Bacterial compositions for metabolizing uremic toxins

Abstract

Described herein are bacterial compositions of modified probiotic bacteria having tolerance to one or more uremic toxins. Compositions of the disclosure can be used in the treatment of subjects having a condition or who are at risk of developing a condition associated with uremic toxin build up. Also provided herein are methods of preparing bacterial compositions of the disclosure.

Description

BACTERIAL COMPOSITIONS FOR METABOLIZING UREMIC TOXINSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 471,844, entitled “BACTERIAL COMPOSITIONS FOR METABOLIZING UREMIC TOXINS,” filed June 8, 2023, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] One of the main functions of the normal, healthy kidney, besides its regulatory, endocrine, and metabolic functions, is the disposal of waste products. Any impairment of excretory function can lead to the accumulation of a variety of nitrogenous waste products including, urea, creatinine, and uric acid. As concentration of these toxins builds up in the blood, a significant portion of the toxins can be excreted into the gut and the colon becomes the primary organ for toxin-excretion in the absence of functioning kidneys. In addition, the gut can also be a source of uremic toxin-precursors. Dietary aromatic amino acids such as tyrosine, tryptophan, and phenylalanine, after being deaminated and decarboxylated by colonic microbes, become phenolic compounds and can also serve as uremic-toxin precursors that are subsequently processed into multiple uremic toxins in the liver. Thus, there is a need for therapeutic strategies and compositions that can reduce uremic toxin levels in patients.SUMMARY

[0003] In various aspects, the present disclosure provides a bacterial composition comprising one or more modified probiotic bacteria having tolerance to a uremic toxin.

[0004] In some aspects, the one or more modified probiotic bacteria independently include a uremic toxin tolerance modification. In some aspects, the uremic toxin tolerance modification is stable for at least one of the one or more modified probiotic bacteria. In some aspects, the uremic toxin tolerance modification is stable for at least one of the one or more modified probiotic bacteria in the absence of a uremic toxin. In some aspects, the uremic toxin tolerance modification includes heterologous expression of an enzyme that hydrolyzes or decomposes a uremic toxin. In some aspects, the enzyme includes urease, urea amidolyase, oxygenase, carboxylase, allophanate hydrolase, or flavin reductase. In some aspects, the uremic toxin tolerance modification includes expression of at least one of the genes iifC, iifD, ureC, UreB, UreA, tnaA, iaaM, iaaaH, iifC, hbsAB, or pcmh. In some aspects, the uremic toxin tolerance modification includes production of short-chain fatty acids. In some aspects, the uremic toxin tolerance modification includes expression of butyrate kinase or butyryl-Co A: acetate CoA-transferase. In some aspects, the one or more modified probiotic bacteria are genetically modified.

[0005] In some aspects, the one or more modified probiotic bacteria are stable in a gastric fluid or an intestinal fluid. In some aspects the gastric fluid includes a pH of 1.0 to 3.5. In some aspects, the intestinal fluid includes a pH of 5.5 to 7.5. In some aspects, the one or more modified probiotic bacteria are stable for at least one hour to 10 days. In some aspects, the one or more modified probiotic bacteria include modified Lactobacillus rhamnosus, modified Bifidobacterium longum, modified Bacillus pasteurii, modified Lactobacillus sporogenes, modified Streptococcus thermophilus, modified Erwinia herbicola, modified Lactobacillus casei, modified Lactobacillus acidophilus, modified Lactobacillus bulgaricus, modified Bifidobacterium breve, modified Lactobacillus plantarum, modified Lactobacillus gasseri, modified Bifidobacterium infantis, modified Lactobacillus salivarius, modified Streptococcus faecium, modified Bacillus subtilis, modified Bacillus coagulans, modified Akkermansia muciniphila, or a combination thereof. In some aspects, the one or more modified probiotic bacteria are selected from the group consisting of Lactobacillus rhamnosus, Bifidobacterium longum, Bacillus pasteurii, and combinations thereof.

[0006] In some aspects, the uremic toxin is a water-soluble uremic toxin or a protein-bound uremic toxin. In some aspects, the water-soluble uremic toxin is urea, Creatinine, 1- methyladenosine, N2,N2-dimethylguanosine, N4-acetylcytidine, Xanthosine, 1- methylguanosine, Inosine, 1 -methylinosine, N6-methyladenosine, N6- threonylcarbamoyladenosine, Pseudouridine, S-adenosylhomocysteine, Monomethylamine, Dimethylamine, Ethylamine, Trimethylamine, Trimethylamine-N-oxide, 2-heptenal, 2-hexenal, Nonanal, 2-nonenal, 2-octenal, 4-decenal, Heptanal, Decanal, Hexanal, Dimethylglycine, 4-HO- decenal, 4-HO-hexenal, 4-HO-nonenal, 4-HO-octenal, P-lipotropin, 4-pyri done-3 -carboxamide- 1 -P-D-ribonucleoside, N-methyl-2-pyridone-5-carboxamide, N-methyl-4-pyridone-3- carboxamide, Nicotinamide, 8-hydroxy-2'-deoxyguanosine, Cytidine, Hypoxanthine, Neopterin, Uric acid, Xanthine, Arab(in)itol, Mannitol, Erythritol, Myoinositol, Sorbitol, Threitol, Argininic acid, Asymetric dimethylarginine (ADMA), Creatine, Guanidine, Guanidinosuccinic acid, Methylguanidine, Symmetric dimethylarginine (SDMA), Taurocyamine, a-keto-5- Guanidinovaleric acid, a-N-acetylarginine, P-guanidinopropionic acid, y-guanidinobutyric acid, Malondialdehyde, Orotic acid, Orotidine, Uridine, Oxalate, or Phenylacetylglutamine.

[0007] In some aspects, the protein-bound uremic toxin is p-cresol, indole, Angiogenin, Insulin-like growth factor 1 (IGF-1), Leptin, Osteocalcin, Retinol binding protein (RBP), Vascular endothelial growth factor, al -acid glycoprotein, Hippuric acid (total), Pentosidine, 3- deoxyglucosone, Fructoselysine, Glyoxal, Methylglyoxal, N(6)-Carboxymethyllysine (CML),Dihydroxyphenylalanine (PB-DOPA), Homocysteine, Kinurenine, Kynurenic acid, Indican, Indole-3 -acetic acid, Indoxyl sulfate, Indoxyl-fyD-glucoronidc, Melatonin, Quinolinic acid, Interleukin- 10, Phenol, p-Cresylsulfate, Putrescine, 3-carboxy-4-methyl-5-propyl-2- furanpropanoic acid, or Phenylacetic acid. In some aspects, the uremic toxin includes urea, p- cresol, or indole.

[0008] In some aspects, the present disclosure provides a bacterial composition, including a first and second bacterial composition, wherein the first bacterial composition includes modified bacteria and the second bacterial composition includes unmodified bacteria. In some aspects, the bacterial composition has an ODeoo value of at least 0.1. In some aspects, the bacterial composition has an ODeoo value of from 1 to 3. In some aspects, the bacterial composition has an ODeoo measurement of from about 0.2 to 3, from about 0.5 to 3, from about 0.5 to 2, from about 1 to 3, from about 1 to 2, or from about 2 to 3. In some aspects, the bacterial composition has an ODeoo measurement is from about 0.2 to 1.0, from about 1.0 to 1.5, from about 1.5 to 2.0, from about 2.0 to 2.5, from about 2.5 to 3, or from about 2.0 to 3.0. In some aspects, the bacterial composition has an ODeoo measurement is at least 0.2, at least 0.3, at least 0.4, at least0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, or at least 2.9. In some aspects, the bacterial composition has an ammonium concentration after at least one day in culture in the presence of a uremic toxin of at least 1 OOpg / ml. In some aspects, the bacterial composition after at least one day in culture in the presence of a uremic toxin comprises urea, p-cresol, indole, or a combination thereof.

[0009] In some aspects, the modified probiotic bacterium is genetically modified to express a urease gene and / or a urea transporter gene, and the modified probiotic bacterium is capable of metabolizing urea into ammonia and carbon dioxide in a medium with a pH of 5.0 to 8.0.

[0010] In some aspects, the present disclosure provides a pharmaceutical composition including the bacterial composition, and a pharmaceutically acceptable carrier or excipient. In some aspects, the pharmaceutically acceptable carrier or excipient is a binder, a lubricant, a glidant, a carrier, an antioxidant. In some aspects, the pharmaceutical composition further including an active ingredient, wherein the active ingredient is a prebiotic or a vitamin.

[0011] In some aspects, the present disclosure provides a method for producing one or more modified probiotic bacteria having tolerance to a uremic toxin, the method including: (i) modifying one or more probiotic bacteria; (ii) culturing the one or more modified probiotic bacteria in medium including an increasing concentration gradient of a uremic toxin, optionally a decreasing concentration gradient of nutrients; and (iii) identifying one or more modifiedprobiotic bacteria that can grow in the medium including an increasing concentration gradient of the uremic toxin, thereby preparing one or more modified probiotic bacteria having tolerance to a uremic toxin.

[0012] In some aspects, the method includes lyophilizing and / or freezing the one or more modified probiotic bacteria. In some aspects, the method includes combining the one or more modified probiotic bacteria with non-modified probiotic bacteria. In some aspects, the method includes analyzing the one or more modified probiotic bacteria by one or more of genomic analysis, transcriptomic analysis, proteomic analysis and metabolomic analysis. In some aspects, the one or more modified probiotic bacteria further include a modification that maintains the tolerance to the uremic toxin. In some aspects, the one or more modified probiotic bacteria are further modified to be stable in a gastric fluid or an intestinal fluid. In some aspects, the gastric fluid includes a pH of 1.0 to 3.5. In some aspects, the intestinal fluid includes a pH of 5.5 to 7.5.

[0013] In some aspects, the techniques described herein relate to a method, wherein the one or more modified probiotic bacteria are stable for at least one hour to 10 days. In some aspects, the present disclosure provides a method, wherein in step i) modifying one or more probiotic bacteria includes genetically modifying the one or more probiotic bacteria. In some aspects, the one or more probiotic bacteria include Lactobacillus rhamnosus, Bifidobacterium longum, Bacillus pasteurii, Lactobacillus sporogenes, Streptococcus thermophilus, Erwinia herbicola, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus gasseri, Bifidobacterium infantis, Lactobacillus salivarius, Streptococcus faecium, Bacillus subtilis, Bacillus coagulans or Akkermansia muciniphila. In some aspects, the one or more modified probiotic bacteria are obtained from a biological sample. In some aspects, the biological sample is a fecal sample. In some aspects, the uremic toxin is a water-soluble uremic toxin, or a protein-bound uremic toxin. In some aspects, the uremic toxin is a water-soluble uremic toxin, and wherein the water-soluble uremic toxin is urea, 1 -methyladenosine, N2,N2-dimethylguanosine, N4-acetylcytidine, Xanthosine, 1- methylguanosine, Inosine, 1 -methylinosine, N6-methyladenosine, N6- threonylcarbamoyladenosine, Pseudouridine, S-adenosylhomocysteine, Monomethylamine, Dimethylamine, Ethylamine, Trimethylamine, Trimethylamine-N-oxide, 2-heptenal, 2-hexenal, Nonanal, 2-nonenal, 2-octenal, 4-decenal, Heptanal, Decanal, Hexanal, Dimethylglycine, 4-HO- decenal, 4-HO-hexenal, 4-HO-nonenal, 4-HO-octenal, P-lipotropin, 4-pyri done-3 -carboxamide- 1 -P-D-ribonucleoside, N-methyl-2-pyridone-5-carboxamide, N-methyl-4-pyridone-3- carboxamide, Nicotinamide, 8-hydroxy-2'-deoxyguanosine, Cytidine, Hypoxanthine, Neopterin, Uric acid, Xanthine, Arab(in)itol, Mannitol, Erythritol, Myoinositol, Sorbitol, Threitol, Argininic acid, Asymetric dimethylarginine (ADMA), Creatine, Creatinine, Guanidine,Guanidinosuccinic acid, Methylguanidine, Symmetric dimethylarginine (SDMA), Taurocyamine, a-keto-5-Guanidinovaleric acid, a-N-acetylarginine, P-guanidinopropionic acid, y-guanidinobutyric acid, Malondialdehyde, Orotic acid, Orotidine, Uridine, Oxalate, or Phenylacetylglutamine. In some aspects, the uremic toxin is a protein-bound uremic toxin, and the protein-bound uremic toxin is p-cresol, indole, Angiogenin, Insulin-like growth factor 1 (IGF-1), Leptin, Osteocalcin, Retinol binding protein (RBP), Vascular endothelial growth factor, al -acid glycoprotein, Hippuric acid (total), Pentosidine, 3-deoxyglucosone, Fructoselysine, Glyoxal, Methylglyoxal, N(6)-Carboxymethyllysine (CML), Dihydroxyphenylalanine (PB- DOPA), Homocysteine, Kinurenine, Kynurenic acid, Indican, Indole-3 -acetic acid, Indoxyl sulfate, Indoxyl-P-D-glucoronidc, Melatonin, Quinolinic acid, Interleukin- 10, Phenol, p- Cresylsulfate, Putrescine, 3 -carboxy -4-methyl-5-propyl-2-furanpropanoic acid, or Phenylacetic acid. In some aspects, the uremic toxin includes urea, p-cresol, or indole.

[0014] In various aspects, the present disclosure provides a method of treating a condition associated with uremic toxin build up in a subject in need thereof, the method comprising administering a bacterial composition as described herein comprising one or more probiotic bacteria having tolerance to a uremic toxin, thereby treating the condition associated with uremic toxin build up. In some aspects, the condition is a kidney disease. In some aspects, the kidney disease is chronic kidney disease, Fabry disease, cystinosis, glomerulonephritis, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, polycystic kidney disease, diabetic kidney disease, or uremia. In some aspects, the condition is associated with reduced kidney function. In some aspects, the condition is diabetes, high blood pressure, heart disease, obesity, kidney damage, blood or fluid loss, heart attack, heart disease, an infection, liver failure, an allergic reaction, severe burns, severe dehydration, surgery indued kidney strain, a condition with high anabolic need, blood clots in and around the kidneys, cholesterol deposits blocking blood flow in the kidneys, glomerulonephritis, hemolytic uremic syndrome, COVID-19, lupus, scleroderma, thrombotic thrombocytopenic purpura, toxins, muscle tissue breakdown, breakdown of tumor cells, or old age.

[0015] In some aspects, the condition is associated with consumption of or exposure to a blood pressure medication, aspirin, ibuprofen, naproxen sodium, a chemotherapy drug, an antibiotic, an imaging dye, alcohol, a heavy metal, or a stimulant drug. In some aspects, the condition is associated with impaired passage of urine leading to impaired kidney or renal function. In some aspects, the condition is bladder cancer, blood clots in the urinary tract, cervical cancer, colon cancer, enlarged prostate, kidney stones, nerve damage, or prostate cancer.

[0016] In various aspects, the present disclosure provides a method of treating a subject at risk for developing uremic toxin build up, the method comprising administering a bacterial composition as described herein comprising one or more probiotic bacteria having tolerance to a uremic toxin, thereby treating the subject at risk of developing uremic toxin build up. In some aspects, the subject is at risk of developing kidney disease or having impaired kidney function. In some aspects, the subject has advanced age, a blockage in the blood vessels in the arms or legs, diabetes, high blood pressure, heart failure, kidney disease, liver disease, cancer, or has been hospitalized.

[0017] In various aspects, the present disclosure provides a method of producing one or more modified probiotic bacteria that are tolerant of uremic toxins, the method including: (i) exposing one or more modified probiotic bacteria to a uremic toxin; (ii) measuring at least one of the survival rate, growth rate, or metabolic activity of the one or more modified probiotic bacteria; (iii) comparing the results with those of unexposed probiotic bacteria or probiotic bacteria exposed to a lower concentration of the uremic toxin; and producing one or more modified probiotic bacteria that are tolerant of uremic toxins.

[0018] . In some aspects, the uremic toxin is selected from the group consisting of urea, creatinine, uric acid, indoxyl sulfate, p-cresol sulfate, hippuric acid, indole, p-cresol, phenol, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid.

[0019] In some aspects, the one or more modified probiotic bacteria is selected from the group consisting of Lactobacillus rhamnosus, Bifidobacterium longum, Bacillus pasteurii, Lactobacillus sporogenes, Streptococcus thermophilus, Erwinia herbicola, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus gasseri, Bifidobacterium infantis, Lactobacillus salivarius, Streptococcus faecium, Bacillus subtilis, Bacillus coagulans or Akkermansia muciniphila, and combinations thereof.

[0020] In some aspects, the method further comprises measuring an ODeoo value. In some aspects, the one or more modified probiotic bacteria have an ODeoo value of at least 0.1. In some aspects, the one or more modified probiotic bacteria have an ODeoo value of from 1 to 3. In some aspects, one or more modified probiotic bacteria have an ODeoo value of from about 0.2 to 3, from about 0.5 to 3, from about 0.5 to 2, from about 1 to 3, from about 1 to 2, or from about 2 to 3. In some aspects, the one or more modified probiotic bacteria have an OD600 value of from about 0.2 to 1.0, from about 1.0 to 1.5, from about 1.5 to 2.0, from about 2.0 to 2.5, from about 2.5 to 3, or from about 2.0 to 3.0. In some aspects, the one or more modified probiotic bacteria have an OD600 value of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, or at least 2.9. In some aspects, the method further comprises measuring a concentration of ammonium. In some aspects, measuring the metabolic activity comprises measuring a concentration of ammonium of at least lOOpg / ml In some aspects, the method further comprises measuring a concentration of urea, p-cresol, indole, or a combination thereof. In some aspects, measuring the metabolic activity comprises measuring a concentration of urea, p-cresol or indole.

[0021] In some aspects, the present disclosure provides a bacterial composition, including a first and second bacterial composition, wherein the first bacterial composition includes modified bacteria and the second bacterial composition includes unmodified bacteria. In some aspects, the bacterial composition has an ODeoo value of at least 0.1. In some aspects, the bacterial composition has an ODeoo value of from 1 to 3. In some aspects, the bacterial composition has an ODeoo measurement of from about 0.2 to 3, from about 0.5 to 3, from about 0.5 to 2, from about 1 to 3, from about 1 to 2, or from about 2 to 3. In some aspects, the bacterial composition has an ODeoo measurement is from about 0.2 to 1.0, from about 1.0 to 1.5, from about 1.5 to 2.0, from about 2.0 to 2.5, from about 2.5 to 3, or from about 2.0 to 3.0. In some aspects, the bacterial composition has an ODeoo measurement is at least 0.2, at least 0.3, at least 0.4, at least0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, or at least 2.9. In some aspects, the bacterial composition has an ammonium concentration after at least one day in culture in the presence of a uremic toxin of at least 1 OOpg / ml. In some aspects, the bacterial composition after at least one day in culture in the presence of a uremic toxin comprises urea, p-cresol, indole, or a combination thereof.INCORPORATION BY REFERENCE

[0022] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrativeembodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0024] FIG. 1 is a graphical image illustrating the plating approach to generate mutants that can utilize uremic toxins as sole carbon and nitrogen sources. The concentration gradients within the plates of nutrient media, minimal-media metabolites and uremic toxins are represented graphically below the plates. Top plate selects for mutants that can metabolize nitrogen rich toxins first and sequentially applies selection pressure to metabolize carbon rich aromatic uremic toxins, while the bottom plate applies the selection pressure in reverse order.

[0025] FIG. 2 is a graphical image illustrating concentrations of ammonia at day 0 for strains grown on MM2+5mM of urea, including 8 mutated strains from Example 15.

[0026] FIG. 3 is a graphical image illustrating concentrations of ammonia at day 2 for strains grown on MM2+5mM of urea, including 8 mutated strains from Example 15.

[0027] FIG. 4 is a graphical image illustrating concentrations of ammonia at day 4 for strains grown on MM2+5mM of urea, including 8 mutated strains from Example 15.

[0028] FIG. 5 is a graphical image illustrating concentrations of ammonia at day 6 for strains grown on MM2+5mM of urea, including 8 mutated strains from Example 15.

[0029] FIG. 6 is a graphical image illustrating urea metabolization profiles of 16 strains of probiotic species. Pure cultures of probiotics were grown in Christianson’s broth supplemented with 50mM urea and measured daily for concentration of ammonium ions through Nessler’s method. The cell densities within the cultures over a period of 6 days are given in TABLE 30.

[0030] FIG. 7 is a graphical image illustrating growth and viability for each of 16 strains grown in Christensen’s broth, with low nitrogen, for 6 days.

[0031] FIG. 8 is a graphical image illustrating concentrations of ammonia from strains grown to log phase followed by incubation in the presence of urea without any growth nutrients, as in Example 20.

[0032] FIG. 9 is a graphical image illustrating OD measurements at 600nm for 8 strains for 6 days performed in Christensen's broth (without phenol red).

[0033] FIGs. 10A-10F are graphical images illustrating calculated concentrations of calibration curves (CC) and quality control curves (QC) for indole and p-cresol, including indole CC (FIG. 10A), indole QC1 (FIG. 10B), indole QC2 (FIG. 10C), p-cresol CC (FIG. 10D), p- cresol QC1 (FIG. 10E) and p-cresol QC2 (FIG. 10F).

[0034] FIG. 11 is a graphical image illustrating concentration of indole for control and test media.

[0035] FIG. 12 is a graphical image illustrating concentration of p-cresol for control and test media, where the concentration of the test media was too high and was repeated, with results shown in FIG. 14.

[0036] FIGs. 13A-13D are graphical images illustrating a repeat analysis of calibration curves and quality control curves for p-cresol, including p-cresol CC (FIG. 13A), p-cresol QC1 (FIG. 13B), p-cresol QC2 (FIG. 13C) and p-cresol QC3 (FIG. 13D).

[0037] FIG. 14 is a graphical image illustrating concentrations of p-cresol for control and test media.

[0038] FIG. 15 is a graphical image illustrating a LCMS quantification plot of indole metabolized by individual probiotic strains. Each probiotic species was incubated at 37°C in respective ATTC recommended growth media supplemented with a final concentration of 5mM indole . Samples for LCMS analysis were sent at the end of weeks 1 and 2.

[0039] FIG. 16 is a graphical image illustrating a LCMS quantification plot of p-cresol metabolized by individual probiotic strains. Each probiotic species was incubated at 37°C in respective ATTC recommended growth media supplemented with a final concentration of 5mM p-Cresol. Samples for LCMS analysis were sent at the end of weeks 1, 2, 3 and 4.

[0040] FIG. 17 is a graphical image illustrating a LCMS analysis plot of indole metabolized by probiotic mixtures.DETAILED DESCRIPTION

[0041] Described herein are bacterial compositions of modified probiotic bacteria that can metabolize uremic toxin and are therefore tolerant to the uremic toxins. There is increasing evidence that gut microbiome can affect multiple organ systems and also lead to numerous chronic diseases, such as chronic kidney disease (CKD). Thus, the connection between the kidneys and gut microbiome, i.e., the gut-kidney axis, is bidirectional. Accumulation of urea and volume overload may affect the composition of gut microbiome and increase permeability of the gut epithelial barrier, which in turn can boost the production of gut-derived uremic toxins with significant renal and vascular toxic effects. This can create a feedback loop exacerbating uremic toxin levels in patients. A subject may be treated with these bacterial compositions to prevent or treat conditions associated with uremic toxin build up.

[0042] The present disclosure provides compositions that can metabolize uremic toxins. As used herein, the term “uremic toxin” can refer to a substance, organic or inorganic, that accumulates in the body fluids of subjects when kidney function is impaired. In healthy individuals such substances are cleared or filtered out by the action of the kidneys.

[0043] The ongoing project is aimed at generating probiotic based formulations for alleviating the uremic toxicity in kidney disease (KD) patients. The chief uremic toxins under current investigation are urea, ammonia, indole and p-cresol. The project is based on the hypothesis that high uremic toxicity in blood can significantly be lowered in the patient through the colonization of his / her gut with probiotic bacteria that were previously conditioned invitro to metabolize uremic toxins. Emerging research in this area places great promise on the potential benefits of probiotics tailored to KD patients for reducing the systemic concentration of uremic toxins. It is to this effect that experiments were performed invitro to validate the capacity of various probiotic strains in removing uremic toxins from the growth milieu. The uremic toxin metabolization capacity of sixteen probiotic species were quantified as described below, with Lactobacillus species demonstrating a higher capacity than others in efficiently metabolizing both urea and indole. High urease expression was found in the probiotics upon pretreatment with urea (5mM to 300mM) in minimal media conditions. The described pretreatment of probiotics is an efficient method for priming commercial quantities of probiotics for enabling the maximal removal of uremic toxins from KD patients.

[0044] Prerequisite for urease activation in probiotics: By exposing probiotics to environments of high urea and low nutrients for short periods (1- 12 hrs), probiotics were trained to quickly metabolize urea and retain that memory for many successive generations as bacteria proliferate. All tested probiotic strains metabolized urea poorly in environments rich in both urea and nutrients. However, most probiotic strains exhibited efficient urea metabolizing behavior upon short exposure (1-12 hrs) to urea under low nutrient conditions, which was a prerequisite condition for the activation of urease activity in the experiments. In the absence of starvation in urea rich environment, the probiotics exhibited urease activities lower than an order of magnitude compared to probiotics exposed to high urea under low nutrient conditions. While microbiologists have used Christianson’s agar and Stuart’s broth for eliciting urease expression in bacteria, these traditional methods take many days before urease expression can be verified. However, applying a short urea exposure in minimal media may save time and resources in commercial formulation of the probiotics. Briefly, individual probiotic strains were slowly adapted to urea with a decreasing gradient of nutrients over periods of ~10 days. Rapid changes from rich nutrient media to low nutrient media with urea also yielded urease activation. Urea metabolization was measured using Nessler’s reagent by quantifying the byproduct NH3 / NH4+ions. The urea concentrations in experiments ranged from 1 mM to 300mM, and with starting bacterial inoculum of O.Deoo 0.1 to 0.6. The low nutrient media utilized were Suart’s broth / agar, Christianson’s broth / agar, and buffers to maintain osmolarity (buffers Ml to M3).

[0045] Probiotic species for kidney patients, including urea metabolizing probiotics: After exposure to high urea and low-nutrient environments, the following probiotic species exhibited robust urea metabolization activity under varying conditions: Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Bifidobacterium breve, Lactobacillus gasseri, Bacillus pasteurii, and Bacillus subtilis. The tested urea concentrations in experiments ranged from 5mM to 300mM. And the starting bacterial inoculum ranged from 0.1 to 1.0 at O.Deoo. Additionally, growth rates of the probiotics were documented in 96 well plates with orthogonal concentration gradients of nutrients vs uremic toxins.

[0046] Gut bacteria metabolize tryptophan to produce indole. Indole is modified to form indoxyl sulfate, a uremic toxin that accumulates in chronic kidney disease. Bacteria that can utilize indole as a nutrient source are better adapted to the intestinal environment. The ability to grow in the presence of indole allows these bacteria to contribute to gut homeostasis and treatment of diseases such as kidney disease. The present disclosure provides bacteria modified to metabolize indole, and to treat diseases such as kidney disease.

[0047] P-Cresol metabolizing probiotics: Similar to indole, p-cresol is also produced in the gut due to microbiome metabolism, and also converts downstream into p-cresol sulphate which is a uremic toxin. Erwinia herbicola was the only species in the tested probiotics that showed significant metabolization capacity for p-cresol.

[0048] In some embodiments, uremic toxins may be the product of protein metabolism and may be influenced by colonic microbiota and enterohepatic circulation. The composition of the diet, particularly the consumption of animal versus vegetable proteins, can also influence the generation of certain uremic toxins. In some embodiments, uremic toxins can be removed from the body by glomerular filtration. In some embodiments, protein-bound uremic toxins can be removed by tubular secretion.

[0049] In some embodiments, the uremic toxin can be a carbon-rich uremic toxin or a nitrogen-rich uremic toxin (e.g., urea).

[0050] In some embodiments the uremic toxin can be urea, p-cresol, and / or indole.

[0051] In some embodiments, the uremic toxin can be a water-soluble uremic toxin. In some embodiments, the molecular weight of water-soluble uremic toxins can be less than about 500 Da. As a non-limiting example, water-soluble uremic toxins can be urea and / or creatinine.

[0052] Non-limiting uremic toxins include urea, creatinine, 1 -methyladenosine, N2,N2- dimethylguanosine, N4-acetylcytidine, Xanthosine, 1 -methylguanosine, Inosine, 1- methylinosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, Pseudouridine, S- adenosylhomocysteine, Monomethylamine, Dimethylamine, Ethylamine, Trimethylamine, Trimethylamine-N-oxide, 2-heptenal, 2-hexenal, Nonanal, 2-nonenal, 2-octenal, 4-decenal,Heptanal, Decanal, Hexanal, Dimethylglycine, 4-HO-decenal, 4-HO-hexenal, 4-HO-nonenal, 4- HO-octenal, P-lipotropin, 4-pyridone-3-carboxamide-l-P-D-ribonucleoside, N-methyl-2- pyridone-5 -carboxamide, N-methyl-4-pyridone-3 -carboxamide, Nicotinamide, 8-hydroxy-2'- deoxy guanosine, Cytidine, Hypoxanthine, Neopterin, Uric acid, Xanthine, Arab(in)itol, Mannitol, Erythritol, Myoinositol, Sorbitol, Threitol, Argininic acid, Asymetric dimethylarginine (ADMA), Creatine, Guanidine, Guanidinosuccinic acid, Methylguanidine, Symmetric dimethylarginine (SDMA), Taurocyamine, a-keto-5-Guanidinovaleric acid, a-N- acetylarginine, P-guanidinopropionic acid, y-guanidinobutyric acid, Malondialdehyde, Orotic acid, Orotidine, Uridine, Oxalate, and / or Phenylacetylglutamine.

[0053] In some embodiments, uremic toxins can be protein-bound uremic toxins. These compounds can originate from the metabolism of amino acids of the diet. In some embodiments, removal of protein-bound uremic toxins can occur in a healthy kidney via tubular secretion.

[0054] In some embodiments, protein-bound uremic toxins can include phenols and indoles. Non-limiting examples of uremic toxins include, p-cresol, indole, Angiogenin, Insulin-like growth factor 1 (IGF-1), Leptin, Osteocalcin, Retinol binding protein (RBP), Vascular endothelial growth factor, al -acid glycoprotein, Hippuric acid (total), Pentosidine, 3- deoxyglucosone, Fructoselysine, Glyoxal, Methylglyoxal, N(6)-Carboxymethyllysine (CML), Dihydroxyphenylalanine (PB-DOPA), Homocysteine, Kinurenine, Kynurenic acid, Indican, Indole-3 -acetic acid, Indoxyl sulfate, Indoxyl-P-D-glucoronide, Melatonin, Quinolinic acid, Interleukin- 10, Phenol, p-Cresylsulfate, Putrescine, 3-carboxy-4-methyl-5-propyl-2- furanpropanoic acid, and / or Phenylacetic acid.

[0055] In some embodiments, uremic toxins can be medium compounds. As a non-limiting example, uremic toxins can be peptides with a molecular weight of greater than about 500 Da.

[0056] Non-limiting examples of medium compounds include, Adiponectin, Basic fibroblast growth factor (BFGF), Complement factor D, Guanylin, Uroguanylin, Vasoactive intestinal peptide (VIP), Adrenomedullin, Atrial natriuretic peptide (ANP), Calcitonin gene-related peptide (CGRP), Methionine-enkephalin, Motiline, Cholecystokinin, Clara cell protein (CC16), Cystatin C, Hyaluronic acid (Hyaluronan), Neuropeptide Y, Resistin, Vasopressin (ADH), Endothelin, Degranulation inhibiting Protein I, Delta-sleep inducing Peptide, Parathyroid hormone, Substance P, P-2-Microglobulin, fl-endorphin, X-Ig light chain, Interleukin- 18, Interleukin- ip, Interleukin-6, and / or Tumor necrosis factor alpha (TNF).Bacterial Compositions

[0057] The present disclosure provides compositions that are tolerant to a uremic toxin. In some embodiments, the composition may be a bacterial composition. In some embodiments, thebacterial composition may include one or more probiotic bacteria. In some embodiments, the probiotic bacteria can be modified to be tolerant to one or more uremic toxins.

[0058] In various aspects, the present disclosure provides a bacterial composition comprising one or more modified probiotic bacteria having tolerance to a uremic toxin.

[0059] In some aspects, the one or more modified probiotic bacteria independently include a uremic toxin tolerance modification. In some aspects, the uremic toxin tolerance modification is stable for at least one of the one or more modified probiotic bacteria. In some aspects, the uremic toxin tolerance modification is stable for at least one of the one or more modified probiotic bacteria in the absence of a uremic toxin. In some aspects, the uremic toxin tolerance modification includes heterologous expression of an enzyme that hydrolyzes or decomposes a uremic toxin. In some aspects, the enzyme includes urease, urea amidolyase, oxygenase, carboxylase, allophanate hydrolase, or flavin reductase. In some aspects, the uremic toxin tolerance modification includes expression of at least one of the genes iifC, iifD, ureC, UreB, UreA, tnaA, iaaM, iaaaH, iifC, hbsAB, or pcmh. In some aspects, the uremic toxin tolerance modification includes production of short-chain fatty acids. In some aspects, the uremic toxin tolerance modification includes expression of butyrate kinase or butyryl-Co A: acetate CoA- transferase. In some aspects, the one or more modified probiotic bacteria are genetically modified.

[0060] As used herein the term “tolerance” can refer to the ability of bacteria to survive, grow, and / or metabolize a particular substance or a compound. In some embodiments, the substance or the compound may be a uremic toxin. In some embodiments, tolerance can be promoted by environmental or genetic factors. In some embodiments, individual bacteria in a population are or become tolerant to one or more uremic toxins. In some embodiments, an entire population of bacteria are or can become tolerant to one or more uremic toxins.

[0061] In some embodiments, the bacterial compositions can include one probiotic bacterium, that is the bacterial composition includes a monoculture of the probiotic bacterium. In some embodiments, the bacterial composition can include at least two probiotic bacteria with each probiotic bacterium constituting about 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the bacterial composition.

[0062] In some embodiments, the bacterial compositions of the disclosure can include one or more probiotic bacteria. As used herein, the term “probiotic bacteria” refers to live microorganisms which when administered to a host may confer a health benefit on the host.

[0063] In some embodiments, the probiotic bacteria may be stable in the gastrointestinal tract of a subject. In some embodiments, the probiotic bacteria may be stable in the mouth, pharynx,esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestines (cecum and colon) and / or rectum of the subject. In some embodiments, the probiotic bacteria are stable in an intestinal fluid and / or a gastric fluid. In some embodiments, the probiotic bacteria are stable at a pH of about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or higher. In some embodiments, the probiotic bacteria may be stable in a gastric fluid having a pH of about 1 to about 3.5. In some embodiments, the probiotic bacteria may be stable in an intestinal fluid having a pH of about 5.5 to 7.5.

[0064] In some embodiments, the probiotic bacteria may be stable in the gastrointestinal tract e.g., gastric fluid and / or intestinal fluid for about 1 hour to about 1 week. In some embodiments, the probiotic bacteria may be stable in the gastrointestinal tract e.g., gastric fluid and / or intestinal fluid for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or more. In some embodiments, the probiotic bacteria may be stable in the gastrointestinal tract e.g., gastric fluid and / or intestinal fluid for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days (1 week), 8 days, 9 days, 10 days or more. In some embodiments, the probiotic bacteria may be stable in the gastrointestinal tract e.g., gastric fluid and / or intestinal fluid for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more.

[0065] In some embodiments, the probiotic bacteria can be at least one of Lactobacillus rhamnosus, Bifidobacterium longum, Bacillus pasteurii, Lactobacillus sporogenes, Streptococcus thermophilus, Erwinia herbicola, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus gasseri, Bifidobacterium infantis, Lactobacillus salivarius, Streptococcus faecium, Bacillus coagulans, Akkermansia muciniphila, Abiotrophia defective, Acetobacter okinawensis, Achromobacter arsenitoxydans, Achromobacter xylosoxidans, Acidovorax sp., Actinoplanes friuliensis, Aeromonas enteropelogenes, Aeromonas hydrophila, Afipia birgiae, Afipia clevelandensis, Afipia sp., Agrobacterium albertimagni, Agrobacterium tumefaciens, Agrobacterium sp., Akkermansia muciniphila, Alcaligenes faecalis, Alicychphilus denitrificans, Alistipes finegoldii, Alistipes indistinctus, Alistipes onderdonkii, Alistipes putredinis, Alistipes shahii, Aquamicrobium defluvii, Arenimonas donghaensis, Arthrobacter sp., Azospirillum sp., Bacillus bataviensis, Bacillus cereus, Bacillus endophyticus, Bacillus weihenstephanensis, Bacillus subtilis, Bacteroidaceae bacterium, Bacteroides acidifaciens, Bacteroides caccae, Bacteroides cellulosilyticus, Bacteroides clarus, Bacteroides dorei, Bacteroides eggerthii, Bacteroides finegoldii, Bacteroides fragilis, Bacteroides gallinarum, Bacteroides intestinalis, Bacteroides massiliensis, Bacteroides oleiciplenus, Bacteroides ovatus, Bacteroides rodentium,Bacteroides salyersiae, Bacteroides sartorii, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides vulgatus, Bacteroides xylanisolvens, Bacteroides xylanisolvens, Bacteroides sp., Barnesiella intestinihominis, Bhargavaea cecembensis, Bifidobacterium adolescentis, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium ruminantium, Blastococcus sp., Blautia obeum, Blautia wexlerae, Bordetella bronchiseptica, Bordetella trematum, Bosea sp., Bradyrhizobium sp., Brevibacillus borstelensis, Brevundimonas diminuta, Brevundimonas naejangsanensis, Brucella abortus, Brucella melitensis, Brucella neotomae, Burkholderia mallei, Burkholderia multivorans, Butyricimonas virosa, Carnobacterium gallinarum, Caulobacter crescentus, Caulobacter sp., Cellulomonas flavigena, Cellulomonas sp., Cellulosimicrobium cellulans, Cetobacterium somerae, Citrobacter amalonaticus, Cloacibacillus evryensis, Clostridium acetobutylicum, Clostridium perfringens, Clostridium perfringens, Clostridium sordellii, Clostridium sp., Clostridium sp., Comamonas granuli, Coprobacillus sp., Coprococcus sp., Corynebacterium variabile, Cupriavidus basilensis, Cupriavidus sp., Dechloromonas agitata, Deinococcus geothermalis, Delftia tsuruhatensis, Desulfovibrio desulfur icans, Desulfovibrio sp., Devosia riboflavina, Dorea longicatena, Eggerthella lenta, Eggerthella sp., Ensifer adhaerens, Enterococcus casseliflavus, Enterococcus flavescens, Escherichia coli, Eubacterium limosum, Eubacterium nodatum, Eubacterium rectale, Eubacterium saphenum, Fusobacterium periodonticum, Gordonia sputi, Gordonia terrae, Gordonibacter pamelaeae, Gordonibacter pamelaeae, Halomonas stevensii, Halomonas titanicae, Hoeflea sp., Intrasporangium calvum, Janibacter hoylei, Kaistia granuli, Kineococcus radiotolerans, Lactobacillus coleohominis, Lactobacillus reuteri, Lactococcus garvieae, Lactococcus lactis, Lautropia mirabilis, Leucobacter salsicius, Luteimonas huabeiensis, Magnetospirillum magnetotacticum, Marinobacter lipotyticus, Marmoricola sp., Megasphaera micronuciformis, Megasphaera sp., Mesorhizobium sp., Methanobrevibacter arboriphilus, Methylobacterium radiotolerans, Methylobacterium sp., Microbacterium sp., Micromonospora aurantiaca, Mogibacterium, Morganella morganii, Mycobacterium smegmatis, Mycobacterium vanbaalenii, Mycobacterium sp., Neisseria sicca, Neorhizobium galegae, Nocardia rhamnosiphila, Nocardiopsis alkaliphila, Nocardiopsis ganjiahuensis, Nocardiopsis synnemataf ormans, Nocardiopsis valliformis, Novosphingobium nitrogenifigens, Ochrobactrum intermedium, Odoribacter laneus, Odoribacter splanchnicus, Oerskovia turbata, Oscillibacter sp., Pannonibacter phragmitetus, Pantoea vagans, Parabacteroides distasonis, Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides johnsonii, Parabacteroides merdae, Parabacteroides merdae, Parabacteroides sp., Paracoccus denitrificans, Paracoccus yeei, Paracoccus sp., Parvimonas sp., P ectobacterium carotovorum,Phyllobacterium sp., Polaromonas sp., Porphyromonas bennonis, Proteus mirabilis, Providencia alcalifaciens, Providencia burhodogranariea, Providencia rettgeri, Pseudacidovorax intermedins, Pseudoalteromonas sp., Pseudochrobactrum sp., Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas chloritidismutans, Pseudomonas chlororaphis, Pseudomonas japonica, Pseudomonas knackmussii, Pseudomonas mendocina, Pseudomonas monteilii, Pseudomonas oleovorans, Pseudomonas putida, Pseudomonas savastanoi, Pseudomonas stutzeri, Pseudomonas synxantha, Pseudomonas syringae, Pseudomonas sp., Pseudonocardia sp., Ralstonia solanacearum, Raoultella planticola, Rhizobium leguminosarum, Rhizobium sp., Rhodococcus defluvii, Rhodococcus pyridinivorans, Rikenella microfusus, Robinsoniella sp., Roseomonas cervicalis, Roseomonas sp., Ruminococcus gnavus, Salmonella enterica, Sanguibacter keddieii, Shewanella baltica, Shewanella sp., Shinella zoogloeoides, Sphingopyxis alaskensis, Starkeya novella, Stenotrophomonas maltophilia, Stenotrophomonas rhizophila, Streptomyces atroolivaceus, Streptomyces coelicollavus, Streptomyces olindensis, Streptomyces rimosus, Streptomyces roseoverticillatus, Streptomyces toyocaensis, Streptomyces turgidiscabies, Streptomyces sp., Synergistes sp., Tannerella sp., Thauera terpenica, Turicibacter sanguinis, Variovorax paradoxus, Variovorax sp., and / or Xanthomonas axonopodis, or a mixture thereof.

[0066] In some embodiments, the probiotic bacteria can be any bacteria present in the gastrointestinal tract of a subject. The subject may be a human or a non-human. In some embodiments, the probiotic bacteria can be gut microbiome bacteria. In some embodiments, the probiotic bacteria can be obtained from a fecal sample of a subject.Modified Probiotic Bacteria Having Tolerance to Uremic Toxins

[0067] In some embodiments, bacteria can be modified or engineered to have tolerance to one or more uremic toxins. The probiotic bacteria can be engineered using techniques of molecular biology and / or they can be evolved using the process of selection to have tolerance to the uremic toxins. In some embodiments, the modifications can be heritable modifications, i.e., are passed on to the progeny bacteria or non-heritable modifications.

[0068] In some embodiments, the probiotic bacteria can efficiently utilize uremic toxins as nutritional sources, thereby turning them into non-toxin components required for survival, growth and propagation.

[0069] In some embodiments, modified probiotic bacteria can include further modifications to maintain tolerance to the uremic toxin. In some embodiments, modified probiotic bacteria can include modifications to maintain tolerance to the uremic toxin even when other nutrients are available to the probiotic bacteria.

[0070] In some embodiments, the probiotic bacteria can also be modified to promote colonization in the gastrointestinal tract, immune modulation, modification of host metabolism, and / or to enhance the therapeutic effect of the probiotic bacteria.Adapting Probiotic Bacteria for Tolerance to Uremic Toxins

[0071] Probiotic bacteria can be prepared to be tolerant to one or more uremic toxins by applying one or more selective pressures. In some embodiments, probiotic bacteria exhibiting elevated levels of catabolic enzyme secretion can be selected or trained by exposing selected bacteria to increasing amounts of the uremic toxin of interest. In some embodiments, the bacteria are cultured in increasing concentration gradient of uremic toxins. In some embodiments, the probiotic bacteria can be adapted by sequential passage of the probiotic bacteria on increasing amounts of the uremic toxin, e.g., a single colony growing on 0.5% uremic toxin is selected and applied to medium containing 1.0% uremic toxin, a single colony growing on 1.0% uremic toxin is selected and applied to medium containing 2.0% uremic toxin or higher.

[0072] Once probiotic bacteria that are adapted for tolerance to uremic toxins are identified, they can be further analyzed by genomic analysis, transcriptomic analysis, proteomic analysis and / or metabolomic analysis. Any genes or proteins identified through such analysis can be used to further genetically modify the probiotic bacteria as described herein.

[0073] In various aspects, the present disclosure provides a method of producing one or more modified probiotic bacteria that are tolerant of uremic toxins, the method including: (i) exposing one or more modified probiotic bacteria to a uremic toxin; (ii) measuring at least one of the survival rate, growth rate, or metabolic activity of the one or more modified probiotic bacteria; (iii) comparing the results with those of unexposed probiotic bacteria or probiotic bacteria exposed to a lower concentration of the uremic toxin; and producing one or more modified probiotic bacteria that are tolerant of uremic toxins.

[0074] . In some aspects, the uremic toxin is selected from the group consisting of urea, creatinine, uric acid, indoxyl sulfate, p-cresol sulfate, hippuric acid, indole, p-cresol, phenol, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid.

[0075] In some aspects, the one or more modified probiotic bacteria is selected from the group consisting of Lactobacillus rhamnosus, Bifidobacterium longum, Bacillus pasteurii, Lactobacillus sporogenes, Streptococcus thermophilus, Erwinia herbicola, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus gasseri, Bifidobacterium infantis, Lactobacillus salivarius,Streptococcus faecium, Bacillus subtilis, Bacillus coagulans or Akkermansia muciniphila, and combinations thereof.Genetically Modified Probiotic Bacteria

[0076] Synthetic biology approaches can be used to perform targeted genome modifications to prepare bacterial compositions of the disclosure. Synthetic biology-based tools including construction of bacterial chassis, genome editing, expression systems, and gene circuits can be utilized for preparing modified probiotic bacteria. The use of synthetic gene circuits may allow precise regulation of gene expression and the assembly of operons containing synthetic transcriptional and translational control.

[0077] In some embodiments, probiotic bacteria are modified by knocking-out genes, incorporating new genes, and / or by introducing mutations. The incorporation of foreign DNA for modification can be typically in the form of a plasmid which is transferred into probiotic bacteria via transformation, electroporation, conjugation, and / or phage transduction.

[0078] Chromosomal modification strategies can also be used for modifying probiotic bacteria using non-replicative plasmids and insertion sequences. Modification of probiotic bacteria by genome engineering can also be accomplished using genome engineering methods such as homologous recombination, recombineering, and CRISPR-Cas9 mediated genome editing.

[0079] In some embodiments, probiotic bacteria can also be modified for heterologous expression of proteins. Expression systems used in heterologous expression of proteins by modified probiotic bacteria may be constitutive or inducible. In some embodiments, heterologous protein expression may be operably linked to factors such as temperature, pH, and / or bile salt concentration. Elevated levels of secretion can also be obtained by overexpressing the gene of interest (e.g., via multiple copies or a promoter driving high levels of expression) in the probiotic bacteria. The gene of interest can be under the regulatory control of an inducible or constitutive promoter. Promoters for use in recombinant prokaryotic expression vectors are well-established in the art and can include the beta-lactamase (penicillinase) and lactose promoter systems. The promoter and Shine -Dalgamo sequence (for prokaryotic host expression) are operably linked to the DNA encoding the gene of interest, i.e., they are positioned so as to promote transcription of the messenger RNA from the DNA, and subsequently introduced into a suitable host cell.

[0080] In some embodiments, probiotic bacteria can be modified to metabolize any of the uremic toxins described herein. In some embodiments, probiotic bacteria can be modified to metabolize urea, p-cresol, and / or indole. In some embodiments, probiotic bacteria can bemodified to metabolize urea by heterologous expression of an enzyme that hydrolyzes or decomposes urea e.g., urease or urea amidolyase. In another embodiment, probiotic bacteria can be modified to metabolize indole by heterologous expression of an enzyme that metabolizes indole such as, but not limited to, oxygenase and flavin reductase units encoded by genes iifC and / or iifD. In some embodiments, the mutations can be uremic toxin tolerance modifications.Methods of Preparing Probiotic Bacteria

[0081] The present disclosure provides methods of preparing one or more modified probiotic bacteria having tolerance to one or more uremic toxins. Such methods can include modifying one or more probiotic bacteria. In some embodiments, the probiotic bacteria can be modified by any of the methods described herein. Modified probiotic bacteria can then be cultured in a medium that includes an increasing concentration gradient of a uremic toxin. In some embodiments, the medium can also include a decreasing concentration gradient of nutrients that are known in the art to be suitable for culturing a particular probiotic bacterial strain. One or more modified probiotic bacteria that can grow in the increasing concentration gradient of the uremic toxin can then be selected for inclusion in the bacterial compositions of the disclosure. In some embodiments, probiotic bacteria that can grow in at least one increased concentration of uremic toxins are selected.

[0082] In some embodiments, the modified probiotic bacteria can be analyzed by genomic, transcriptomic, proteomic and / or metabolomic methods to identify and / or characterize the additional changes to the probiotic bacteria. In some embodiments, the modified probiotic bacteria can include further modifications that allow the maintenance of the tolerance to the uremic toxins. Such modifications can be selected, for example, by culturing the modified probiotic bacteria in a medium comprising increasing concentration of a uremic toxin, followed by culturing the probiotic bacteria without uremic toxin i.e., in medium containing nutrients followed by culturing the probiotic bacteria in increasing concentration of the uremic toxins.

[0083] In some embodiments, the modified probiotic bacteria can be further modified to be stable in a gastric fluid and / or an intestinal fluid. In some embodiments, the gastric fluid can have a pH of about 1.0 to about 3.5. In some embodiments, the intestinal fluid can have a pH of about 5.5 to about 7.5.

[0084] In some embodiments, the probiotic bacteria can be obtained from a biological sample. As a non-limiting example, the probiotic bacteria can be obtained from a fecal sample. When fecal samples are utilized, the identity of the probiotic bacteria in the fecal sample that are tolerant to the uremic toxins can be identified at the genus and species level through a PCR or through sequencing their ribosomal RNA.

[0085] Modified probiotic bacteria identified by the methods described herein can be further characterized to determine their ability to metabolize the uremic toxins. Modified probiotic bacteria that are able to reduce the levels of uremic toxins may be preferentially selected for inclusion in the compositions described herein.

[0086] In the present disclosure, the death phase of a sample of probiotic bacteria is when ODeoo is greater than 2.0; the stationary phase is when ODeoo is greater than 1.0 and less than 2.0; the log-growth phase is when ODeoo is greater than 0.2 and less than 1.0; and the lag-growth phase is when ODeoo is greater than 0.05 and less than 0.2. In some embodiments, a probiotic bacterium is modified when it is able to reach a log-growth phase or greater. In the present disclosure, high amounts of uremic toxin degradation for all the strains occurs when the OD is greater than 1.0 and less than 3.0. The death phase, stationary phase, log-growth phase, and laggrowth phase do not always follow these exact OD600 measurements for each strain, for each medium used, or for each measurement protocol; different strains may grow at different rates under various uremic toxin concentration gradients, different stains may have different ratios of dead and lie cells above an ODeoo of 1.0, and different incubation and shaking protocols can change the ODeoo measurements, bacterial growth can also be quantified using cell counters or through traditional methods of plating and subsequent colony counting. Other methods can be used to select modified bacteria, including spectrophotometric analysis for the quantification of urea, ammonia, indole and p-cresol. Spectrophotometric methods can be used by introducing an upstream extraction step where the toxin is extracted into a polar solvent, for example, acetonitrile, before the spectroscopic measurement. In addition, it is beneficial to have bacteria metabolizing the toxins at high ODs, as that would result in higher yields with less raw materials and in shorter time periods.

[0087] In some embodiments, a probiotic bacterium is selected for inclusion in a pharmacological composition when the bacterium reaches an OD of greater than 0.2 after incubation with a toxin for at least 1 day. In some embodiments, a probiotic bacterium is selected for inclusion in a pharmacological composition when measurements of ammonia are greater than zero after incubation with a toxin for at least 1 day. In some embodiments, a probiotic bacterium is selected for inclusion in a pharmacological composition when the concentration of indole is decreased after incubation with the probiotic bacterium for at least one week. In some embodiments, a probiotic bacterium is selected for inclusion in a pharmacological composition when the concentration of p-cresol is decreased after incubation for at least one week.Methods of Use

[0088] The present disclosure provides methods of treatment using the bacterial compositions described herein.

[0089] In some embodiments, the bacterial composition can be a component or additive to a food product or to an enterically coated tablet, a tablet, a capsule, powder, soft gel, gelcap, film, coated beads, spray-coated granules, microbeads, or a liquid. In some embodiments, the bacterial compositions can include probiotic bacteria at concentration of about 108CFU / mL, 109CFU / mL, IO10CFU / mL, 1011CFU / mL, 1012CFU / mL or more when added as a liquid. When added as a freeze-dried or lyophilized powder, the probiotic bacteria can be at a concentration of about 108CFU / g, 109CFU / g, IO10CFU / g, 1011CFU / g, 1012CFU / g or more.

[0090] In some embodiments, the bacterial compositions can be administered orally. Alternate routes of administration can include, topical, transdermal, rectal, intravenous, intraarterial, vaginal, parenteral routes of administration, or via inhalation.

[0091] The bacterial composition of the disclosure can be provided to a subject for treating a condition associated with uremic toxin build up. In some embodiments, the subject has or is at risk of kidney disease. A consumable composition (e.g., a medical food) of the present disclosure may be consumed for reducing uremic toxins. A consumable composition (e.g., a medical food) of the present disclosure may be administered under the guidance of a medical professional or as a dietary supplement. In various aspects the consumable composition disclosed herein may be provided to subjects with high levels of uremic toxins (e.g., urea, p- cresol, or indole).

[0092] In various aspects, the present disclosure provides a method of treating a condition associated with uremic toxin build up in a subject in need thereof, the method comprising administering a bacterial composition as described herein comprising one or more probiotic bacteria having tolerance to a uremic toxin, thereby treating the condition associated with uremic toxin build up.

[0093] In various aspects, the present disclosure provides a method of treating a subject at risk for developing uremic toxin build up, the method comprising administering a bacterial composition as described herein comprising one or more probiotic bacteria having tolerance to a uremic toxin, thereby treating the subject at risk of developing uremic toxin build up. In some aspects, the subject is at risk of developing kidney disease or having impaired kidney function. In some aspects, the subject has advanced age, a blockage in the blood vessels in the arms or legs, diabetes, high blood pressure, heart failure, kidney disease, liver disease, cancer, or has been hospitalized.

[0094] A consumable composition (e.g., a medical food) of the present disclosure may be provided as a dietary supplement for a patient with high levels of uremic toxins (e.g., urea, p- cresol, or indole). The composition may be used to treat or prevent a kidney disorder. For example, the composition may provide a uremic-metabolizing probiotic composition which reduces the concentration of uremic toxins (e.g., urea, p-cresol, or indole) in the blood following administration, and which may therefore be amenable for administration to kidney disease patients. In some embodiments, the composition is administered to a subject having or at risk of having a kidney disease such as, but not limited to, chronic kidney disease, Fabry disease, cystinosis, glomerulonephritis, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, polycystic kidney disease, diabetic kidney disease, or uremia.

[0095] In some embodiments, the subject may have a condition associated with reduced kidney function. Non-limiting examples of the condition associated with reduced kidney function include, diabetes, high blood pressure, heart disease, obesity, kidney damage, blood or fluid loss, heart attack, heart disease, an infection, liver failure, an allergic reaction, severe bums, severe dehydration, surgery indued kidney strain, a condition with high anabolic need, blood clots in and around the kidneys, cholesterol deposits blocking blood flow in the kidneys, glomerulonephritis, hemolytic uremic syndrome, COVID-19, lupus, scleroderma, thrombotic thrombocytopenic purpura, toxins, muscle tissue breakdown, breakdown of tumor cells, or old age.

[0096] In some embodiments, the compositions of the disclosure can be used to treat uremic toxin build up associated with consumption of or exposure to a blood pressure medication, aspirin, ibuprofen, naproxen sodium, a chemotherapy drug, an antibiotic, an imaging dye, alcohol, a heavy metal, or a stimulant drug.

[0097] In some embodiments, the compositions of the disclosure can be used to treat uremic toxin build up associated with impaired passage of urine leading to impaired kidney or renal function. Non-limiting examples of such conditions include, bladder cancer, blood clots in the urinary tract, cervical cancer, colon cancer, enlarged prostate, kidney stones, nerve damage, or prostate cancer.

[0098] In some embodiments, the subject may be at risk for developing uremic toxin build up. Such subjects can also be treated with the compositions of the disclosure. In some embodiments, the subject may be at risk of developing kidney disease or having impaired kidney function. In some embodiments, the subject may have advanced age, a blockage in the blood vessels in the arms or legs, diabetes, high blood pressure, heart failure, kidney disease, liver disease, cancer, or has been previously hospitalized.

[0099] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0100] As used herein, the terms “about” and “approximately,” in reference to a number, is used herein to include numbers that fall within a range of 10%, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0101] As used herein, the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, may refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0102] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0103] For purposes herein, percent identity and sequence similarity may be performed using the BLAST algorithm, which is described in Altschul et al. (J. Mol. Biol. 215:403-410 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0104] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0105] As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).

[0106] As used herein, the term “effective amount” refers to the amount of a composition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0107] As used herein, the term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a“prophylactically effective amount” as prophylaxis can be considered therapy. In some embodiments, the therapeutically effective amount can be 1 to 2 billion CFU, 2 to 3 billion CFU, 3 to 4 billion CFU, 4 to 5 billion CFU, 5 to 6 billion CFU, 6 to 7 billion CFU, 7 to 8 billion CFU, 8 to 9 billion CFU, or 9 to 10 billion CFU. In some embodiments, the therapeutically effective amount can be 1 billion CFU, 2 billion CFU, 3 billion CFU, 4 billion CFU, 5 billion CFU, 6 billion CFU, 7 billion CFU, 8 billion CFU, 9 billion CFU, 10 billion CFU, 11 billion CFU, 12 billion CFU, 13 billion CFU, 14 billion CFU, 15 billion CFU, 16 billion CFU, 17 billion CFU, 18 billion CFU, 19 billion CFU, or 20 billion CFU. As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal or lingual), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0108] As used herein, the term “treatment” means an approach to obtaining a beneficial or intended clinical result. The beneficial or intended clinical result can include alleviation of symptoms, a reduction in the severity of the disease, inhibiting an underlying cause of a disease or condition, steadying diseases in a non-advanced state, delaying the progress of a disease, and / or improvement or alleviation of disease conditions.

[0109] As used herein, the term “pharmaceutical composition” refers to the combination of an active ingredient with a carrier, inert or active, making the composition especially suitable for therapeutic or diagnostic use in vitro, in vivo or ex vivo.

[0110] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

[0111] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), glycerol, liquid polyethylene glycols, aprotic solvents such as dimethylsulfoxide, N-methylpyrrolidone and mixtures thereof, and various types of wetting agents, solubilizing agents, binders, lubricants, anti-oxidants, bulking agents, protein carriers such as albumins, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g.,Martin, Remington’s Pharmaceutical Sciences, 21st Ed., MackPubl. Co., Easton, Pa. (2005), incorporated herein by reference in its entirety.

[0112] Excipients may be used to provide bulk, shape, texture, flavor, sweetness, pH adjustment, lubrication, wetting, absorption, retardation, film formation, enteric coating, gelling, plasticizing, anti-caking, or topcoating to the probiotic composition. Excipients may include lactose, mannitol, sorbitol, microcrystalline cellulose, sucrose, sodium citrate, dicalcium phosphate, magnesium stearate, calcium stearate, talc, solid polyethylene glycols, sodium lauryl sulfate, colloidal silicon dioxide, agar-agar, calcium carbonate, sodium carbonate, silicates, alginic acid, corn starch, potato tapioca starch, primogel, soy lectin, medium-chain triglycerides, whey protein concentrate, non-fat dry milk, milk protein concentrate, flavoring agents, sweeteners, organic acids, phospholipids, fats, fatty acid esters, waxes, film formers, enteric coating materials, pH adjusting agents, gelling aids, plasticizers, anti-caking agents, and topcoat materials.

[0113] As used herein, the term “uremic toxin tolerance modifications” refers to modifications that allow a bacterium to metabolize any of the uremic toxins described herein. In some embodiments, the modifications include genes that metabolize urea, p-cresol, and / or indole. In some embodiments, the modifications include heterologous expression of an enzyme that hydrolyzes or decomposes urea e.g., urease or urea amidolyase. The modifications can also include epigenetic modifications. In some embodiments, the epigenetic modifications increase expression of a gene that metabolizes a uremic toxin, or increase levels of an enzyme that hydrolyzes or decomposes urea. In some embodiments, the epigenetic modifications decrease expression of an enzyme. In another embodiment, toxin tolerance modifications include bacteria that metabolize indole by heterologous expression of an enzyme that metabolizes indole such as, but not limited to, oxygenase and flavin reductase units encoded by genes iifC and / or iifD. Modifications can include expression of a ureC gene, a UreB gene, a UreA gene, a tnaA gene, an iaaM gene, an iaaaH gene, an iifC gene a hbsAB gene, or a pcmh gene. Modifications can also include production of short-chain fatty acids through expression of genes such as butyrate kinase and butyryl-CoA:acetate CoA-transferase.

[0114] As used herein, the term “stable” refers to a bacterium that metabolizes toxins in the presence of varying amounts of nutrients for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least 10 days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, or at least six weeks. In some embodiments, the term “stable” refers to a bacterium including a modification that allows the bacterium to metabolize toxins in the presence of varying amounts of nutrients for at least one day, at leasttwo days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least 10 days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, or at least six weeks. In some embodiments, a stable modification can allow the bacterium to grow in gastric or intestinal environments in the presence of a toxin for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, or at least six weeks. In some embodiments, a stable bacterium can metabolize a uremic toxin for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, a stable bacterium can metabolize a uremic toxin for one week, two weeks, three weeks, four weeks, five weeks, or six weeks.

[0115] In some embodiments, the bacterial composition can include a modified bacterium. In some embodiments, the bacterial composition can contain a non-modified bacterium. In some embodiments, the bacterial composition can contain a modified bacterium and a non-modified bacterium.

[0116] In some embodiments, the composition can include 1% to 5%, 5% to 10%, 10% to15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% of modified bacteria. In some embodiments, the composition can include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% modified bacteria.

[0117] In some embodiments, the composition can include 1% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25’% to 30%, 30’% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, or 90% to 95%, non-modified bacteria. In some embodiments, the compositions can include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98%, non-modified bacteria.

[0118] In some embodiments, the composition can include a ratio of at least two strains of modified probiotic bacteria of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1 :48, 1 :49, or 1 :50. In some embodiments, the composition can include a ratio of at least three strains of modified probiotic bacteria of 1:1 :1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:2:1, 1:2:2, 1:2:3, 1:2:4, 1:2:5, 1:2:6, 1:2:7, 1:2:8, 1:2:9, 1:2:10, 1:3:1, 1:3:2, 1:3:3, 1:3:4, 1:3:5, 1:3:6. 1:3:7. 1:3:8. 1:3:9. 1:3:10. 1:4:1, 1:4:2, 1:4:3, 1:4:4, 1:4:5, 1:4:6, 1:4:7, 1:4:8, 1:4:9, 1:4:10, 1:5:1, 1:5:2, 1:5:3, 1:5:4, 1:5:5, 1:5:6, 1:5:7, 1:5:8, 1:5:9, 1:5:10, 1:6:1, 1:6:2. 1:6:3. 1:6:4. 1:6:5, 1:6:6, 1:6:7, 1:6:8, 1:6:9, 1:6:10, 1:7:1, 1:7:2, 1:7:3, 1:7:4, 1:7:5, 1:7:6, 1:7:7, 1:7:8, 1:7:9, 1:7:10, 1:8:1, 1:8:2, 1:8:3, 1:8:4, 1:8:5, 1:8:6, 1:8:7, 1:8:8, 1:8:9, 1:8:10, 1:9:1, 1:9:2, 1:9:3, 1:9:4, 1:9:5, 1:9:6, 1:9:7, 1:9:8, 1:9:9, 1:9:10, 1:10:1, 1:10:2, 1:10:3, 1:10:4, 1:10:5, 1:10:6, 1:10:7, 1:10:8, 1:10:9, or 1:10:10.

[0119] In some embodiments, the selecting one or more modified probiotic bacteria that are tolerant of uremic toxins includes measuring at least one of the survival rate, growth rate, or metabolic activity of the probiotic bacteria. In some embodiments, the measurements include an OD600measurement from a culture of the one or more modified bacteria. In some embodiments, the OD600measurement can be from about 0.2 to 3, from about 0.5 to 3, from about 0.5 to 2, from about 1 to 3, from about 1 to 2, or from about 2 to 3. In some embodiments, the OD600measurement can be from about 0.2 to 1.0, from about 1.0 to 1.5, from about 1.5 to 2.0, from about 2.0 to 2.5, from about 2.5 to 3, or from about 2.0 to 3.0. In some embodiments, the OD600measurement can be at least at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, or at least 2.9. In some embodiments, the OD600measurement can be from about 0.1 to 0.2, from about 0.2 to 0.4, from about 0.4 to 0.8, from about 0.8 to 1, from about 1-1.5, from about 1.5-2, from about 2-2.5, or from about 2.5-3. In some embodiments, the OD600measurement can be about about 0.10, about 0.20, about 0.30, about 0.40, about 0.50, about 0.60, about 0.70, about 0.80, about 0.90, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, or about3.5. In some embodiments, the measurements include a measurement of the concentration of urea, ammonia or p-cresol. In some embodiments, the concentration is between 1-10 mM. In some embodiments, the concentration is between 1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 5-7 mM,or 7-10 mM. In some embodiments, the concentration is about ImM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.

[0120] In some embodiments, the selecting one or more modified probiotic bacteria that are tolerant of uremic toxins includes growing the one or more modified probiotic bacteria in a gradient plate containing varying concentrations of toxins, varying concentrations of nutrients, or varying concentrations of toxins and nutrients.

[0121] In some embodiments, the concentration of toxin is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In some embodiments, the concentration of toxin is about ImM, 2mM, 3mM, 4mM, 5mM, 6mM, or 7mM. In some embodiments, the concentration of toxins is from about 1 mM to 7mM, from about 1 mM to 6 mM, from about 1 mM to 5 mM, from about 2 mM to 4 mM, from about 2 mM to 4 mM, from about 3 mM to 5 mM, from about 4 mM to 6 mM, from about 2 mM to 6 mM, from about 3 mM to 6 mM, or from about 4 mM to 6 mM.

[0122] In some embodiments, the concentration of nutrients is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%. In some embodiments, the concentration of nutrients is the concentration of nutrient growth media. In some embodiments, the concentration of nutrients is from about 45 mM to 50mM, from about 50 mM to 60 mM, from about 60 mM to 70 mM, from about 70 mM to 80 mM, from about 80mM to 90 mM, from about 90 mM to 100 mM, from about 50 mM to 80 mM, from about 60 mM to 90 mM, from about 30 mM to70 mM, or from about 20 mM to 50 mM. In some embodiments, the concentration of nutrients is from about 45 to 100 mM. In some embodiments, the concentration of nutrients is about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.

[0123] In some embodiments, the one or more modified probiotic bacteria that are tolerant of uremic toxins are selected by exposing the bacteria to varying concentrations of a toxin for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the measurements are taken after exposing the bacteria to varying concentrations of a toxin for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the bacteria are exposed to a gradient of nutrient media concentration and to a gradient of toxin concentration. In some embodiments, the bacteria are exposed to increased concentrations of toxin each day. In some embodiments, the concentration of toxin is increased by 0.5%, by 1%, by 1.5%, by 2%, by 2.5%, by 3%, by 3.5%, by 4%, by 4.5%, or by 5% each increment. In some embodiments, the concentration of toxin is increased by about 0.5 mM, by about ImM, by about 1.5 mM, or by about 2 mM each increment. In some embodiments, the increment is a row or a column on a multi-well growth plate. In some embodiments, the increment is a period of time. In some embodiments, the period of time is 12-24 hours.

[0124] In some embodiments, the bacterial composition can be administered once a day, twice a day, three times a day, tour times a day, five times a day, six times a day, seven times a day, or eight times a day. In some embodiments, the administration continues for one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, or longer.

[0125] In some embodiments, the bacterial composition includes a prebio tic or a vitamin, In some embodiments, the prebiotic can be a fiber. In some embodiments, the vitamin can be folate, vitamin C, or zoledronic acid.EXAMPLES

[0126] The invention is further illustrated by the following non-limiting examples.EXAMPLE 1Conditioning Bacteria to Metabolize Uremic Toxins

[0127] This example describes the conditioning of probiotic bacteria including Lactobacillus rhamnosus (ATCC 21052) and Bifidobacterium longum (ATCC 15707) to metabolize uremic toxins. The bacterial strains were tested for their ability to grow on and metabolize uremic toxinsunder nutrient depleting conditions. Each bacterial strain was plated on a gradient agar plate where the ratio of nutrients to uremic toxins was varied from 1:0 to 0:1, respectively over a 15x10 cm petri dish. The uremic toxins tested included urea, p-cresol, and indole. The plates were then incubated for bacterial growth for 24, 48, 96, and 120 hours.

[0128] Lactobacillus rhamnosus (L. rhamnosus) was shown to be able to grow in high urea concentrations (10 mM) and depleted nutrient broth (>90% depletion; v / v) when supplemented with 20% dextrose. A growth rate of over 1.0 ODeoo was achieved within 24 hours upon inoculation of 100 CFU (collected from log phase with over 0.4 ODeoo) into a total volume of 5 mL under 5% CO2 at 37°C (ATCC recommended incubation conditions). When dextrose was replaced with p-cresol (10 mM) and indole (10 mM) in the above conditions, the growth rate of L. rhamnosus significantly decreased and was observed to achieve >1.0 ODeoo after 48 hours under 5% CO2 at 37°C (ATCC recommended incubation conditions). Thus, L. rhamnosus was shown to be able to grow in high urea concentrations (lOmM) and high p-cresol and indole concentrations (lOmM) in these experiments.

[0129] Bifidobacterium longum (B. longum) was shown to be able to grow in conditions of high urea concentrations (10 mM) and depleted nutrient broth (>90% depletion; v / v) when supplemented with 20% dextrose. A growth rate of over 1.0 ODeoo was observed after 96 hours upon inoculation of 100 CFU (collected from log phase with over 0.4 ODeoo) into a total volume of 5 mL under an anaerobic atmosphere at 37°C (ATCC recommended incubation conditions). Thus, B. longum was shown to be able to grow in high urea concentrations (lOmM) in these experiments. The slower growth rate of B. longum compared to L. rhamnosus was expected since B. longum is anaerobic.EXAMPLE 2Bacterial Compositions Tolerant to High Urea and Low Nutrient Environment: Growth on MM2 without Ammonium chloride with 5mM of urea (Tube method)

[0130] This example describes the design of bacterial compositions that can efficiently utilize uremic toxins, specifically urea as the nutritional source, thereby turning them into non-toxin components required for survival, growth and propagation.

[0131] The experimental design for evaluating growth rates across selection pressure landscape is shown in TABLE 1. Pure cultures established in log-tracks of growth were used as starting inocula. On day 0 100 CFU / well were inoculated from log-phase culture.Comprehensive growth profiles under wide gradients of depleting nutrients and increasing uremic toxins (urea, p-cresol, indole) were collected in Agar and broth (96 wells) experiments.

[0132] Sixteen strains of bacteria were grown in a toxin environment (5 mM of urea) with different media and dilutions. 1 The study used four types of growth media: Lactobacillus MRS broth, Brain Heart Infusion Broth, Modified Reinforced Clostridia broth, and NH4YE media. The study also used two types of minimal media: MM1 without ammonium chloride plus 20% dextrose, and MM2 toxin, which was MM1 plus 5 mM of urea. The study involved 15 ATCC strains of bacteria and one unknown strain.

[0133] The procedure of the study was as follows: First, the strains were subcultured in their respective growth media and incubated overnight in their respective growth conditions. Second, the optical density (OD) of the cultures was adjusted to 0.1 at 600 nm and incubated again until they reached the log phase (0.4 to 0.6 OD). Third, a fresh Falcone tube (50mL) was prepared with 2.5 mL of MM1 + 5mM of Urea and inoculated with 2.5 mL of the log phase culture, and incubated for 24 hours. A control tube was also prepared with 2.5 mL of growth media and 2.5 mL of MM1, and used as a blank. After one day, the OD of the tubes was measured at 600 nm and 1 uL of the culture was spread on a growth media plate and incubated. Then, a fresh tube was prepared with 2.5 mL of MM1 +5mM of Urea and inoculated with 2.5 mL of the previous tube (Day 1), and incubated for 24 hours. This step was repeated for consecutive days.

[0134] The study performed a urease test to assess the urease gene expression of the strains in response to the toxin environment. The test involved inoculating 10 uL of the culture from day 6 and day 10 tubes into a urease slant, which is a medium that changes color in the presence of urease activity. The slants were incubated at the respective temperature conditions, and were observed for color change every 24 hours.TABLE 1: Experimental Design for Evaluating Growth Rates Across Selection Pressure Landscape (Measurements in CFU, Nutrient broth / MMl (v / v) (across)ZUremic Toxin (mM) (down).

[0135] A targeted list of bacteria utilized included Lactobacillus rhamnosus, Lactobacillus plantarum, Streptococcus thermophilus, Streptococcus faecium, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus gasseri, Akkermansia muciniphila, Bifidobacterium infantis, Bacillus coagulans, Erwinia herbicola, and Bacillus pasteurii.

[0136] All bacterial strains tested survived high uremic toxin conditions in minimal media conditions but required at least >1% (v / v) of nutrients for growth. The results are shown in TABLE 2A AND TABLE 2B.TABLE 2A: Growth of Bacteria Across Selection Pressure Landscape, Days 0-6.TABLE 2B: Growth of Bacteria Across Selection Pressure Landscape, Days 7-10.

[0137] Six probiotic strains that metabolized urea were identified: namely, Bacillus pasteurii, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Bifidobacterium breve, and Bacillus subtilis.

[0138] A high optical density (OD) was observed in the tube from day 6, and a urease test was performed on both the day 6 and day 10 tubes. Upon conducting these tests, it was found that six strains were urease positive from the day 6 tubes, while two strains exhibited a positive urease reaction from the day 10 tubes. In light of these results, the original ATCC strains were then subjected to a urease test to verify the presence and expression of the urease gene within the ATCC strains.EXAMPLE 3Designing Bacterial Compositions Tolerant to Uremic Toxins

[0139] This example describes the design of bacterial compositions that can efficiently utilize uremic toxins as nutritional sources, thereby turning them into non-toxin components required for survival, growth and propagation. Such probiotic bacteria are administered to patients for eliminating the uremic toxins and toxin-precursors in their guts.

[0140] In a first approach a targeted list of bacteria is utilized: Bacillus pasteurii, Lactobacillus sporogenes, Streptococcus thermophilus, Bifidobacterium longum, Erwinia herbicola, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus gasseri, Bifidobacterium inf antis, Lactobacillus salivarius, and Streptococcus f aecium. Pure cultures established in log-tracks of growth are used as starting inocula.Step 1: Mother plate screening

[0141] Each species from the targeted list is inoculated on a separate agar plate with a starting inoculum of fresh grown culture at an optical density of 0.4 measured at 600 nm. Each rectangular agar plate (~25 cm x ~50 cm) is divided into ~10 zones with and an increasing and decreasing gradients of uremic toxins and nutrient growth media respectively (FIG. 1). After inoculation each plate is incubated appropriately depending upon the aerobic and anaerobic growth conditions. Timelapse images of each plate are documented in intervals of ~12 hours, and the position of the moving bacterial front is noted. The plates are under passive observation until the bacterial fronts reach a high concentration of uremic toxins, or if the movement of the bacterial fronts are arrested for at least 48 hours. After which the mother plates are processed - the leading bacterial fronts with overlapped mutants on the mother plates are re-streaked on fresh agar plates to separate the mutants. The agar plates are of identical composition ofnutrients / toxins found to support the growth of the bacterial front of the mother plate. The largest colonies are picked and re-streaked on fresh plates and subsequently the pure cultures are through the generation of glycerol stocks. The assay is expected to be performed for around 10 days with addition of MM1 media made with Na2HPO4 7H2O (423mM), KH2PO4(220Mm), NaCl (86mM), MgSCU (IM), and CaCh (0.1M). The media conditions from day 0 to day 10 are as follows:Day 0 = 100% growth media (GM, specific for each strain),Day 1 = 50% GM + 50% MM1 + 5mM urea,Day 2 = 25% GM + 75% MM1 + 5mM urea,Day 3 = 12.5% GM + 87.5% MM1 + 5mM urea,Day 4 = 6.25% GM + 93.75% MM1 + 5mM urea,Day 5 = 3.13% GM + 96.87% MM1 + 5mM urea,Day 6 = 1.56% GM + 98.44% MM1 + 5mM urea,Day 7 = 0.78% GM + 99.22% MM1 + 5mM urea,Day 8 = 0.39% GM + 99.61% MM1 + 5mM urea,Day 9 = 0.20% GM + 99.80% MM1 + 5mM urea, andDay 10 = 0.10% GM + 99.90% MM1 + 5mM urea.Step 2: Uremic toxin metabolization assay

[0142] Each cataloged bacterial strain is assayed for its growth rate and break down efficiency of uremic toxins in 96 well plates. Each data point is collected in triplicates. For each plate, a concentration gradient for two uremic toxins is established across two axes and with a depleting gradient of nutrients on one axis. After a predetermined period of incubation (12 - 24 hours), the optical density of the wells is noted, and the plate is biochemically processed where uremic toxin levels in the wells are measured and quantified.Step 3: Genomic characterization

[0143] All mutant strains that breakdown uremic toxins above a certain threshold are sequenced and their genomes compared to the genomes of starting strains used as inoculums. The threshold will be set based on time and % of nutrients bacteria are grown in. The threshold for genomic evaluation is expected to occur after Day 7. The data are analyzed, and the mutated genes / regions are annotated.EXAMPLE 4Designing Bacterial Compositions Tolerant to Uremic Toxins Using Whole Microbiome

[0144] This example describes the design of bacterial compositions that can efficiently utilize uremic toxins using raw fecal samples as the starting material. In this approach, raw fecal samples of humans and animals are processed to extract the fraction containing the bacterial microbiomes, which serve as the starting inoculum.Step 1: Mother plate screening

[0145] The fraction of raw fecal sample containing the microbial microbiome is extracted and inoculated on a separate agar plate at a CFU / ml equivalent to that of a pure culture of optical density 0.4. Each rectangular agar plate (~25 cm x ~50 cm) is divided into ~10 zones with and an increasing and decreasing gradients of uremic toxins and nutrient growth media respectively (as shown in FIG. 1). After inoculation each plate is incubated appropriately depending upon the aerobic and anaerobic growth conditions. Timelapse images of each plate are documented in intervals of ~12 hours, and the position of the moving bacterial front is noted. The plates are under passive observation until the bacterial fronts reach a high concentration of uremic toxins, or if the movement of the bacterial fronts are arrested for at least 48 hours. After which the mother plates are processed - the leading bacterial fronts with overlapped mutants on the mother plates is re-streaked on fresh agar plates to separate the mutants. These agar plates are of identical composition of nutrients / toxins found supporting the growth of the bacterial front of the mother plate. The largest colonies are picked and re-streaked on fresh plates and subsequently the pure cultures are cataloged through the generation of glycerol stocks.

[0146] All isolated strains are genetically identified at the genus and species level through a PCR or through sequencing their ribosomal RNA.Step 2: Uremic toxin metabolization assay

[0147] Each isolated bacterial strain is assayed for its growth rate and break down efficiency of uremic toxins in 96 well plates. Each data point is collected in triplicates. For each plate, a concentration gradient for two uremic toxins is established across two axes and with a depleting gradient of nutrients on one axis. After a predetermined period of incubation (12 - 24 hours), the optical density of the wells is noted, and the plate is biochemically processed where uremic toxin levels in the wells are measured and quantified.Step 3: Genomic characterization

[0148] All mutant strains that breakdown uremic toxins above a certain threshold are sequenced and their genomes compared to the genomes of starting strains used as inoculums. The data are analyzed, and the mutated genes / regions are annotated.

[0149] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.EXAMPLE 5Assay Optimization Gradient Plate Preparation

[0150] The objective of the following experiment was to isolate uremic-toxin metabolizing strains by growing all parent strains as mono-cultures on gradient agar plates, with depleting nutrients and increasing toxins (urea, p-cresol, indole). A double layered agar plate consisting of 2 different wedge-like layers of medium was used, where the upper agar surface intersects with the surface of the bottom agar layer, that contains toxin, and the concentration on the upper surface forms a linear gradient.

[0151] The bacterial inoculum included Lactobacillus rhamnose and Bifidobacterium longum. The growth media included Lactobacillus MRS Agar - Ml 163-500G - HiMedia, Lactobacilli MRS Broth - 288130-500G - HiMedia, BSM medium - 88517-250F - Sigma, BSM-Broth - 90273-500G - Sigma, and BSM supplement - 83055-1G-F - Sigma. Growth medium was prepared as per ATTC instructions for each strain.TABLE 3: Minimal media 1 (MM1) M9 salt solutionTABLE 4: Minimal media 2 (MM2) includes MM1+10 mM Urea+ 20% Dextrose (w / v)1.5% agar, prepared in the listed quantity and sterilized in the autoclaveTABLE 5: Minimal media 3 (MM3) includes MM1+10 mM Urea + lOmM -cresol + lOmM Indole +1.5% agar, prepared in the listed quantity and sterilized in the autoclaveEXAMPLE 6 Growth Curve Experiments

[0152] The growth profile of the probiotic strains was studied in order to identify the growth curve, the lag phase, the exponential or log phase, the stationary phase, and the death or decline phase of a particular strain. Each strain was grown in MM2 / 3 toxin media with different gradients of growth media 0.0, 0.5, 1.0, 2.5, 5.0 & 7.5 % (v / v) to identify a percentage of nutrients (v / v) above which the strain can grow in the presence of toxin.

[0153] This experiment included selecting a single isolated colony from zone 11 of a gradient plate for each strain, from all four plates used in this growth curve experiment; inoculating the colony in an MM2 / 3 toxin medium containing 10% of growth nutrients; incubating the inoculated medium for 48 hours under the respective ATTC growth conditions for each strain; transferring 1 OOpiL of the culture (grown in 10% growth media+MM2 / 3) to new tubes with varying concentrations of growth nutrients ranging from 0.0 to 7.5 % in MM2 / 3 toxin medium; incubating the new tubes in respective growth conditions; and measuring the optical density at 600 nm of the tubes every 24 hours to monitor the growth of the strains.TABLE 6: Growth Curve ResultMM2 BSM MM3 BSM

[0154] In this study of growth curves, various observations were made across different media broths. For the MM2 MRS media, there was a notable growth starting from the 2.5% growth media concentration, and this growth continued to increase as the incubation period was extended. In contrast, the MM3 MRS media exhibited growth in the 5% and 7.5% concentrations on the first day, but no further growth was detected thereafter. The MM3 tubes displayed a pink coloration. Both L. rhamnosus and B. longum exhibited growth in the presence of toxins for MM2 media, but with MM3 media only L. rhamnosus exhibited growth. The presence of a pink color indicates a lack of growth.

[0155] For the MM2 BSM media, there was a discernible slow growth that commenced from the 1% concentration, becoming apparent from the third day of observation. However, the MM3 BSM media did not show any growth across all tested concentrations, suggesting a stark difference in the growth-promoting capabilities of the MM3 BSM media.

[0156] An additional note of interest was the change in color observed in the MM3 toxin media tubes after 24 hours of incubation. The tubes turned pink, with a darker pink hue in the MM3 tubes containing MRS and a lighter pink in the MM3 tubes with BSM. This color change might be attributed to the presence of indole, a compound that can cause such a reaction under certain conditions.EXAMPLE 7Evaluating Growth Rates Across Selection Pressure Landscape for L. rhamnosus and B. longum

[0157] The aim of this study was to assess the growth rates of two bacterial strains in the presence of different concentrations of nutrients and toxins, specifically using MM2 / MM3 media. The experiment was designed in a 96 well plate which allows the use of 12 different concentration for nutrients (growth media and minimal media) in the presence of different concentrations of toxin (0 to 7 mM). Minimal media with different concentrations of urea and Indole+p-cresol were prepared. This study evaluated the growth rate of each strain with respect to nutrient and toxin concentrations.

[0158] For the experiment, the growth media included Lactobacillus MRS broth and BSM broth. MM1 media was modified by omitting ammonium chloride and adding 20% dextrose to create MM2 plates, while MM3 plates simply used MM1 without ammonium chloride. Toxins for MM2 were prepared as lOOmM Urea in MM1 with an additional 20% dextrose, whereas for MM3, a combination of lOOmM Urea, Indole, and p-cresol was prepared in MM1. The inoculum for MM2 was prepared in MM1 with 20% dextrose, and for MM3, it was prepared in MM1.

[0159] The procedure used sterile flat-bottom 96-well plates for each culture. The respective growth media (MRSB for £. rhamnosus MM2 & MM3; B. longum MM2 & MM3) were added to the wells, with concentrations decreasing from 55 to 0 mM across wells 1 to 12 (55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 0), as depicted in FIG. 1. MM1 was added in increasing concentrations from 45 to 100 mM across columns 1 to 12 (45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100), also as shown in FIG. 1. Toxins were added in increasing concentrations along each row, with row A starting at 0 mM and row H ending at 7 mM (A-0, B-l, C-2, D-3, E-4, F-5, G- 6, H-7). Each well received 100 CFU of the respective culture, and the plates were incubated at 37°C. Lactobacillus rhamnosus cultures were shaken at 150 RPM in aerobic conditions, while Bifidobacterium longum cultures were incubated anaerobically. The growth was monitored over a period of 10 days, with readings taken every 24 hours. Optical density (OD) was measured at 600nm, and the plates were shaken for 60 seconds before each reading to ensure consistency.

[0160] For example, well Al contained a total volume of 200 pL, made up of 110 pL of growth media, 70 pL of MM1, and 20 pL of culture. In contrast, well A2 also had a total volume of 200 pL but was composed of 110 pL of growth media, 68 pL of MM1, 20 pL of culture, and an additional 2 pL of toxin. This systematic approach allowed for a detailed analysis of the growth patterns of the bacterial strains under various nutrient and toxin conditions.TABLE 7: Plat Map and Analysis at Day 0.

[0161] The top row includes percent of nutrient broth / MMl (without ammonium chloride) while the first column includes levels of toxins as MM2 / MM3 with toxin (mM, N=2). At day 0,100 CFU is inoculated in each well from log-phase culture. In the present analyses, the death phase is when ODeoo is greater than 2.0, the stationary phase is when ODeoo is greater than 1.0 and less than 2.0, the log-growth phase is when ODeoo is greater than 0.2 and less than 1.0, and the lag-growth phase is when ODeoo is greater than 0.05 and less than 0.2.TABLE 8: N1 Result OD for Lactobacillus rhamnose.TABLE 9: N2 Result OD for Lactobacillus rhamnose.TABLE 10: N1 Result OD for Bifidobacterium longum.TABLE 11: N2 Result OD for Bifidobacterium longum.

[0162] The experimental design was aimed al evaluating the growth rate under selection pressure, which entailed a reduction in growth nutrients and an increase in minimal media, along with varying concentrations of toxins. During the observation phase, it became apparent that no significant growth occurred in column 12, which was devoid of growth nutri ents and consisted solely of 100% minimal media, despite the presence of different toxin concentrations.Furthermore, it was noted that the strains Lactobacillus rhamnose and Bifidobacterium longum grew in media with a low amount of growth nutrients. Across all the plates, a lag phase optical density (OD) was recorded for Lactobacillus rhamnose and Bifidobacterium longum and two toxins, (MM2 containing urea, and MM3 containing urea, indole and p-cresol). The lag growth OD ranged from greater than 0.05 to less than 0.2 when measured at a wavelength of 600nm.EXAMPLE 8Growth Curve for All 15 Probiotic ATCC Strains

[0163] The objective is to evaluate the growth profiles of all 15 probiotic strains in the presence of different concentration of nutrients and toxins (MM2 / MM3). Four distinct phases of the growth curve: lag, exponential (log), stationary, and death were noted for each strain. Each strain has a distinct growth curve based on the doubling time. The experiment used log phase culture (0.4 to 0.6 OD read at 600), and was conducted to identify the time used for each strain to reach the OD 0.4-0.6 when the identical concentration of inocula was used.TABLE 12: ATCC Strain List with Growth Conditions.

[0164] The procedure for the growth curve was carried out with all the strains having been sub-cultured from the glycerol stocks, which were originally made from the revival of ATCC lyophilized cultures. The objective was to obtain growth curves for all ATCC strains to ensure that log phase cultures were achieved, with an optical density (OD) between 0.4 and 0.6, corresponding to a target concentration of 100 colony- forming units (CFU) per 100 pL.

[0165] Each strain was sub-cultured in its respective media under their specific growth conditions. Freshly prepared growth media was then taken for each and adjusted with the broth culture to an OD of 0.1, as read at 600 nm. These adjusted tubes were then incubated under their respective growth conditions, with OD values being read at 600nm every hour.

[0166] The incubation continued until the cultures attained the desired log phase, with an OD between 0.4 and 0.6. Upon reaching this phase, a serial dilution and plating process was conducted using a 100 pL pour plate to ensure the CFU count was in accordance with the OD readings. Subsequently, the plates were incubated under the appropriate growth conditions, and after 18 to 24 hours, the colonies were counted.

[0167] The table provided details the time each strain took to progress from an initial OD of 0.1 to the final log phase OD of 0.4 to 0.6, along with the corresponding CFU counts.TABLE 13: Growth Curve Result for ATCC Strains.

[0168] Growth curve for all 15 were evaluated and CFU counts with corresponding OD values at 600 nm were captured. All the strains were incubated at least 6 hours to reach log phase (0.4-0.6); for the slow growing strains the initial inoculum was increased to avoid more than 6 hours of incubation. Hence, based the measured growth rates of individual probiotic strains, it becomes feasible to synchronize the incubation periods such that all strains reach OD values of 0.4 to 0.6 simultaneously for formulating probiotic combinations.EXAMPLE 9Evaluating Growth Rates Across Selection Pressure Landscape for Six Strains (Without Toxin)

[0169] The objective was to evaluate the growth rate of each strain by varying the concentration of nutrients. The experiment was designed in a 96 well plate which allowed for 12 different concentrations of nutrients (growth media and minimal media), to identify the lowest nutrient concentrations used in this experiment for each strain to grow in the absence of toxin and also the time taken for each strain to grow in minimal media.

[0170] The media used included various growth media such as Lactobacillus MRS broth, Brain Heart Infusion Broth, and Modified Reinforced Clostridial broth. Additionally, a modified medium, MM1 without ammonium chloride, was utilized. The inoculum was prepared in MM1. The ATCC strains used in the study comprised Lactobacillus rhamnosus (21052), Lactobacillus plantarum (BAA-793), Streptococcus thermophilus (19258), Streptococcus faecium (BAA- 2320), Bifidobacterium longum (15707), and Bifidobacterium breve (15700).

[0171] During the procedure, 96 well plates with sterile flat bottoms were employed for culturing all strains. The respective growth media were added to the wells in decreasing concentrations ranging from 55 to 0 (55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 and 0) from columns 1 to 12 respectively, following the sequence provided in FIG. 1. Conversely, MM1 was added in increasing concentrations from 45 to 100 (45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100) from columns 1 to 12 respectively. TABLES 14 to 25 capture the OD values of bacterial growth post 24 hours. Each well received 100 CFU of the respective culture. The plates were then incubated under the appropriate growth conditions for each strain. Observations were made by reading the plate every 24 hours over a period of six days. Before taking triplicate readings of the optical density at 600nm, the plates were shaken for 300 seconds.

[0172] The incubation conditions varied slightly for each strain. Lactobacillus rhamnosus (21052) and Lactobacillus plantarum (BAA-793) were incubated at 37°C for 24 to 48 hours in an aerobic environment with 5% CO2. Streptococcus thermophilus (19258) and Streptococcus faecium (BAA-2320) were also incubated at 37°C but for 24 hours in an aerobic environment with shaking at 150 RPM. Bifidobacterium longum (15707) and Bifidobacterium breve (15700) were incubated at 37°C for 24 to 48 hours under anaerobic conditions.

[0173] An example of the well composition is provided for well Al, which contained a total volume of 200uL made up of 110 uL of growth media, 70uL of MM1, and 20uL of culture. See TABLE 7 for a description of the plat map and analysis used.

[0174] Results of experiment for Lactobacillus rhamnosus (21052): Growth was observed in both the plates, Stationary phase OD was observed from day 3 onwards.TABLE 14: N1 Plate OD for Lactobacillus rhamnosus (21052).TABLE 15: N2 Plate OD for Lactobacillus rhamnosus (21052).

[0175] Lactobacillus plantarum (BAA-793): Growth was observed in both the plates, and stationary phase OD was observed from day 1 onwards (24 Hrs of incubation).TABLE 16: N1 Plate OD for Lactobacillus plantarum.TABLE 17: N2 Plate OD Lactobacillus plantarum.

[0176] Streptococcus thermophilus (19258): Growth was observed in both the plates, stationary phase OD was observed from day 5 on N1 plate. N2 plate OD was not changed from 48 hrs. onwards.TABLE 18: N1 Plate OD for Streptococcus thermophilus (19258).TABLE 19: N2 Plate OD for Streptococcus thermophilus (19258).

[0177] Streptococcus faecium (BAA-2320): Growth was observed in both the plates,Stationary phase OD was observed from day 5 on both the plates.TABLE 20: N1 Plate OD for Streptococcus faecium (BAA-2320).TABLE 21: N2 Plate OD for Streptococcus faecium (BAA-2320).

[0178] Bifidobacterium longum (15707): Growth was observed in both the plates, Lag phase OD observed from day 1 onwards, OD did not increase past 24 hrs (OD was constant between 0.3 to 0.5).TABLE 22: N1 Plate OD for Bifidobacterium longum (15707).TABLE 23: N2 Plate OD for Bifidobacterium longum (15707).

[0179] Bifidobacterium breve (15700): Growth was observed in both the plates, Lag phase OD observed from day 1 onwards, OD was not increased past 24 hrs (OD was constant between 0.3 to 0.5).TABLE 24: N1 Plate OD for Bifidobacterium breve (15700).TABLE 25: N2 Plate OD for Bifidobacterium breve (15700).

[0180] The evaluation of growth rates in the past indicated that the six strains under study used low concentrations of growth nutrients to grow. It was noted that there was no growth in column 12, which represented a condition of 100% minimal media. Additionally, when the Bifidobacterium strains were exposed to the right concentration of growth nutrients, specifically 55% as found in the Al well, no growth was observed. To further investigate these findings, a trial assay had been conducted using Falcon tubes, each containing 10 mL of culture. This assay successfully produced growth of the six strains at the highest concentration used in the study, which was a mixture of 55% growth media and 45% minimal media.EXAMPLE 10Growth Curve in Tubes (Replicate of Al Wells for All Strains)

[0181] The objective of this experiment was to evaluate the growth rate of Al well (55 % of growth nutrients and 45 % of minimal media) for each strain. The experiment was designed in 50mL Falcone tube, where 5 mL of media (3.5mL MM1 + 5.5mL GM + ImL Inoculum from 10A5 Dilution) incubated for 6 days and OD read every 24 hours. In this study the growth curve was evaluated of each strain, measuring the time used to reach log / stationary / death phase in the presence of high concentrations of growth nutrients.

[0182] Six strains were selected for a growth curve analysis using a tube assay, which was conducted in a 96-well plate format. Each tube was prepared with a mixture consisting of 5.5 mL of growth media and 3.5 mL of MM1, and 1 mL of culture previously adjusted in MM1. This composition mirrored the total concentration found in the Al well of the previous test format, with 55% of the mixture being growth media and 45% minimal media. The tubes were then placed under their respective growth conditions. Optical density (OD) readings at 600 nm were recorded every 24 hours over a period of six days.TABLE 26: Result of Growth Curve in Tube (Replicate of Al Well).

[0183] Growth was observed by measuring the optical density (OD) at 600nm daily up until the sixth day. An increase in OD was noted after the first 24 hours, indicating that a mixture consisting of 55% growth nutrients and 45% minimal media was sufficient to support robust growth. For all six strains, the OD values reached up to 0.9, which is indicative of the log phase of growth.EXAMPLE 11Evaluating Growth Rates Across Selection Pressure Landscape

[0184] The objective was to evaluate the growth rate of each strain in the presence of different concentration of nutrients and toxins (MM2 / MM3). The experiments were designed in a 96 well plate which allows 12 different concentrations of nutrients (growth media and minimal media) in the presence of 8 different concentration of toxin (0 to 7 Mm). MM2 was prepared with urea under varying concentrations and MM3 with varying concentrations of Indole and p- cresol. This study evaluates the growth rate of each strain with respect to nutrient toxin concentrations.

[0185] Various growth media were used, including Lactobacillus MRS broth, Brain Heart Infusion Broth, and Modified Reinforced Clostridial broth. Modifications to the MM1 medium were prepared by omitting ammonium chloride and adding 20% dextrose to create MM2 plates, while MM3 plates were prepared with MM1 lacking ammonium chloride. Toxins for MM2 and MM3 were prepared with lOOMm Urea in MM1 with 20% dextrose and lOOMm of a combination of Urea, Indole, and p-Cresol in MM1, respectively. Inoculum for MM2 was prepared in MM 1+20% dextrose, and inoculum for MM3 was prepared in MM1 media.

[0186] A variety of ATCC strains were utilized, each requiring specific growth conditions. Lactobacillus plantarum (BAA-793) was grown in Lactobacillus MRS broth, while both Streptococcus thermophilus (19258) and Streptococcus faecium (BAA-2320) were cultured in Brain Heart Infusion Broth. Bifidobacterium breve (15700) was cultivated in Modified Reinforced Clostridial broth, and several other Lactobacillus strains, including Lactobacillus casei (15008), Lactobacillus gasseri and Lactobacillus acidophilus (BAA-2845), were grown in Lactobacilli MRS Broth. Additional strains such z\ Akkermansia muciniphila (BAA-835) and Bacillus coagulans (7050) were grown in Brain Heart Infusion Broth and Nutrient broth, respectively. Bifidobacterium infantis (15697) was grown in Modified Reinforced Clostridial, Erwinia herbicola (33243) was grown in Nutrient broth, and Bacillus pasteurii (11859) was grown in NH4-YE medium.

[0187] The experimental procedure involved taking a sterile 96 well plate with a flat bottom and adding the respective growth media in decreasing concentrations from wells 1 to 12 (55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 and 0) as depicted in FIG. 1. MM1 was added in increasing concentrations across columns 1 to 12 (45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100), as in FIG. 1. Toxins were added in increasing concentrations in each row (A-0, B-l, C-2, D-3, E-4, F-5, G- 6, H-7). Each well received 100 CFU of the respective culture, and the plate was then incubated under the appropriate growth conditions.

[0188] Incubation times and conditions varied for each strain. Lactobacillus plantarum (BAA-793) was incubated at 37°C for 24 to 48 hours under aerobic conditions with 5% CO2, while Streptococcus thermophilus (19258) and Streptococcus faecium (BAA-2320) were incubated in aerobic conditions at 37°C for 24 hours with shaking at 150 RPM. Bifidobacterium breve (15700) was incubated for 24 to 48 hours in anaerobic conditions at 37°C. Lactobacillus casei (15008) was incubated for 24 to 48 hours in anaerobic conditions at 37°C with 5% CO2. And Lactobacillus bulgaricus (11842) was incubated at 37°C for 24 to 48 hours under aerobic conditions with 5% CO2. Lactobacillus gasseri (33323) was incubated at 37°C for 24 to 48 hours under aerobic conditions while Akkermansia muciniphila (BAA-835) was shaken and incubated anaerobically at 37°C for 2 to 7 days under 100% N2. The incubation conditions for Bifidobacterium infantis (15697) included 48 to 72 hours incubation at 37°C anaerobically, and Bacillus coagulans was incubated aerobically at 37°C for 24 hours. Erwinia herbicola was incubated at 26°C for 24 hours in aerobic conditions, and Bacillus pasteurii was incubated at 30°C for 48 to 72 hours in aerobic conditions.

[0189] The plates were read every 24 hours for a period of 10 days. Optical density readings were taken at 600nm after shaking the plates for 60 seconds. For example, well Al contained a total volume of 200pL, comprising 110 pL of growth media, 70 pL of MM1, and 20 pL of culture. In contrast, well A2 had the same total volume but included 78 pL of MM 1+20 pL of culture and an additional 2 pL of toxin. See TABLE 7 for a description of the plate map.

[0190] Lactobacillus plantarum (BAA-793): Growth was observed in both the plates, stationary phase OD was observed in most of the wells in day 8 and reduced on day 9 & 10 for both MM2 & MM3TABLE 27: Day 10 results for Lactobacillus plantarum (BAA-793) in MM2.TABLE 28: Day 10 results for Lactobacillus plantarum (BAA-793) in MM3.

[0191] Streptococcus thermophilus (19258): Growth was observed in both the plates, with a high OD reached on Day 2 (0.4 to 0.5). Evaporation was observed on Day 4 in column 1, likely due to an edge effect.TABLE 29: Streptococcus thermophilus (19258) Growth on MM2 Media.TABLE 30: Streptococcus thermophilus (19258) Growth on MM3 Media.

[0192] Streptococcus faecium (BAA-2320): Growth was observed in both the plates; highestOD (0.4 to 0.5) was observed on day 2 onwards. Evaporation observed on Day 4 in column 1 (Edge effect) in both the plates, while OD had reduced on day 10.TABLE 31: Streptococcus f aecium (BAA-2320) Growth on MM2 Media.TABLE 32: Streptococcus f aecium (BAA-2320) Growth on MM3 Media.

[0193] Bifidobacterium breve (15700): Growth was observed in both the plates, Lag phase OD was observed from day 1 onwards, while OD did not increased post 24 hrs. (OD was constant between 0.3 to 0.5). Highest OD was observed on day 8 in most of the wells and was reduced on days 9 and 10.TABLE 33: Bifidobacterium breve (15700) Growth at Day 10 on MM2 Media.TABLE 34: Bifidobacterium breve (15700) Growth at Day 10 on MM3 Media.TABLE 35: Lactobacillus casei (15008) on MM2 Media.TABLE 36: Lactobacillus casei (15008) on MM3 Media.TABLE 37: Lactobacillus acidophilus (BAA-2845) on MM2 Media.TABLE 38: Lactobacillus acidophilus (BAA-2845) on MM3 Media.TABLE 39: Lactobacillus bulgaricus (11842) on MM2 Media.TABLE 40: Lactobacillus bulgaricus (11842) on MM3 Media.TABLE 41: Lactobacillus gasseri (33323) on MM2 Media at Day 10 (OD Reduced with Day 0).TABLE 42: Lactobacillus gasseri (33323) on MM3 Media at Day 10 (OD Reduced with Day 0).TABLE 43: A. muciniphilia (BAA-835) on MM2 Media, Day 10.TABLE 44: A. muciniphilia (BAA-835) on MM3 Media, Day 10.TABLE 45: B. coagulans (1050) on MM2 Media, Day 10.TABLE 46: B. coagulans (1050) on MM3 Media, Day 10.TABLE 47: B. infantis (15691) on MM2 Media, Day 10.TABLE 48: B. infantis (15691) on MM3 Media, Day 10.TABLE 49: E. herbicola (33243) on MM2 Media, Day 10.TABLE 50: E. herbicola (33243) on MM3 Media, Day 10.TABLE 51: B. pasteurii (11859) on MM2 Media, Day 10.TABLE 52: B. pasteurii (11859) on MM3 Media, Day 10.

[0194] Growth was observed in wells with growth nutrients and not in wells with low levels of media for all the strains. It was observed that some level of growth nutrients were used by all the stains to allow growth irrespective of toxin concentrations.EXAMPLE 12Evaluating Growth Rate for 6 Strains in MM2 (5Mm Urea) with Different Concentration of Culture

[0195] This study evaluated the growth rate of strains in MM2 toxin (5Mm of Urea) with different cell densities ranging from 0 to 10,240 CFU per well in the absence of growth nutrients. The experiment was designed in a 96 well plate which allows 12 different ranges for cell density in the presence of standard 5Mm Urea toxin concentration. This study evaluates the growth rate of each strain with respect to cell density in standard toxin concentration in the presence of minimal media (without growth media / nutrients).

[0196] MM1 was prepared without the addition of ammonium chloride but was supplemented with 20% dextrose. Meanwhile, MM2 was a solution of lOMm Urea that hadbeen prepared in MM1 and also contained a 20% dextrose concentration, resulting in a solution that was twice the standard concentration.

[0197] A selection of ATCC strains was utilized in the experiment. These included Lactobacillus rhamnosus (21052), Lactobacillus plantarum identified as BAA-793), Streptococcus thermophilus with the number (19258), Streptococcus faecium known as (BAA- 2320), Bifidobacterium longum with the number (15707), and Bifidobacterium breve identified as (15700).

[0198] The procedure followed involved the use of a sterile 96 well plate with a flat bottom. Each well received 100 microliters of the 2x concentrated MM2. Following this, 100 microliters of inoculum, which had been prepared in MM1 supplemented with 20% dextrose, were added to each column according to a specified table. For the purpose of serving as a negative control, 100 microliters of MM2 were added to wells Al through Hl. The plate was incubated at respective growth conditions.TABLE 53: Inoculum Concentration, Culture CFU per Well.

[0199] Lactobacillus rhamnosus (21052) was incubated at 37°C for a period ranging from 24 to 48 hours under aerobic conditions with 5% CO2. Similarly, Lactobacillus plantarum strain BAA-793 underwent incubation at the same temperature and for the same duration, also in an aerobic environment supplemented with 5% CO2. Streptococcus thermophilus strain 19258 was cultured at 37°C for 24 hours in an aerobic setting and was shaken at 150 RPM. The same conditions were applied to Streptococcus faecium strain BAA-2320, which was incubated with shaking at 150 RPM during its 24-hour incubation period at 37°C.

[0200] Bifidobacterium longum strain 15707 and Bifidobacterium breve strain 15700 were both incubated anaerobically at 37°C, with their incubation times ranging from 24 to 48 hours. Following the incubation, the plates were thoroughly mixed by pipetting. The optical density (OD) at 600nm was then measured every 48 hours to monitor the growth of the cultures. Throughout these experiments, wells from column Al to Hl on the plate were designated as the negative control to ensure the accuracy and reliability of the experimental results.

[0201] In the conducted experiment, MM2 was utilized with a urea concentration of 5Mm, and various concentrations of culture were taken for the assay. The first column of the assay, which contained no culture, served as the negative control, while columns two through twelve varied in colony-forming unit (CFU) count. The plates were then incubated under theirrespective growth conditions. The optical density (OD) at 600nm was measured every 48 hours over a total incubation period of 10 days. It was observed that growth did not occur in any of the plates across all six strains. This outcome indicated that all strains require minimal growth nutrients for their development.EXAMPLE 13Evaluating Growth Rate for Six Strains in MM2 with Different Concentration of Culture and Toxin

[0202] The objective of the experiment was to evaluate the growth rate of six bacterial strains in the presence of MM2 toxin, which lacked growth nutrients but had varying concentrations of the toxin. The strains were cultured in a range of cell densities from 0 to 10,240 CFU per well. A 96-well plate was utilized, allowing for 12 different cell densities to be tested alongside varying urea toxin concentrations. The growth rate of each strain was assessed in relation to the cell density within a specific toxin concentration.

[0203] MM1 medium, which consisted of ammonium chloride and 20% dextrose, was prepared. The MM2 toxin was then created by mixing 0 to 7 Mm of urea with the MM1 medium, also containing 20% dextrose.

[0204] The bacterial strains used in the study were Lactobacillus rhamnosus (21052), Lactobacillus plantarum (BAA-793), Streptococcus thermophilus (19258), Streptococcus faecium (BAA-2320), Bifidobacterium longum (15707), and Bifidobacterium breve (15700).

[0205] The procedure began with the preparation of 96-well plates that had sterile flat bottoms. Each well from B to H received 100 pL of 2X toxin, which was prepared in MM1 with ammonium chloride and 20% dextrose. The toxin concentration increased with each row, starting from 0 Mm in row A to 7 Mm in row H (A-0, B-l, C-2, D-3, E-4, F-5, G-6, H-7Mm).Additionally, 200 pL of MM1 was added to wells Al to Hl to serve as a negative control. The inoculum was prepared in MM1 with ammonium chloride and 20% dextrose to achieve the desired culture CFU per well, as in the previous Example.

[0206] The incubation conditions varied for each strain. Lactobacillus rhamnosus (21052) and Lactobacillus plantarum (BAA-793) were incubated at 37°C for 24 to 48 hours in an aerobic environment with 5% CO2. Streptococcus thermophilus (19258) and Streptococcus faecium (BAA-2320) were also incubated at 37°C for 24 hours but were shaken at 150 RPM to maintain aerobic conditions. Bifidobacterium longum (15707) and Bifidobacterium breve (15700) were incubated at 37°C for 24 to 48 hours under anaerobic conditions. During incubation, the plates were mixed well by pipetting, and the optical density (OD) at 600 nm wasread every 48 hours. Columns Al to Hl were used as the negative control. See TABLE 7 for the plate map and analysis.

[0207] After 10 days of incubation, cell viability was assessed. OD readings at 600 nm were recorded, and 100 pL samples from wells Al 2 and H12 were spread onto agar plates containing growth media. These plates were then incubated under the respective growth conditions for each strain. Post-incubation, the growth on the agar plates was observed over a gradient of growth from A12, which had 0% toxin, to H12, which had a high toxin concentration of 7 Mm urea.

[0208] Results are shown in TABLE 54 below.TABLE 54: Results of Growth Rate Experiment for S. thermophilus.TABLE 55: Results of Growth Rate Experiment for S. faecium.TABLE 56: Results of Growth Rate Experiment for L. rhamnosusTABLE 57: Results of Growth Rate Experiment for L. Plantarum.TABLE 58: Results of Growth Rate Experiment for B. breve.

[0209] Growth was observed in random wells, although the optical density (OD) readings were minimal, indicating that the cultures were in the lag phase with OD values greater than 0.05 but less than 0.2. To verify the cell viability, 100 microliters were taken from wells A12 and H12 and were spread onto agar plates that contained growth media. Upon examination, row 12 displayed a high colony- forming unit (CFU) count. Specifically, well Al 2 showed a 0%toxin presence, while well H12 contained a high concentration of toxin, with 7 Mm urea being detected.TABLE 59: Growth Observed in Different Cells for Different Strains.

[0210] It was observed that Bifidobacterium longum did not exhibit growth in MM1 media that was devoid of toxin, indicating that these cells remained in a state of stasis within a minimal media environment. In contrast, the remaining six strains demonstrated growth in MM1 media with a 0% toxin concentration, suggesting that while the cells were capable of surviving in minimal media, their growth rate was not as robust as it would be in the presence of a more nutrient-rich growth media.

[0211] Furthermore, when exposed to a high toxin concentration, specifically 7 Mm of urea, both Lactobacillus plantarum and Bifidobacterium longum cells were found to be non-viable. This lack of viability was particularly notable for Bifidobacterium longum, as it failed to survive in both the absence and 7Mm concentration of urea, underscoring the need for a low nutrient concentration to support the growth and viability of these cells. The findings highlight the sensitivity of Bifidobacterium longum to both nutrient availability and toxin presence, and the importance of optimizing growth conditions for the cultivation of such bacterial strains.EXAMPLE 14Growth on Christensen’s Urea Broth and Stuart’s Urea Broth

[0212] The objective of the study was to identify the strains which express urease genes which facilitate the breakdown of urea for nitrogen source. Christiansen’s and Stuart’s urea broth were used to determine whether the strains were urease positive or negative.

[0213] The procedure began with the subculturing of strains into their respective broth media, which were then incubated overnight under the appropriate growth conditions. The optical density (OD) of these cultures was subsequently adjusted to 0.1 at 600nm in their respective growth conditions. This step ensured that the cultures reached the log phase, indicated by an OD of 0.4 to 0.6. Once the log phase was achieved, the cultures were further adjusted toobtain a concentration of 5000 colony- forming units (CFU) per 100 microliters. This precise volume was then transferred into the corresponding urease test tubes.

[0214] In addition to the cultures, 15 ml Falcon tubes were prepared by inoculating them with the respective probiotic strains in 5 ml of urease broth, which included both Christensen’s Urea Broth and Stuart’s Urea Broth. These tubes were then incubated under the strains’ respective growth conditions for a period of 24 hours. To ensure the accuracy of the test, a negative control was set up using 5 ml of urease broth without any inoculation. Conversely, for the positive control, 5 ml of urease broth was inoculated with 100 microliters of Klebsiella pneumoniae 35657.

[0215] The first day following incubation included capturing images of the tubes. Over the subsequent six days, the tubes were regularly observed for color change, which was expected to transition from yellow or light orange to pink, indicating urease activity. This observation was a part of the procedure to determine the presence and activity of urease in the probiotic strains under study.TABLE 60: Results of Experiment Days 1-3.TABLE 61: Results of Experiment Days 4-6.

[0216] All 16 strains were inoculated into both Christensen’s and Stuart’s Urea Broth under their respective growth conditions and were incubated for a period of 6 days. After this incubation period, it was observed that none of the strains exhibited a positive urease reaction in either of the broths. In contrast, Klebsiella pneumoniae 35657, which was utilized as a positive control for urease activity, demonstrated a positive expression of urease from the second day of incubation onward. This stark difference highlighted the urease-producing capabilities of Klebsiella pneumoniae 35657 in comparison to the other strains under study.EXAMPLE 15Growth on Minimal Media with 5 Mm of Urea (MM2) Followed by Christensen’s Slant

[0217] The objective of the study was to activate the urease gene in each strain by growing the cells to log phase and followed by incubation in the presence in MM2 (5Mm of Urea) without any growth nutrients. Incubation the cells in MM2 for 6 days was followed by inoculation in Christensen’s slant to test for urease activity.

[0218] The procedure began with the subculturing of strains in their respective broth media, which were then incubated overnight under appropriate growth conditions. The optical density (OD) of these cultures was adjusted in their respective growth media to 0.1 at 600nm. The cultures were further incubated under the same growth conditions until they reached the log phase, indicated by an OD between 0.4 and 0.6. Upon reaching this phase, the cultures were centrifuged at 4500 RPM for 5 minutes, and the supernatant was subsequently discarded.

[0219] The cells were then washed with 5ml of MM1, pelleted again at 4500 RPM for another 5 minutes, and the supernatant was discarded once more. The pelleted cells were resuspended in 5ml of MM2, which contained 5Mm urea and 20% dextrose. Following resuspension, the tubes were placed back into their respective growth conditions to incubate. After an hour of incubation, 10U1 samples were taken from each tube and streaked onto Christensen’s agar slant, which was labeled as day 0.

[0220] The Stuart’s broth tubes, containing MM2 with 5 mM urea and 20% dextrose, along with the Urease slant, were incubated under respective growth conditions. The broth tubes were left to incubate for a total of 6 days. During this period, samples were streaked onto the urease slant after the first hour of incubation on day 0, and then again on days 2, 4, and 6 to confirm growth.

[0221] Throughout the 6-day period, observations of the urease slant were carried out every 24 hours. Each observation included recording an image of the slant to document the progress and results of the experiment.TABLE 62: Results from Day 0, Days 1-3.TABLE 63 Results from Day 0, Days 4-6.

[0222] Urease for day 0 (Post 1 hrs. in MM2 media) showed 6 strains positive from day 2 onwards, and 8 strains from day 3 onwards.TABLE 64: Results from Day 2, Days 1-3.TABLE 65: Results from Day 2, Days 4-6.

[0223] Post 48 hrs of incubation in MM2, 8 strains showed urease positive from day 1 onwards.TABLE 66: Results from Day 4, Days 1-3.TABLE 67: Results from Day 4, Days 4-6.

[0224] Post 96 hrs of incubation in MM2, 8 strains showed urease positive from day 1 onwards.TABLE 68: Results from Day 6, Days 1-3.TABLE 69: Results from Day 6, Days 4-6.

[0225] After six days of incubation in MM2, it was observed that six strains exhibited urease positive reactions starting from the second day onwards. However, Lactobacillus rhamnosus (21052) and Lactobacillus acidophilus (BAA-2845) remained urease negative throughout the six-day period, despite being incubated in MM2 supplemented with 5Mm of urea and 20% dextrose. In addition to these observations, glycerol stocks were prepared from the ureasepositive tubes on the second day of incubation. These stocks were then carefully stored at -80 degrees Celsius for future utilization.

[0226] Out of 16 strains, 7 strains were urease positive from day 0, and 8 strains were urease positive from the day 2, 4 & 6 tubes.EXAMPLE 16Growth on MM2+5mM of urea for Strains (8 mutated strains from Week 17 experiment)

[0227] The objective of the study was to re-evaluate (quantitative analysis) the urease activity for the 8 mutated strains obtained from Example 15 by performing ammonia quantification, inaddition to growth profile measurements for all strains through dilution and plating. Ammonia quantification was performed to evaluate the percentage of urea converted into ammonia by each strain and the viability of each strain was assessed in Christensen's broth.

[0228] In Example 15, nine mutated strains were observed. These included Bacillus pasteurii (11859), Bacillus subtilis (6633), Bifidobacterium breve (15700), Lactobacillus bulgaricus (11842), Lactobacillus casei (15008), Lactobacillus gasseri (33323), Lactobacillus rhamnosus

[0229] Strains were subcultured in their respective broth media, and were then incubated overnight under their respective growth conditions. The optical density (OD) of these cultures was adjusted to 0.1 at 600nm in their respective growth conditions, and they were further incubated to reach the log phase, with an OD ranging from 0.4 to 0.6. Upon reaching the desired OD, the cultures were centrifuged at 4500 RPM for 5 minutes, and the supernatant was discarded. The cells were then washed with 5ml of MM1, centrifuged again at the same speed and duration, and the supernatant was once more discarded. The cells were subsequently resuspended in 5ml of MM2, which contained 5mM urea and 20% dextrose.

[0230] Following the resuspension, the tubes were incubated under their respective growth conditions. After an hour, lOuL from each tube was streaked onto Christensen's agar slant, labeled as day 0, and lOOuL was inoculated into Christensen's broth. Both the broth culture and the Christensen's agar and broth were then incubated according to their specific growth conditions. The broth tubes were incubated for six days, after which they were streaked onto a urease slant and inoculated into broth, with these samples were also labeled as day 0 (1 hour of incubation), day 2, day 4, and day 6. The urease slant was observed every 24 hours, images were recorded, and ammonia quantification was performed for urease positive tubes as previously described.

[0231] The incubation conditions for various strains were as follows: Bacillus pasteurii (11859) was incubated at 30°C for 48 to 72 hours under aerobic conditions; Bacillus subtilis (6633) at 37°C for 24 hours aerobically; Bifidobacterium breve (15700) at 37°C for 24 to 48 hours anaerobically; Lactobacillus bulgaricus (11842) and Lactobacillus casei (15008) at 37°C for 24 to 48 hours under aerobic conditions with 5% CO2, and Lactobacillus rhamnosus (21052) at 37°C for 24 to 48 hours under aerobic conditions plus 5%CO2; Lactobacillus gasseri (33323) at 37°C for 24 to 48 hours aerobically; Streptococcus thermophilus (19258) at 37°C for 24 hours aerobically with shaking at 150 RPM; and Klebsiella pneumoniae (35657) at 37°C for 24 hours under aerobic conditions.

[0232] For the determination of ammonia, a standard curve was prepared by creating standard ammonia solutions at different concentrations, with the highest being 100 pg / mL. Atwo-fold dilution was performed up to a 10-point concentration. From each concentration, ImL was taken, 20 pL of sodium potassium tartrate solution was added and mixed well, followed by the addition of 30 pL of Nessler's reagent. The mixture was allowed to stand at 25 °C for 10 minutes, during which time any precipitate was observed. The absorbance was read at 420nm using water as a blank, and the OD values were plotted against the concentration of the solution, with the concentration of ammonia (pg / ml) on the x-axis and OD values at 420nm on the y-axis.

[0233] The quantification of ammonia in the sample followed a similar procedure. From the test sample, ImL was taken, 20 pL of sodium potassium tartrate solution was added and mixed well, followed by the addition of 30 pL of Nessler's reagent. After allowing the mix to stand at 25 °C for 10 minutes and observing for any precipitate, the absorbance was read at 420nm using water as a blank. The OD values were plotted against the concentration of the solution, with the concentration of ammonia (pg / ml) on the x-axis and OD values at 420nm on the y-axis. It was noted that if the OD was high, the sample was diluted 1:10 or 1:50 before performing the quantification assay.TABLE 70: Result from Urea, Christensen’s slant, Day 0.TABLE 71: Concentration of NH4CI (pg / ml), Christensen’s slant, Day 0.

[0234] Day 0 tubes showed urease positive from day 2 onwards, the ammonia quantification increased from day 4 and the highest observed was in day 5 tubes, after 120 hrs of incubation. Results are displayed in FIG. 2 and the below Tables.TABLE 72: Results of Urease Analysis Day 2.TABLE 73: Ammonia Quantification Results Day 2.

[0235] In day 2 tubes, 6 strains showed urease positive expression and the quantification of ammonia was carried out from day 1 onwards for the positive strains. It was observed that the ammonia increased rapidly from day 1 onwards for all positive strains. Highest ammonia was observed on day 5 & 6 (see FIG. 3) and the below tables.TABLE 74: Results of Urease Expression Day 4.TABLE 75: Ammonia Concentration Measurements Day 4.

[0236] Day 4 tubes showed more ammonia concentration on day 1 onwards compared to day 0 & 2. Results are shown in FIG. 4. The ammonia concentration fluctuated between day 1 and 6, it increased from day 1 onwards and eventually decreased on day 4 & 5, followed by an increase that was recorded on day 6.TABLE 76: Results of Urease Expression Day 6.TABLE 77 Ammonia Concentration Results Day 6.

[0237] Three strains showed urease positive on day 6 tubes, ammonia concentration for these3 strains was less than those compared to tubes on day 2 & 4 (see FIG. 5).EXAMPLE 17Growth on MM1 without Ammonium Chloride with 5mM of Urea

[0238] The objective of the of the experiment was to mutate the strains in MM1 broth containing 5mM of urea with least growth nutrients. The strains were grown in their respective conditions and were subsequently sub-cultured into minimal media until reaching the dilution of 0.09%GM + 99.91%MM2. Once the strains grew in the minimal media with 5mM concentrationof Urea, they were sub-cultured onto urease slant and broth to find out the quantitative and qualitative expression of the urease enzyme. Strain viability was evaluated by reading the OD at 600 and followed by dilution and plating which showed the number of viable cells.

[0239] The experiment used various growth media, including Lactobacillus MRS broth, Brain Heart Infusion Broth, Modified Reinforced Clostridia broth, and NH4YE media. Modifications to the MM1 media were made by omitting ammonium chloride and adding 20% (v / v) dextrose, while the MM2 toxin was prepared with 5 mM urea in MM1 supplemented with 20% (v / v) dextrose.

[0240] A range of ATCC strains were cultured under specific growth conditions. Lactobacillus rhamnosus (21052), Lactobacillus plantarum (BAA-793), Lactobacillus casei (15008), and Lactobacillus bulgaricus (11842) were incubated at 37°C for 24 to 48 hours in an aerobic environment with 5% CO2. Streptococcus thermophilus (19258) and Streptococcus faecium (BAA-2320) were also incubated at 37°C for 24 hours but were shaken at 150 RPM. Bifidobacterium longum (15707) and Bifidobacterium breve (15700) were cultured anaerobically at 37°C for 24 to 48 hours, while Lactobacillus acidophilus (BAA-2845) was incubated at 37°C under anaerobic conditions for 24 hours. Lactobacillus gasseri (33323) and Bacillus coagulans (7050) were grown aerobically at 37°C for 24 to 48 hours and 24 hours, respectively. Akkermansia muciniphila (BAA-835) was incubated with shaking at 37°C for 2 to 7 days under anaerobic conditions with 100% N2. Bifidobacterium inf antis (15697) was incubated anaerobically at 37°C for 48 to 72 hours. Erwinia herbicola (33243) and Bacillus pasteurii (11859) were grown aerobically at 26°C for 24 hours and 30°C for 48 to 72 hours, respectively. Bacillus subtilis (6633) was incubated at 37°C for 24 hours under aerobic conditions.

[0241] The procedure involved subculturing the strains in their respective broth media and incubating them overnight under the respective growth conditions. The optical density (OD) was adjusted to 0.1 at 600nm in the respective growth media, and the cultures were incubated until they reached the log phase, with an OD between 0.4 and 0.6. Once the log phase was achieved, fresh Falcon tubes (50mL) containing 2.5mL MM1 with 5mM urea were inoculated with 2.5mL of the log phase culture and incubated. A control was also prepared using 2.5mL growth media and 2.6mL MM1. The tubes were incubated for 24 hours, and 'Dilution 1' tubes contained a 50 / 50 mix of growth media and MM2 (MMl+5mM of Urea+20 % dextrose). The OD for Dilution 1 was measured at 600nm post-incubation, and if it was more than half of the previous day's OD, it indicated continued growth. If the OD was less than 50% of the previous day’s log phase OD, the tubes were incubated for an additional 48 hours. For example: if Log phase OD was 0.440, Dilution 1 OD was >0.220. If OD was <0.220, incubate for 48 Hrs. If there was noincrease in OD after 48 hours, the tubes were spun down, the supernatant was discarded, and fresh media (of the same dilution of growth media and MM2) were added before further incubation. If the OD is >0.220, a fresh tube was labeled 'Dilution 2' and inoculated with 2.5mL from Dilution 1 and further supplemented with 2.5mL of MM2 (MMl+5mM Urea+ 20% dextrose). The tubes were incubated in respective growth conditions for 24 hours, and the procedure was repeated for consecutive dilutions. This process was repeated for up to 10 dilutions or until the growth media concentration reached 0.10%.

[0242] For the urease procedure, 1 OpiL of strains that survived 5mM of urea in MM2 with 20% dextrose, in dilution 10, were streaked onto Christensen's agar slant and 1 OOpiL was inoculated into Christensen's broth. The urease slant was observed and imaged every 24 hours for urease activity, and positive results led to ammonia quantification and growth curve analysis.

[0243] Growth verification involved streaking 10|lL from 'Dilution 10' tubes onto respective growth media and a positive control strain. The plates were incubated under their respective conditions and the colony morphology was observed. The OD of Christensen's broth was read at 600nm, and based on this, dilutions were made for spread plating with 100|lL for each day. The plates were incubated and the results recorded.

[0244] For ammonia determination, a standard curve was prepared using different concentrations of ammonia nitrogen solution (100 pg / mL would be the highest concentration in this study, but other concentrations can be used). A two-fold dilution was performed up to 10 point concentration. ImL of each concentration was treated with 2O|1L of sodium potassium tartrate solution and mixed. 30pL of Nessler's reagent was added and mixed, allowed to stand at 25°C for lOmin (and observed for precipitate), and then read at 420nm using water as a blank. The OD values at 420nm (y-axis) were plotted against the ammonia concentrations (pg / ml) (x- axis). The same procedure was applied to the test samples, and if the OD was high, the sample was diluted 1:10 or 1:50 before quantification.

[0245] This meticulous process allowed the accurate measurement of bacterial growth and urease activity, contributing to the understanding of bacterial metabolism under specific conditions.

[0246] It was observed that each strain had a different growth rate which decreased in relation to the depletion of nutrients in the broth. Dilutions post 1.5 % of growth media, some strains took more than 48 hours to grow in the broth.TABLE 78: Results for Growth in MM2 in the Presence of 5mM of Urea with Depletion, Dilutions 1-3.TABLE 79: Results for Growth in MM2 in the Presence of 5mm of Urea with Depletion, Dilutions 4-7.TABLE 80: Results for Growth in MM2 in the Presence of 5mm of Urea with Depletion, Dilutions 8-10.TABLE 81: Result for Urease, Ammonia quantification, OD and CFU count: Qualitative (Urease activity from slant) and Quantitative (Ammonia quantification, OD at 600nm and CFU count for cell viability in Christensen’s broth).TABLE 82: Results for Ammonia Quantification Day 2.TABLE 83: Results for Ammonia Quantification Day 3.TABLE 84: Results for Ammonia Quantification Day 4.TABLE 85: Results for Ammonia Quantification Day 5.TABLE 86: Results for Ammonia Quantification Day 6.EXAMPLE 18Ammonia Quantification for 16 Strains for Six Days

[0247] All 16 strains were inoculated into Christensen’s broth and incubated for 6 days prior to ammonia quantification. Pure cultures of probiotics were grown in Christianson’s broth supplemented with 50mM urea and measured daily for concentration of ammonium ions through Nessler’s method. The cell densities within the cultures over a period of 6 days are given in TABLE 87. The breakdown of urea into ammonia was quantified by Nessler’s method and graphed in FIG. 6. The below chart depicts comparative data of ammonia formation for all the strains for 6 days consecutively.TABLE 87: Data Comparison: Ammonia Quantification for 16 Strains for 6 Days.EXAMPLE 19Christensen’s Broth (Without Phenol Red) OD at 600nm for 16 strains for 6 days

[0248] All 16 strains were inoculated into Christensen’s broth and incubated for 6 days. Christensen’s broth has low levels of nitrogen content and hence the challenge of urea utilization as sole nitrogen source could be monitored. The cell density and viability for each strain was evaluated by taking OD readings at 600 nm and plating carried out to verify the viability of the cells. Results are presented in the below table and in FIG. 7.

[0249] TABLE 88 includes measurements in Christensen's broth (without phenol red) OD at 600nm for 16 strains for 6 days.TABLE 88: OD Readings After Six Days.EXAMPLE 20Repeat Assay for Growth on MM1 Without Ammonium Chloride with 5mM of Urea

[0250] The objective of the study was to re-evaluate (quantitative analysis) the urease activity for all the strains obtained from Example 15 by performing ammonia quantification and growth profile experiments.

[0251] In this experiment, nine mutated strains from Example 15 were utilized, including Bacillus pasteurii (11859), Bacillus subtilis (6633), Bifidobacterium breve (15700), Lactobacillus bulgaricus (11842), Lactobacillus casei (15008), Lactobacillus gasseri (33323), Lactobacillus rhamnosus (21052), Streptococcus thermophilus (19258), and Klebsiella pneumoniae (35657). The procedure began with the subculturing of these strains in their respective broth media, followed by overnight incubation under their respective growth conditions. The optical density (OD) in the growth media was then adjusted to 0.1 at 600nm, and the cultures were incubated until they reached the log phase, with an OD between 0.4 and 0.6. Upon reaching this phase, the cultures were centrifuged at 4500 RPM for 5 minutes, and the supernatant was discarded. The cells were washed with 5ml of MM1, centrifuged again at 4500 RPM for 5 minutes, and the supernatant was discarded. The cells were then resuspended in 5ml of MM2 containing 5mM urea and 20% dextrose.

[0252] After one hour of incubation under the respective growth conditions, 1 OpL from each tube was streaked onto Christensen's agar slant, labeled as day 0, and I OOpL was inoculated into Christensen's broth. Both the broth culture and the Christensen's agar and broth were incubated under the appropriate conditions. The broth tubes were incubated for six days, and samples were streaked onto urease slants and broth on days 0 (1 hr of incubation), 2, 4, and 6. The urease slants were observed and imaged every 24 hours. If the result was urease positive, ammonia quantification was performed as described below.

[0253] The incubation conditions for each strain were as follows: Bacillus pasteurii (11859) was incubated at 30°C for 48 to 72 hours aerobically; Bacillus subtilis (6633) at 37°C for 24 hours aerobically; Bifidobacterium breve (15700) at 37°C for 24 to 48 hours anaerobically; Lactobacillus bulgaricus (11842), Lactobacillus casei (15008), and Lactobacillus rhamnosus (21052) at 37°C for 24 to 48 hours in an aerobic environment with 5% CO2; Lactobacillus gasseri (33323) at 37°C for 24 to 48 hours aerobically; Streptococcus thermophilus (19258) at 37°C for 24 hours aerobically with shaking at 150 RPM; and Klebsiella pneumoniae (35657) at 37°C for 24 hours aerobically.

[0254] For the determination of ammonia, a standard curve was prepared using different concentrations of standard ammonia nitrogen solution, with the highest concentration in this study being 100 pg / mL, though other concentrations can be used. A two-fold dilution was performed up to a 10-point concentration. From each concentration, ImL was taken, 20 pL of sodium potassium tartrate solution was added, and the mixture was well mixed. Then, 30 pL of Nessler's reagent was added, and the mixture was again well mixed. The mix was allowed to stand at 25 °C for 10 minutes, during which time any precipitate was observed. The absorbance was read at 420nm using water as a blank, and the OD values were plotted against theconcentration of the solution, with the concentration of ammonia (pg / ml) on the x-axis and the OD values at 420nm on the y-axis.

[0255] For the quantification of ammonia in the sample, ImL from the test sample was taken, 20 pL of sodium potassium tartrate solution was added, and the mixture was well mixed. Then, 30 pL of Nessler's reagent was added, and the mixture was again well mixed. The mix was allowed to stand at 25 °C for 10 minutes, and any precipitate was observed. The absorbance was read at 420nm using water as a blank, and the OD values were plotted against the concentration of the solution, with the concentration of ammonia (pg / ml) on the x-axis and the OD values at 420nm on the y-axis. It was noted that if the OD was high, the sample was diluted 1 : 10 or 1 :50, and the quantification assay was be performed.

[0256] Out of 8 mutated strains, 5 showed urease positive expression, L. bulgaricus, L. casei and Bacillus pasteurii was found to be negative. And re-streaking from Christensen's broth confirmed urease negative results.TABLE 89: Result Assay for Urease Expression on Christensen’s Urea Slant on MM1 without Ammonium Chloride with 5mM Urea.TABLE 90: Restreak Results of Urea Expression Analysis: Christensen’s Urea Slant.TABLE 91: Result of Urease, Ammonia Quantification, OD and CFU Count.TABLE 92: Data Comparison: Ammonia Quantification.

[0257] Results are also presented in FIG. 8, showing ammonia concentration increased from day 1 to day 3, with the highest concentration observed on day 6.TABLE 93: Christensen’s Broth (Without Phenol Red) OD at 600nm for Eight Strains for Six Days.

[0258] FIG. 9 shows results including OD measurements at 600nm for 8 strains for 6 days performed in Christensen's broth (Without phenol red), the OD values were constant and no rapid changes in the OD were observed probably due to low nutrient content of Christensen's broth.EXAMPLE 22Growth on MM1 with 0.5 mM of Indole and p-Cresol

[0259] The objective of the experiment was to develop a method to quantify indole and p- cresol by analytical liquid chromatography and mass spectrometry (LC-MS). This involved growing the strains in minimal media containing 0.5mM of Indole / p-cresol followed by quantification by the LC-MS method.

[0260] The experiment included various growth media, such as Lactobacillus MRS broth, Brain Heart Infusion Broth, Modified Reinforced Clostridia broth, and NH4YE media. Four different toxins were prepared: Toxin 1 consisted of 0.5 mM of Indole in MM1 with an addition of 20% dextrose, Toxin 2 was made of 0.5 mM of Indole in MM1, Toxin 3 contained 0.5 mM of p-cresol in MM1 plus 20% dextrose, and Toxin 4 was 0.5 mM of p-cresol in MM1.

[0261] A variety of ATCC strains were cultured under specific growth conditions.Lactobacillus rhamnosus (21052), Lactobacillus plantarum (BAA-793), Lactobacillus casei (15008), and Lactobacillus bulgaricus (11842), were all incubated at 37°C for 24 to 48 hours in an aerobic environment with 5% CO2. Lactobacillus gasseri was incubated at 37°C for 24 to 48 hours in an aerobic environment. Streptococcus thermophilus (19258) and Streptococcus faecium (BAA-2320) were incubated aerobically at 37°C for 24 hours and shaken at 150 RPM. Bifidobacterium longum (15707) and Bifidobacterium breve (15700) were cultured anaerobically at 37°C for 24 to 48 hours, while Lactobacillus acidophilus (BAA-2845) was incubated anaerobically at 37°C for 24 hours. Akkermansia muciniphila (BAA-835) was shaken at 37°C for 2 to 7 days under anaerobic conditions with 100% N2. Bifidobacterium infantis (15697) was incubated anaerobically at 37°C for 48 to 72 hours. Bacillus coagulans (7050) was incubated aerobically for 24 hours at 37°C, Erwinia herbicola (33243) was incubated aerobically at 26°C for 24 hours, Bacillus pasteurii (11859) was incubated aerobically at 30°C for 48 to 72 hours, and Bacillus subtilis (6633) was incubated aerobically at 37°C for 24 hours.

[0262] The procedure began with the preparation of log phase cultures. The strains were subcultured overnight in their respective growth conditions. The following day, the optical density (OD) in the respective growth media was adjusted to 0.1 at 600nm and incubated to achieve log phase cultures with an OD between 0.4-0.6 in four tubes. Once the desired OD was reached, the tubes were centrifuged at 4500 rpm for 10 minutes, the supernatant was discarded, and the pellet was resuspended in 2 mL of MM1. Two identical sets of sterile Falcon tubes were then prepared by adding 1 mL of inocula from each tube, pooling all 16 strains. These 8 tubes (2 sets of duplicates) were then centrifuged at 4500 rpm for 10 minutes, the supernatant was discarded, and the pellets served as the inoculum for the next phase.

[0263] For the growth media and toxin media preparation, the growth media for each strain was combined in equal measure to reflect its proportion in the pooled inoculum. For example, in a sterile 500mL flask, 10 mL of each strain's growth media was added to create a total of 160 mL of pooled growth media. The toxin media were prepared as follows: Toxin 1 with MM1, 20% dextrose, and 0.5 mM of Indole; Toxin 2 with MM1 and 0.5 mM of Indole; Toxin 3 with MM1, 20% dextrose, and 0.5 mM of p-cresol; and Toxin 4 with MM1 and 0.5 mM of p-cresol. Control media were also prepared with the same components as the toxin media but without the addition of the pooled cultures.

[0264] The growth on toxin media assay was performed in duplicates. Two tubes of pooled culture labeled T1 A and TIB (Toxin 1 A and Toxin IB) were taken, and 15 mL of Toxin 1 was added to 5 mL of the pooled growth media, resulting in a mixture of 25% growth media and 75% toxin media. This step was replicated for Toxins 2, 3, and 4, for all 8 tubes of the pooled culture, as well as for the four control tubes, which contained no culture. All tubes were incubated at 37°C for 24 hours with shaking at 150-200 RPM under aerobic conditions on day 0. On day 1, a 1:1 dilution was performed with the respective toxin media. For example, from the Day 0 T1 A tube containing 20 mL (25% growth media and 75% toxin media), 10 mL was transferred to a new tube labeled Day 1, and 10 mL of Toxin 1 media was added. The tubes were then incubated at 37°C with aerobic shaking at 200 RPM. This dilution process was repeated for 6 days. The remaining 10 mL in the original tube was adjusted to a concentration of 25% growth media and incubated for an additional 72 hours. After 72 hours, the tubes were centrifuged at 4500 rpm, the supernatant was collected in two vials, flash-frozen, and stored at -80°C.TABLE 94: Methods of Preparation of Tubes.

[0265] **To the leftover mixture (lOmL), makeup to 12.5 % of GM & Incubate at 37C for 72 Hrs. spin at 1000 RPM for 10 min and aliquot the supernatant into 3 vials 3 mL each and to pellet add 50 % of glycerol and store at -80.TABLE 95: Total Number of Samples for Analysis.HPLC AnalysisTABLE 96: Bioanalytical Details.

[0266] In the process of sample preparation, 4 pL of aqueous calibration curve (CC) and quality control (QC) samples were combined with 96 pL of blank growth media. This mixture was then vortexed to ensure thorough mixing. The test samples underwent a dilution process where the Indole samples were diluted 2-fold and the p-cresol samples 5 -fold. Specifically, 4 pL of the test sample was added to 16 pL of blank growth media, followed by vortexing for proper mixing.

[0267] For the analysis of Indole, 50 pL of the previously spiked CC & QC samples, as well as the diluted test samples, were extracted using 200 pL of Acetonitrile. This Acetonitrile contained an internal standard with concentrations of Telmisartan and Tolbutamide at lOOng / mL each. A similar extraction process was carried out for p-cresol, but with a smaller volume of 20 pL of the spiked and diluted CC and QC samples, which were also extracted with 200 pL of Acetonitrile containing the same internal standards (Telmisartan & Tolbutamide lOOng / mL each).

[0268] After the addition of Acetonitrile, the samples were vortexed for 10 minutes at a speed of 1000 rpm. This was followed by centrifugation at 4000 rpm for another 10 minutes. Postcentrifugation, 130 pL of the supernatant was carefully transferred into a 96-well plate and analyzed using LC-MS / MS.

[0269] The analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The chromatographic conditions included the following. The mass spectra were obtained using a SCIEX TRIPLE QUAD 4500 system, operated with Analyst software version 1.7.0. High-performance liquid chromatography (HPLC) was performed on a Shimadzu Nexera X2 series system. The column used for the chromatography was a Kinetex C18 with dimensions of 100*3.0 mm and 5pm. The ionisation source was a heated nebulizer.TABLE 97: Chromatography Conditions.TABLE 98: HPLC Result: Standard and Quality Control.

[0270] Indole was analyzed within a calibration curve (CC) range that spanned from 0.4 to 400 micromolar (pM). The low-quality control (LQC) for indole was established at 1.8 pM, while the medium-quality control (MQC) was set at 160 pM, and the high-quality control (HQC) was determined to be 320 pM. In the case of p-cresol, the substance was subjected to a calibration curve range that was broader, extending from 2 to 2000 pM. The LQC for p-cresol was noted at 9.6 pM, the MQC was considerably higher at 640 pM, and the HQC was set near the upper limit of the range at 1600 pM. Results are shown in FIGs. 10A-10F.TABLE 99: Results of Quantification of Indole.

[0271] The analysis of pooled bacterial strains was conducted using a mixture of 12.5% pooled growth media and 87.5% MM1, which was supplemented with and without 20% Dextrose. These strains were exposed to a 0.5 mM concentration of toxins, specifically Indole and p-cresol, and the resulting interactions were scrutinized through Liquid Chromatography- Mass Spectrometry (LC-MS). To ensure accuracy, a standard curve was established by running it once before the actual sample injection into the mass spectrometer. This precaution was taken to guarantee that the data collected fell within the linear range. Quality control (QC) samples were run twice - once before and once after the sample run. The data from both QC runs confirmed that the results were within the expected range. To achieve a linear range for accuratemeasurement, samples were diluted accordingly. This dilution aimed to reach a linear range of 0.4-400 pM for Indole and 2-2000 pM for p-Cresol.

[0272] In the control tubes, an average concentration of 386 pM and 421 pM of Indole was detected when 20% Dextrose was present. In contrast, the test group showed lower average concentrations of 258 pM and 216 pM of Indole under the same conditions with 20% Dextrose. The reduction in Indole concentration was notable when comparing the MM1 media alone to the MM1 media supplemented with 20% Dextrose. Specifically, 127.5 pM of Indole was reduced in MM1, and 205 pM was reduced in the MM1 media with the addition of 20% Dextrose. Results are shown in FIG. 11. In addition to metabolization of indole at concentration of 0.5 mM, a more pronounced effect of indole metabolization was observed with a 10 fold higher concentration of 5mM (FIG 15).TABLE 100: Results of Quantification of p-Cresol.

[0273] Concentration of p-cresol was lOx high (see also FIG. 12); this might be due to addition of high concentration in the broth. A repeat experiment was performed for p-cresol followed by quantification (see FIG. 14).EXAMPLE 23Repeat Analysis of p-C resol

[0274] Initially, 4 pL of aqueous calibration curve (CC) and quality control (QC) samples were combined with 96 pL of blank growth media. This mixture was then thoroughly vortexed to ensure proper mixing. In parallel, a 5X dilution of the test sample was prepared by adding 4 pL of the test sample to 16 pL of blank growth media, which was vortexed to mix.

[0275] Following the preparation, 20 pL of the spiked CC and QC samples, as well as the diluted test samples, were extracted using 200 pL of Acetonitrile. This extraction solventcontained an internal standard with concentrations of Telmisartan and Tolbutamide at 100 ng / mL each. The mixture was then vortexed for 10 minutes at a speed of 1000 rpm, ensuring that the samples were thoroughly mixed.

[0276] After vortexing, the samples were centrifuged for 10 minutes at a speed of 4000 rpm. Carefully, 130 pL of the supernatant was transferred to a 96-well plate. The samples on the plate were then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS).TABLE 101: Analyses Conditions Used in LC-MS / MS.TABLE 102: Method Settings for LC-MS / MS.

[0277] The method employed was a binary gradient. The buffer consisted of 5mM ammonium acetate in water. For the composition of the mobile phase, Pump-A was filled with 5mM ammonium acetate in water, while Pump-B utilized methanol. The flow rate was set at 500 pL / min. The total run time for the process was 3.0 minutes. To maintain the integrity of the samples, the cooler temperature was kept at 8°C. The injection volume for the chromatography was 5 pL. The column oven was regulated to a constant temperature of 40 °C. Throughout the procedure, the polarity was maintained in the negative mode.TABLE 103 HPLC Result, Standard and Quality Control.

[0278] QC and CC results are shown in FIGs. 13A-13D.TABLE 104 HPLC Result: Standard and quality control.

[0279] Bacterial strains had been pooled in a mixture of 12.5% pooled growth media and 87.5% MM1, which was supplemented with and without 20% Dextrose. These strains were then exposed to a concentration of 0.5 mM of the toxin p-cresol. The analysis of these samples was conducted using Liquid Chromatography-Mass Spectrometry (LC-MS). Prior to running the samples, a standard curve was established to ensure the accuracy of the measurements. This preliminary step confirmed that the data fell within the expected linear range. Quality control (QC) was rigorously performed twice before and after the sample runs, to further validate that the results were consistent and within the linear range. To achieve this linear range, specificallybetween 2-2000 pM for p-cresol, the samples underwent a dilution process. Upon examination of all the tubes, it was noted that the concentration of p-cresol consistently exceeded 0.5 mM. A significant difference was not observed in test samples.TABLE 105: Indole and p-Cresol Assay Summary.EXAMPLE 24Growth of Bacteria in the Presence on 0.5 - 5 mM of Indole / p-Cresol and Quantification by LCMS Method

[0280] The study involved the growth of bacteria in the presence of Indole and p-cresol, with the growth quantified using the LCMS method. We performed two sets of experiments with 10 fold variation of concentration for indole and p-cresol, ranging from 0.5mM to 5mM for both toxins. Because levels of p-cresol were high in Example 22, the study was repeated. Quality control checks of p-cresol and indole are presented in Table 106 and Table 107.TABLE 106: LCMS QC Check of p-Cresol (Repeated Experiment).TABLE 107: LCMS QC Check of Indole (Repeated Experiment).EXAMPLE 25Growth of probiotic strain in Lactobacillus MRS broth with 5 mM of Indole / p-cresol

[0281] Indole degradation experiments: Bacterial cultures were incubated for a total of two weeks, and LC-MS analysis was performed to determine indole concentration at the end of each week. Indole is a percussor to indoxyl sulfate which is a uremic toxin in blood. However a significant portion of indole is produced due to metabolism of the microbiome in the gut. Therefore experiments were performed to access the capacity of the probiotics to metabolize indole. All experiments to analyze indole metabolization activity were performed under 7mM which proved to be the threshold concentration of probiotic cytotoxicity. However indole experiments were performed under rich nutrient conditions. The following species consumed more than 85% of the indole in the culture medium within two weeks: Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus gasseri, Bacillus pasteurii, and Bacillus subtilis (FIG. 15).

[0282] A total of 16 different strains were individually grown and brought to a log phase culture using their respective broths. For the preparation of toxins, two specific compounds were used: Indole and p-cresol. Indole and p-cresol were prepared at concentrations of lOOmM in Dimethyl Sulfoxide (DMSO), while 5mM solutions of Indole or p-cresol were also prepared in Lactobacillus MRS broth. The media used for these experiments contained a 0.5% concentration of DMSO. Toxin 1 included a 5 mM solution of Indole in Lactobacillus MRS broth, and Toxin 2 was a 5 mM solution of p-cresol prepared in Lactobacillus MRS broth. For the incubation, 5mLof the toxin media, containing Indole or p-cresol, were transferred into 15mL Falcon tubes. To this, 50 pL of the log phase culture was added. The tubes were then incubated under the respective growth conditions for a period of 24 hours.

[0283] Following the incubation period, the Falcon tubes were removed from the incubator and subjected to centrifugation at a speed of 10,000 RPM for 10 minutes. After centrifugation, the supernatant was carefully aliquoted and stored at a temperature of -80 degrees Celsius. The samples were stored for subsequent Drug Metabolism and Pharmacokinetics (DMPK) analysis, which would provide insights into the metabolic and toxicological profile of the toxins in relation to the bacterial cultures.

[0284] Results are shown below and in FIG. 15 (quantification of indole) and FIG. 16 (quantification of p-cresol). For FIG. 15, each probiotic species was incubated at 37°C in respective ATTC recommended growth media supplemented with a final concentration of 5mM indole . Samples for LCMS analysis were sent at the end of week 1 and 2.TABLE 108: LCMS Indole Quantification.TABLE 109: LCMS p-Cresol Quantification.

[0285] Summary of results: Indole is a percussor to indoxyl sulfate which is a uremic toxin in blood. However a significant portion of indole is produced due to metabolism of the microbiome in the gut. Therefore we performed experiments to access the capacity of the probiotics to metabolize indole. All experiments to analyze indole metabolization activity were performed under 7mM which proved to be the threshold concentration of probiotic cytotoxicity. However indole experiments were performed under rich nutrient conditions. The following species tested positive for high metabolization of indole: Lactobacillus casei, Lactobacillus acidophilus,Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus gasseri, Enterococcus faecium, Lactobacillus fermentum; while moderate metabolization of indole was exhibited by Sterptococcus thermophilus, Bacillus pasteurii, and Bacillus subtilis. Bifidobacterium breve, Bifidobacterium adolescentis, and Bifidobacterium longum. Similar to indole, p-cresol is also produced in the gut due to microbiome metabolism, and also converts downstream into p-cresol sulfate which is a uremic toxin. We found that Erwinia herbicola was a species in the tested probiotics that showed significant metabolization capacity for p-cresol.EXAMPLE 26Probiotic Mixtures

[0286] Several probiotic mixtures were created for evaluation of indole metabolization (FIG. 15). Lactobacillus mixture 2 showed the high reduction after 8 days (FIG. 17). The growth conditions are listed in TABLES 110 and 111. Each probiotic mixture was incubated at 37°C in respective growth media (TABLE 110) supplemented with 5mM indole. Samples for LCMS analysis were sent on day 0, 1, 2, 3, 5 and 8.TABLE 110: Growth Conditions of the Probiotic Mixtures.TABLE 111: Aerobic and Anaerobic Probiotic Mixtures Used for Evaluating Indole Degradation.

[0287] LCMS analysis plot of indole metabolized by probiotic mixtures. Each probiotic mixture was incubated at 37°C in respective growth media (TABLE 110) supplemented with 5mM indole. Samples for LCMS analysis were sent on day 0, 1, 2, 3, 5 and 8. Results are shown in FIG. 17.EXAMPLE 27Treatment of Kidney Disease with Modified Probiotic Bacteria

[0288] A clinical trial will be conducted to evaluate the safety and efficacy of a pharmaceutical composition comprising a mixture of genetically modified probiotic bacteria for treating chronic kidney disease (CKD) patients. The composition includes modified strains of Lactobacillus rhamnosus (21052), Lactobacillus plantarum (BAA-793), Streptococcus thermophilus (19258), Streptococcus faecium (BAA-2320), Bifidobacterium longum (15707), and Bifidobacterium breve (15700), which were engineered to have tolerance to uremic toxins, such as urea, creatinine, p-cresol, and indole. The composition includes 10 billion CFU of lyophilized or frozen bacteria, a mixture of the four modified strains listed above. The trial will enroll 100 CKD patients with stage 3 or 4 kidney function impairment, who are randomized to receive either the composition or a placebo twice daily for 12 weeks administered orally, as part of a therapeutic treatment regime. The primary outcome will be the change in serum levels of uremic toxins from baseline to the end of the treatment period. The secondary outcomes will include the change in kidney function parameters, such as estimated glomerular filtration rate (eGFR) and serum creatinine, and quality of life measures, such as fatigue, nausea, and pruritus. The trial results will show that the composition significantly reduces serum levels of uremic toxins compared to the placebo, indicating a beneficial effect on the metabolic burden of CKD. The composition will also improve kidney function parameters and quality of life measures, producing a positive impact on the renal and systemic health of CKD patients. Due to the beneficial effects of these bacteria and of the composition, the composition will be well tolerated, with no serious adverse events or gastrointestinal disturbances.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A bacterial composition comprising one or more modified probiotic bacteria, wherein the one or more modified probiotic bacteria independently comprise a uremic toxin tolerance modification.

2. The bacterial composition of claim 1, wherein the uremic toxin tolerance modification is stable for at least one of the one or more modified probiotic bacteria.

3. The bacterial composition of any one of claims 1 or 2, wherein the uremic toxin tolerance modification is stable for at least one of the one or more modified probiotic bacteria in the absence of a uremic toxin.

4. The bacterial composition of any one of claims 1-3, wherein the uremic toxin tolerance modification comprises heterologous expression of an enzyme that hydrolyzes or decomposes a uremic toxin.

5. The bacterial composition of claim 4, wherein the enzyme comprises urease, urea amidolyase, oxygenase, carboxylase, allophanate hydrolase, or flavin reductase.

6. The bacterial composition of any one of claims 1-5, wherein the uremic toxin tolerance modification includes expression of at least one of the genes iifC, iifD, ureC, UreB, UreA, tnaA, iaaM, iaaaH, iifC, hbsAB, or pcmh.

7. The bacterial composition of any one of claims 1-6, wherein the uremic toxin tolerance modification comprises production of short-chain fatty acids.

8. The bacterial composition of any one of claims 1-7, wherein the uremic toxin tolerance modification comprises expression of butyrate kinase or butyryl-CoA:acetate Co A- transferase.

9. The bacterial composition of any one of claims 1-8, wherein the one or more modified probiotic bacteria are genetically modified.

10. The bacterial composition of any one of claims 1-9, wherein the one or more modified probiotic bacteria are stable in a gastric fluid or an intestinal fluid.

11. The bacterial composition of claim 10, wherein the gastric fluid comprises a pH of 1.0 to3.5.

12. The bacterial composition of claim 10 or 11, wherein the intestinal fluid comprises a pH of 5.5 to 7.5.

13. The bacterial composition of any one of claims 1-12, wherein the one or more modified probiotic bacteria are stable for at least one hour to 10 days.

14. The bacterial composition of any one of claims 1-13, wherein the one or more modified probiotic bacteria comprise modified Lactobacillus rhamnosus, modified Bifidobacterium longum, modified Bacillus pasteurii, modified Lactobacillus sporogenes, modified Streptococcus thermophilus, modified Erwinia herbicola, modified Lactobacillus casei, modified Lactobacillus acidophilus, modified Lactobacillus bulgaricus, modified Bifidobacterium breve, modified Lactobacillus plantarum, modified Lactobacillus gasseri, modified Bifidobacterium infantis, modified Lactobacillus salivarius, modified Streptococcus faecium, modified Bacillus subtilis, modified Bacillus coagulans, modified Akkermansia muciniphila, or a combination thereof.

15. The bacterial composition of any one of claims 1-14, wherein the one or more modified probiotic bacteria are selected from the group consisting of Lactobacillus rhamnosus, Bifidobacterium longum, Bacillus pasteurii, and combinations thereof.

16. The bacterial composition of any one of claims 1-15, wherein the uremic toxin is a water-soluble uremic toxin or a protein-bound uremic toxin.

17. The bacterial composition of any one of claims 1-16, wherein the uremic toxin is a water-soluble uremic toxin, and wherein the water-soluble uremic toxin is urea, Creatinine, 1- methyladenosine, N2,N2-dimethylguanosine, N4-acetylcytidine, Xanthosine, 1- methylguanosine, Inosine, 1 -methylinosine, N6-methyladenosine, N6- threonylcarbamoyladenosine, Pseudouridine, S-adenosylhomocysteine, Monomethylamine, Dimethylamine, Ethylamine, Trimethylamine, Trimethylamine-N-oxide, 2-heptenal, 2-hexenal, Nonanal, 2-nonenal, 2-octenal, 4-decenal, Heptanal, Decanal, Hexanal, Dimethylglycine, 4-HO- decenal, 4-HO-hexenal, 4-HO-nonenal, 4-HO-octenal, P-lipotropin, 4-pyri done-3 -carboxamide- 1 -P-D-ribonucleoside, N-methyl-2-pyridone-5-carboxamide, N-methyl-4-pyridone-3- carboxamide, Nicotinamide, 8-hydroxy-2'-deoxyguanosine, Cytidine, Hypoxanthine, Neopterin, Uric acid, Xanthine, Arab(in)itol, Mannitol, Erythritol, Myoinositol, Sorbitol, Threitol, Argininic acid, Asymetric dimethylarginine (ADMA), Creatine, Guanidine, Guanidinosuccinic acid, Methylguanidine, Symmetric dimethylarginine (SDMA), Taurocyamine, a-keto-5-Guanidinovaleric acid, a-N-acetylarginine, P-guanidinopropionic acid, y-guanidinobutyric acid, Malondialdehyde, Orotic acid, Orotidine, Uridine, Oxalate, or Phenylacetylglutamine.

18. The bacterial composition of any one of claims 1-17, wherein the uremic toxin is a protein-bound uremic toxin, and wherein the protein-bound uremic toxin is p-cresol, indole, Angiogenin, Insulin-like growth factor 1 (IGF-1), Leptin, Osteocalcin, Retinol binding protein (RBP), Vascular endothelial growth factor, al -acid glycoprotein, Hippuric acid (total), Pentosidine, 3-deoxyglucosone, Fructoselysine, Glyoxal, Methylglyoxal, N(6)- Carboxymethyllysine (CML), Dihydroxyphenylalanine (PB-DOPA), Homocysteine, Kinurenine, Kynurenic acid, Indican, Indole-3 -acetic acid, Indoxyl sulfate, Indoxyl-fl-D- glucoronide, Melatonin, Quinolinic acid, Interleukin- 10, Phenol, p-Cresylsulfate, Putrescine, 3- carboxy-4-methyl-5-propyl-2-furanpropanoic acid, or Phenylacetic acid.

19. The bacterial composition of any one of claims 1-18, wherein the uremic toxin comprises urea, p-cresol, or indole.

20. The bacterial composition of any one of claims 1-19, comprising a first and second bacterial composition, wherein the first bacterial composition comprises modified bacteria and the second bacterial composition comprises unmodified bacteria.

21. The bacterial composition of any one of claims 1-20, wherein the bacterial composition has an ODeoo value of at least 0.1.

22. The bacterial composition of any one of claims 1-21, wherein the bacterial composition has an ODeoo value of from 1 to 3.

23. The bacterial composition of any one of claims 1-22, wherein the bacterial composition has an ODeoo measurement of from about 0.2 to 3, from about 0.5 to 3, from about 0.5 to 2, from about 1 to 3, from about 1 to 2, or from about 2 to 3.

24. The bacterial composition of any one of claims 1-23, wherein the bacterial composition has an ODeoo measurement is from about 0.2 to 1.0, from about 1.0 to 1.5, from about 1.5 to 2.0, from about 2.0 to 2.5, from about 2.5 to 3, or from about 2.0 to 3.0.

25. The bacterial composition of any one of claims 1-24, wherein the bacterial composition has an ODeoo measurement is at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, or at least 2.9.

26. The bacterial composition of any one of claims 1-25, wherein the bacterial composition has an ammonium concentration after at least one day in culture in the presence of a uremic toxin of at least lOOpg / ml.

27. The bacterial composition of any one of claims 1-26, wherein the bacterial composition after at least one day in culture in the presence of a uremic toxin comprises urea, p-cresol, indole, or a combination thereof.

28. A bacterial composition comprising a modified probiotic bacterium that is genetically modified to express a urease gene and / or a urea transporter gene, wherein the modified probiotic bacterium is capable of metabolizing urea into ammonia and carbon dioxide in a medium with a pH of 5.0 to 8.0.

29. A pharmaceutical composition comprising the bacterial composition of any one of claims 1-27, and a pharmaceutically acceptable carrier or excipient.

30. The pharmaceutical composition of claim 29, wherein the pharmaceutically acceptable carrier or excipient is a binder, a lubricant, a glidant, a carrier, an antioxidant.

31. The pharmaceutical composition of claim 29 or 30, further comprising an active ingredient, wherein the active ingredient is a prebiotic or a vitamin.

32. A method for producing one or more modified probiotic bacteria having tolerance to a uremic toxin, the method comprising: i) modifying one or more probiotic bacteria; ii) culturing the one or more modified probiotic bacteria in medium comprising an increasing concentration gradient of a uremic toxin, optionally a decreasing concentration gradient of nutrients; and iii) identifying one or more modified probiotic bacteria that can grow in the medium comprising an increasing concentration gradient of the uremic toxin, thereby preparing one or more modified probiotic bacteria having tolerance to a uremic toxin.

33. The method of claim 32, further comprising lyophilizing and / or freezing the one or more modified probiotic bacteria.

34. The method of claim 32 or 33, comprising combining the one or more modified probiotic bacteria with non-modified probiotic bacteria.

35. The method of any one of claims 32-34, comprising analyzing the one or more modified probiotic bacteria by one or more of genomic analysis, transcriptomic analysis, proteomic analysis and metabolomic analysis.

36. The method of any one of claims 32-35, wherein the one or more modified probiotic bacteria further comprising a modification that maintains the tolerance to the uremic toxin.

37. The method of any one of claims 32-36, wherein the one or more modified probiotic bacteria are further modified to be stable in a gastric fluid or an intestinal fluid.

38. The method of claim 37, wherein the gastric fluid comprises a pH of 1.0 to 3.5.

39. The method of claim 37 or 38, wherein the intestinal fluid comprises a pH of 5.5 to 7.5.

40. The method of any one of claims 32-39, wherein the one or more modified probiotic bacteria are stable for at least one hour to 10 days.

41. The method of any one of claims 32-41, wherein in step i) modifying one or more probiotic bacteria comprises genetically modifying the one or more probiotic bacteria.

42. The method of any one of claims 32-41, wherein the one or more probiotic bacteria comprise Lactobacillus rhamnosus, Bifidobacterium longum, Bacillus pasteurii, Lactobacillus sporogenes, Streptococcus thermophilus, Erwinia herbicola, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus gasseri, Bifidobacterium infantis, Lactobacillus salivarius, Streptococcus faecium, Bacillus subtilis, Bacillus coagulans or Akkermansia muciniphila.

43. The method of any one of claims 32-42, wherein the one or more modified probiotic bacteria are obtained from a biological sample.

44. The method of claim 43, wherein the biological sample is a fecal sample.

45. The method of any one of claims 32-44, wherein the uremic toxin is a water-soluble uremic toxin, or a protein-bound uremic toxin.

46. The method of any one of claims 32-45, wherein the uremic toxin is a water-soluble uremic toxin, and wherein the water-soluble uremic toxin is urea, 1 -methyladenosine, N2,N2-dimethylguanosine, N4-acetylcytidine, Xanthosine, 1 -methylguanosine, Inosine, 1- methylinosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, Pseudouridine, S- adenosylhomocysteine, Monomethylamine, Dimethylamine, Ethylamine, Trimethylamine, Trimethylamine-N-oxide, 2-heptenal, 2-hexenal, Nonanal, 2-nonenal, 2-octenal, 4-decenal, Heptanal, Decanal, Hexanal, Dimethylglycine, 4-HO-decenal, 4-HO-hexenal, 4-HO-nonenal, 4- HO-octenal, P-lipotropin, 4-pyridone-3-carboxamide-l-P-D-ribonucleoside, N-methyl-2- pyridone-5 -carboxamide, N-methyl-4-pyridone-3 -carboxamide, Nicotinamide, 8-hydroxy-2'- deoxy guanosine, Cytidine, Hypoxanthine, Neopterin, Uric acid, Xanthine, Arab(in)itol, Mannitol, Erythritol, Myoinositol, Sorbitol, Threitol, Argininic acid, Asymetric dimethylarginine (ADMA), Creatine, Creatinine, Guanidine, Guanidinosuccinic acid, Methylguanidine, Symmetric dimethylarginine (SDMA), Taurocyamine, a-keto-5- Guanidinovaleric acid, a-N-acetylarginine, P-guanidinopropionic acid, y-guanidinobutyric acid, Malondialdehyde, Orotic acid, Orotidine, Uridine, Oxalate, or Phenylacetylglutamine.

47. The method of any one of claims 32-46, wherein the uremic toxin is a protein-bound uremic toxin, and wherein the protein-bound uremic toxin is p-cresol, indole, Angiogenin, Insulin-like growth factor 1 (IGF-1), Leptin, Osteocalcin, Retinol binding protein (RBP), Vascular endothelial growth factor, al -acid glycoprotein, Hippuric acid (total), Pentosidine, 3- deoxyglucosone, Fructoselysine, Glyoxal, Methylglyoxal, N(6)-Carboxymethyllysine (CML), Dihydroxyphenylalanine (PB-DOPA), Homocysteine, Kinurenine, Kynurenic acid, Indican, Indole-3 -acetic acid, Indoxyl sulfate, Indoxyl-P-D-glucoronide, Melatonin, Quinolinic acid, Interleukin- 10, Phenol, p-Cresylsulfate, Putrescine, 3-carboxy-4-methyl-5-propyl-2- furanpropanoic acid, or Phenylacetic acid.

48. The method of any one of claims 32-47, wherein the uremic toxin comprises urea, p- cresol, or indole.

49. A method of treating a condition associated with uremic toxin build up in a subject in need thereof, the method comprising administering a therapeutically effective amount of the bacterial composition of any one of claims 1-27 or the pharmaceutical composition of any one of claims 18-20, thereby treating the condition associated with uremic toxin build up.

50. The method of claim 49, wherein the condition is a kidney disease.

51. The method of claim 50, wherein the kidney disease is chronic kidney disease, Fabry disease, cystinosis, glomerulonephritis, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, polycystic kidney disease, diabetic kidney disease, or uremia.

52. The method of any one of claims 49-51, wherein the condition is associated with reduced kidney function.

53. The method of any one of claims 49-52, wherein the condition is diabetes, high blood pressure, heart disease, obesity, kidney damage, blood or fluid loss, heart attack, heart disease, an infection, liver failure, an allergic reaction, severe burns, severe dehydration, surgery indued kidney strain, a condition with high anabolic need, blood clots in and around the kidneys, cholesterol deposits blocking blood flow in the kidneys, glomerulonephritis, hemolytic uremic syndrome, COVID-19, lupus, scleroderma, thrombotic thrombocytopenic purpura, toxins, muscle tissue breakdown, breakdown of tumor cells, or old age.

54. The method of any one of claims 49-53, wherein the condition is associated with consumption of or exposure to a blood pressure medication, aspirin, ibuprofen, naproxen sodium, a chemotherapy drug, an antibiotic, an imaging dye, alcohol, a heavy metal, or a stimulant drug.

55. The method of any one of claims 49-54, wherein the condition is associated with impaired passage of urine leading to impaired kidney or renal function.

56. The method of any one of claims 49-55, wherein the condition is bladder cancer, blood clots in the urinary tract, cervical cancer, colon cancer, enlarged prostate, kidney stones, nerve damage, or prostate cancer.

57. A method of treating a subject at risk for developing uremic toxin build up, the method comprising administering the bacterial composition of any one of claims 1-27 comprising one or more probiotic bacteria having tolerance to a uremic toxin, thereby treating the subject at risk of developing uremic toxin build up.

58. The method of claim 57, wherein the subject is at risk of developing kidney disease or having impaired kidney function.

59. The method of claim 57 or 58, wherein the subject has advanced age, a blockage in the blood vessels in the arms or legs, diabetes, high blood pressure, heart failure, kidney disease, liver disease, cancer, or has been hospitalized.

60. A method of producing one or more modified probiotic bacteria that are tolerant of uremic toxins, the method comprising: i) exposing one or more modified probiotic bacteria to a uremic toxin;ii) measuring at least one of the survival rate, growth rate, or metabolic activity of the one or more modified probiotic bacteria; iii) comparing the results with those of unexposed probiotic bacteria or probiotic bacteria exposed to a lower concentration of the uremic toxin; and iv) producing one or more modified probiotic bacteria that are tolerant of uremic toxins.

61. The method of claim 60, wherein the uremic toxin is selected from the group consisting of urea, creatinine, uric acid, indoxyl sulfate, p-cresol sulfate, hippuric acid, indole, p-cresol, phenol, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid.

62. The method of claim 60 or 61, wherein the probiotic bacteria is selected from the group consisting of Lactobacillus rhamnosus, Bifidobacterium longum, Bacillus pasteurii, Lactobacillus sporogenes, Streptococcus thermophilus, Erwinia herbicola, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus gasseri, Bifidobacterium infantis, Lactobacillus salivarius, Streptococcus faecium, Bacillus subtilis, Bacillus coagulans or Akkermansia muciniphila, and combinations thereof.

63. The method of any one of claims 60-62, further comprising measuring an ODeoo value.

64. The method of any one of claims 60-63, wherein the one or more modified probiotic bacteria have an ODeoo value of at least 0.1.

65. The method of claim 63 or 64, wherein the one or more modified probiotic bacteria have an ODeoo value of from 1 to 3.

66. The method of any one of claims 63-65, wherein the one or more modified probiotic bacteria have an ODeoo value of from about 0.2 to 3, from about 0.5 to 3, from about 0.5 to 2, from about 1 to 3, from about 1 to 2, or from about 2 to 3.

67. The method of any one of claims 63-66, wherein the one or more modified probiotic bacteria have an ODeoo value of from about 0.2 to 1.0, from about 1.0 to 1.5, from about 1.5 to 2.0, from about 2.0 to 2.5, from about 2.5 to 3, or from about 2.0 to 3.0.

68. The method of any one of claims 63-67, wherein the one or more modified probiotic bacteria have an ODeoo value of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, or at least 2.9.

69. The method of any one of claims 60-68 further comprising measuring a concentration of ammonium.

70. The method of any one of claims 60-69, wherein measuring the metabolic activity comprises measuring a concentration of ammonium of at least lOOpg / ml.

71. The method of any one of claims 60-70, further comprising measuring a concentration of urea, p-cresol, indole, or a combination thereof.

72. The method of any one of claims 60-71, wherein measuring the metabolic activity comprises measuring the concentration of urea, p-cresol, indole, or a combination thereof.

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