but one potential mechanism involves membrane protein stabilization essential for survival of the rehydration process. This hypothesis is supported by the observation that a stationary-phase pssA null E. coli mutant, lacking phosphatidylethanolamine and resulting in membrane protein misfolding, also develops this specific membrane defect. 23、24 .

[0145] Example 5: Compatibility of synthetic extremophiles with pharmaceutical manufacturing processes These synthetic extremophiles open up a field of potential use cases for microbial products to treat disease, enhance agricultural yields, and support exploratory space travel (Figure 4A). One opportunity for human and animal health would be the ability to create dosage forms with tailored release parameters (e.g., delayed release, enteric coating, etc.). However, this requires applying pharmaceutical manufacturing techniques such as milling (exposure to shear forces), wet granulation (exposure to isopropanol and baking), tableting (exposure to high pressure), and spray coating (exposure to acetone and baking), which are typically too harsh for living cells. Therefore, the E. coli Nissle 1917 synthetic extremophile was subjected to this series of processes (Figure 4B) and its robustness compared to current commercial alternatives (maltodextrin formulations) was characterized (Figure 4C). It was found that synthetic extremophiles could be milled, mixed with excipients (i.e., binders, lubricants, fillers), and compressed at room temperature in open air and still maintain >15% viability (Figure 4C), 2.4 orders of magnitude higher than the commercial control (0.06% viability). Wet granulation was also well tolerated (>28% viability), and compression pressure was found to modulate ultimate storage stability (Figure 15).

[0146] Once tableted, the synthetic extremophiles also survived spray coating with an acetone solution of an enteric polymer (Eudragit L100-55), whereas the commercial alternative lost all viability (Figure 2C). In this condition previously inaccessible to E. coli Nissle 1917, the synthetic extremophiles were found to maintain 100% viability through a 1-hour exposure to simulated gastric fluid at pH 1.2 at 37°C (Figure 17).

[0147] Example 6: Compatibility of synthetic extremophiles with ionizing radiation Next, to verify the suitability of the synthetic extremophile organism for use in exploratory spaceflight, its robustness was characterized by exposure to various ionizing radiation doses ranging from 100 to 10,000 Gy (typical radiation dose values: approximately 15 uGy / day at Earth's surface).11 , approximately 200 uGy / day on the moon 12 , approximately 250 uGy / day on the Martian surface 13 The total cumulative dose for the 500-day journey to Mars was 0.32 Gy 14 , and 0.41 Gy for 444 days outside the ISS 15 The maximum value of 10 kGy represents the generally accepted dose for radiation-based sterilization of food. 16 The synthetic extremophile was able to withstand exposures up to 1000 Gy (Figure 4E). At this radiation level, a liquid suspension of the same bacteria lost all measurable viability.

[0148] Example 7: Functional characterization of synthetic extremophiles. Next, we determined whether the synthetic extremophilic organisms could maintain functionality in the tableted state and whether their functionality could be tailored through formulation design. To test this, we incorporated a bioluminescence biosynthetic pathway (luxCDABE) that produces luminescence upon reconstitution. While the dry tablets did not produce measurable luminescence, 48% of the final luminescence level was achieved within 15 minutes of reconstitution (Figure 4D). Replacing the tablet filler with the matrix-forming material hydroxypropyl methylcellulose (HPMC) resulted in the sustained release of bacteria over a 24-hour period and the tailoring of luminescence function kinetics (Figure 4D). Combining immediate-release and sustained-release tablet layers can be used to create mixed-microbial pills that seed different bacteria at different rates in the host microbiome.

[0149] Next, it was determined whether the ability of E. coli Nissle 1917 to inhibit enteric pathogens was affected by the formulation. 25 Specifically, Shigella flexneri, the leading cause of diarrhea-related deaths in low- and middle-income countries, 26Inhibition of S. flexneri was characterized. Both E. coli Nissle 1917 and its synthetic extremophile counterpart (Formulation D) inhibited S. flexneri growth by >99.8% in coculture, whereas the commercial comparator (maltodextrin) formulation showed no increased efficacy compared to a non-inhibited strain of E. coli (Figure 4F). Control experiments showed that the stabilizers alone did not cause inhibition or alter the inhibitory effect when added to fresh E. coli Nissle 1917 cells (Figures 20A-B).

[0150] Finally, due to its potential to increase the sustainability of agricultural systems with reduced need for chemical fertilizers, it was determined whether the disclosed approach to creating synthetic extremophiles would interfere with the complex biological process of symbiotic nitrogen fixation in plant roots. 3、17 This symbiotic process occurs between legumes and soil bacteria (rhizobia), through which the formation of nodules that house the bacteria allows the bacteria to fix atmospheric nitrogen (N2) into a form that can be used by the plant. 17 Specifically, the nodulation of the nitrogen-fixing bacterium E. meliloti on the model legume Medicago truncatula, which was used to study this process, was evaluated. 17、27 .

[0151] Although E. meliloti is currently used as a seed inoculant prior to planting, there remains a need for improved stability of microbial products during formulation, storage, and seed inoculation, particularly with regard to tolerance to temperatures above 40°C. 28、29Therefore, to generate synthetic extremophilic versions of E. meliloti, a heat shock screen was repeated at 50°C to identify materials that would confer heat resistance to the synthetic E. meliloti (Figure 18). From these, three optimal materials (sucrose, soytone, and maltose) and two suboptimal materials (maltodextrin and polydextrose) were selected. E. meliloti was formulated in each, subjected to a 50°C shock for 24 hours, reconstituted, and applied to M. truncatula A17 seedlings (Figure 4G). Functional symbiosis was measured as the percentage of seedlings that successfully nodulated. Synthetic E. meliloti extremophiles made with sucrose and maltose successfully nodulated, whereas extremophiles made with soytone caused plant stunting (Figure 4G-H). These results demonstrate that the high-throughput pipeline described herein can define a set of materials that are not only ideal for a target microbial species, but can also be further selected for species suited to specific target applications, which may involve complex biological processes such as nodulation.

[0152] In summary, presented herein is an approach to identify materials that can confer tolerance to extreme environments to microorganisms. These synthetic extremophiles will transform the power of bioactive organisms to propagate across human applications, from shelves around the world to agricultural practices to space shuttles.

[0153] Example 8 Viability survey of commercial probiotic and microbial products (Figure 1, Figures 5A-5B).

[0154] All products were purchased and analyzed before their expiration date. Specific product names, lot numbers, expiration dates, analysis dates, and dosage forms are summarized in Table 1. All procedures were analyzed under sterile conditions. The dosage forms encountered were two-piece capsules filled with loose powder, packets filled with loose powder, or tablets. In each instance, only the microbial fraction was analyzed. Specifically, for capsules, the two pieces were carefully separated, and the contents were emptied onto paper for further analysis. For packets, the packets were carefully torn open, and the contents were emptied onto paper for further analysis. For tablets, the tablets were placed in a Ziploc bag and crushed by manually rolling a plastic tube over the tablets, and the resulting powder was emptied onto paper for further analysis.

[0155] The expected colony-forming unit (CFU) count per dose was recorded from the product packaging and converted to CFU per gram by dividing by the average mass of the four doses. For Florastor, the amount of yeast cells is reported as milligrams per dose, which is the approximate cell dry weight of the yeast (approximately 20 pg, 32% of 60 pg) [BNID 101795, BNID 105094]. 18 The dose mass was converted to CFU per dose by dividing by . The dose mass only includes the recovered microbial fraction (i.e., the contents of the dosage form), as described above. If a product reports both "as manufactured" and "until expiration" CFU counts, only the "until expiration" CFU count was used. This data is summarized in Table 1.

[0156] The viable CFU count per dose was determined by reconstituting one dose on ice at the specified reconstitution ratio in phosphate-buffered saline (PBS, ThermoFisher 10010049), making 10-fold serial dilutions in PBS on ice, plating 100 μL of each dilution onto the appropriate solid medium in a 100 mm round Petri dish with 4.5 mm plating beads (Zymo), incubating under appropriate conditions, and counting the colonies on the dilution that yielded approximately 200–1,000 individual colonies. Four or five separate doses of each product were prepared as described. As noted in Table 1, the reconstitution ratio was typically one dose in 50 mL; however, in a few cases, the reconstitution volume was reduced when low cell counts were found during initial analysis. The media and culture conditions used for each product were selected based on the microorganisms listed on the package and are listed in Table 1. All media components were purchased from BD Life Sciences as specified in Table 3, solidified with 1.5% w / v Bacto Agar (BD, 214010), and prepared according to label instructions. For aerobic conditions, plates were incubated in a static incubator. For anaerobic conditions, plates were first placed in a BD GasPak EZ Gas Generating Systems container (BD, 260002) with an anaerobic pouch (BD, 260001) and then incubated in a static incubator. Colony counts were first obtained by imaging plates in a gel imager (ChemiDoc XRS+, BioRad) using UV transillumination and a standard emission filter (580 / 120), followed by FIJI (imageJ). 19The viable CFU counts per dose were then converted to CFU counts per gram by dividing by the mass of the particular dose analyzed. The dose only encompasses the microbial fraction recovered (i.e., the contents of the dosage form), as described above.

[0157] Total cell counts per dose were determined using an automated microscopic cytometer (QUANTOM Tx Microbial Cell Counter, Logos Biosystems) according to the manufacturer's protocol. Specifically, a 10-µL sample of the reconstituted dose used for viable CFU counts (above) was mixed on ice with 2 µL of a 1:1 mixture of whole cell dye and enhancer (Logos Biosystems, Q13501). Then, 8 µL of cell loading buffer (Logos Biosystems, Q13501) and 6 µL of the mixture were loaded onto a hemocytometer slide (Logos Biosystems, Q12001). The slide was centrifuged at 300 rcf for 10 minutes in a slide microcentrifuge (Logos Biosystems, Q10002) and then loaded into the cell counter. Four or five separate doses of each product were prepared as described, and each sample was loaded and quantified in duplicate. For products that gave counts greater than 1e9 cells per mL during the initial analysis, the reconstituted dose was first diluted 10-fold in PBS before preparing the sample. The total cell count per dose was calculated by multiplying the cells per mL as reported by the instrument by the dilution factor (if any) and the total reconstituted volume as specified in Table 1. The total cell count per dose was converted to a cell count per gram by dividing by the mass of the dose analyzed. The dose only encompasses the microbial fraction recovered (i.e., the contents of the dosage form), as described above.

[0158] The percentage of live cells relative to the expected cell count was calculated by dividing the viable CFU count per gram by the expected cell count per gram. The percentage of live cells relative to the total cell count was calculated by dividing the total CFU count per gram by the total cell count per gram.

[0159] Heat stress testing of commercial probiotic and microbial products (Figure 6A-6B)

[0160] Three or four doses of each product as listed in Table 1 were incubated in their original dosage form in a static incubator at 50°C for 24 hours. Three or four control doses were stored at the manufacturer's recommended storage temperature (4°C or 23°C, as noted in Figures 6A-6B) for the same period. Viable CFU counts per gram for the heat-stressed and control doses were determined as described above ("Viability Studies of Commercial Probiotic and Microbial Products"). Percent viability retention was calculated by dividing each of the heat-stressed CFU counts per gram by the average of the control CFU counts per gram.

[0161] Microbial strains used for stabilization and general culture conditions

[0162] Details of the microbial strains used are listed in Table 4, and details of the medium components are listed in Table 3.

[0163] E. coli Nissle 1917 was isolated from the commercial product Mutaflor and routinely cultured on LB agar at 37°C in a static incubator or on LB broth in 14 mL culture tubes or aerated baffled culture flasks shaken at 250 rpm at 37°C in a shaking incubator.

[0164] S. boulardii was isolated from the commercial product Florastor and routinely cultured on YPD agar at 30°C in a static incubator or on YPD broth in 14 mL culture tubes or aerated baffled culture flasks shaken at 250 rpm at 30°C in a shaking incubator.

[0165] E. meliloti Rm1021 was purchased from the American Type Culture Collection (ATCC) and routinely cultured on TY agar at 30°C in a static incubator or on TY broth in 14 mL culture tubes or aerated baffled culture flasks shaken at 250 rpm at 30°C in a shaking incubator.

[0166] L. plantarum NC8 was purchased from the Culture Collection University of Gothenburg (CCUG) and routinely cultivated on MRS agar or MRS broth in sealed 14 mL culture tubes or sealed culture flasks at 37°C in a static incubator.

[0167] S. flexneri was purchased from the American Type Culture Collection (ATCC) and cultured as described for E. coli Nissle 1917.

[0168] High-throughput pipeline for microbial material stabilizers (Figure 2A-2C, Figure 3A, Figure 4H, Figure 7A-7C, Figure 8, Figure 18)

[0169] Microbial strains were pre-cultured overnight in 5 mL liquid medium under appropriate conditions (see above). The overnight cultures were diluted 1:1000 into 3 replicates of 125 mL fresh liquid medium in flasks and incubated for 24 hours. The optical density (OD) of each replicate culture was measured. 600) was recorded (typically 2-2.5 for E. coli Nissle 1917, E. meliloti, and L. plantarum, and 5-6 for S. boulardii), and the cells were pelleted at 3220 rcf for 15 minutes. The cell pellet was then measured for a final OD 600 They were resuspended in PBS to a concentration of 2.25 ( E. meliloti , L. plantarum , S. boulardii ) or in spent medium ( E. coli Nissle 1917 ) and used immediately.

[0170] Material libraries were prepared by mixing each material with ultrapure water to the specified concentration (1X, 5X) in Table 2. Vendors and product numbers for all materials are listed in Table 2. Material solutions were arrayed in sealed deep-well plates and stored at -20°C until needed. On the day of use, material plates were thawed at room temperature, vortexed thoroughly, and briefly centrifuged.

[0171] For two-material combinations with melibiose, the arrayed material library was first mixed 1:1 with melibiose or water as a control at a 1X concentration of all components (Table 2). This gives a 0.5X final concentration of each component in Figure 3A relative to the one-material library results reported in Figure 2C and Figure 8.

[0172] For freeze-drying, microbial cell suspensions (25 μL) from each replicate were mixed in batches with arrayed material (75 μL) into flat-bottom 96-well plates using a liquid-handling robot (Tecan, EVO 150). The arrayed microorganism-material plates were immediately placed into a tray-type freeze dryer (Labconco, FreeZone Stoppering Tray Dryer) with shelves pre-cooled to -40°C and on a custom-made 0.25-inch aluminum platform to ensure heat transfer to the plates. Samples were annealed at -20°C and then dried at -20°C and 0.1 mBar for 12 hours (nominal), followed by 37°C for 3 hours (nominal). The chamber was backfilled with nitrogen and vented to atmosphere, after which the plates were loosely wrapped in parafilm, capped, and placed in nitrogen-flushed zip-top bags with desiccant (Drierite, granular, ∼8 mesh). The bagged lyophilized plates were stored in the dark at the designated temperature (23°C or 50°C) for 24 hours.

[0173] The viability of the stored microorganism-material combinations was determined by reconstitution in water, making 10-fold serial dilutions in PBS, plating on appropriate solid media, and quantifying the resulting colonies. Specifically, using a liquid-handling robot (Tecan, EVO 150), each replicate plate was reconstituted with ultrapure water (200 μL / well) and diluted 1:10 (relative to the initial volume before drying) in PBS. 2 , 1:10 3 , 1:10 4 Dilutions were made, and 4 μL of each dilution was spotted onto solid medium in 1-well rectangular plates. After incubation under appropriate culture conditions (see above), the plates were imaged in a gel imager (ChemiDoc XRS+, BioRad) using UV transillumination and a standard emission filter (580 / 120).

[0174] To assign viability scores, FIJI (imageJ) was used to programmatically separate 16-bit tiff images of plates into individual spot images, segment each spot, and measure several image attributes (raw particle count, mean background, mean segmented area, and segmented area area). These were used to classify spot images into empty, countable, and lawn using the mean segmented area to background ratio and the segmented area attributes (Figures 7A-7C). Raw particle counts for the empty classification were set to 0. Raw particle counts for the countable classification were interpreted as true colony counts. Raw particle counts for the lawn classification were set to the organism-specific colony count, determined by extrapolating the observed data for raw counts versus segmented area plots (Figures 7A-7C). For E. coli Nissle 1917 and E. meliloti, lawn was set to 60 counts. For S. boulardii, lawn was set to 50 counts. For L. plantarum, the lawn was set to 120 counts. These values ​​are subject to colony size at the time of analysis. Finally, for each microorganism-material combination, a viability score was calculated by dividing the cumulative sum of colony counts across the three dilutions by the maximum possible total count (i.e., the entire lawn). Viability scores therefore range from 0 to 1. This method allows for the use of data across all three dilutions analyzed (i.e., the three dilutions serve as technical replicates) to average out sampling noise due to plating and counting minority colonies. The method also allows for agnosticism regarding the actual number of countable colonies for any dilution and for the assignment of robust values ​​to microorganism-material combinations that yield lawns in some dilutions and biological replicates (i.e., by using only countable spots, values ​​can be assigned to a wider range of viabilities than may be possible). This analysis was applied to three independent biological replicates for each substance-microorganism combination.

[0175] A correlation analysis of the top-performing materials for each organism (Figure 2D) was performed by first normalizing the viability score for each specific material and concentration to the maximum viability score observed for each organism. This yielded a list of materials for each organism (at a specific concentration) with normalized scores spanning the full range of 0 to 1. All materials with a normalized score above 0.75 were then defined as the top materials for that organism. Based on which organism set each of these top-performing materials (at a specific concentration) belonged to, they were placed into 15 possible sets of 1-organism duplicates, 2-organism duplicates, 3-organism duplicates, and 4-organism duplicates.

[0176] Validation of vial-based viability of microbial material stabilizer (Figure 2E)

[0177] The top-performing microorganism-material combinations in the high-throughput pipeline were assayed on a larger scale to determine more accurate relative viability values ​​(CFU / vial) and to assess their short-term (1 day) and long-term (30 day) stabilization potential at room temperature. The analyses described below were repeated for 7-8 replicates for the 1 day time point and 4-5 replicates for the 30 day time point.

[0178] The microbial strains were pre-cultured overnight in 5 mL liquid medium under appropriate conditions (see above). The overnight cultures were grown at OD in 25 mL liquid medium in flasks. 600 The cells were diluted to 0.00225 and incubated for 24 hours under appropriate conditions (see above). Cells were harvested by centrifugation and diluted to a final OD of 0.00225 in cold PBS. 600 It was resuspended at 2.25 and used immediately.

[0179] For freeze-drying, each microbial cell suspension (100 μL) was mixed with 300 μL of target material using a micropipette in an autoclaved screw-threaded tube vial (Electron Microscopy Sciences, 60304-04) and capped up to the first stop with a two-prong freeze-drying stopper (Electron Microscopy Sciences, 60304-41). Within 10 minutes of mixing, the vials were placed in a tray freeze dryer with shelves pre-cooled to -40°C and in an aluminum StableTemp vial block (Cole-Parmer, EW-36600-44). Samples were dried as described above (see "High-Throughput Pipeline"). After drying, the chamber was backfilled with nitrogen and vented to atmosphere, and the vials were fully stoppered, sealed with screw caps, and stored in the dark at 23°C for the specified time.

[0180] The viability of stored samples was assessed by rehydrating the samples with 4 mL of cold PBS (to give a 1:10 dilution relative to the original volume), making 10-fold serial dilutions, plating 100 μL of each dilution onto appropriate solid medium in 100 mm round Petri dishes with 4.5 mm plating beads (Zymo), incubating under appropriate conditions, and counting colonies on dilutions that gave approximately 200 to 1000 individual colonies. CFU counts per vial were systematically determined using a gel imager and FIJI as described above (see "Viability study of commercial probiotics").

[0181] Verification of two-material combination (Fig. 10)

[0182] E. coli Nissle 1917 was prepared as described above (see "Verification of Viability in Vial Bases") with minor modifications: flask cultures were 2 L and incubated for 17–18 h, and the final cell suspension in PBS reached an OD 600 It was set to 10-12.

[0183] To freeze-dry, the cell suspension (12.5 mL) was mixed with the target material combination (37.5 mL), poured into a rectangular 1-well plate, and placed into a tray freeze-dryer with shelves pre-cooled to -40°C and on a custom-made 0.25-inch aluminum platform to ensure heat transfer to the plate. Samples were dried as described above (see "High-Throughput Pipeline"). After drying, the chamber was backfilled with nitrogen and vented to atmosphere, the plate was transferred to a zippered bag, the dried material was scraped into the bag, and crushed by rolling a plastic tube from the outside of the bag. The resulting powder was stored in the bag with a desiccant pack and stored in the dark at 4°C until needed.

[0184] To prepare the "with excipient" sample, dried bacterial powder was mixed with 1% w / w magnesium stearate, 5% w / w polyvinylpyrrolidone, and 64% w / w lactose to give a 30% w / w bacterial material loading.

[0185] Long-term storage of samples was performed by placing approximately 20 mg (for bacterial powder only) or 60 mg (for bacterial powder with excipients) aliquots into 12-well plates, capping the plates, and placing them in nitrogen-filled zip-top bags with desiccant (Drierite, granular, ∼8 mesh). The bagged plates were stored at 37°C in the dark for the indicated times.

[0186] The viability of stored samples was assessed by rehydrating the samples with 1 mL of PBS, making 10-fold serial dilutions, spotting approximately 1.5 μL of each dilution in triplicate onto LB agar in 1-well rectangular plates using a pin replicator (V&P Scientific, VP 407A), incubating at 37°C, and counting colonies on the strongest dilution that gave >1 colony per spot. The total CFU count per sample was calculated by first dividing the spot count by the spotted volume, multiplying by the dilution factor and total reconstituted volume, and then averaging across the three spotting replicates. The total CFU count per mass was calculated by dividing the CFU count per sample by the mass of the stored sample. Three independently stored replicate samples were analyzed as described. The percent viability at each time point was calculated by dividing the total CFU count per mass by the average total CFU count per mass of the three replicate samples on day 0. This method provides a robust measure of relative viability.

[0187] 2. Optimization of Material Formulation (Figure 3B)

[0188] To find the optimal concentration of each component in the two-material formulation selected for E. coli Nissle 1917, a combinatorial library of component concentrations was screened using the high-throughput pipeline described above (see "High-throughput pipeline for microbial material stabilizers") with minor modifications: flask cultures were 2 L and incubated for 17–18 h, and the final cell suspension in PBS reached an OD 600 The plate was stored at 37°C for 23 days and viability was assessed by a 1:10 ratio where only empty or countable spots were present. 5 Dilutions were assessed and raw counts were converted directly to CFU / mL by dividing the spot count by the spotted volume (4 uL) and multiplying by the dilution factor.

[0189] The top performing combinations were designated Formulation D (1 / 9X melibiose + 1X yeast extract) and Formulation E (1 / 9X melibiose + 1 / 125X yeast extract).

[0190] Direct comparison of the synthetic extremophile E. coli Nissle 1917 with Mutaflor (Figure 3C, Figure 15)

[0191] Stabilized E. coli Nissle 1917 was prepared as described above (see "Validation of Two-Material Combinations") using either melibiose (1X) or formulation D (see "Optimization of Two-Material Formulations"). For 100X cultures, 2 L cultures were concentrated 100X in PBS before mixing with the stabilizer. Mutaflor capsules remained intact as manufactured during storage.

[0192] Samples were stored in nitrogen-purged zip-top bags with desiccant (Drierite, granules, ∼8 mesh) at 23°C in the dark for the indicated times.

[0193] The viability of the stored samples was determined as described above (see "Viability studies of commercial probiotic and microbial products").

[0194] Assessment of ultra-long-term viability (Figure 3D, Figure 11)

[0195] Stabilized E. coli Nissle 1917 was prepared as described above (see "Validation of Two-Material Combinations") using either melibiose, Formulation D, Formulation E, or 5% w / w maltodextrin (16.5-19.5 DE) as a commercial control. From the list of inactive ingredients, maltodextrin was determined to be the only stabilizer used in the E. coli Nissle 1917 product Mutaflor.

[0196] Multigram samples of the dried powder were stored in 20 mL glasses with desiccant and kept in the dark at the specified temperature. Viability was assessed by withdrawing a small amount (approximately 60 mg) of the stock powder and following the procedure described above (see "Validation of Two-Material Combinations"), except the sample was rehydrated in ultrapure water instead of PBS. In addition to pin replicator spotting, one sample was also conventionally plated onto a 100 mm circular Petri dish as described above (see "Validation of Two-Material Combinations") to determine the scaling factor between the spotting method and Petri dish plating. When CFU / mg is reported, scaled values ​​were used. Percent viability is a relative value and is therefore not affected by this scaling factor.

[0197] Viability after wet granulation, tableting, and exposure to enteric coating (Figures 4B-4C, Figure 16)

[0198] Stabilized E. coli Nissle 1917 was prepared as described above (see "Validation of Two-Material Combination") using either Formulation D or 5% w / w maltodextrin (16.5-19.5 DE) as a commercial control. The dry powder was mixed with the following excipients: 1% w / w magnesium stearate, 5% w / w polyvinylpyrrolidone, and 64% w / w lactose (which gave a 30% w / w bacterial material loading).

[0199] Wet granulation was performed by adding 600 μL of isopropanol to 2 g of the bacteria-excipient mixture with continuous stirring at room temperature using a stand mixer (Sunbeam, B000COC69C). Stirring was continued for an additional 5 minutes, and the resulting paste was passed through a benchtop vibratory granulator (ERWEKA, FGS II) equipped with a 1 mm mesh screen. The collected granules were dried at 45°C for 20 minutes in a food dehydrator (Magic Mill, MFD-9100) retrofitted with a HEPA filter (Vornado, MD1-0022) to maintain sterility. Granules were stored in tubes with desiccant at 4°C until use.

[0200] Tableting was performed in air on a tablet press (Natoli, NP-RD10A) equipped with 5x5mm circular punches and dies, set to a depth of 8mm, and compressed using a force of 17kN (3.4kN equivalent to 173uPa pressure per tablet, or the indicated pressure). The bacteria-excipient mixture was either compressed directly ("direct tableting") or the granulated mixture was compressed ("granulation tableting"). Tablets were stored in tubes with desiccant at 4°C until use.

[0201] The tablets were enteric-coated by spray coating with a 3.6% w / w Eudragit S100 (Evonik) solution in a 1:1 cosolvent of acetone and isopropanol, along with 0.36% w / w triethyl citrate as a plasticizer. Spray coating was carried out for 1 hour at 23°C through a spray nozzle with a 0.5 mm opening, with the tablets placed in a rotary pan coater (ERWEKA, DKE). The coated tablets were dried for 2 hours at 40°C in a food dehydrator as described above. The coated tablets were stored in tubes with desiccant at 4°C until use.

[0202] The viability of each processed sample was assessed as described above (see "Validation of Two-Material Combinations"), except that samples were rehydrated in water instead of PBS, and CFU per mass was calculated by dividing by the mass of the bacterial fraction only (i.e., 30% of the total mass for samples including excipients). Coated tablets were first cut in half to expose the center before rehydration. Three independent samples of each type were assessed as described.

[0203] Viability after exposure to simulated gastric fluid (Figure 17)

[0204] Enteric-coated tablets of E. coli Nissle 1917 were prepared as described above (see "Enteric Coating"), placed in 40 mL of simulated gastric fluid (USP, pH 1.2, without enzymes) in 50 mL conical-bottom tubes, and incubated in a shaking tube revolver (Thermo Scientific, 88881001) in a static incubator at 37°C for 1 hour. After exposure to simulated gastric fluid, the tablets were dried, cut in half, and viability assessed as described above (see "Validation of Two-Material Combinations").

[0205] Viability after exposure to ionizing radiation (Figure 4E)

[0206] Stabilized E. coli Nissle 1917 was prepared as described above (see "High-Throughput Pipeline for Microbial Material Stabilization") with minor modifications: only melibiose (1x) was used as the stabilizer, 32 replicate samples were arrayed on eight 96-well plates (4 replicates per plate), and the bagged plates were stored at 4°C until use. On the day of irradiation, 5 mL of a fresh overnight culture of E. coli Nissle 1917 was arrayed onto the same plates as a control (4 replicates per plate).

[0207] The plates were sequentially placed into a Gammacell irradiator (Best Theratronics, 40 Exactor) and exposed to a Cobalt-60 source for the time required to achieve the specified dose of ionizing radiation (dose rate = 37.12 Gy / min). One plate was held together with the others but was not exposed to any radiation.

[0208] The viability of each sample was assessed as described above (see "Validation of two-material combinations"), except that samples were directly reconstituted with 200 uL of PBS in the treatment plate to provide an initial 1:2 dilution for subsequent serial dilutions.

[0209] Tuning release kinetics via matrix-forming agents (Figure 4D)

[0210] E. coli Nissle 1917 was transformed via electroporation with a plasmid encoding a constitutively expressed pathway for bioluminescence (luxCDAB). Plasmid and strain details are listed in Table 4.

[0211] Luminescent E. coli Nissle 1917 stabilized with Formulation D was prepared as described above (see "Validation of Two-Material Combination") using cultures supplemented with 100 μg / mL ampicillin. The resulting bacterial powder was mixed with either standard excipients (1% w / w magnesium stearate, 5% w / w polyvinylpyrrolidone, and 64% w / w lactose) or matrix-forming agents (50% w / w hydroxypropylmethylcellulose, 1% w / w magnesium stearate, 5% w / w polyvinylpyrrolidone, and 14% w / w lactose). In both instances, the bacterial material retained a 30% w / w loading.

[0212] A portion of each of these two powder mixtures was tableted as described above for the "direct tablet" method. Then, either one tablet or an equivalent mass of bulk powder (with excipients) was placed in 30 mL of ultrapure water in a conical-bottom tube and incubated at 37°C for 24 hours in a static incubator with a shaking tube revolver (Thermo Scientific, 88881001). At designated time points, 200 μL samples were withdrawn, arrayed in a white-walled, clear-bottom 96-well plate, and luminescence quantified in a luminometer (Tecan, Infinite 200). Each of the four sample types (standard excipients or matrix-forming excipients in bulk or tablet form) was analyzed in duplicate as described.

[0213] To calculate the percent luminescence released, the luminescence value of the tableted sample was divided by the average luminescence value of the corresponding bulk powder sample at each time point.

[0214] Shigella flexneri inhibition assay (Figure 4F)

[0215] Dried stabilized E. coli Nissle 1917 was prepared as described above (see "Validation of Two-Material Combinations") using either Formulation D or 5% w / w maltodextrin (16.5-19.5 DE) as a commercial control. Control "material-only" samples were prepared similarly, but omitting the bacterial cells. The dried powder was stored at 4°C until use. Fresh S. flexneri, E. coli Nissle 1917, and E. coli DH5a were grown overnight in LB at 37°C, pelleted, and analyzed to determine the OD. 600 The suspension was prepared by resuspending in fresh LB at 3°C.

[0216] On the day of the experiment, the dry powder was reconstituted in 500 μL of deionized water (20 mg / mL), diluted to a total volume of 1.5 mL in water, and mixed with 1.5 mL of 2 X LB. For fresh cell samples (E. coli Nissle 1917 and DH5a), 3 μL of concentrated overnight culture was mixed with 1.5 mL of water, followed by 1.5 mL of 2 X LB, to balance the reconstituted dry sample. A vehicle control was prepared by mixing 1.5 mL of water with 1.5 mL of 2 X LB.

[0217] These cultures were incubated in triplicate at 37°C for 2 hours, then challenged with 3 μL of an enriched overnight culture of S. flexneri and co-cultured for 10 hours at 37°C. To quantify the remaining viable fraction of S. flexneri, the co-cultures were diluted 1:100 into fresh LB (3 mL), grown at 37°C for 12 hours, and total DNA was extracted (Promega Wizard DNA Purification Kit). Shigella DNA was quantified by qPCR on a Roche LightCycler 96 using FastStart Essential DNA Green Master Mix (Roche, 06924204001) and the following primers: forward CCTTTTCCGCGTTCCTTGA, reverse CGGAATCCGGAGGTATTGC.

[0218] The obtained C t Values ​​were converted to DNA concentrations using a standard curve of purified S. flexneri DNA. For each experiment, DNA concentrations were normalized to the mean of vehicle control replicates, which was set at 100%.

[0219] Plant nodulation assay using stabilized E. meliloti (Figure 4G-4H)

[0220] E. meliloti cultures were prepared as described above (see "High-Throughput Pipeline for Microbial Material Stabilization") and lyophilized with the indicated stabilizers according to the one-well plate method described in "Validation of Two-Material Combinations." The pouched stabilized powders were exposed to 50°C in a static incubator for 24 hours and then stored at 4°C in the dark until use.

[0221] Nodulation of Medicago truncatula A17 was assayed according to a protocol from Sadowsky et al., modified for growth in pouches. 20~22Specifically, M. truncatula A17 seeds (Noble Research Institute) were sacrificed by soaking in concentrated sulfuric acid for 8–9 minutes, washed eight times with sterile water, sterilized by soaking in 8.25% sodium hypochlorite for 90 seconds, washed eight times with sterile water, infiltrated in sterile water for approximately 1 hour, plated on 1% agar plates, sealed with parafilm and foil, vernalized at 4°C in the dark for 5 days, and germinated at 23°C in the dark for 20 hours. Germinated seeds with roots approximately 1.5 cm long were transferred to sterile propagation pouches (Mega International, CYG) pre-wetted with 5 mL of ½XBNM medium (see Table 3). Five seeds were placed in each pouch. The pouches were placed between 1-inch foam blocks in a dome-shaped seed propagator (EarlyGrow, B07HHR5DGN) and incubated in a plant growth chamber (Fisher Scientific, PR505755L) set on a 16-hour day / night cycle at 25°C and 8-hour day / night at 21°C. The chamber was kept humid using a large water tray.

[0222] Two days after the seeds were transferred to the pouches, each of the resulting seedlings was inoculated with 1 mL of bacterial sample in 1 / 2XBMN. Specifically, heat-stress stabilized E. meliloti powder was added to 10 mL of the initial bacterial culture before freeze-drying. 8 Similarly, freshly grown E. meliloti cells were pelleted and reconstituted in 1 / 2XBNN at a common concentration equal to 10 CFU / mL. 7 The cells were resuspended in 1 / 2XBNM to a concentration of CFU / mL.

[0223] The pouches were left undisturbed for 6 days, watered with 5 mL of 1 / 2XBNM as needed, and inspected for nodules every other day. On day 12, the number of nodulated seedlings in each pouch was counted. The percentage of nodulated seedlings was calculated by dividing the number of nodulated seedlings by the total number of seedlings in that pouch.

[0224] Transmission electron microscopy of dried stabilized microbial material (Figure 3E, Figures 19A-19D)

[0225] Stabilized E. coli Nissle 1917 was prepared as described above (see "Validation of Two-Material Combinations") using either Formulation D or 5% w / w maltodextrin (16.5-19.5 DE) as a commercial control. The dried powder was either stored at 4°C ("As-Dried") or incubated in a sealed bag at 50°C for 6.5 hours before being stored at 4°C ("Dried + 50°C").

[0226] To fix the cells, 10 mg of each sample was reconstituted in 500 μL of deionized water and pelleted at 21 k rcf for 3.5 minutes. All but 20 μL of the bulk supernatant was removed, and the resulting pellet was resuspended in 30 μL of fixative (2.5% w / v paraformaldehyde, 5% w / v glutaraldehyde, 0.06% picric acid in 0.2 M cacodylate buffer). The cell suspension was pelleted at 21 k rcf for 10 minutes at room temperature, then transferred to 4°C and fixed overnight.

[0227] To prepare samples for imaging, fixed cells were washed in 0.1 M cacodylate buffer, postfixed with 1% osmium tetroxide (OsO4) / 1.5% potassium ferrocyanide (KFeCN6) for 1 hour, washed twice in water, washed once in 1x maleic acid buffer (MB), incubated in 1% uranyl acetate in MB for 1 hour, washed twice in water, and subsequently dehydrated in different grades of alcohol (10 min each; 50%, 70%, 90%, 2x 100%). The samples were then placed in propylene oxide for 1 hour and infiltrated overnight in a 1:1 mixture of propylene oxide and Spurr resin (EMS). The next day, the samples were embedded in Spurr resin and polymerized at 60°C for 48 hours.

[0228] To image the samples, ultrathin sections (approximately 80 nm) were cut on a Reichert Ultracut-S microtome, mounted on copper grids, stained with lead citrate, and examined in a JEOL 1200EX transmission electron microscope. Images were recorded using an AMT 2k CCD camera.

[0229] Three image planes from each sample type containing approximately 65-95 cells were used to quantify the observed cell classifications. Cells were manually classified into three categories: "cell debris (no envelope)," "denuded intimal membrane," and "intact envelope" (representative examples of each are shown in Figure 19C). Totals were quantified using the Cell Counter plugin in FIJI (ImageJ).

[0230] Scanning electron microscopy of dried stabilized microbial material (Figure 4B)

[0231] Various dried microbial materials were imaged in high-vacuum mode using a secondary electron detector from a Hitachi FlexSEM TM-1000 II (Tokyo, Japan). Low-voltage imaging (3 kV) was used to provide high surface detail while preventing damage from electron bombardment. Powders were mounted using double-sided carbon tape (Ted Pella Inc.). They were gold-coated (approximately 4 nm of gold was deposited) for approximately 2.5 minutes using a JEOL USA Smart Coater (Peabody, Massachusetts, USA). This conductive coating prevented excessive surface charging artifacts in the images. Typical imaging conditions included a spot intensity of 50 (based on the instrument's unitless scale of 1 to 100) and a working distance of less than 7 mm.

[0232] Data analysis

[0233] Numerical data were analyzed and plotted using Prism 9 (GraphPad). Statistical tests, number of replicates, and error bars are indicated for each relevant figure panel.

[0234] Table 1. Commercial probiotics and microbial materials analyzed

[0235] "Days until exp." is the number of days until exp. on the day the assessment was performed. "Mass per dose" is the average mass of the microbial fraction of the dose used in the assessment (see above). "Hyd. vol. per dose" is the volume used to rehydrate each dose. All culture media were solidified with 1.5% agar (details listed in Table 3). [Table 1-1] [Table 1-2]

[0236] Table 2. Library of materials used for stabilizer screening

[0237] "Solution" indicates when material was purchased or obtained as a synthetic solution and used as is. A 1X concentration was made by diluting the 5X solution 1:4 by volume in ultrapure water. ATCC reagent recipes are listed at the end of the table. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] ATCC Reagent 18: 0.75g Trypticase Soy Broth 10g sucrose 5g Bovine Serum Albumin Fraction V 100mL distilled water Filter sterilize through a 0.2 μm filter. ATCC Reagent 20: 20g sucrose 10g Bovine Serum Albumin Fraction V 100mL distilled water Filter sterilize through a 0.2 μm filter.

[0238] Table 3. Growth media used in each example

[0239] Where recipes are listed, details of the individual subcomponents are listed at the end of the table. Media used as solids for plating assays were always solidified with 1.5% w / v agar. [Table 3]

[0240] Table 4. Strains and plasmids used in each example [Table 4] References [Table 5-1] [Table 5-2] [Table 5-3]

[0241] Incorporation by Reference This application references various issued patents, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. Details of one or more aspects of the invention are set forth herein. Other features, objects, and advantages of the invention will become apparent from the detailed description, figures, examples, and claims.

[0242] Equivalents and Scope Articles such as "a," "an," and "the" may mean one or more unless indicated to the contrary or otherwise clear from the context. An embodiment or description including "or" between one or more members of a group satisfies whether one, more than one, or all of the group members are present in, employed in, or otherwise related to a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention encompasses embodiments in which exactly one member of the group is present in, employed in, or otherwise related to a given product or process. The invention encompasses embodiments in which more than one, or all of the group members are present in, employed in, or otherwise related to a given product or process.

[0243] Furthermore, the present disclosure covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more enumerated claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends from the same base claim. Where elements are presented as enumerated in Markush group format, by way of example, each subgroup of elements is also disclosed, and any element(s) may be removed from the group. In general, when the invention, or aspects of the invention, are described as including particular elements and / or features, it should be understood that certain aspects of the invention or certain embodiments of the invention consist of or consist essentially of such elements and / or features. For purposes of brevity, those embodiments have not been specifically recited herein in haec verba. It should also be noted that the terms "comprising" and "containing" are intended to be open and permit the inclusion of additional elements or steps. When ranges are given, endpoints are also included. Furthermore, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any specific value, or subrange within the ranges set forth in different aspects of the invention, up to 10 times the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0244] This application references various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. In addition, any particular aspect of the invention that falls within the prior art may be expressly excluded from any one or more of the aspects. Because such aspects would be known to those of skill in the art, they may be excluded even if the exclusion is not expressly stated herein. Any particular aspect of the invention may be excluded from any aspect for any reason, whether or not related to the existence of prior art.

[0245] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather as set forth in the accompanying embodiments. Those skilled in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit or scope of the invention, as defined in the following claims.

Claims

1. A composition comprising Escherichia coli (E. coli) and a first stabilizing excipient selected from an enzymatic digest of soybeans (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, potassium gluconate, melibiose, sucrose, animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), L-rhamnose monohydrate, (+)-L-sodium ascorbate, animal-origin pancreatic digest of casein (e.g., Bacto tryptone), trehalose dihydrate, D-(+)-galactose, the water-soluble portion of malted barley (e.g., Bacto malt extract), D-(+)-melezitose monohydrate, β-lactose, D(-)-fructose, glucose, 1-kestose, or a combination thereof.

2. 2. The composition of claim 1, wherein the first stabilizing excipient is selected from an enzymatic digest of soybean (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, melibiose, sodium (+)-L-ascorbate, trehalose dihydrate, β-lactose, glucose, potassium gluconate, or a combination thereof.

3. 3. The composition of claim 1, wherein the first stabilizing excipient is selected from an enzymatic digest of soybean (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, melibiose, sodium (+)-L-ascorbate, trehalose dihydrate, β-lactose, glucose, or a combination thereof.

4. 4. The composition of claim 1, wherein the first stabilizing excipient is selected from an enzymatic digest of soybean (e.g., Bacto soytone), palatinose hydrate, D-(+)-turanose, maltitol, melibiose, sodium (+)-L-ascorbate, trehalose dihydrate, β-lactose, glucose, or a combination thereof.

5. The composition of any one of claims 1 to 4, wherein the first stabilizing excipient is melibiose.

6. 6. The composition of claim 1, further comprising a second stabilizing excipient selected from short-chain inulin, a concentrate of water-soluble portions of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract), an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), porcine mucin type III, 1,4-benzenedimethanol, caffeine, an enzymatic digest of soybean (e.g., Bacto soytone), sucralose, dioctyl sulfosuccinate, 2-methyl-1-propanol, propyl gallate, β-glycerophosphate disodium salt hydrate, DL-β-(2-thienyl)serine, melibiose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), sodium citrate dihydrate, N-phenylthiourea, 4-guanidinobutyric acid, calcium D-gluconate, or a combination thereof.

7. 7. The composition of claim 6, wherein the second stabilizing excipient is selected from short-chain inulin, a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract), caffeine, or a combination thereof.

8. 8. The composition of claim 6, wherein the second stabilizing excipient is selected from a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract), caffeine, or a combination thereof.

9. 9. The composition of any one of claims 6 to 8, wherein the first stabilizing excipient is melibiose and the second stabilizing excipient is a concentrate of the water-soluble part of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract) or caffeine.

10. 10. The composition of any one of claims 1 to 9, wherein the first stabilizing excipient is present in a concentration of about 7% to about 50% (w / w).

11. 11. The composition of any one of claims 1 to 10, wherein the first stabilizing excipient is melibiose present at a concentration of about 0.3% to about 9% (w / w).

12. 12. The composition of any one of claims 6 to 11, wherein the second stabilizing excipient is present at a concentration of about 0.01% to about 7% (w / w).

13. 13. The composition of any one of claims 6 to 12, wherein the second stabilizing excipient is a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract) present at a concentration of about 0.04% to about 7% (w / w).

14. 14. The composition of any one of claims 6 to 9 or 11 to 13, wherein the first stabilizing excipient is melibiose present at a concentration of about 1% (w / w) and the second stabilizing excipient is a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract) present at a concentration of about 0.04% (w / w) or about 5% (w / w).

15. 13. The composition of any one of claims 6 to 12, wherein the second stabilizing excipient is caffeine present in a concentration of about 0.01% to about 0.4% (w / w).

16. 16. The composition of any one of claims 6-9, 11, 12, or 15, wherein the first stabilizing excipient is melibiose present at a concentration of about 1% (w / w) and the second stabilizing excipient is caffeine present at a concentration of about 0.01% (w / w).

17. The composition according to any one of claims 1 to 16, wherein the E. coli is E. coli Nissle 1917.

18. A composition comprising Saccharomyces boulardii (S. boulardii) and a first stabilizing excipient selected from L-glutamic acid monosodium salt monohydrate, skim milk powder, D-(+)-turanose, the water-soluble portion of malted barley (e.g., Bacto malt extract), maltitol, melibiose, lactulose, D-(+)-raffinose pentahydrate, palatinose hydrate, sucrose, animal-origin pancreatic digest of casein (e.g., Bacto tryptone), glucose, enzymatic digest of soybean (e.g., Bacto soytone), potassium gluconate, polydextrose, sodium gluconate, or a combination thereof.

19. 19. The composition of claim 18, wherein the first stabilizing excipient is selected from L-glutamic acid monosodium salt monohydrate, skim milk powder, D-(+)-turanose, the water-soluble portion of malted barley (e.g., Bacto malt extract), maltitol, melibiose, D-(+)-raffinose pentahydrate, palatinose hydrate, sucrose, pancreatic digest of casein of animal origin (e.g., Bacto tryptone), glucose, potassium gluconate, or a combination thereof.

20. 20. The composition of claim 18, wherein the first stabilizing excipient is selected from the water-soluble portion of malted barley (e.g., Bacto malt extract), D-(+)-turanose, D-(+)-raffinose pentahydrate, L-glutamic acid monosodium salt monohydrate, maltitol, palatinose hydrate, skim milk powder, melibiose, sucrose, glucose, pancreatic digest of casein of animal origin (e.g., Bacto tryptone), or a combination thereof.

21. A composition comprising Lactobacillus plantarum (L. plantarum) and a first stabilizing excipient selected from pancreatic digests of gelatin (e.g., Gelysate peptone), the water-soluble portion of malted barley (e.g., Bacto malt extract), animal-origin pancreatic digests of casein (e.g., Bacto tryptone), sucrose, animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), D-sorbitol, polydextrose, trehalose dihydrate, L-glutamic acid monosodium salt monohydrate, β-lactose, maltodextrin, L-rhamnose monohydrate, 1-kestose, animal-origin enzymatic digests of bovine and porcine animal proteins (e.g., Bacto peptone), chondroitin sulfate A, or combinations thereof.

22. 22. The composition of claim 21, wherein the first stabilizing excipient is selected from the water-soluble portion of malted barley (e.g., Bacto malt extract), a pancreatic digest of gelatin (e.g., Gelysate peptone), an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), sucrose, an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), D-sorbitol, or a combination thereof.

23. 23. The composition of claim 21, wherein the first stabilizing excipient is selected from an animal-origin peptone derived from beef infusion (e.g., Bacto beef extract), sucrose, a pancreatic digest of gelatin (e.g., Gelysate peptone), D-sorbitol, an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), or a combination thereof.

24. A composition comprising Ensifer meliloti (E. meliloti) and a first stabilizing excipient selected from an enzymatic digest of soybean (e.g., Bacto soytone), skim milk powder, polydextrose, the water-soluble portion of malted barley (e.g., Bacto malt extract), L-glutamic acid monosodium salt monohydrate, maltodextrin, palatinose hydrate, D-(+)-melezitose monohydrate, trehalose dihydrate, 1-kestose, maltose monohydrate, α-lactose monohydrate, sucrose, D-cellobiose, D-(+)-raffinose pentahydrate, melibiose, or a combination thereof.

25. 25. The composition of claim 24, wherein the first stabilizing excipient is selected from an enzymatic digest of soybean (e.g., Bacto soytone), skim milk powder, polydextrose, the water-soluble portion of malted barley (e.g., Bacto malt extract), L-glutamic acid monosodium salt monohydrate, maltodextrin, palatinose hydrate, D-(+)-melezitose monohydrate, or a combination thereof.

26. 26. The composition of claim 24, wherein the first stabilizing excipient is selected from an enzymatic digest of soybeans (e.g., Bacto soytone), skim milk powder, polydextrose, the water-soluble portion of malted barley (e.g., Bacto malt extract), L-glutamic acid monosodium salt monohydrate, or a combination thereof.

27. 27. The composition of any one of claims 24 to 26, wherein the first stabilizing excipient is selected from an enzymatic digest of soybean (e.g., Bacto soytone), skim milk powder, L-glutamic acid monosodium salt monohydrate, or a combination thereof.

28. 25. The composition of claim 24, wherein the first stabilizing excipient is selected from trehalose dihydrate, 1-kestose, palatinose hydrate, maltose monohydrate, α-lactose monohydrate, an enzymatic digest of soybean (e.g., Bacto soytone), D-(+)-melezitose monohydrate, sucrose, D-cellobiose, D-(+)-raffinose pentahydrate, melibiose, polydextrose, maltodextrin, or a combination thereof.

29. 29. The composition of claim 24, wherein the first stabilizing excipient is selected from trehalose dihydrate, 1-kestose, palatinose hydrate, maltose monohydrate, α-lactose monohydrate, an enzymatic digest of soybean (e.g., Bacto soytone), D-(+)-melezitose monohydrate, sucrose, D-cellobiose, D-(+)-raffinose pentahydrate, melibiose, or a combination thereof.

30. 30. The composition of any one of claims 24, 28, or 29, wherein the first stabilizing excipient is selected from sucrose, maltose monohydrate, polydextrose, maltodextrin, an enzymatic digest of soybean (e.g., Bacto soytone), or a combination thereof.

31. 31. The composition of any one of claims 24 or 28-30, wherein the first stabilizing excipient is selected from sucrose, maltose monohydrate, polydextrose, or a combination thereof.

32. The composition has a solubility of at least about 1 x 10 after 30 days at 23°C. 6 , at least about 1 x 10 7 , at least about 1 x 10 8 , at least about 1 x 10 9 , or at least about 1 × 10 10 32. The composition of any one of claims 1 to 31, exhibiting colony forming units (CFU) / gram of

33. 33. The composition of any one of claims 1 to 32, wherein the composition is stable at about 23°C, about 37°C, or about 50°C for at least one day.

34. 34. The composition of any one of claims 1 to 33, wherein the composition is stable at about 23°C, about 37°C, or about 50°C for at least one month.

35. 35. The composition of any one of claims 1 to 34, wherein the composition is stable at a relative humidity of less than about 5% for at least 1 day.

36. 36. The composition of any one of claims 1 to 35, wherein the composition is stable at a relative humidity of less than about 5% for at least one month.

37. 37. The composition of any one of claims 1 to 36, wherein the composition is stable at about 23°C, about 37°C, or about 50°C and at a relative humidity of less than about 5% for at least 1 day.

38. 38. The composition of any one of claims 1 to 37, wherein the composition is stable at about 23°C, about 37°C, or about 50°C at a relative humidity of less than about 5% for at least one month.

39. 39. The composition of any one of claims 1-38, wherein the composition maintains at least about 10% viability at about 23°C, about 37°C, or about 50°C for at least about 6.5 months.

40. 40. The composition of any one of claims 1 to 39, wherein the composition is stable upon exposure to organic solvents.

41. 41. The composition of claim 40, wherein the organic solvent is acetone or isopropanol.

42. 42. The composition of any one of claims 1-41, wherein the composition maintains at least about 5%, at least about 10%, or at least about 15% viability upon grinding.

43. 43. The composition of claim 42, wherein the grinding comprises exposure to shear forces.

44. 44. The composition of any one of claims 1 to 43, wherein the composition maintains at least about 5%, at least about 10%, or at least about 15% viability upon tableting.

45. 45. The composition of claim 44, wherein tableting involves application of a force of about 3.4 kN per tablet.

46. 46. ​​The composition of any one of claims 44 or 45, wherein tableting involves the application of a force of up to about 6 kN per tablet.

47. 47. The composition of any one of claims 44 to 46, wherein tableting comprises exposure to increased pressure.

48. 48. The composition of claim 47, wherein the increased pressure is about 173 uPa per tablet.

49. 49. The composition of any one of claims 47 or 48, wherein the increased pressure is up to about 650 uPa per tablet.

50. 50. The composition of any one of claims 1 to 49, wherein the composition maintains at least about 20% or at least about 28% viability upon wet granulation.

51. 51. The composition of claim 50, wherein the wet granulation comprises exposure to an organic solvent and baking.

52. 52. The composition of claim 51, wherein the organic solvent is isopropanol.

53. 52. The composition of claim 51, wherein baking comprises exposure to a temperature of at least about 45°C.

54. 54. The composition of any one of claims 1-53, wherein the composition maintains at least about 0.2%, at least about 1%, or at least about 3% viability upon spray coating.

55. 55. The composition of claim 54, wherein the spray coating comprises exposure to an organic solvent and a polymer, and baking.

56. 56. The composition of claim 55, wherein the organic solvent is acetone and / or isopropanol.

57. 56. The composition of claim 55, wherein the polymer is an enteric polymer.

58. 58. The composition of claim 57, wherein the enteric polymer is Eudragit L100-55, Eudragit L100, Eudragit S100, or a combination thereof.

59. 59. The composition of any one of claims 55 to 58, wherein baking comprises exposure to a temperature of at least about 40°C.

60. 60. The composition of any one of claims 1 to 59, wherein the composition is stable upon exposure to acid.

61. 61. The composition of any one of claims 1 to 60, wherein the composition is stable after exposure to a solution having a pH of about 1.2 for at least about 1 hour at about 37°C.

62. 62. The composition of any one of claims 1-61, wherein the composition maintains at least about 90% or at least about 95% viability after exposure to a solution having a pH of about 1.2 for at least about 1 hour at about 37°C.

63. 63. The composition of any one of claims 1 to 62, wherein the composition is stable upon exposure to ionizing radiation.

64. 64. The composition of claim 63, wherein the ionizing radiation is at least about 0.01 kGy, at least about 0.1 kGy, or at least about 1 kGy.

65. 65. The composition of any one of claims 1 to 64, wherein the composition is capable of inducing bacterial growth in a plant.

66. 66. The composition of claim 65, wherein inducing bacterial growth comprises inducing nodule formation in a plant.

67. 67. The composition of any one of claims 1 to 66, wherein the composition is capable of inhibiting enteric pathogens.

68. 68. The composition of claim 67, wherein the enteric pathogen is Shigella flexneri.

69. 69. The composition of any one of claims 1 to 68, comprising a solid dosage form.

70. 70. The composition of claim 69, wherein the solid dosage form is a tablet.

71. 71. The composition of any one of claims 69 or 70, wherein the solid dosage form comprises an immediate release layer.

72. 72. The composition of any one of claims 69 to 71, wherein the solid dosage form comprises a sustained release layer.

73. 73. The composition of claim 72, wherein the sustained-release layer comprises a matrix-forming polymer.

74. 74. The composition of claim 73, wherein the matrix-forming polymer is hydroxypropyl methylcellulose (HPMC).

75. 75. The composition of any one of claims 72 to 74, further comprising an additional microorganism.

76. 76. The composition of any one of claims 1 to 75, wherein E. coli, S. boulardii, L. plantarum, or E. meliloti is pre-cultured in a medium containing peptone, extract, and salt concentration.

77. The composition of claim 76, wherein the peptone is an animal-origin pancreatic digest of casein (e.g., Bacto tryptone), an enzymatic digest of soybean (e.g., Bacto soytone), a pancreatic digest of gelatin (e.g., Gelysate peptone), an animal-origin enzymatic digest of bovine and porcine animal proteins (e.g., Bacto peptone), or a combination thereof.

78. 78. The composition of any one of claims 76 or 77, wherein the extract is a peptone of animal origin derived from beef infusion (e.g., Bacto beef extract), a concentrate of the water-soluble portion of autolyzed Saccharomyces cerevisiae cells (e.g., Bacto yeast extract), the water-soluble portion of malted barley (e.g., Bacto malt extract), or a combination thereof.

79. 79. The composition of any one of claims 76-78, wherein the salt concentration is from about 50 mM to about 800 mM NaCl.

80. A method for delivering E. coli to a subject in need thereof, comprising administering to the subject a composition described in any one of claims 1-17 or 32-79.

81. 80. A method for delivering S. boulardii to a subject in need thereof, comprising administering to the subject a composition described in any one of claims 18-20 or 32-79.

82. 80. A method for delivering L. plantarum to a subject in need thereof, comprising administering to the subject a composition of any one of claims 21-23 or 32-79.

83. 80. A method for delivering E. meliloti to a subject in need thereof, comprising administering to the subject a composition according to any one of claims 24 to 79.

84. 80. A method of inducing bacterial growth in a subject or in a cell, tissue, or biological sample, comprising administering to a subject or contacting with a cell, tissue, or biological sample a composition of any one of claims 1 to 79.

85. 85. The method of claim 84, wherein the cell, tissue, or biological sample is a plant.

86. 86. The method of claim 85, comprising inducing nodulation in a plant.

87. 80. A method of inhibiting an enteric pathogen in a subject or in a cell, tissue, or biological sample, comprising administering to a subject or contacting with a cell, tissue, or biological sample a composition of any one of claims 1-79.

88. 88. The method of claim 87, wherein the enteric pathogen is Shigella flexneri.

89. 80. A method of treating a dysbiosis in a subject in need thereof, comprising administering to the subject a composition according to any one of claims 1 to 79.

90. 90. The method of claim 89, wherein the subject in need of treatment for a dysbiosis has a gastrointestinal disorder.

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