WO2024130119A2

WO2024130119A2 - Synbiotic compositions for short chain fatty acid production

Info

Publication number: WO2024130119A2
Application number: PCT/US2023/084290
Authority: WO
Inventors: Gregory Mckenzie; Julie E. Button; Abigail REENS; Casey COSETTA; Aislinn ROWAN-NASH
Original assignee: Prolacta Bioscience, Inc.
Priority date: 2022-12-16
Filing date: 2023-12-15
Publication date: 2024-06-20
Also published as: WO2024130119A3

Abstract

Provided herein are compositions, methods, strategies, kits, and articles of manufacture that are useful, inter alia, in the treatment or prevention of diseases, disorders, or conditions that may be associated with inflammation, infection, allergy, immune dysfunction, or dysbiosis of the intestinal microbiome. In some aspects, the invention provides a synergistic combination of a prebiotic, e.g., a mixture of human milk oligosaccharides, and probiotic strains of bacteria, such as one or more strains capable of internalizing and consuming the prebiotic, e.g., Bifidobacterium longum subsp. infantis, and one or more strains capable of producing short chain fatty acids such as propionate, e.g., Veillonella sp.

Description

SYNBIOTIC COMPOSITIONS FOR SHORT CHAIN FATTY ACID PRODUCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application No. 63/387,807 filed December 16, 2022, entitled “SYNBIOTIC COMPOSITIONS FOR SHORT CHAIN FATTY ACID PRODUCTION” and U.S. provisional application No. 63/490,283 filed March 15, 2023, entitled “SYNBIOTIC COMPOSITIONS FOR SHORT CHAIN FATTY ACID PRODUCTION,” the contents of each of which is incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled PROL04604WOSEQLIST ST25, created December 12, 2023, which is 113 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] Provided herein are compositions, methods, strategies, kits, and articles of manufacture that are useful, inter alia, in the treatment or prevention of diseases, disorders, or conditions that may be associated with inflammation, infection, allergy, immune dysfunction, or dysbiosis of the intestinal microbiome. In some aspects, the invention provides a synergistic combination of a prebiotic, e.g., a mixture of human milk oligosaccharides, and probiotic strains of bacteria, such as one or more strains capable of internalizing and consuming the prebiotic, e.g., Bifidobacterium longum subsp. infantis, and one or more strains capable of producing short chain fatty acids such as propionate, e.g., Veillonella sp.

BACKGROUND OF THE INVENTION

[0004] The microorganisms that colonize the human gastrointestinal tract, also referred to as the gut microbiota, comprise a complex community that plays an important role in the maintenance of health and resistance to disease. A multitude of factors, including host diet, environmental exposures, and antibiotic usage, influence the composition of the microbiome, sometimes leading to dysbiosis and negative health consequences. One promising approach to treat or prevent disease is direct manipulation of the dysbiotic microbiome. Such therapies include live biotherapeutic products (LBPs) that may “engraft” or durably persist in the recipient gastrointestinal tract and thereby lead to positive outcomes. While LBPs conceptually hold promise as potentially effective therapeutic tools, control of their engraftment and accurate prediction of their efficacy and effects on co-resident microbes remain relatively unpredictable. Not only is selection of microbial species still a challenge, but questions remain about the duration and success of engraftment of introduced strains into the human intestinal microbiome.

[0005] What is needed in the art are defined live biotherapeutic products capable of predictably, controllably, and reversibly engrafting within the host intestinal microbiome that reliably benefit the host and treat or prevent disease.

SUMMARY OF THE INVENTION

[0001] Provided herein compositions, kits, articles of manufacture, and methods of use thereof, that are or include a prebiotic mixture, e.g., of human milk oligosaccharides, at least one Bifidobacterium, e.g., B. longum subsp. inf antis, and at least one propionate producing bacterium. The provided prebiotic mixture, Bifidobacteria, and propionate producing bacteria are, inter alia, surprisingly effective for the treatment or prevention of diseases involving an infectious or inflammatory component, or for preventing negative health effects of a dysbiotic intestinal microbiome.

[0002] Provided herein is a method of treating or preventing a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium.

[0003] Also provided herein are uses for a prebiotic mixture and probiotic strains of bacteria for the treatment or prevention of a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof. In certain embodiments, the prebiotic mixture comprises one or more human milk oligosaccharides. In certain embodiments, the probiotic strains of bacteria comprise at least one Bifidobacterium capable of consuming one or more human milk oligosaccharides and at least one propionate producing bacterium. [0004] Additionally provided herein are prebiotic mixtures and probiotic strains of bacteria for use in the manufacture of medicaments for the treatment or prevention of a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof. In particular embodiments, provided herein are probiotic strains of bacteria for use in the manufacture of medicaments for the treatment or prevention of a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome to a subject in need thereof in conjunction with the administration of a prebiotic mixture. In certain embodiments, the prebiotic mixture comprises one or more human milk oligosaccharides. In particular embodiments, the probiotic strains of bacteria comprise at least one Bifidobacterium capable of consuming one or more human milk oligosaccharides and at least one propionate producing bacterium.

[0005] Provided herein are methods of treating or preventing a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium. Also provided are methods of ameliorating a symptom of a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium.

[0006] In particular embodiments, the subject has received or will receive an allogenic hematopoietic stem cell transplant, and wherein the disease, condition, or disorder comprises graft versus host disease. In some embodiments, the disease, disorder, or condition comprises one or more of obesity, type II diabetes, a chronic inflammatory disease, an autoimmune disease, an infection, an infectious disease domination, bowel resection, or a condition associated with chronic diarrhea. In certain embodiments, the disease, disorder, or condition comprises one or more of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), short bowel syndrome (SBS), celiac disease, small intestinal bacterial overgrowth (SIBO), gastroenteritis, leaky gut syndrome, pouchitis, or gastric lymphoma. In particular embodiments, the disease, disorder, or condition comprises an atopic disease. In some embodiments, the atopic disease comprises atopic dermatitis, food allergy, and/or asthma.

[0007] In certain embodiments, the disease, condition, or disorder is associated with an infection. In particular embodiments, the infection comprises a bacterial infection or gut domination. In some embodiments, the bacterial infection or gut domination comprises an infection or gut domination by one or more species, subspecies, or strains of Aeromonas, Bacillus, Blautia, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterobacteriaceae, Enterococcus, Escherichia, Faecalicatena, Francisella, Haemophilus, Helicobacter, Hungatella, Klebsiella, Lachnospiraceae, Legionella, Leptospira, Listeria, Morganella, Mycobacterium, Mycoplasma, Neisseria, Orientia, Plesiomonas, Proteus, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, or Yersinia, optionally one or more of Aeromonas hydrophila, Bacillus cereus, Campylobacter fetus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, enteroaggregative Escherichia coli, enter ohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enteropathogenic E. coli, enterotoxigenic Escherichia coli, Escherichia coli 0157:H7, Helicobacter pylori, Klebsiellia pneumonia, Lysteria monocytogenes, Salmonella paratyphi, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, or Yersinia enter ocolitica.

[0008] In certain embodiments, the disease, condition, or disorder is associated with an infection. In particular embodiments, the infection comprises a bacterial infection or gut domination. In some embodiments, the bacterial infection or gut domination comprises an infection or gut domination by one or more species, subspecies, or strains of Aeromonas, Bacillus, Bordetella, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterobacteriaceae, Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella, Legionella, Leptospira, Listeria, Morganella, Mycobacterium, Mycoplasma, Neisseria, Orientia, Plesiomonas, Proteus, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, or Yersinia, optionally one or more of Aeromonas hydrophila, Bacillus cereus, Campylobacter fetus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enteropathogenic E. coli, enterotoxigenic Escherichia coli, Escherichia coli 0157:H7, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Salmonella paratyphi, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, or Yersinia enterocolitica.

[0009] In certain embodiments, the bacterial infection or gut domination comprises an infection or gut domination by one or more of Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus anginosus, Streptococcus australis, Streptococcus constellatus, Streptococcus cristatus, Streptococcus gordonii, Streptococcus infantis, Streptococcus intermedins, Streptococcus mitis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus tigurinus, or Streptococcus vestibularis.

[0010] In particular embodiments, wherein the bacterial infection or gut domination comprises an infection or gut domination by drug -resistant bacteria. In some embodiments, the drug-resistant bacteria comprise one or more of antibiotic-resistant bacterium (ARB), Antibiotic-resistant Proteobacteria, Carbapenem-resistant Ewterotocterzaceae (CRE), Extended Spectrum Beta-Lactamase producing Enterobacteriaceae (ESBL-E), fluoroquinolone-resistant Enter obacteriaceae, extended spectrum beta-lactam resistant Enterococci (ESBL), vancomycin-resistant Enterococci (VRE), multi-drug resistant A. coli, or multi-drug resistant Klebsiella.

[0011] In certain embodiments, the bacterial infection or gut domination comprises an infection or gut domination by drug-resistant bacteria. In particular embodiments, the drugresistant bacteria comprises one or more of antibiotic-resistant bacterium (ARB), Antibioticresistant Proteobacteria, Carbapenem-resistant Enterobacteriaceae (CRE), Extended Spectrum Beta-Lactamase producing Enterobacterales (ESBL-E), fluoroquinolone-resistant Enterobacteriaceae, vancomycin-resistant Enterococci (VRE), multi-drug resistant A. coli, or multi-drug resistant Klebsiella. [0012] In some embodiments, the subject has undergone or will undergo an ileal pouch-anal anastomosis (IPAA) surgery, and wherein the disease, condition, or disorder comprises pouchitis.

[0013] In certain embodiments, provided herein is a method for increasing propionate in the gut of a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium. In certain embodiments, the at least one propionate producing bacterium comprises a strain that is isolated from a human intestinal microbiome and/or is capable of engrafting within the human intestinal microbiome.

[0014] In particular embodiments, the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella. In some embodiments, the at least one propionate producing bacterium comprises one or more of Veillonella atypica (V. atypica), Veillonella dispar (V. dispar), Veillonella infantium (V. infantium), Veillonella nakazawae (V. nakazawae), Veillonella parvula (V. parvula), Veillonella ratti (V. ratti), Veillonella rogosae (V. rogosae), Veillonella seminalis (V. seminalis), and/or Veillonella tobetsuensis (V. tobetsuensis). In certain embodiments, the at least one propionate producing bacterium comprises V. atypica, V. dispar, V. infantium, V. nakazawae, V. parvula, and/or V. rogosae. In particular embodiments, the at least one propionate producing bacterium comprises V. infantium, V. nakazawae, V. parvula, and/or V. rogosae. In some embodiments, the at least one propionate producing bacterium comprises V. infantium, V. dispar, and/or V. nakazawae . In certain embodiments, the at least one propionate producing bacterium comprises V. parvula. In particular embodiments, the at least one propionate producing bacterium comprises V. rogosae.

[0015] In some embodiments, the at least one propionate producing bacterium comprises one or more strains of the genus Megasphaera. In certain embodiments, the at least one propionate producing bacterium comprises one or more of Megasphaera elsdenii (M. elsdenii), Megasphaera hominis (M. hominis), Megasphaera indica (M. indica), Megasphaera massiliensis (M. massiliensis), and/ or Megasphaera micronuciformis (M. micronuciformis). In particular embodiments, the at least one propionate producing bacterium comprises one or more of M. elsdenii ndJo M. massiliensis. In some embodiments, the at least one propionate producing bacterium comprises one or more strains of the genus Anaerotignum. In certain embodiments, the at least one propionate producing bacterium comprises Anaerotignum lactatifermentans (A. lactatifermentans). In particular embodiments, the at least one propionate producing bacterium comprises one or more strains of the genus Bacteroides. In some embodiments, the at least one propionate producing bacterium comprises Bacteroides fragilis (B. fragilis o Bacteroides caccae (B. caccae). In certain embodiments, the at least one propionate producing bacterium comprises one or more strains of the genus Coprococcus. In particular embodiments, the at least one propionate producing bacterium comprises Coprococcus catus (C. catus). In some embodiments, the at least one propionate producing bacterium comprises one or more strains of the genus Merdimmobilis . In certain embodiments, the at least one propionate producing bacterium comprises Merdimmobilis hominis.

[0016] In particular embodiments, the propionate producing bacterium comprises a nucleotide sequence with at least 97%, 98%, or 99% sequence identity to any of SEQ ID NOS: 40-49 or 52-58 and/or an amino acid sequence with at least 97%, 98%, or 99% sequence identity to SEQ ID NO: 50 or 51. In some embodiments, the Bifidobacterium comprises B. breve, B. bifidum, or B. longum subsp. infantis. In certain embodiments, the Bifidobacterium comprises B. longum subsp. infantis.

[0017] In particular embodiments, the method further comprising at least one butyrate producing strain of bacteria. In some embodiments, the at least one butyrate producing strain comprises a strain of Clostridium Cluster IV or Clostridium Cluster XlVa bacteria. In certain embodiments, the at least one butyrate producing strain comprises one or more of Agathobacter rectalis, Anaerobutyricum hallii, Anaerostipes caccae, Blautia producta, Clostridium leptum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Roseburia hominis, or Roseburia intestinalis. In particular embodiments, the at least one butyrate producing strain comprises one or more of Anaerostipes caccae, Clostridium innocuum, Roseburia hominis, or Roseburia intestinalis.

[0018] In some embodiments, the prebiotic mixture comprises one or more of 2'- fucosyllactose, 3-fucosyllactose, difucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N- tetraose, lacto-N-neo-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c, lacto-N-difuco-hexaose I, lacto-N-difuco-hexaose II, lacto-N-hexaose, para-lacto-N-hexaose, disialyllacto-N-tetraose, Fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose a, difucosyl- lacto-N-hexaose b, lactodifucotetraose, 6’galactosyllactose, 3 ’galactosyllactose, 3-sialyl-3- fucosyllactose, sialylfucosyllacto-N-tetraose, sialyllacto-N-fucopentaose V, disialyl-lacto-n- fucopentaose II, disialyl-lacto-n-fucopentaose V, lacto-N-neo-difucohexaose II, 3-Fucosyl- sialylacto-N-tetraose c, para-lacto-N-neohexose, lacto-N-octaose, lacto-N-neooctaose, lacto- N-neohexaose, lacto-N-fucopentaose V, iso-lacto-N-octaose, para-lacto-N-octaose, lacto- decaose, or sialyl-lacto-N-fucopentaose I.

[0019] In certain embodiments, the prebiotic mixture comprises one or more of 2'- fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N- difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, or disialyllacto-N-tetraose. In particular embodiments, the prebiotic mixture comprises one or more of 2'-fucosyl-lactose, 3-fucosyllactose, 3’-sialyllactose, 6'- sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, or difucosyllactose. In some embodiments, the prebiotic mixture comprises one or more of 2'-fucosyllactose, 3- fucosyllactose, lacto-N-tetraose, or lacto-N-neotetraose. In certain embodiments, the prebiotic mixture comprises one or both of 2'-fucosyllactose and lacto-N-neotetraose. In particular embodiments, the prebiotic mixture comprises at least 10, at least 25, at least 50, at least 100, or at least 150 human milk oligosaccharides. In some embodiments, the prebiotic mixture comprises 2'-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N- tetraose, lacto-N-difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N- tetraose c, sialylacto-N-tetraose b, and disialyllacto-N-tetraose.

[0020] In certain embodiments, the prebiotic mixture is, is derived from, or comprises a concentrated human milk permeate, wherein the concentrated human milk permeate is obtained by a process comprising the steps of ultra-filtering human skim milk to obtain human milk permeate and concentrating the human milk oligosaccharide content of the human milk permeate. In particular embodiments, the human skim milk is obtained from human milk pooled from at least 25, 50, or 100 individual donors.

[0021] Also provided herein is a kit comprising i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium. In some embodiments, the at least one propionate producing bacterium comprises a strain that is isolated from a human intestinal microbiome and/or is capable of engrafting within the human intestinal microbiome. In certain embodiments, the kit further comprises at least one butyrate producing strain of bacteria.

[0022] Further provided herein is a method for increasing propionate concentration and/or propionate production in the gut of a subject in need thereof, the method comprising administering to the subject the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium any of the kits described herein.

[0023] Also provided herein is a method of treating or preventing a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium of any of the kits described herein.

[0024] Additionally provided herein is a method of ameliorating a symptom of a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium of any of the kits described herein.

[0025] Also provided is a pharmaceutical composition comprising i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium. In particular embodiments, the at least one Bifidobacterium comprises B. longum subsp. inf antis and the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella. In some embodiments, the pharmaceutical composition further comprises at least one butyrate producing strain of bacteria, optionally wherein the at least one butyrate producing strain comprises one or more of Agathobacter rectalis, Anaerobutyricum hallii, Anaerostipes caccae, Blautia producta, Clostridium leptum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Roseburia hominis, or Roseburia intestinalis.

[0026] Also provided herein is a method for treating one or more symptoms of acute radiation syndrome in a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides and ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides, thereby treating the symptoms of acute radiation syndrome. In certain embodiments, the at least one Bifidobacterium comprises B. longum subsp. infantis. In particular embodiments, the method further comprises administering to the subject in need thereof iii) at least one propionate producing bacterium. In some embodiments, the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella. In certain embodiments, the one or more symptoms of acute radiation syndrome comprise gastrointestinal damage. In particular embodiments, the subject was exposed to an ionizing radiation dose of at least 0.3 Gray (Gy), optionally at least 0.7 Gy, 1 Gy, 2 Gy, 5 Gy, 6 Gy, or 10 Gy over a period of time lasting under 60 minutes, optionally under 30 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, 2 minutes, or 1 minute.

[0027] Also provided herein is an article of manufacture, comprising any of the kits described herein and instructions for use describing any of the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 provides a schematic of the study described in Example 2.

[0029] FIGS. 2A-2D show graphs displaying time courses of B. longum subsp. infantis abundance during a clinical study. FIG. 2A shows B. infantis abundance over time as determined by qPCR and plotted as the average for subjects in each of the three cohorts. Circles indicate Cohort 1, triangles indicate Cohort 2, and squares indicate Cohort 3. Data are geometric means with 95% confidence intervals. The line labeled LOD represents the limit of detection (27 copies/ng DNA). Significant differences were calculated using mixed effects analysis for repeated measures of log-transformed data; significance between kinetic curves were determined using the time versus treatment factor and significances at individual timepoints were determined using Sidak’s post-test. FIG. 2B shows B. infantis abundance over time as determined by qPCR for subjects in the cohort that received B. infantis and HMO (Cohort 3) comparing those who were engrafted and not engrafted. Subjects were deemed “engrafted” (dark lines) if B. infantis signal was two geometric standard deviations above the geometric mean of signal on days 3-5 (equivalent to 5.4 x 10³ copies/ng DNA) for at least two consecutive time points; subjects below the geometric mean of the B. infantis only cohort on any one of those days were deemed “not engrafted” (light lines). Traces represent individual subjects. FIG. 2C shows B. infantis abundance over time as determined by qPCR for subjects in the cohort that received B. infantis alone (Cohort 2) comparing those who were engrafted (dark lines) and not engrafted (light lines), using the definitions of “engrafted” and “not engrafted” in FIG. 2B. FIG. 2D shows the abundance of B. infantis within the microbiome as determined by whole metagenome sequencing (WMS) using a strain tracking algorithm. Circles indicate Cohort 1, triangles indicate Cohort 2, and squares indicate Cohort 3. Each point represents the median of the indicated cohort subpopulation, and error bars represent the interquartile range. Undetected values were imputed with the LOD. The LOD represents the lowest detected value.

[0030] FIGS. 3A-3F show graphs summarizing whole metagenomic sequencing (WMS) from genomic DNA extracted from stool samples collected from subjects as described in Example 2. FIG. 3A displays Shannon Entropy of observed reads rarefied to 76,000 reads used as a measure of the alpha diversity of samples over time. Data are median and 95% confidence interval. Significant differences were calculated using mixed effects analysis for repeated measures; significance between kinetic curves was determined using the time versus treatment factor. Significances between individual timepoints were determined using Sidak’s post-test for each individual cohort and asterisks indicate significance across both cohorts. FIG. 3B shows Bray-Curtis dissimilarity for each sample with rarefied sequences aggregated to the genus taxonomic level and displayed by cohort and day in a principal coordinate analysis (PcoA) plot noting the trajectory from pre-treatment (day 1) and end (day 5) of antibiotic treatment (arrow). FIG. 3C contains stacked bar charts displaying aggregated families (and two at the order level) present at >1% abundance for each cohort on each day of the study. Taxa <1% in each cohort were aggregated to “Other”. For the antibiotics + B. infantis + HMO cohort (Cohort 3), subjects are separated into Engrafted and Not Engrafted (NE). FIG. 3D shows PcoA plots of Bray-Curtis dissimilarity calculated with rarefied sequences aggregated to the genus taxonomic level for days 1, 5 and 14 only. The antibiotics only cohort was compared to engrafted subjects in the antibiotics + B. infantis + HMO cohort, including (top row) and excluding (bottom row) taxa assigned to the genus Bifidobacterium. FIG. 3E shows a volcano plot of aggregated genera >1% across cohorts comparing abundance in Cohort 3 with Cohort 1. Log2 fold change (CLR transformed, sampled 150 times from a Dirichlet model) and p-values (Wilcoxon’s Rank Sum Test with Bejamini -Hochberg correction for multiple comparisons) were calculated using ALDEx2 (v 1.29.1). Values with an adjusted p value < 0.05 and greater than 0.6-fold change are highlighted and colored by taxon. FIG. 3F shows the relative abundance of Veillonella across study days comparing Cohort 1 (left) and Cohort 3 (right) showing statistical differences calculated using the CLR transformed abundances in ALDEx2.

[0031] FIGS. 4A-4F show graphs summarizing gut metabolites in stool samples of antibiotic-treated subjects. FIGS. 4A and 4C show levels of acetic acid (FIG. 4A) and lactic acid (FIG. 4C) as measured in stool samples from subjects in the antibiotics only cohort (Cohort 1, circles) and engrafted subjects in the antibiotics + B. infantis + HMO cohort (Cohort 3, squares). Dotted lines represent limit of detection. Significant differences were calculated using mixed effects analysis for repeated measures of log-transformed data; significance between kinetic curves was determined using the time versus treatment factor. In FIG. 4A, significance between day 1 and day 5 was determined using Sidak’s post-test for each individual cohort and was significant across both cohorts. In FIG. 4C, significances between cohorts at individual timepoints were determined using Sidak’s post-test. FIG. 4B shows Kaplan-Meier curve plotting the day on which stool acetate for each subject returned to baseline levels, where baseline is the average of subjects at Day 1. Significance between groups was calculated using log-rank test. FIG. 4D provides a Volcano plot showing log2 fold change in fecal metabolites on day 14 between engrafted subjects in the antibiotics + B. infantis + HMO cohort (Cohort 3) and the antibiotics only cohort (Cohort 1) plotted against significance of the observed change, calculated via two-way repeated measures ANOVA. The dotted line represents the threshold of p = 0.05. Metabolites at a significantly different abundance are colored to indicate categorization. Notable metabolites are labeled. FIG. 4E shows levels of indolelactate in stool at day 14 (left) and in serum at days 1, 5, 14, 28, and 35 (right). FIG. 4F shows levels of p-cresol sulfate in stool at day 14 (left) and in serum at days 1, 5, 14, 28, and 35 (right). In FIGS. 4E and 4F scatterplot data, horizontal lines are geometric mean; dotted lines are the imputed value for samples without detected signal, and significances are derived from two-way repeated measures ANOVA calculated for the entire global metabolomics dataset as described in FIG. 4D. Timecourse data in FIGS. 4E and 4F are geometric mean and 95% confidence interval; dotted lines are limit of detection; significances are calculated as in FIG. 4B.

[0032] FIGS. 5A-5D show graphs summarizing in vitro cross-feeding of three different Veillonella species by B. infantis + HMO. The indicated propionate-producing species of Veillonella were grown with no addition, B. infantis, HMO, both B. infantis and HMO, or lactate alone as indicated. Dotted lines labeled “LOQ” indicate the limits of quantitation. FIG. 5A shows propionate quantified using liquid chromatography with tandem mass spectrometry from media collected after 30 hours of culture with media alone or the noted concentration of lactate. FIG 5B shows propionate quantified from media collected after 30 hours using liquid chromatography with tandem mass spectrometry. FIGS. 5C and 5D show abundance of B. infantis (FIG. 5C) or Veillonella spp. (FIG. 5D) quantified using qPCR after 30 hours of culture. Data are geometric mean and standard deviation of 3 independent experiments. Statistics reflect a two-way ANOVA with Tukey’s test for multiple comparisons within each Veillonella condition, using log-transformed data (* indicates adjusted p <0.05, ns indicates p > 0.05). Within each experiment, triplicate culture wells were pooled for propionate analysis and qPCR.

[0033] FIGS. 6A-6D depict in vivo cross-feeding of Veillonella species by B. infantis + HMO. FIG. 6A provides a study schematic. Germ-free mice were divided into three groups, two of which were inoculated with Veillonella parvula and the third with B. infantis as a control. After one week, the two V. parvula-associated groups were given a single gavage of either PBS or B. infantis + HMO; B. infantis-associated animals were gavaged with HMO only. Over the subsequent three days, animals received once-daily gavages of PBS or HMO. FIG. 6B shows quantification of Veillonella in feces. Cage-bottom fecal pellets collected over time were used to quantify the levels of Veillonella in cage-bottom fecal pellets using qPCR. Each pair of connected symbols represents an individual mouse. FIG. 6C shows the levels of propionate quantified in contents of the indicated intestinal segments using LC-MS/MS. FIG. 6D shows the levels of lactate quantified in contents of the indicated intestinal segments using LC-MS/MS. Significance of differences was calculated by Sidak’s multiple comparison test from mixed effects analysis for repeated measures of log- transformed data.

[0034] FIGS. 7A and 7B show relative abundance of B. infantis as determined by qPCR. FIG. 7A shows data from FIG. 2 A replotted to exclude the non-engrafted subjects in the B. infantis + HMO cohort; significances were calculated as in FIG. 2A. FIG. 7B shows B. infantis relative abundance in stool samples determined using qPCR to measure B. infantis (shown in FIG. 2 A) and 16S rRNA and then normalizing B. infantis levels to 16S rRNA levels. Each point represents the median, and error bars represent the interquartile regions. The dotted line represents limit of detection (LOD) calculated across the entire study using the following calculation: [LOD for B. infantis copy number per reaction] / [median 16S rRNA gene copy number per reaction across all samples],

[0035] FIGS. 8A-8H depict microbiome changes after antibiotic perturbation. FIG. 8A shows number of observed reads whole metagenomic sequencing (WMS) data from stool samples over time; significances were calculated as in FIG. 3 A. FIG. 8B shows the geometric mean of 16S rRNA gene copy number per gram of stool for each cohort over time. Each dot represents the geometric mean and error bars represent the 95% confidence intervals. Significances were calculated as in FIG. 3 A except that data were log-transformed prior to statistical analysis. FIG. 8C provides stacked bar charts of metagenomic sequencing at the family level for subjects receiving B. infantis only (Cohort 2) over time. Each study day is separated into engrafted (E) or not engrafted. FIGS. 8D-I are box and whisker plots of the relative abundance of bacteria highlighted in the ALDEx2 analysis shown in FIG. 3E and Bacteroides over time comparing subjects in the antibiotics only cohort (Cohort 1, left) and the B. Infantis + HMO (engrafted only) cohort (Cohort 3, right).

[0036] FIGS. 9A-9N depict changes in gut metabolites after antibiotic perturbation. FIGS. 9A and 9B show levels of butyrate (FIG. 9A) and propionate (FIG. 9B) measured in stool samples collected on days 1, 5, 9, 11, 14, 17, 28, and 35 from subjects that received antibiotics only (Cohort 1, circles) and engrafted subjects who also received B. infantis and HMO (Cohort 3, squares). Dots represent the geometric mean and error bars represent the 95% confidence intervals. Significances were calculated as in FIG 4A. FIG. 9C shows pH measurements of fecal samples within cohorts over time. Data show average pH with error bars representing standard error. A mixed effects model with Sidak’s multiple comparisons test was used for comparisons. FIGS. 9D-9I show volcano plots of fecal global metabolomic data comparing cohorts (FIG. 9F and FIG. 91) or study days within each cohort (FIGS. 9D, 9E, 9G, and 9H). FIG. 9J shows Bray-Curtis dissimilarity calculated for each sample and displayed by cohort and day in a PcoA plot. FIG. 9K shows HMOs from the stool metabolomics analysis on day 5 represented in batch-normalized imputed peak area values. Each dot represents a subject in the Antibiotics only cohort (Cohort 1, left) or the engrafted subjects in the B. infantis + HMO cohort (Cohort 3, right), with a line at the median. The observed minimum value was imputed for each individual metabolite if a metabolite was undetected in any sample. Significances are derived from two-way repeated measures ANOVA calculated for the entire global metabolomics dataset as described in FIG 4D. FIG. 9L is a volcano plot showing fold change in serum metabolites in the Antibiotics Only cohort (Cohort 1) between Day 5 and Day 1 against significance of the observed change. Significances were calculated with a repeated measures test on log-transformed data and Sidak’s post-test to correct for multiple comparisons. Relevant metabolites are noted in color and labeled. FIG. 9M is a volcano plot showing fold change in serum metabolites on Day 14 between engrafted subjects (Cohort 3) and subjects that only received antibiotics (Cohort 1) plotted against significance of the observed change, calculated as in FIG. 9L. FIG. 9N shows levels of phenol sulfate measured in serum samples over time for each cohort. Data and significances are presented as in FIG. 9A. [0037] FIGS. 10A-10G show in vitro cross-feeding of Veillonella species by B. infantis + HMO. The indicated species of Veillonella were grown in the absence or presence of B. infantis, HMO, both, or lactate. FIGS. 10A-10C are growth curves of the three different Veillonella strains presented in FIG. 5 A with varying concentrations of lactate. Data are mean and standard deviation of 9 replicates from 3 independent experiments. FIGS. 10D-10G show quantification of acetate (FIGS. 10D and 10F) and lactate (FIGS. 10E and 10G) in cultures presented in FIG. 5. Data represent geometric mean and standard deviation of 3 independent experiments. Within each experiment, triplicate culture wells were pooled for analysis. Dotted lines indicate the limit of quantitation. Statistics reflect a two-way ANOVA with Tukey’s test for multiple comparisons to assess acetate production (FIGS. 10D and 10F) or lactate consumption (FIGS. 10E and 10G) under each set of growth conditions, in all cases using log-transformed data (* indicates adjusted p <0.05, “ns” indicates adjusted p >0.05). Lines in FIG. 10E and 10G indicate the displayed comparisons between levels of lactate in cultures containing Veillonella and the corresponding Veillonella-V culture.

[0038] FIGS. 11A-11C depict in vivo cross-feeding of Veillonella species with B. infantis + HMO in mice described in Example 12. FIGS. HA and 11B show quantification of Veillonella (FIG. 11 A) and B. infantis (FIG. 11B) in mice using qPCR. Data are plotted as the geometric mean and standard deviation. FIG. 11C shows concentration of acetate isolated intestinal sections and feces from mice of the experiments shown in FIG. 6.

[0039] FIG. 12 shows a scatterplot depicting concentrations of propionate (Y axis) and lactate (x axis) of various test strains following a 48 hour incubation with lactate as a carbon source. Starting lactate concentration is indicated by the vertical dotted line.

[0040] FIGS. 13A-13C show quantification of Veillonella sp. (left) and B. longum subsp. infantis (B. infantis,' right) in stool collected at various timepoints from germ free mice inoculated with Veillonella sp., B. infantis, or both and administered a human milk oligosaccharide composition (HMO) or a vehicle control. Veillonella sp. included a strain of V. parvula (FIG. 13A) and two Veillonella strains (FIGS. 13B and 13C) isolated as described in Example 11. Strains were quantified using qPCR. Data are plotted as the geometric mean and standard deviation.

[0041] FIGS. 14A-14C show quantification of propionate levels detected in ileal, cecal, rectal, and fecal samples collected from germ free mice inoculated with Veillonella sp. (V sp.), B. infantis (BI), or both and administered a human milk oligosaccharide composition (HMO) or a vehicle control. Veillonella sp. included a strain of V. parvula (FIG. 14A) and two Veillonella strains (FIGS. 14B and 14C) isolated as described in Example 11. Data are plotted as the geometric mean and standard deviation. Data are plotted as the geometric mean and standard deviation.

[0042] FIGS. 15A-15C show quantification of lactate levels detected in ileal, cecal, rectal, and fecal samples collected from germ free mice inoculated with Veillonella sp. (V sp.), B. infantis (BI), or both and administered a human milk oligosaccharide composition (HMO) or a vehicle control. Veillonella sp. included a strain of V. parvula (FIG. 15A) and two Veillonella strains (FIGS. 15B and 15C) isolated as described in Example 11. Data are plotted as the geometric mean and standard deviation. Data are plotted as the geometric mean and standard deviation.

[0043] FIGS. 16A-16C show quantification of acetate levels detected in ileal, cecal, rectal, and fecal samples collected from germ free mice inoculated with Veillonella sp. (V sp.), B. infantis (BI), or both and administered a human milk oligosaccharide composition (HMO) or a vehicle control. Veillonella sp. included a strain of V. parvula (FIG. 16A) and two Veillonella strains (FIGS. 16B and 16C) isolated as described in Example 11. Data are plotted as the geometric mean and standard deviation. Data are plotted as the geometric mean and standard deviation.

[0044] FIGS. 17A-17F depict experimental results following inoculation of strains in germ free mice. FIG. 17A show quantification of B. longum subsp. infantis (B. infantis; left) and Megasphaera elsdenii (M. elsdenii; right) in stool collected at various timepoints from germ free mice inoculated with A/, elsdenii, B. infantis, or both and administered a human milk oligosaccharide composition (HMO) or a vehicle control. Strains were quantified using qPCR. FIGS. 17B-17F show quantification of butyrate (FIG. 17B), propionate (FIG. 17C), valerate (FIG. 17D), lactate (FIG. 17E), and acetate (FIG. 17F) levels detected in cecal samples collected from these mice. Data are plotted as the geometric mean and standard deviation.

DETAILED DESCRIPTION

[0045] Provided herein are compositions, kits, and articles of manufacture as well as methods of use thereof. In certain embodiments, the provided compositions, kits, and articles of manufacture are or include prebiotics and at least two probiotic strains of bacteria. In particular embodiments, the prebiotics are or include a mixture, e.g., of oligosaccharides such as human milk oligosaccharides. In some embodiments, the at least two probiotic strains of bacteria are or include one or more strains of Bifidobacteria, for example a strain of Bifibacterium capable of consuming the prebiotics and/or human milk oligosaccharides, and a strain of bacteria capable of producing the short chain fatty acid propionate, e.g., a strain of Veillonella sp. In some aspects, the provided compositions, kits, and articles of manufacture may be administered to a subject to treat or prevent dysbiosis, e.g., of the intestinal microbiome, as well as conditions, diseases, or disorders that may originate from or cause dysbiosis. In some aspects, the provided compositions, kits, and articles of manufacture may be administered to a subject to treat or prevent conditions, diseases, or disorders that are, include, or contribute to inflammation, infection, allergy, or immune dysfunction.

[0046] In certain aspects, the maintenance of a healthy human metabolism depends on a symbiotic consortium among bacteria, archaea, viruses, fungi, and host eukaryotic cells throughout the human gastrointestinal tract. For example, microbial communities may provide enzymatic machinery and metabolic pathways that contribute to food digestion, xenobiotic metabolism, and production of a variety of bioactive molecules. Disturbances to the microbiome may result in a microbial imbalance (dysbiosis) characterized by phylumlevel changes in the microbiota composition, including a marked decrease in the representation of obligate anaerobic bacteria and an increased relative abundance of facultative anaerobic bacteria. While dysbiosis is associated with numerous diseases and conditions, successfully treating dysbiosis is difficult, particularly in vulnerable or immunocompromised patients.

[0047] The provided compositions, methods, kits, and articles of manufacture address these needs. In particular, the present invention includes specific combinations of prebiotics, such as human milk oligosaccharides, and probiotics, such as Bifidobacterium including B. longum subspecies (subsp.) infantis and bacteria capable of producing propionate, e.g., Veillonella sp., that are particularly safe and effective for treating, ameliorating, or reducing dysbiosis in the gut microbiome as well as effective in treating, ameliorating, or preventing diseases or disorders that may be accompanied by dysbiosis, such including but not limited to diseases associated with immune disorders, inflammatory disorders, or infection. This synbiotic approach effectively allows for the beneficial Bifidobacteria and Veillonella to grow, expand, and engraft within the gut, thereby regulating pH, suppressing growth of opportunistic and/or pathogenic microbes, and producing short chain fatty acids (SCFAs) such as propionate to reduce inflammation in the host. [0006] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications, and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0007] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. COMPOSITIONS, KITS, AND ARTICLES OF MANUFACTURE

[0008] Provided herein are compositions, kits, and articles of manufacture that are or include i) a prebiotic mixture that are or include one or more oligosaccharides, ii) at least one strain of bacteria from the genus Bifidobacterium that is capable of consuming oligosaccharides, e.g., the oligosaccharides of the mixture and/or human milk oligosaccharides; and iii) one or more strains of bacteria capable of producing propionate. In some embodiments, the prebiotic mixture is or includes human milk oligosaccharides. In certain embodiments, the Bifidobacteria is or includes B. longum subsp. infantis. In particular embodiments, the one or more strains of bacteria capable of producing propionate are or include strains of bacteria from the genus Veillonella.

[0009] In certain embodiments, the Bifidobacteria capable of consuming human milk oligosaccharides, the strain capable of producing propionate, and the prebiotic mixture are or are included in separate compositions, e.g., are administered separately to a subject. Thus, in certain embodiments, provided herein are kits and articles of manufacture that include (i) a composition that is or includes a strain from the genus Bifidobacteria capable of consuming human milk oligosaccharides (ii) a composition that is or includes at least one propionate producing strain of bacterium (also referred to herein and used interchangeably with “strain of bacteria capable of producing propionate” or “propionate producing bacterium” unless otherwise indicated), and (iii) a composition that is or includes the prebiotic mixture, e.g., of human milk oligosaccharides. Also provided are kits and articles of manufacture that are or include two compositions, a probiotic composition that is or includes the Bifidobacteria, e.g., B. longum subsp. infantis, and bacteria capable of producing propionate, e.g., Veillonella, and a composition that is or includes the prebiotic mixture, e.g., of human milk oligosaccharides. Thus, in some embodiments, additionally provided are kits and articles of manufacture that include a composition that is or includes all of the Bifidobacteria, e.g., B. longum subsp. infantis, the strain of bacteria capable of producing propionate, e.g., Veillonella, and the prebiotic mixture, e.g., of human milk oligosaccharides.

[0010] In certain embodiments, the provided compositions, kits, and articles of manufacture are or include any of the strains of bacteria capable of producing propionate, (also referred to herein as a “propionate producing strain of bacteria,” “propionate producing strain” or “propionate producing bacterium”) described throughout the application, including in Section I-A. In particular embodiments, the provided compositions, kits, and articles of manufacture are or include any of the one or more strains of the genus Bifidobacterium, e.g., a strain of Bifidobacterium capable of consuming human milk oligosaccharides, described throughout the application including in Section I-B. In some embodiments, the provided compositions, kits, and articles of manufacture are or include any of the prebiotic mixtures, e.g., of human milk oligosaccharides, described throughout the application, including at least at Section I-C. The provided kits and articles of manufacture may also include labels or instructions for use. In some embodiments, such labels or instructions for use may describe any of the uses or methods provided herein, such as those described in Section II.

A. Propionate producing bacteria

[0011] In certain embodiments, the at least one propionate producing bacterium is capable of consuming lactate and/or acetate. In certain embodiments, the at least one propionate producing bacterium is capable of producing, generating, and/or creating propionate in the presence of lactate and/or acetate. In certain embodiments, the at least one propionate producing bacterium is capable of growing or expanding in the presence of a strain of Bifidobacterium, e.g., a strain of Bifidobacterium probiotic strain described herein such as in Section I-B. In some embodiments, the at least one propionate producing strain of bacterium is capable of growing or expanding in the presence of B. longum subsp. infantis.

[0012] In some embodiments, the at least one propionate producing bacterium is or includes at least one, two, three, four, five, six, seven, eight, nine, ten, or more species, subspecies, or strains of bacteria capable of producing propionate. In particular embodiments, the propionate producing bacterium is or includes at least three strains of bacteria capable of producing propionate. In some embodiments, the propionate producing strain is or includes between one and five species, subspecies, or strains of bacteria capable of producing propionate.

[0013] In certain embodiments, the at least one propionate producing bacterium has or contains one or more functional genes that contribute to the production, generation, synthesis, or making of propionate.

[0014] In particular embodiments, the at least one propionate producing bacterium is or includes a strain of bacterium from the genus Veillonella. In certain embodiments, the at least one propionate producing bacterium is or includes Veillonella sp. In particular embodiments, the at least one strain of bacterium from the genus Veillonella is capable of converting lactate to propionate. In some embodiments, the one or more propionate producing strain of bacteria is or includes Veillonella agrestimuris, Veillonella alcalescens, Veillonella atypica, Veillonella caviae, Veillonella cricetid, Veillonella denticariosi, Veillonella dispar, Veillonella hominis, Veillonella infantium, Veillonella intestinalis, Veillonella magna, Veillonella montpellierensis, Veillonella nakazawae, Veillonella parvula, Veillonella ratti, Veillonella rodentium, Veillonella rogosae, Veillonella seminalis, or Veillonella tobetsuensis.

[0015] In certain embodiments, the propionate producing bacterium is or includes Veillonella atypica (V. atypica), Veillonella dispar (V. dispar), Veillonella infantium (V. infantium), Veillonella nakazawae (V. nakazawae), or Veillonella parvula (V. parvula). In some embodiments, the propionate producing bacterium is or includes V. infantium, V. dispar, or V. nakazawae. In some embodiments, the propionate producing bacterium is or includes V. parvula. In some embodiments, the propionate producing bacterium is or includes V. rogosae.

[0016] In some embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 85%, 90%, 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 40-49. In particular embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 85%, 90%, 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 40-43. In certain embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence with at least 85%, 90%, 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 40-49. In certain embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence with at least 85%, 90%, 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 40-43.

[0017] In some embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence encoding an rpoB gene having a sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40 or 41. In particular embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence encoding an rpoB gene with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40 or 41.

[0018] In particular embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence encoding an 16S gene having a sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 42-49. In certain embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence encoding an 16S gene with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 42-49. In some embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence encoding an 16S gene having a sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42 or 43. In certain embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence encoding an 16S gene with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42 or 43.

[0019] In certain embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence encoding an rpoB gene with at least 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40 and a nucleic acid sequence encoding a 16S gene with at least 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42. In some embodiments, the propionate producing bacterium is or includes a strain of Veillonella sp. that has or includes a nucleic acid sequence encoding an rpoB gene with at least 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 41 and a nucleic acid sequence encoding a 16S gene with at least 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 43.

[0020] In some embodiments, the propionate producing bacterium is or includes a strain of bacteria that has, includes, or expresses a methylmalonyl-CoA decarboxylase subunit alpha and/or a methylmalonyl-CoA decarboxylase subunit beta protein and/or has or includes one or more genes encoding a methylmalonyl-CoA decarboxylase subunit alpha and/or a methylmalonyl-CoA decarboxylase subunit beta. In particular embodiments, the methylmalonyl-CoA decarboxylase alpha and beta subunits are from and/or are identical to alpha and beta subunits found in and/or expressed by a Veillonella sp. In certain embodiments, the propionate producing bacterium is or includes a strain of bacteria that has, includes, or expresses a protein having an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 50 or 51. In particular embodiments, the propionate producing bacterium is or includes a strain of bacteria that has, includes, or expresses a protein having an amino acid sequence with 100% identity to SEQ ID NO: 50 or 51.

[0021] In certain embodiments, the at least one propionate producing bacteria is or includes at least one strain of bacterium from the genus Megasphaera. In particular embodiments, the at least one strain of bacterium from the genus Megasphaera is capable of converting lactate to propionate. In some embodiments, the at least one strain of bacterium from the genus Megasphaera is capable of converting lactate to propionate and butyrate. In particular embodiments, the at least one propionate producing bacterium is or includes at least one strain of Megasphaera sp. In some embodiments, the propionate producing bacterium is or includes Megasphaera elsdenii (M. elsdenii), Megasphaera hominis (M hominis), Megasphaera indica (M indica), Megasphaera massiliensis (M.massiliensis), and/ or Megasphaera micronuciformis (M micromiciformis). In certain embodiments, the propionate producing bacterium is or includes Megasphaera elsdenii and/ or Megasphaera massiliensis.

[0022] In some embodiments, the propionate producing bacterium is or includes a strain of Megasphaera sp. that has or includes a nucleic acid sequence encoding an 16S gene having a sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 52 or 53. In particular embodiments, the propionate producing bacterium is or includes a strain of Megasphaera sp. that has or includes a nucleic acid sequence encoding an 16S gene with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 52 or 53.

[0023] In some embodiments, the at least one propionate producing bacteria is or includes at least one strain of bacterium from the genus Anaerotignum. In some embodiments, the at least one strain of bacterium from the genus Anaerotignum is capable of converting lactate to propionate and butyrate. In particular embodiments, the at least one propionate producing bacterium is or includes at least one strain of Anaerotignum sp. In some embodiments, the propionate producing bacterium is or includes Anaerotignum lactatifermentans (A. lactatifermentans).

[0024] In some embodiments, the propionate producing bacterium is or includes a strain of Anaerotignum sp. that has or includes a nucleic acid sequence encoding an 16S gene having a sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 54. In particular embodiments, the propionate producing bacterium is or includes a strain of Anaerotignum sp. that has or includes a nucleic acid sequence encoding an 16S gene with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 54.

[0025] In some embodiments, the at least one propionate producing bacteria is or includes at least one strain of bacterium from the genus Bacteroides. In some embodiments, the at least one strain of bacterium from the genus Bacteroides is capable of converting lactate to propionate and butyrate. In particular embodiments, the at least one propionate producing bacterium is or includes at least one strain of Bacteroides sp. In some embodiments, the propionate producing bacterium is or includes Bacteroides fragilis (B. fragilis and/ or Bacteroides caccae (B. caccae).

[0026] In some embodiments, the propionate producing bacterium is or includes a strain of Bacteroides sp. that has or includes a nucleic acid sequence encoding an 16S gene having a sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 55 and/or 57. In particular embodiments, the propionate producing bacterium is or includes a strain of Bacteroides sp. that has or includes a nucleic acid sequence encoding an 16S gene with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 55 and/or 57.

[0027] In some embodiments, the at least one propionate producing bacteria is or includes at least one strain of bacterium from the genus Coprococcus. In some embodiments, the at least one strain of bacterium from the genus Coprococcus is capable of converting lactate to propionate. In particular embodiments, the at least one propionate producing bacterium is or includes at least one strain of Coprococcus sp. In some embodiments, the propionate producing bacterium is or includes Coprococcus catus (C catus).

[0028] In some embodiments, the propionate producing bacterium is or includes a strain of Coprococcus sp. that has or includes a nucleic acid sequence encoding an 16S gene having a sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 56. In particular embodiments, the propionate producing bacterium is or includes a strain of Coprococcus sp. that has or includes a nucleic acid sequence encoding an 16S gene with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 56.

[0029] In certain embodiments, the at least one propionate producing bacteria is or includes at least one strain of bacterium from the genus Merdimmobilis. In some embodiments, the at least one strain of bacterium from the genus Merdimmobilis is capable of converting lactate to propionate. In particular embodiments, the at least one propionate producing bacterium is or includes at least one strain of Merdimmobilis sp. In some embodiments, the propionate producing bacterium is or includes Merdimmobilis hominis).

[0030] In some embodiments, the propionate producing bacterium is or includes a strain of Merdimmobilis sp. that has or includes a nucleic acid sequence encoding an 16S gene having a sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,400 nucleotides in length with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 58. In particular embodiments, the propionate producing bacterium is or includes a strain of Merdimmobilis sp. that has or includes a nucleic acid sequence encoding an 16S gene with at least 95%, 97%, 97.5%, 98%, 99% or 100% sequence identity to SEQ ID NO: 58.

[0031] In particular embodiments, the at least one propionate producing bacteria is or includes at least one strain of bacterium from the genus Blautia. In some embodiments, the at least one strain of bacterium from the genus Blautia is capable of converting human milk oligosaccharides and/or 1,2-propanediol to propionate and/or butyrate. In particular embodiments, the at least one propionate producing bacterium is or includes at least one strain of Blautia sp. In some embodiments, the propionate producing bacterium is or includes Blautia obeum.

[0032] In various embodiments, the at least one propionate producing bacteria is or includes at least one strain of bacterium from the genus Ruminococcus. In certain embodiments, the at least one strain of bacterium from the genus Ruminococcus is capable of converting human milk oligosaccharides and/or 1,2-propanediol to propionate and/or butyrate. In particular embodiments, the at least one propionate producing bacterium is or includes at least one strain of Ruminococcus sp. In some embodiments, the propionate producing bacterium is or includes Ruminococcus torques.

[0033] In particular embodiments, the at least one propionate producing bacteria is or includes at least one strain of bacterium from the genus Roseburia. In certain embodiments, the at least one strain of bacterium from the genus Roseburia is capable of converting human milk oligosaccharides and/or 1,2-propanediol to propionate and/or butyrate. In particular embodiments, the at least one propionate producing bacterium is or includes at least one strain of Roseburia sp. In some embodiments, the propionate producing bacterium is or includes Roseburia inulinivorans .

B. Bifidobacteria

[0048] In particular embodiments, provided herein are compositions that are or include at least one strain of the genus Bifidobacteria, e.g., a strain of Bifidobacteria capable of consuming human milk oligosaccharides such as B. longum subsp. infantis. In certain embodiments, Bifidobacteria is or includes two or more strains or species of Bifidobacteria, such as any two or more of those described herein, e.g., in Section I-B. Some suitable Bifidobacteria strains may be described in PCT App. Pub. No. W02021061991, hereby incorporated by reference in its entirety.

[0049] In particular embodiments, the at least one strain of Bifidobacterium is capable of consuming or metabolizing non-digestible carbohydrates, e.g., oligosaccharides such as human milk oligosaccharides. In particular embodiments, at least one strain of Bifidobacterium is capable of consuming or metabolizing oligosaccharides such as human milk oligosaccharides. In some embodiments, at least one strain of Bifidobacterium is capable of utilizing the prebiotics of the prebiotic mixture, e.g., human milk oligosaccharides, as a carbon source. In particular embodiments, human milk oligosaccharides are preferentially consumed or metabolized by the at least one strain of Bifidobacterium, e.g., as compared to other microbes or bacteria present in the gut or microbiome. In certain embodiments, the at least one strain of Bifidobacterium is capable of consuming or metabolizing one or more prebiotics of the mixture, including those of any of the prebiotic mixtures described herein, e.g., in Section I-C. In certain embodiments, the at least one strain of Bifidobacterium is capable of consuming or metabolizing all or essentially all of the oligosaccharides of the prebiotic mixture. In certain embodiments, the at least one strain of Bifidobacterium is capable of consuming or metabolizing human milk oligosaccharides.

Particular embodiments contemplate that strains of Bifidobacteria that consume or metabolize human milk oligosaccharides are known and may be identified by routine techniques such as those described in Gotoh et al. Sci Rep. 2018 Sep 18;8(1): 13958, incorporated by reference herein in its entirety.

[0050] In some embodiments, the at least one strain of Bifidobacterium contains one or more enzymes capable of hydrolyzing the prebiotics of the mixture. In certain embodiments, the at least one strain of Bifidobacterium contains one or more enzymes capable of hydrolyzing the human milk oligosaccharides. In particular embodiments, the one or more enzymes hydrolyze external oligosaccharides, e.g., oligosaccharides such as human milk oligosaccharides that are outside of the Bifidobacterial cell. In some embodiments, the one or more enzymes hydrolyze oligosaccharides such as human milk oligosaccharides internally or within the Bifidobacterial cell. In certain embodiments, the one or more enzymes hydrolyze internalized human milk oligosaccharides.

[0051] In particular embodiments, the at least one strain of Bifidobacterium contains one or more enzymes capable of hydrolyzing one or more human milk oligosaccharides. In particular embodiments, the one or more enzymes hydrolyze external human milk oligosaccharides. In some embodiments, the one or more enzymes hydrolyze human milk oligosaccharides that are outside of the Bifidobacterial cell. In some embodiments, the one or more enzymes hydrolyze human milk oligosaccharides internally. In particular embodiments, the one or more enzymes hydrolyze human milk oligosaccharides within the probiotic cell. In certain embodiments, the one or more enzymes hydrolyze internalized human milk oligosaccharides.

[0052] In some embodiments, the at least one strain of Bifidobacterium is capable of internalizing human milk oligosaccharides. In certain embodiments, the at least one strain of Bifidobacterium internalizes human milk oligosaccharides prior to hydrolyzing the human milk oligosaccharides. In various embodiments, the at least one strain of Bifidobacterium selectively or exclusively utilizes human milk oligosaccharides as a carbon source. Thus, in certain embodiments, if the at least one strain of Bifidobacterium is administered to the subject and/or has engrafted, e.g., within the subject’s microbiome (such as the intestinal microbiome), the at least one strain of Bifidobacterium is present, expands, or increases in amount within the subject’s microbiome when human milk oligosaccharides are administered to and/or ingested by the subject, and, in certain embodiments, the at least one strain of Bifidobacterium is no longer present and/or decreases in amount within the subject’s microbiome when the human milk oligosaccharides are no longer ingested or administered.

[0053] In some embodiments, the at least one strain of Bifidobacterium is capable of internalizing oligosaccharides, such as to consume or metabolize the oligosaccharides. In certain embodiments, the at least one strain of Bifidobacterium is capable of internalizing one or more oligosaccharides of the mixture, including those of any of the oligosaccharides or mixtures described herein, e.g., in Section I-C. In certain embodiments, the probiotic strain is capable of internalizing human milk oligosaccharides.

[0054] In particular embodiments, the at least one strain of Bifidobacterium is or includes a strain of B. longum subsp. infantis, B. adolescentis, B. animalis subsp. animalis, B. animalis subsp. lactis B. bifidum, B. breve, B. catenulatum, B. longum subsp. longum, B. pseudocatanulatum, or B. pseudoIongum.

[0055] In particular embodiments, the species or subspecies of a given strain of Bifidobacteria may be identified by routine techniques. For example, in some embodiments, the species or subspecies is identified by assessing the sequence similarity of one or more genes to corresponding sequences of known members of bacterial species or subspecies. In certain embodiments, a probiotic strain falls within a species or subspecies if all or a portion of its 16S gene has at least 97% sequence identity to all or a portion of a known 16S sequence of a known strain falling within the species. In particular embodiments, a probiotic strain falls within a species or subspecies if all or a portion of its 16S gene has at least 97% sequence identity to all or a portion of a known 16S sequence of a known strain falling within the species. Exemplary full or partial 16S sequences are summarized in Table 1.

Table 1: Exemplary 16S sequences

[0056] In certain embodiments, the at least one strain of Bifidobacterium has or includes a nucleic acid sequence with at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a nucleic acid sequence set forth in any of SEQ ID NOS: 1-16 or 20-39. In particular embodiments, the at least one strain of Bifidobacterium. In certain embodiments, the at least one strain of Bifidobacterium has or includes a nucleic acid sequence with at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a nucleic acid sequence set forth in any of SEQ ID NOS: 1-7 or 20-39. In some embodiments, the at least one strain of Bifidobacterium has or includes a nucleic acid sequence with at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a nucleic acid sequence set forth in any of SEQ ID NOS: 1-7 or 11. In certain embodiments, the at least one strain of Bifidobacterium has or includes a nucleic acid sequence with at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a nucleic acid sequence set forth in any of SEQ ID NOS: 1-16. In particular embodiments, the at least one strain of Bifidobacterium has or includes a nucleic acid sequence with at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a nucleic acid sequence set forth in any of SEQ ID NOS: 1-7.

[0057] In particular embodiments, the at least one strain of Bifidobacterium is or includes a strain of B. longum subsp. infantis. In particular embodiments, the strain of B. longum subsp. infantis has or includes a nucleic acid sequence with at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a nucleic acid sequence set forth in any of SEQ ID NOS: 1-7 or 20-39. In particular embodiments, the strain of B. longum subsp. infantis has or includes a nucleic acid sequence of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or 1,500 nucleotides in length with at least 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% sequence identity to a nucleic acid sequence set forth in SEQ ID NOS: 1-7 or 20-39. In some embodiments, the strain of B. longum subsp. infantis has or includes nucleic acid sequences having at least 90%, 95%, or 99% sequence identity to one or more of the nucleic acid sequences set forth in SEQ ID NOS: 20-39. In some embodiments, the strain of B. longum subsp. infantis has or includes the nucleic acid sequences set forth in one or more of SEQ ID NOS: 20-39. In particular embodiments, the strain of B. longum subsp. infantis has or includes nucleic acid sequences having at least 90%, 95%, or 99% sequence identity to all of the nucleic acid sequences set forth in SEQ ID NOS: 20-39. In various embodiments, the strain of B. longum subsp. infantis has or includes the nucleic acid sequences set forth in SEQ ID NOS: 20-39.

[0058] In some embodiments, at least one strain of Bifidobacterium capable of consuming, metabolizing, and/or internalizing human milk oligosaccharides. In some aspects, HMO cannot be metabolized by the host, e.g., mammals such as humans, or most bacteria, including most bacteria commonly found in the microbiome of adult humans. In particular aspects, some strains, species, or subspecies of Bifidobacterium, such as B. longum subsp. infantis, have enzymatic activity able to degrade specific alpha and beta bonds of human milk oligosaccharides. Five monosaccharides can be found in different HMO structures, glucose, galactose, N-acetyl glucosamine, fucose, and sialic acid (also referred to herein as N-acetyl neuraminic acid). Some strains, species, or subspecies of Bifidobacterium are able to fully degrade HMO intracellularly. Such Bifidobacterium possess genes encoding specific transporters (e.g., ABC transporters such as those described in Sela et al. PNAS (2008) 105 (48) 18964-18969; Schell, et al. PNAS. (2002) 99(22): 14422-14427 and LoCascio et al. Appl Environ Microbiol. (2010) 76(22):7373-81), incorporated by reference herein, that selectively transport or import HMO and enzymes necessary for HMO degradation (alpha-fucosidase, alpha-sialidase, beta-galactosidase, and beta-N- hexosaminidase). Other Bifidobacterium strains, such as for example !?, bifidum, degrades HMO externally or extracellularly, such as for example by lacto-N-biosidase, which cleaves LNB from HMO. The LNB is then internalized by a transporter and degraded by LNB- phosphorylase. In some embodiments, the probiotic strain is at least one strain of bacterium having one or more genes encoding all or a portion of a transporter, e.g., an ABC transporter, capable of internalizing an oligosaccharide such as an HMO. In particular embodiments, the probiotic strain is a bacterium having one or more genes encoding one or more enzymes, e.g., alpha-fucosidase, alpha-sialidase, beta-galactosidase, and beta-N-hexosaminidase, capable degrading an oligosaccharide such as an HMO. In certain embodiments, the probiotic strain is at least one strain of Bifidobacterium or Bacteroides having one or more genes encoding all or a portion of a transporter, e.g., an ABC transporter, capable of internalizing an oligosaccharide, e.g., an HMO.

[0059] In some embodiments, the at least one strain of Bifidobacterium is B. longum subsp. infantis. Particular embodiments contemplate that B. longum subsp. infantis is known and readily identifiable by those of skill in the art using routine techniques. In some embodiments, B. longum subsp. infantis, including its genome and biology, are known and for example have been described, including in Sela et al. PNAS (2008) 105 (48) 18964- 18969; Underwood et al., Pediatr Res. (2015) 77(0): 229-235, incorporated by reference herein. In certain embodiments, Bifidobacterium, e.g., B. longum subsp. infantis, may be isolated using known selective microbiological media, e.g., De Man, Rogosa and Sharpe agar (MRS), optionally in combination with mupirocin, or those described in O’Sullivan et al., J Appl Microbiol. 2011 Aug;l 11(2):467-73, incorporated by reference herein. In some embodiments, suitable sources for isolating Bifidobacterium, e.g., B. longum subsp. infantis, are known, and include stool samples obtained from breast fed infants. In certain embodiments, bacterial colonies may be identified or characterized by routine biochemical techniques, such as PCR. In some embodiments, B. longum subsp. infantis is identified by taqman qPCR, such as described in Lawley et al., PeerJ. 2017 May 25;5:e3375. e.g., as performed with forward primer sequence ATACAGCAGAACCTTGGCCT (SEQ ID NO: 17), reverse primer sequence GCGATCACATGGACGAGAAC (SEQ ID NO: 18) and probe sequence [FAM dye] -TTTCACGGA - [ZEN quencher] - TCACCGGACCATACG - [3IABkFQ quencher] (SEQ ID NO: 19). In some aspects, a strain may be confirmed as B. longum subsp. infantis by observing growth when human milk oligosaccharides are provided as the sole carbon source, such as with an assay described in Gotoh et al. Sci Rep. 2018 Sep 18;8(1): 13958, incorporated by reference herein. C. Prebiotic mixtures

[0034] In some embodiments, the prebiotic mixture is a mixture of non-digestible carbohydrates, e.g., oligosaccharides such as human milk oligosaccharides (HMOs), that promotes the growth or expansion of the probiotic strain, e.g., in vivo such as in the human gut and/or within the human gut microbiome. In certain embodiments, the prebiotic mixture, e.g., of non-digestible carbohydrates such as human milk oligosaccharides, promotes, e.g., selectively or exclusively, the colonization, expansion, extension, or increased presence of the probiotic strain within the microbiome. In particular embodiments, the mixture of non- digestible carbohydrates, e.g., human milk oligosaccharides, promotes the growth or expansion of a probiotic strain of Bifidobacterium such as B. longum subsp. infantis, e.g., in vivo such as in the human gut. In certain embodiments, the prebiotic mixture is a mixture of oligosaccharides, e.g., human milk oligosaccharides, that promote, e.g., selectively or exclusively, the colonization, expansion, extension, or increased presence of one or more strains of Bifidobacterium, e.g., B. longum subsp. infanlis, within the microbiome.

[0035] In some embodiments, the prebiotic mixture is or includes a mixture of non- digestible carbohydrates. In various embodiments, the prebiotic mixture is or includes a mixture of oligosaccharides. In particular embodiments, the prebiotic mixture is a mixture of one or more human milk oligosaccharides.

[0036] In some embodiments, the non-digestible carbohydrates are or include oligosaccharides. In particular embodiments, the non-digestible carbohydrates are or include milk oligosaccharides. In certain embodiments, the non-digestible carbohydrates are or include human milk oligosaccharides (human milk oligosaccharides). In some embodiments, the prebiotic mixture is a mixture of non-digestible carbohydrates that are or include human milk oligosaccharides. In particular embodiments, the prebiotic mixture is a mixture of human milk oligosaccharides, such as those that are obtained or derived from permeate, e.g., permeate derived or obtained from pooled human milk.

[0037] In some embodiments, the provided mixture may contain any oligosaccharide that may be internalized by one or more strains of Bifidobacterium such as a strain of B. longum subsp. infantis. In some embodiments, the oligosaccharides of the mixture may include one or more of a fructo-oligosacharide (FOS), galactooligosaccharide (GOS), transgalactooligosaccharide (TOS), gluco-oligosaccharide, xylo-oligosaccharide (XOS), chitosan oligosaccharide (COS), soy oligosaccharide (SOS), isomalto-oligosaccharide (IMOS), or derivatives thereof. In certain embodiments, such derivatives include those with modifications that may increase the likelihood or probability of consumption, metabolism, and/or internalization (such as by transport or import) of the oligosaccharide by the probiotic strain, e.g., B. longum subsp. infantis. Such modifications may include but are not limited to fucosylation or sialylation. In some embodiments, the oligosaccharides of the mixture may include one or more of a FOS, GOS, TOS, gluco-oligosaccharide, XOS, COS, SOS, IMOS, or derivatives or any or all of the foregoing, that are capable of being metabolized, consumed, and/or internalized by one or more strains, species, or subspecies of Bifidobacterium, e.g., B. longum subsp. infantis. In certain embodiments, the oligosaccharides of the mixture include one or more oligosaccharides that are obtained or derived from a resistant starch, pectin, psyllium, arabinogalactan, glucomannan, galactomannan, xylan, lactosucrose, lactulose, lactitol and various other types of gums such as tara gum, acacia, carob, oat, bamboo, citrus fibers, such as by treatment with enzymes that hydrolyze fiber or polysaccharides. In some embodiments, the one or more oligosaccharides of the mixture that are obtained by these means are capable of being consumed, metabolized, and/or internalized by at least one strain of Bifidobacterium such as B. longum subsp. infantis.

[0038] In certain embodiments, the prebiotic mixture is a mixture that is or includes at least one HMO. In some embodiments, the prebiotic mixture is a mixture of human milk oligosaccharides that is or includes a plurality of human milk oligosaccharides. In some embodiments, the prebiotic mixture is or includes a plurality of, of about, or at least 2, 3, 5, 10, 25, 50, 75, 100, 125, 150 different individual human milk oligosaccharides, e.g., human milk oligosaccharides with different individual chemical formulas or chemical structures. In certain embodiments, the prebiotic mixture is or includes a plurality of, of about, or at least 10, 25, 50, 75, 100, 125, 150 different individual human milk oligosaccharides. In some embodiments, the prebiotic mixture is or includes a plurality of, of about, or at least 25 different individual human milk oligosaccharides. In some embodiments, the prebiotic mixture is or includes a plurality of, of about, or of at least 80 different individual human milk oligosaccharides. Particular embodiments contemplate that one of skill may determine if an oligosaccharide is an HMO, such as if the oligosaccharide has a chemical formula and structure that is identical to an oligosaccharide that is found in human milk, as a matter of routine.

[39] In certain embodiments, the prebiotic mixture contains one or more synthetic human milk oligosaccharides, e.g., human milk oligosaccharides that are obtained, purified, or synthesized from a source other than human milk. In some aspects, synthetic human milk oligosaccharides, as well as methods for synthesizing oligosaccharides and human milk oligosaccharides, are known, and include but are not limited to those described in PCT Publication Nos.: W02017101958, WO2015197082, WO2015032413, WO2014167538, WO2014167537, WO2014135167, W02013190531, W02013190530, WO2013139344, WO2013182206, WO2013044928, W02019043029, W02019008133, WO2018077892, WO2017042382, WO2015150328, WO2015106943, WO2015049331, WO2015036138, and W02012097950, each of which is incorporated by reference herein in its entirety.

[40] In some embodiments, the prebiotic mixture includes some or all of 2'- fucosyllactose, 3’ -fucosy llactose, 3’-sialyllactose, 6'-sialyllactose, Lacto-N-tetraose, lacto-N- difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, and disialyllacto-N-tetraose. In particular embodiments, the mixture includes all of 2'-fucosyllactose, 3’-fucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N- tetraose, lacto-N-difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N- tetraose c, sialylacto-N-tetraose b, and disialyllacto-N-tetraose.

[41] In certain embodiments, the prebiotic mixture includes some or all of 2- fucosyllactose, lacto-N-tetraorose, 3-sialyllactose, 3-fucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, and 6’sialyllactose. In particular embodiments, the prebiotic mixture includes some or all of 2'-fucosyl-lactose, 3’-fucosyl-lactose, 3’-sialyl-lactose, 6'- sialyl-lactose, lacto-N-tetraose, lacto-N-neo-tetraose, lacto-N-fucopentaose I, lacto-N- fucopentaose II, lacto-N-fucopentaose III, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c, lacto-N-difuco-hexaose I, lacto-N-difuco-hexaose II, lacto-N-hexaose, para-lacto-N-hexaose, disialyllacto-N-tetraose, Fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose a, and difucosyl-lacto-N-hexaose b.

[0042] In certain embodiments, the prebiotic mixture contains at least 25, 50, 100, 125, or 150 human milk oligosaccharides which include all of 2-fucosyllactose, lacto-N- tetraorose, 3-sialyllactose, 3-fucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, and 6’sialyllactose. In particular embodiments, the prebiotic mixture contains at least 25, 50, 100, 125, or 150 human milk oligosaccharides which include all of 2'-fucosyl-lactose, 3’- fucosyl-lactose, 3’-sialyl-lactose, 6'-sialyl-lactose, lacto-N-tetraose, lacto-N-neo-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, sialyl-lacto-N- tetraose b, sialyl-lacto-N-tetraose c, lacto-N-difuco-hexaose I, lacto-N-difuco-hexaose II, lacto-N-hexaose, para-lacto-N-hexaose, disialyllacto-N-tetraose, Fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose a, and difucosyl-lacto-N-hexaose b. In some embodiments, the prebiotic mixture is a mixture of human milk oligosaccharides that is, includes, or is obtained or derived from human milk or a fraction thereof. In certain embodiments, the prebiotic mixture is or includes a mixture of human milk oligosaccharides that is or is obtained from an ultra-filtered permeate from human skim milk. In various embodiments, the prebiotic mixture is, includes, or is derived or produced from a concentrated human milk permeate. In some embodiments, the mixture of human milk oligosaccharides or the concentrated human milk permeate is or is obtained from a process described herein, e.g., in Section I-C-i. In certain embodiments, the prebiotic mixture is a concentrated human milk permeate, such as those described in U.S. Pat. No. 8,927,027 or in PCT Application No. WO 2018053535, incorporated herein by reference. In some embodiment, the prebiotic mixture is a prebiotic mixture produced or resulting from any of the methods described herein, e.g., in Section I-C- (i).

[0043] In some embodiments, the prebiotic mixture is or includes a concentrated human milk permeate that contains a plurality of human milk oligosaccharides. In particular aspects, the human milk permeate is obtained by filtering human skim milk that was obtained by separating cream from whole human milk. In some embodiments, the permeate was ultrafiltered from the human skim milk. In particular embodiments, the permeate is further concentrated, e.g., by reverse osmosis or nanofiltration, to increase the HMO content within the permeate. In some embodiments, the human milk permeate, e.g., the concentrated human milk permeate, has a concentration of at least 0.5%, 1%, 2.5%, 5%, or 10% w/v HMO. The human milk permeate may undergo additional processing steps, such as to digest or remove sugars, e.g., lactose, prior to its formulation or incorporate as a prebiotic mixture.

[0044] In certain embodiments, the concentrated human milk permeate includes a plurality of, of about, or of at least 1, 2, 3, 5, 10, 25, 50, 75, 100, 125, 150 different individual human milk oligosaccharides, e.g., human milk oligosaccharides with different individual chemical formulas or chemical structures. In certain embodiments, the prebiotic mixture is or includes a plurality of, of about, or of at least 10, 25, 50, 75, 100, 125, 150 different individual human milk oligosaccharides. In some embodiments, the prebiotic mixture is or includes a plurality of, of about, or of at least 25 different individual human milk oligosaccharides. In some embodiments, the prebiotic mixture is or includes a plurality of, of about, or of at least 80 different individual human milk oligosaccharides. Thus, in some embodiments, the prebiotic mixture is or includes a concentrated human milk permeate and contains at least 10, 25, 50, 75, 100, 125, 150 different individual human milk oligosaccharides. In particular embodiments, the concentrated human milk permeate contains at least 10, 25, 50, 100, 125, or 150 human milk oligosaccharides which include all of 2- fucosyllactose, lacto-N-tetraorose, 3-sialyllactose, 3-fucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, and 6’sialyllactose.

[0045] In certain embodiments, the concentrated human milk permeate and/or the prebiotic mixture has an increased amount, level, or concentration of one or more human milk oligosaccharides as compared to what is typically found human milk. In particular embodiments, the prebiotic mixture has an increased amount, level, or concentration of one or more human milk oligosaccharides as compared to what is typically found in untreated human milk permeate, e.g., permeate resulting from ultrafiltration of pooled human skim milk. In particular embodiments, the prebiotic mixture is or includes at least 25, 50, 75, 100, 125, 150, of the different human milk oligosaccharides found, present, or detected in pooled human milk or in permeate (e.g., permeate resulting from ultra-filtering skim) obtained from pooled human milk. In some embodiments, the prebiotic mixture is or includes at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% of the different human milk oligosaccharides found, present, or detected in pooled human milk or in permeate (e.g., permeate resulting from ultra-filtering skim) obtained from pooled human milk. In certain embodiments, the prebiotic mixture is or includes at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% of the individual human milk oligosaccharides that may be found, present, or detected across samples of human milk, e.g., samples of milk obtained from different individuals. In some embodiments, the prebiotic mixture of human milk oligosaccharides is or includes the same or substantially the same human milk oligosaccharides found, present, or detected in pooled human milk or in permeate (e.g., permeate resulting from ultra-filtering skim) obtained from pooled human milk. In certain embodiments the prebiotic mixture is or includes a human milk permeate resulting from the ultrafiltration of human whole or skim milk pooled from at milk collected from at least 10, 25, 50, or 100 individual human milk donors that is further concentrated, e.g., by nanofiltration or reverse osmosis, to increase the concentration of total HMO (e.g., by w/w).

[0046] In certain embodiments, the prebiotic mixture is free or essentially free of oligosaccharides that are not human milk oligosaccharides. In certain embodiments, the prebiotic mixture contains human milk permeate, e.g., concentrated human milk permeate, and one or more synthetic human milk oligosaccharides, e.g., one or more of synthetically derived 2'-fucosyl-lactose, 3’-fucosyl-lactose, 3’-sialyl-lactose, 6'-sialyl-lactose, lacto-N- tetraose, lacto-N-neo-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c, lacto-N-difuco-hexaose I, lacto-N-difuco-hexaose II, lacto-N-hexaose, para-lacto-N-hexaose, disialyllacto-N-tetraose, Fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose a, and difucosyl-lacto-N-hexaose b.

[0047] Prebiotic mixtures containing human milk oligosaccharides for use in the compositions and methods disclosed herein may be obtained according to methods known in the art, including, but not limited to, chemical synthesis and purification from human milk. For example, processes to obtain HMO compositions from human milk are described below and are detailed in PCT Pub. Nos. WO/2010/065652 and WO/2018/053535, the contents of which are hereby incorporated in their entirety.

[0048] In some embodiments, the prebiotic mixture is a mixture of human milk oligosaccharides that are or are derived from a concentrated ultra-filtered human milk permeate, e.g., any ultra-filtered human milk permeate described herein such as in Section I- C-(i).

[0049] In some embodiments, the prebiotic mixtures are mixtures of human milk oligosaccharides having an HMO profile that is substantially similar both structurally and functionally to the profile of human milk oligosaccharides observed across the population of whole human milk. That is to say, in some aspects, since the prebiotic mixtures may be obtained from a source of human milk derived from a pool of donors rather than an individual donor, the array of human milk oligosaccharides will be more diverse than in any one typical individual, and will represent or more closely represent the spectrum of human milk oligosaccharides that are found among human milk across a population as opposed to the spectrum of human milk oligosaccharides that are found or typically found in the human milk produced by any particular individual. Thus, in some embodiments, the prebiotic mixture and the concentrated human milk permeate have more individual HMO species than what can be found in human milk obtained from an individual donor.

[0050] In some instances, the ratio of the amount or concentration of individual human milk oligosaccharide species to total human milk oligosaccharides of the prebiotic mixture or the concentrated human milk permeate may be different from what would be observed in whole human milk or pooled human milk.

[0051] In some embodiments, the prebiotic mixture is or includes a greater amount of different individual human milk oligosaccharides than the number of different individual human milk oligosaccharides found in human milk from an individual donor. In certain embodiments, the prebiotic mixture includes at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 more individual human milk oligosaccharides than the number of different individual human milk oligosaccharides found in human milk from an individual donor. In particular embodiments, a prebiotic mixture is or includes a greater amount of different individual human milk oligosaccharides than the mean or median number of different individual human milk oligosaccharides found in a plurality of human milk samples from individual donors. In certain embodiments, the prebiotic mixture includes at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 more individual human milk oligosaccharides than the number of different individual human milk oligosaccharides found in human milk from an individual donor.

[0052] In some aspects, one of the biggest variables in HMO diversity derives from the mother’s Lewis blood group and specifically whether or not she has an active fucosyltrasferase 2 (FUT2) and/or fucosyltrasferase 3 (FUT3) gene. When there is an active FUT2 gene, an al -2 linked fucose is produced, whereas fucose residues are al -4 linked when the FUT3 gene is active. The result of this “secretor status” is, generally, that “secretors” (i.e. those with an active FUT2 gene) produce a much more diverse profile of human milk oligosaccharides dominated by al-2 linked oligosaccharides, whereas “non-secretors” (i.e. those without an active FUT2 gene) may comprise a more varied array of, for example al, -4 linked oligosaccharides (as compared to secretors), but comprise an overall decrease in diversity since they are unable to synthesize a major component of the secretor’ s HMO repertoire. In some embodiments, the prebiotic mixture includes human milk oligosaccharides that include al-2 linked fucose and human milk oligosaccharides that include al -4 linked fucose.

[0053] In some embodiments, the prebiotic mixture is or includes at least 5% total HMO (w/w). In particular embodiments, the prebiotic mixture is or includes least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25%, or 50% total HMO (w/w). In certain embodiments, the prebiotic mixture is or includes between 5% and 15%, 7.5% and 12.5%, 8% and 12%, 8.5% and 11%, or 8.4% and 10.6% total HMO (w/w). In certain embodiments, the prebiotic mixture is or includes between 8.5% and 11% total HMO (w/w). In some embodiments, the prebiotic mixture is or includes between 8.4% and 10.6% total HMO (w/w).

[0054] In some embodiments, the prebiotic mixture has a pH of between 4.0 and 5.5. In certain embodiments, the prebiotic mixture has less than 10%, 5%, 1%, or 0.1% lactose (w/w). In some embodiments, the prebiotic mixture has less than 10%, 5%, 1%, or 0.1% glucose (w/w). In particular embodiments, the prebiotic mixture has less than 10%, 5%, 1%, or 0.1% galactose (w/w). In certain embodiments, the prebiotic mixture has less than 10% galactose, less than 10% glucose, and less than 0.1% lactose.

[0055] In some embodiments, the prebiotic mixture is a liquid formulation. In some embodiments, the prebiotic mixture is in powdered form, e.g., a lyophilized or spray dried composition. In certain embodiments, the prebiotic mixture is incorporated into a dosage form that is a separate composition from the Bifidobacterium strain. In some embodiments, the prebiotic mixture is incorporated into a dosage form that is a separate composition from the propionate producing strain. i) Exemplary methods for manufacturing prebiotic mixtures

[0056] In some embodiments, the prebiotic mixture is or includes human milk oligosaccharides (HMOs) obtained or purified from ultra-filtered permeate from donated human milk. In certain embodiments, the donated human milk is pooled to provide a pool of human milk. In some embodiments, a pool of human milk comprises milk from two or more (e.g., ten or more) donors. In certain embodiments, the pooled human milk contains milk from at least 50, 75, 100, 150, or 200 individual donors. In certain embodiments, the pooled human milk contains human milk from at least 100 individual donors or between 100 and 300 individual donors. In some embodiments, the pooled human milk contains milk from at least ten, at least twenty -five, at least fifty, at least seventy-five, at least one hundred, or at least one hundred fifty individual human milk donors.

[0057] In some embodiments, the human milk oligosaccharides that are contained or included in the prebiotic mixture are synthetic human milk oligosaccharides, such as those derived from non-human milk sources, e.g., derived or obtained as oligosaccharides or precursors from transgenic microorganisms and/or chemically synthesized. In some aspects, synthetic oligosaccharides and human milk oligosaccharides, as well as methods and techniques for synthesizing oligosaccharides and human milk oligosaccharides, are known, and include but are not limited to those described in PCT Publication Nos.: W02017101958, WO2015197082, WO2015032413, WO2014167538, WO2014167537, WO2014135167, WO2013190531, WO2013190530, WO2013139344, WO2013182206, WO2013044928, W02019043029, W02019008133, WO2018077892, WO2017042382, WO2015150328, WO2015106943, WO2015049331, WO2015036138, and W02012097950, each of which is incorporated by reference herein in its entirety.

[0058] In particular embodiments, one or more synthetically derived human milk oligosaccharides are added to a concentrated human milk permeate to arrive at a prebiotic mixture.

[0059] In certain embodiments, the mixture of oligosaccharides described herein are produced from human milk permeate, e.g., concentrated ultra-filtered permeate from pooled human milk. In some embodiments, the mixture of oligosaccharides described herein contain or are formulated with human milk permeate, e.g., concentrated ultra-filtered permeate from pooled human milk. In some embodiments, the concentrated ultra-filtered permeate may be made according to any suitable method or technique known in the art. In some aspects, suitable methods and techniques include those described in U.S. Pat. No. 8,927,027m PCT Pub. No. WO2018053535, and PCT Pub. No. WO 2021061991, hereby incorporated by reference in their entirety. Exemplary methods and techniques for producing the human milk compositions are briefly summarized herein.

[0060] In some embodiments, the prebiotic mixture is or includes human milk permeate that has been generated or produced by a method described herein. In some embodiments, whole human milk is pooled from multiple donors, and then cream and skim are separated by any suitable technique known in the art, e.g., centrifugation; and then the skim milk is filtered, e.g., ultra-filtered, to obtain human milk permeate and retentate. The human milk permeate may be further processed, such as to remove or digest one or more components, e.g., lactose, or to increase the concentration of human milk oligosaccharides, such as by nanofiltration or reverse osmosis.

[0061] In some embodiments, the donor milk is received frozen, and when desired, is thawed and pooled. In some embodiments, donor milk is then screened, e.g., to identify contaminants, by one or more of the methods discussed herein.

[0062] In some embodiments, the pooled milk is filtered, e.g., through about a 200- micron filter. In some embodiments, the pooled milk is heated, e.g., at about 63°C or greater for about 30 minutes or more. In some embodiments, the milk is transferred to a separator, e.g., a centrifuge, to separate the cream from the skim. In some embodiments, the cream may go through separation once again to yield more skim. In some embodiments, a desired amount of cream is added to the skim prior to ultra-filtration. In certain embodiments, material that that did not pass through the filter is collected as the retentate fraction, and material that passes through the filter is collected as the permeate fraction.

[0063] In some embodiments, the skim fraction undergoes ultra-filtration. In some embodiments, the ultrafiltration is performed with a filter between 1 kDa and 1000 kDa to obtain a protein rich retentate and the HMO-containing permeate. Details of this process can be found, for example, in US 8,545,920; US 7,914,822; 7,943,315; 8,278,046; 8,628,921; and 9,149,052, each of which is hereby incorporated by reference in its entirety. In some embodiments, the ultra-filtration is performed with a filter that is between 1 kDa and 100 kDa, 5 kDa and 50 kDa, or 10 kDa and 25 kDa. In certain embodiments, the filter is about or at least 5 kDa, 10 kDa, 20 kDa, 25 kDa, 50 kDa, or 100 kDa. In some embodiments, the skim fraction undergoes ultrafiltration with a filter that is about 10 kDa. In certain embodiments, the skim fraction undergoes ultrafiltration with a filter that is about 25 kDa. In particular embodiments, the skim fraction undergoes ultrafiltration with a filter that is about 50 kDa.

[0064] In some embodiments, the ultra-filtered permeate undergoes a process for reducing lactose. In certain embodiments, a process for producing a concentrated human milk permeate composition with substantially reduced levels of lactose is provided. In certain embodiments, the substantial reduction includes or requires the biochemical and/or enzymatic removal of lactose from the lactose-rich human milk permeate fraction, without loss of yield or change in molecular profile of the HMO content of human milk permeate. And, in particular embodiments, without leaving residual inactivated foreign protein, if enzymatic digestion is used to reduce lactose. In certain embodiments, about or at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 99.99% of the lactose present in the permeate after ultrafiltration is removed, e.g., enzymatically digested. In certain embodiments, the permeate is free or essentially free of lactose following the enzymatic digestion.

[0065] In certain embodiments, the process for reducing lactose from human milk permeate includes one or more of the steps of a) adjusting the pH of the permeate mixture; b) heating the pH adjusted mixture; c) adding lactase enzyme to the heated permeate mixture to create a permeate/lactase mixture and incubating a period of time; d) removing the lactase from the mixture and filtering the mixture to remove lactase; and e) concentrating human milk oligosaccharides. In some embodiments, the order of when steps (a)-(c) are performed may be varied. Thus, in some aspects, the steps may be performed in the order of (a)-(b)-(c); (a)-(c)-(b); (c)-(b)-(a); (c)-(a)-(b); (b)-(a)-(c); or (b)-(c)-(a), such that, for example, the lactase enzyme may be added prior to heating the mixture, or, alternatively at any point during the heating process. Similarly, and also by way of example only, the mixture may be heated prior to adjustment of the pH. Furthermore, several steps may be grouped into a single step, for example “enzymatically digesting lactose” or “lactases digestion of lactose” involves steps (a)-(c) as described. These steps may be performed concurrently or consecutive in any order. Therefore, as used herein “lactose digestion” refers to the performance of at least these three steps, in any order, consecutively or concurrently.

[0066] In certain embodiments, the pH of the permeate is adjusted to a pH of about 3 to about 7.5. In some embodiments, the pH is adjusted to a pH of about 3.5 to about 7.0. In particular embodiments, the pH is adjusted to a pH of about 3.0 to about 6.0. In certain embodiments, the pH is adjusted to a pH of about 4 to about 6.5. In some embodiments, pH is adjusted to a pH of about 4.5 to about 6.0. In particular embodiments, the pH is adjusted to a pH of about 5.0 to about 5.5. In certain embodiments, the pH is adjusted to a pH of about 4.3 to about 4.7, preferably 4.5. The pH may be adjusted by adding acid or base. In some embodiments, pH is adjusted by adding acid, for example HC1. In particular embodiments, pH is adjusted by adding IN HC1 and mixing for a period of time, e.g., about 15 minutes.

[0067] In some embodiments, the pH-adjusted permeate is heated to a temperature of about of about 25°C to about 60°C. In certain embodiments, the permeate is heated to a temperature of about 30°C to about 55°C. In some embodiments, the permeate is heated to a temperature of about 40°C to about 50°C. In certain embodiments, the permeate is heated to a temperature of about 48°C to about 50°C. In some embodiments, the permeate is heated to a temperature about 50°C. In some embodiments, the permeate is heated to a temperature less than or equal to about 40°C.

[0068] In particular embodiments, lactase enzyme is added to the pH-adjusted, heated permeate to create a permeate/lactase mixture. In certain embodiments, lactose within the permeate/lactase mixture is broken down into monosaccharides. In certain embodiments, lactase enzyme is added at about 0.1% w/w to about 0.5% w/w concentration. In certain embodiments, lactase enzyme is added at about 0.1% w/w, or 0.2% or 0.3% or 0.4% or 0.5% w/w. There are many commercially available lactase enzymes that may be used. As such, the lactase enzyme may be derived from any origin (e.g., fungal or bacterial in origin).

[0069] In some embodiments, the pH-adjusted, heated permeate is incubated with the lactase enzyme for about 5 to about 225 minutes. In certain embodiments, the incubation time is about 15 min to about 90 min. In some embodiments, the incubation time is about 30 minutes to about 90 minutes. In particular embodiments, the incubation time is about 60 minutes. Some aspects contemplate that that incubation time is dependent upon myriad of factors including, but not limited to, the source of the enzyme used, the temperature and pH of the mixture and the concentration of enzyme used. Thus, in some embodiments, incubation time with the lactase enzyme may be adjusted to factor in such variables as a matter of routine. While the pH, temperature, and enzyme incubation conditions provided here are what work optimally for the process described herein, one of skill in the art would understand that modifications may be made to one or more of these variables to achieve similar results. For example, if less enzyme is used than the about 0.1% w/w to about 0.5% w/w described herein, the incubation time may need to be extended to achieve the same level of lactose digestion. Similar adjustments may be made to both the temperature and pH variables as well.

[0070] In certain embodiments, after incubation the permeate/lactase mixture is cooled to a temperature of about 20°C to about 30°C. In a particular embodiment, the permeate/lactase mixture is cooled to a temperature of about 25°C.

[0071] In some embodiments, the permeate/lactase mixture is clarified to remove insoluble constituents. In certain embodiments, insoluble material may form throughout the change in pH and temperature. Thus, in some embodiments, it may be necessary or beneficial to clarify the mixture to remove these insoluble constituents, for example, through a depth filter. The filters may be 0.1 to 10 micron filters. In some embodiments, the filters are about 1 to about 5 micron filters. Alternatively, removal of insoluble constituents can be achieved through a centrifugation process or a combination of centrifugation and membrane filtration. The clarification step is not essential for the preparation of a diverse HMO composition, as described herein, rather, this optional step aids in obtaining a more purified permeate composition. Furthermore, the clarification step is important in the reusability of the filtration membranes and thus to the scalability of the process. Some aspects contemplate that, without adequate clarification, substantially more filter material is required, increasing the difficulty and expense to produce permeate compositions at clinical scale. However, one will understand that more or less stringent clarification may be performed at this stage in order to produce more or less purified permeate compositions, depending on formulation and application. For example, precipitated minerals may be less of a problem for a formulation destined for lyophilization. [0072] In certain embodiments the spent and excess lactase enzyme is removed from the clarified permeate/lactase mixture. There may, however, be some instances where the inactivated foreign protein will carry no biological risk and therefore the added steps of lactase removal or even inactivation may not be necessary. In some embodiments, the spent and excess lactase is inactivated, for example by high temperature, pressure, or both. In some embodiments, the inactivated lactase is not removed from the composition.

[0073] In other embodiments, however, a further purification to remove foreign proteins will be called for. In such embodiments lactase enzyme removal may be accomplished by ultrafiltration. In some embodiments, ultrafiltration is accomplished using an ultrafiltration membrane, for example using a membrane with molecular weight cut-off of < 50,000 Dalton, e.g., a BIOMAX-50K. In some embodiments, the molecular weight cut-off less than or equal to about 10 kDa. In certain embodiments, the molecular weight cut-off less than or equal to about 25 kDa. In particular embodiments, the molecular weight cut-off less than or equal to about 50 kDa.

[0074] In certain embodiments, an additional ultrafiltration is performed through a smaller membrane than the initial membrane with molecular weight cut-off of < 50,000 Dalton. In some embodiments, the additional ultrafiltration is performed with a membrane with a molecular weight cut-off of between 10 kDa and 50 kDa, 1 kDa and 10 kDa, 1 kDa and 5 kDa, or 2 kDa and 3 kDa. In certain embodiments, the additional ultrafiltration is performed with a membrane with a molecular weight cut-off of between 2 kDa and 3 kDa. In certain embodiments, an additional ultrafiltration is not performed. In some embodiments, the additional filtration step is performed, such as to aid in the overall purity of the permeate product, such as by assisting in the removal of smaller potentially bioactive and/or immunogenic factors such as microRNAs and exosomes.

[0075] In some embodiments, the clarified mixture that has undergone at least one, and in some cases two or more rounds of ultrafiltration (or alternative lactase removal means) is further filtered to purify and concentrate human milk oligosaccharides and to reduce the mineral and monosaccharides content.

[0076] In some embodiments, filtration can be accomplished using a nanofiltration membrane. In some embodiments, the membrane has a molecular weight cut-off of < 1,000 Dalton. In certain embodiments, the membrane has a molecular weight cut-off of between 1 kDa and 1,000 kDa. In certain embodiments, the membrane has a molecular weight cut-off of less than 600 Da. In certain embodiments, the membrane has a molecular weight cut-off between 400 Da and 500 Da. In some aspects, the additional nanofiltration removes monosaccharides, minerals, particularly calcium, and smaller molecules to produce the final purified HMO composition.

[0077] In some embodiments, additional or alternative steps may be taken for the removal of minerals. Such an additional step may include, for example, centrifugation, membrane clarification (< 0.6 micron), or combination of centrifugation and membrane filtration of heated (> 40°C) or refrigerated/frozen and thawing of HMO Concentrate. The collected supernatant or filtrate of these additional or alternative steps, in some embodiments, is concentrated further using a nanofiltration membrane. In some embodiments, the nanofiltration comprises filtration through a membrane with a molecular cut-off of < 600 Dalton. In some embodiments, these additional steps may be performed at any stage of the process, including but not limited to prior to or after pasteurization.

[0078] In some embodiments, the physical property of nanofiltration membranes can be modified, such as chemical modification, to selectively concentrate sialylated human milk oligosaccharides, for example, allowing greater efficiency of neutral human milk oligosaccharides removal from HMO concentrate, in instances where concentrated sialylated human milk oligosaccharides are preferred.

[0079] In certain embodiments, the permeate may be further processed, e.g., concentrated or diluted. In some embodiments, the permeate may be concentrated by a suitable process such as nanofiltration, reverse osmosis, or dried, e.g., lyophilized.

[0080] In some embodiments, the permeate is treated to reduce bioburden, such as by any means known in the art. In some embodiments, the purified HMO composition is pasteurized. In some aspects, pasteurization is accomplished at > 63°C for a minimum of 30 minutes. Following pasteurization, the composition is cooled to about 25°C to about 30°C and clarified through a 0.2 micron filter to remove any residual precipitated material.

[0081] In certain embodiments, the mixture of oligosaccharides is formulated with an ultra-filtered permeate obtained from human milk. In some embodiments, the mixture of oligosaccharides is formulated with permeate that has been ultra filtered from the skim fraction of pooled human milk. In certain embodiments, lactose is removed, e.g., enzymatically degraded, prior to formulation into the mixture of oligosaccharides. D. Butyrate producing bacteria

[0082] In certain embodiments, a butyrate producing strain of bacteria is administered along with or in addition to the provided prebiotic mixture, e.g., of human milk oligosaccharides, at least one Bifidobacterium and at least one propionate producing bacterium. In some embodiments, the at least one butyrate producing strain is capable of consuming lactate and/or acetate. In particular embodiments, the at least one butyrate producing strain is capable of producing, generating, and/or creating butyrate in the presence of lactate and/or acetate. In certain embodiments, the at least one butyrate producing strain is capable of producing, generating, and/or creating butyrate in the presence of the at least one Bifidobacterium, e.g., any Bifidobacterium described herein such as in Section I-B, and the prebiotic mixture, e.g., a prebiotic mixture described herein such as in Section I-C. Suitable butyrate producing strains are described in PCT App. Pub. No. WO2022036225, hereby incorporated by reference in its entirety.

[0083] In some embodiments, the at least one butyrate producing strain is or includes at least one, two, three, four, five, six, seven, eight, nine, ten, or more species, subspecies, or strains of bacteria capable of producing butyrate. In particular embodiments, the butyrate producing strain is or includes at least three strains of bacteria capable of producing butyrate. In some embodiments, the butyrate producing strain is or includes between one and five species, subspecies, or strains of bacteria capable of producing butyrate.

[0084] In certain embodiments, the at least one butyrate producing strain has one or more genes that contribute to the production, generation, or making of butyrate. In particular embodiments, the at least one butyrate producing strain has a functional butyryl-Co A: acetate CoA-transferase (but) gene. In certain embodiments, the at least one butyrate producing strain has a functional butyrate kinase (buk) gene. In some embodiments, the at least one butyrate producing strain has a functional butyryl-CoA:4-hydroxybutyrate CoA transferase (4Hbf) gene. In particular embodiments, the at least one butyrate producing strain has a functional butyryl-Co A: acetoacetate CoA transferase (Ato) gene.

[0085] In some embodiments, the at least one butyrate producing strain is or includes one or more species, subspecies, or strains of Clostridium Cluster IV bacteria. In certain embodiments, the at least one butyrate producing strain is or includes one or more species, subspecies, or strains of Clostridium Cluster XlVa bacteria. In certain embodiments, the at least one butyrate producing strain is or includes one or more species, subspecies, or strains of bacteria belonging to the Clostridium, Eubacterium, Ruminococcus, Coprococcus, Dorea, Lachnospira, Roseburia, Butyrivibrio, or Anaerofilum genera. In some embodiments, the at least one butyrate producing strain is or includes one or more species, subspecies, or strains of bacteria belonging to the Clostridium, Eubacterium, Ruminococcus, Coprococcus, Dorea, Lachnospira, Roseburia or Butyrivibrio genera. In particular embodiments, the at least one butyrate producing strain is or includes one or more species, subspecies, or strains of bacteria belonging to the Clostridium, Eubacterium, Ruminococcus ov Anaerofilum genera.

[0086] In some embodiments, the at least one butyrate producing strain is or includes one or more species, subspecies, or strains of Agathobacter rectalis (also referred to as Eubacterium rectale), Anaerobutyricum hallii (also referred to as Eubacterium hallii), Anaerobutyricum soehngenii, Anaerocolumna aminovalerica (also referred to as Clostridium aminovalericum), Anaerostipes butyraticus, Anaerostipes caccae, Anaerostipes hadrus (also referred to as Eubacterium hadrum), Anaerostipes rhamnosivorans, Anaerotruncus colihominis, Blautia argi, Blautia caecimuris, Blautia coccoides (also referred to as Clostridium coccoides), Blautia faecicola, Blautia faecis, Blautia glucerasea, Blautia hansenii (also referred to as Streptococcus hansenii), Blautia schinkii, Blautia ster coris, Blautia wexlerae, Blautia hominis, Blautia hydrogenotrophica (also referred to as Ruminococcus hydrogenotrophicus), Blautia luti (also referred to as Ruminococcus luti), Blautia obeum (also referred to as Ruminococcus obeum), Blautia producta (also referred to as Ruminococcus productus; Streptococcus productus; Peptostreptococcus productus), Blautia schinkii (also referred to as Ruminococcus schinkii), Butyrivibrio crossotus, Clostridium cellulosi, Clostridium hylemonae, Clostridium innocuum, Clostridium leptum, Clostridium nexile, Clostridium orbiscidens, Clostridium scindens, Clostridium sporosphaeroides, Clostridium symbiosum, Clostridium viride, Coprococcus catus, Coprococcus comes, Coprococcus eutactus, Coprococcus phoceensis, Eisenbergiella tayi, Emergencia timonensis, Enterocloster aldenensis (also referred to as Clostridium aldenense), Enterocloster asparagiformis (also referred to as Clostridium asparagiforme), Enterocloster bolteae (also referred to as Clostridium bolteae), Enterocloster citroniae (Clostridium citroniae), Enterocloster clostridioformis (also referred to as Clostridium clostridioforme), Enterocloster lavalensis (Clostridium lavalense), Erysipelatoclostridium ramosum (also referred to as Thomasclavelia ramosa), Eubacterium siraeum, Eubacterium ventriosum, Dorea formicigenerans (also referred to as Eubacterium formicigenerans), Dorea longicatena, Faecalibacterium prausnitzii (also referred to as Fusobacterium prausnitzii), Faecalicatena contorta (also referred to as Eubacterium contortum), Faecalicatena fissicatena (also referred to as Eubacterium fissicatena), Faecalicatena orotica (also referred to as Clostridium oroticum), Faecalimonas umbilicate, Flavonifractor plautii (also referred to as Clostridium orbiscindens), Hungatella effluvii, Hungatella hathewayi (also referred to as Clostridium hathewayi), Hungatella xylanolytica, Lachnospira eligens (also referred to as Eubacterium eligens), Lachnospira multipara, Lachnospira pectinoschiza, Lacrimispora aerotolerans (also referred to as Clostridium aerotolerans), Lacrimispora algidixylanolytica (also referred to as Clostridium algidixylanolyticum), Lacrimispora amygdalina (also referred to as Clostridium amygdalinum), Lacrimispora celerecrescens (also referred to as Clostridium celerecrescens), Lacrimispora indolis (also referred to as Clostridium indolis), Lacrimispora saccharolytica (also referred to as Clostridium saccharolyticum), Lacrimispora sphenoides (also referred to as Bacillus sphenoides), Lacrimispora xylanolytica (also referred to as Clostridium xylanolyticum), Lactonifactor longoviformis, Marvinbryantia formatexigens (also referred to as Bryantella formatexigens), Mediterraneibacter butyricigenes, Mediterraneibacter faecis (also referred to as Ruminococcus faecis), Mediterraneibacter glycyrrhizinilyticus (also referred to as Clostridium glycyrrhizinilyticum), Mediterraneibacter gnavus (also referred to as Ruminococcus gnavus), Mediterraneibacter lactaris (also referred to as Ruminococcus lactaris), Mediterraneibacter massiliensis, Mediterraneibacter torques (also referred to as Ruminococcus torques), Merdimonas faecis, Murimonas intestini, Oscillibacter massiliensis, Papillibacter cinnamivorans, Pseudoflavonifractor capillosus, Robinsoniella peoriensis, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus albus, Ruminococcus bromii, Subdoligranulum variabile, or Syntrophococcus sucromutans.

[0087] In certain embodiments, the at least one butyrate producing strain is or includes one or more species, subspecies, or strains of Agathobacter rectalis, Anaerobutyricum hallii, Anaerostipes caccae, Blautia producta, Clostridium leptum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Roseburia hominis, or Roseburia intestinalis. In some embodiments, the at least one butyrate producing strain is or includes one or more species, subspecies, or strains of Anaerostipes caccae, Clostridium innocuum, Roseburia hominis, or Roseburia intestinalis.

E. Exemplary compositions, kits, and articles of manufacture [0060] In some embodiments, provided herein are compositions, kits, or articles of manufacture that are or include a combination of prebiotics, e.g., prebiotic mixtures of non- digestible carbohydrates such as human milk oligosaccharides, and probiotics that include one or more strains of Bifidobacteria such as B. longum subsp. infantis and one or more strains of propionate producing bacteria, e.g., Veillonella sp. In certain aspects, the prebiotic mixture and probiotics may be formulated as a pharmaceutical composition or a nutritional composition. In some embodiments, the provided composition includes or incorporates the prebiotic mixture and the probiotic strains. In particular embodiments, the probiotic strains may be formulated as a pharmaceutical composition or a nutritional composition. In certain embodiments, the prebiotic mixture and the probiotic are formulated or manufactured as separate compositions. In particular embodiments, provided herein are kits or articles of manufacture that are or include separate prebiotic and probiotic compositions.

[0088] In particular embodiments, provided herein are kits or articles of manufacture that are or include prebiotic mixtures that are or include one or more human milk oligosaccharides, and probiotics that include one or more strains of Bifidobacteria and one or more strains of propionate producing bacteria. In some embodiments, the prebiotic mixture is or includes any of the prebiotic mixtures described herein, e.g., in Section I-C. In particular embodiments, the prebiotic mixture contains at least 2, at least 5, at least 10, at least 25, at least 50, at least 100, or at least 150 human milk oligosaccharides, e.g., at least two, three, four, five, or more than five synthetic human milk oligosaccharides or at least 10, at least 25, at least 50, or at least 100 human milk oligosaccharides from a human milk source such as human milk permeate. In some embodiments, the one or more strains of Bifidobacteria are or include one or more strains of Bifidobacteria described herein, e.g., in Section I-B. In certain embodiments, the one or more strains of Bifidobacteria are capable of consuming, internalizing, and/or hydrolyzing human milk oligosaccharides. In some embodiments, the one or more strains of propionate producing bacteria are or include any of the propionate producing bacteria described herein, e.g., in Section I-A. In some embodiments, the one or more strains of propionate producing bacteria are or include at least one strain from the genus Veillonella or Megasphaera.

[0089] In particular embodiments, the kits or articles of manufacture are or include i) prebiotic mixtures that are or include at least one or more of 2'-fucosyllactose, 3’- fucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, and/or disialyllacto-N-tetraose; ii) at least one Bifidobacterium that is or includes B. longum subsp. infantis, and iii) at least one propionate producing bacterium that is or includes one or more strains of the genus of the genus Veillonella or Megasphaera. In certain embodiments, the kits or articles of manufacture are or include i) prebiotic mixtures that are or include at least one, some, or all of 2'-fucosyllactose, 3’-fucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, and/or disialyllacto-N-tetraose; ii) at least one Bifidobacterium that is or includes B. longum subsp. infanlis. iii) at least one propionate producing bacterium that is or includes one or more of Megaspaera elsdenii, Veillonella atypica (V. atypica), Veillonella dispar (V. dispar), Veillonella infantium (V. infantium), Veillonella nakazawae (V. nakazawae), or Veillonella parvula (V. parvula), and/or Veillonella rogosae (V. rogosae). In some embodiments, the kits or articles of manufacture include one or more strains of butyrate producing bacteria, e.g., any of the butyrate producing bacteria described herein such as in Section I-D. In certain embodiments, the butyrate producing bacteria is or includes one or more of Agathobacter rectalis, Anaerobutyricum hallii, Anaerostipes caccae, Blautia producta, Clostridium leptum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Roseburia hominis, or Roseburia intestinalis.

[0090] In some embodiments, provided herein are articles of manufacture that are or include prebiotic mixtures that are or include one or more human milk oligosaccharides, probiotics that include one or more strains of Bifidobacteria and one or more strains of propionate producing bacteria, and instructions for use, e.g., describing any method herein such as the methods described in Section II. In certain embodiments, the articles of manufacture are or include i) prebiotic mixtures that are or include at least one or more of 2'- fucosyllactose, 3’ -fucosy llactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N- difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, and/or disialyllacto-N-tetraose; ii) at least one Bifidobacterium that is or includes B. longum subsp. infantis, iii) at least one propionate producing bacterium that is or includes one or more strains of the genus of the genus Veillonella or Megasphaera, and iv) instructions for use describing one or more methods included herein in Section II. In particular embodiments, the articles of manufacture are or include i) prebiotic mixtures that are or include at least one, some, or all of 2'-fucosyllactose, 3’-fucosyllactose, 3’- sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, and/or disialyllacto-N-tetraose; ii) at least one Bifidobacterium that is or includes B. longum subsp. infantis, iii) at least one propionate producing bacterium that is or includes one or more oi Megaspaera elsdenii, Veillonella atypica (V. atypica), Veillonella dispar (V. dispar), Veillonella infantium (V. infantium), Veillonella nakazawae (V. nakazawae), or Veillonella parvula (V. parvula), and/or Veillonella rogosae (V. rogosae), and iv) instructions for use describing one or more methods included herein in Section II. In some embodiments, the articles of manufacture include one or more strains of butyrate producing bacteria described herein in Section I-D, such as one or more of Agathobacter rectalis, Anaerobutyricum hallii, Anaerostipes caccae, Blautia producta, Clostridium leptum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Roseburia hominis, or Roseburia intestinalis.

II. USES OF THE PROVIDED COMPOSITONS AND FORMULATIONS

[0091] In particular embodiments, provided herein are methods for treating, preventing, or ameliorating one or more diseases, disorders, or conditions in a subject in need thereof. In certain embodiments, provided herein are methods for generating propionate, and/or increasing the level or amount of propionate, within the gut and/or intestine of a subject in need thereof. In some embodiments, the method is or includes steps for administering to the subject a combination of probiotic bacteria that is or includes at least one strain of Bifidobacterium, e.g., such as any described herein, e.g., in Section I-B, and a strain of bacterium capable of producing propionate, such as any of the propionate producing bacterium described herein, e.g., in Section I-A, and a prebiotic mixture, such as any described herein, e.g., in Section I-C. In some embodiments, the prebiotic mixture, the Bifidobacterium, and the propionate producing bacterium are administered together, such as at the same time, on the same treatment days, and/or within the same dosage formula. In certain embodiments, the prebiotic mixture, the Bifidobacterium, and the propionate producing bacterium are administered separately, such as at different times, on different treatment days, and/or within different dosage formulations. In some embodiments, the treatment may include administering the prebiotic mixture, the Bifidobacterium, and the propionate producing bacterium together during certain days or phases of the treatment and then separately during other certain days or phases of the treatment. [0092] In certain embodiments, provided herein are methods for treating, preventing, or ameliorating one or more diseases, disorders, or conditions that are or may be associated with dysbiosis, e.g., of the intestinal microbiome, in a subject in need thereof. In some aspects, the intestinal microbiome is involved in or associated with a number of physiological functions including digestion, metabolism, extraction of nutrients, synthesis of vitamins, prevention of pathogen colonization, and immune modulation. In some such aspects, alterations or changes in composition and biodiversity of the intestinal microbiome may be associated with or exacerbate various metabolic states, gastrointestinal disorders, and other pathophysiological conditions. In some aspects, conditions, diseases, or disorders with inflammatory components or components relating to infection, allergy, or immune dysfunction may be exacerbated by dysbiosis or may have an underlying contribution of dysbiosis to the pathology. Thus, in certain aspects, targeting the microbiome with the provided prebiotic and probiotic compositions may successfully treat, alleviate, or prevent a wide range of conditions, diseases, and disorders.

[0093] In some embodiments, provided herein is a method for treating, reducing, ameliorating, or preventing dysbiosis. In particular embodiments, the method is or includes steps for administering to the subject at least one strain of Bifidobacterium, e.g., such as any described herein, e.g., in Section I-B, a strain of bacterium capable of producing propionate, such as any of the propionate producing bacterium described herein, e.g., in Section I-A, and a prebiotic mixture, such as any described herein, e.g., in Section I-C. In some embodiments, the one or more diseases, disorders, or conditions is, includes, or is associated with dysbiosis, e.g., of the intestinal microbiome. In certain embodiments, the microbiome is an intestinal microbiome of a human. In various embodiments, the microbiome is the intestinal microbiome of an infant or young child. In certain embodiments, the microbiome is an intestinal microbiome of an adult human.

[0094] In some embodiments, the method is or includes steps for administering to the subject at least one strain of Bifidobacterium, e.g., such as any described herein, e.g., in Section I-B, a strain of bacterium capable of producing propionate, such as any of the propionate producing bacterium described herein, e.g., in Section I-A, and a prebiotic mixture, such as any described herein, e.g., in Section I-C for the treatment of one or more diseases, disorders, or conditions associated with inflammation, infection, allergy, immune dysfunction, or dysbiosis of the intestinal microbiome. In certain embodiments, the one or more conditions, diseases, or disorders is, includes, or is associated with dysbiosis. In certain embodiments, the one or more conditions, diseases, or disorders is, includes, or is associated with inflammation. In particular embodiments, the one or more condition, disease, or disorder is, includes, or is associated with an autoimmune disease. In particular embodiments, the one or more conditions, diseases, or disorders is or is associated with an allergy. In certain embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the Bifidobacterium, e.g., B. longum subsp. infantis, and the propionate producing bacteria, e.g., Veillonella sp., are administered to prevent a disease, disorder, or condition. In some embodiments, the prebiotic mixture and the probiotic strain prevent a condition described herein, e.g, in Section II-B. In particular embodiments, the prebiotic mixture and the probiotic strain reduce the risk, likelihood, or probability of the disease, disorder, or condition, and/or of experiencing one or more symptoms associated with the disease, disorder, or condition. In some embodiments, the risk, likelihood, or probability is reduced by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 99%, or 99.9% as compared to alternative treatments or no treatments, or as compared to administration of the probiotic strain or prebiotic mixture alone.

[0095] As used herein, “subject” and “subject in need thereof’ are used interchangeably. In particular embodiments, the subject is a human. In some embodiments, the subject is an infant, a child, a juvenile, or an adult. In certain embodiments, the subject is at least 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months of age. In certain embodiments, the subject is at least 1 year, 2 years, 5 years, 10 years, 12 years, 16 years, or at least 18 years of age. In some embodiments, the subject is at least 12 years old. In certain embodiments, the subject is at least 18 years old. In some embodiments, the subject is an adult. In certain embodiments, the subject is elderly, e.g, at least 65 or 75 years of age.

A. Administering Provided Compositions to the Subject

[0096] In certain embodiments, the provided methods include one or more treatment phases that are or include administration of one or more of the Bifidobacterium, e.g., B. longum subsp. infantis, the propionate producing bacterium, e.g., Veillonella sp. and the prebiotic mixture, e.g., of human milk oligosaccharides. In some embodiments, the method is or includes a treatment where all three of the propionate producing bacterium, the Bifidobacterium, and the prebiotic mixture are administered. In certain embodiments, the method is or includes a treatment phase where the Bifidobacterium and the prebiotic mixture are administered, e.g., in the absence of the propionate producing bacterium. In certain embodiments, the method is or includes a treatment phase where the prebiotic mixture is administered, e.g., in the absence of the propionate producing bacterium and the Bifidobacterium. In some embodiments, the method includes two or more treatment phases.

[0097] In certain embodiments, the prebiotic mixture is administered daily for at least 2, 3, 4, 5, 7, 10, 14, 21, or 28 days, e.g., consecutive days. In certain embodiments, the prebiotic mixture is administered in an amount of at least 0.001 g, 0.01 g, 0.1 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7.5 g, 8 g, 9 g, 10 g, 12 g, 16 g, 18 g, 20 g, 25 g, or 50 g per day, e.g., total weight of the prebiotics such as non-digestible carbohydrates such as human milk oligosaccharides. In particular embodiments, the prebiotic mixture in an amount of at least 0.001 g, 0.01 g, 0.1 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7.5 g, 8 g, 9 g, 10 g, 12 g, 16 g, 18 g, 20 g, 25 g, or 50 g total human milk oligosaccharides per day. In some embodiments, the prebiotic mixture is administered in an amount of between 0.1 g and 50 g; 0.5 g and 25 g, 1 g and 20 g, 2 g and 18 g, 1 g and 5 g, 2 g and 3 g, 3 g and 6 g, 4 g and 5 g, 5 g and 10 g, 8 g and 10 g, 10 g and 20 g, 15 g and 20 g, or 17 g and 19 g total human milk oligosaccharides per day. In some embodiments, the prebiotic mixture is administered in an amount of, of about, or of at least 2 g, 4.5 g, 6 g, 9 g, 12 g, 16 g, or 18 g total human milk oligosaccharides per day. In some embodiments, the prebiotic mixture is administered in a single dose per day. In various embodiments, the prebiotic mixture is administered in multiple doses per day, e.g., at or at least two, three, four, five, or six or more doses per day. In some embodiments, a daily amount of human milk oligosaccharides, e.g., 2 g, 4.5 g, 6 g, 9 g, 12 g, 16 g, or 18 g total human milk oligosaccharides per day, are administered over two, three, four, five, six, or more than six doses per day to achieve the total amount. In certain embodiments, doses of 9 g of total human milk oligosaccharides are administered twice daily for a total of 18 g/day.

[0098] In certain embodiments, the at least one Bifidobacterium is administered daily for at least 2, 3, 4, 5, 7, 10, 14, 21, or 28 days, e.g., consecutive days. In some embodiments, the at least one Bifidobacterium is administered in an amount of at least 1 x 10¹, 5 x 10¹,! x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 5 x 10⁶, 1 x 10⁷, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, or 5 x 10⁸ colony forming units (CFU) per day. In various embodiments, the at least one Bifidobacterium is administered in an amount of at least 1 x 10¹, 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, l x 10⁸, or 5 x 10⁸ colony forming units (CFU) per dose. In certain embodiments, the at least one Bifidobacterium is administered in an amount of between 1 x 10⁶ and 1 x 10¹², 5 x 10⁶ and 1 x 10¹⁰, 1 x 10⁷ and 1 x 10⁹, or 1 x 10⁷ and 1 x 10⁸ CFU per day. In some embodiments, the Bifidobacterium strain is administered in an amount of, of about, or at least 5 x 10⁶ colony forming units (CFU) per dose or per day. In some embodiments, the Bifidobacterium strain is administered in an amount of, of about, or at least 8 x 10⁷ colony forming units (CFU) per dose or per day.

[0099] In certain embodiments, the at least one propionate producing bacterium is administered daily for at least 2, 3, 4, 5, 7, 10, 14, 21, or 28 days, e.g., consecutive days. In some embodiments, the at least one propionate producing bacterium is administered in an amount of at least 1 x 10¹, 5 x 10¹,! x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵,l x 10⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, or 5 x 10⁸ colony forming units (CFU) per day. In various embodiments, the at least one propionate producing bacterium is administered in an amount of at least 1 x 10¹, 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, or 5 x 10⁸ colony forming units (CFU) per dose. In certain embodiments, the at least one propionate producing bacterium is administered in an amount of between 1 x 10⁶ and 1 x 10¹², 5 x 10⁶ and 1 x IO¹⁰, 1 x 10⁷ and 1 x 10⁹, or 1 x 10⁷ and 1 x 10⁸ CFU per day. In some embodiments, the at least one propionate producing bacterium is administered in an amount of, of about, or at least 5 x 10⁶ colony forming units (CFU) per dose or per day. In some embodiments, the probiotic strain is administered in an amount of, of about, or at least 8 x 10⁷ colony forming units (CFU) per dose or per day.

[0100] In some embodiments, the prebiotic mixture is administered at least once within 3 months, 2 months, 1 month, 60 days, 45 days, 30 days, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 28 days, 21 days, 14 days, 10 days, 7 days, 5 days, 3 days, or 1 day prior to administration of the at least one Bifidobacterium. In certain embodiments, the prebiotic mixture is administered at least once daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 consecutive days prior to the at least one Bifidobacterium. In certain embodiments, the prebiotic mixture is administered at least once within 3 months, 2 months, 1 month, 60 days, 45 days, 30 days, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 28 days, 21 days, 14 days, 10 days, 7 days, 5 days, 3 days, or 1 day after administration of the at least one Bifidobacterium. In certain embodiments, the prebiotic mixture is administered at least once daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 consecutive days after the at least one Bifidobacterium. In various embodiments, the prebiotic mixture is administered prior to and after the at least one Bifidobacterium.

[0101] In certain embodiments, the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium are administered together or separately during the same treatment regimen. In certain embodiments, the treatment regimen has separate treatment phases. The treatment regimen may include separate treatment phases with different combinations, doses, or timing of doses for some or all of the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium are administered to the subject. In some embodiments, all of the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium are administered during a treatment phase that occurs during the treatment regimen, and, in a different treatment phase, only one or two of the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium are administered in a different treatment phase that occurs during the same treatment regimen.

[0102] In certain embodiments, a treatment phase has a duration of at least one, two, three, four, five, six, seven, eight, nine, or ten days, or for at least one, two, three, four, five, or six weeks, or for at least one, two, three, four, five, or six months. In some embodiments, a treatment phase is or is at least seven days. In particular embodiments, a treatment phase is or is at least fourteen days.

[0103] In some embodiments, the treatment regimen includes more than one treatment phase. In particular embodiments, in one treatment phase, e.g., a first treatment phase, the prebiotic mixture and the at least one Bifidobacterium strain are administered. In some embodiments, the prebiotic mixture and the at least one Bifidobacterium strain are administered within the same composition during the treatment phase, e.g., the first treatment phase. In some embodiments, the prebiotic mixture and the at least one Bifidobacterium strain are administered as separate compositions during the treatment phase, e.g., the first treatment phase. In certain embodiments, one or both of the at least one Bifidobacterium strain and the prebiotic mixture is administered at least once, at least twice, at least three times, at least once per week, at least twice per week, at least three time per week, every other day, and/or every day during the treatment phase, e.g., the first treatment phase. In some embodiments, the prebiotic mixture and the at least one Bifidobacterium strain are administered on the same day for at least one, some, or all of the days of the treatment phase, e.g., the first treatment phase. In particular embodiments, the at least one propionate producing strain is also administered during the treatment phase, e.g., the first treatment phase. In some embodiments, the at least one propionate producing strain is administered at least once, at least twice, at least three times, at least once per week, at least twice per week, at least three time per week, every other day, and/or every day during the treatment phase, e.g., the first treatment phase. In particular embodiments, the at least one propionate producing strain is administered on the same days as the at least one Bifidobacterium strain during the treatment phase, e.g., the first treatment phase. In some embodiments, the treatment phase, e.g., the first treatment phase, has a duration of at least one, two, three, four, five, six, seven, ten, fourteen days, or at least one, two, three, four, five, or six weeks, or from one day to fourteen days or from three days to seven days.

[0104] In some embodiments, the treatment regimen includes a treatment phase, e.g., a second treatment phase, where the prebiotic mixture and not the at least one Bifidobacterium strain is administered. In certain embodiments, the prebiotic mixture is administered at least once, at least twice, at least three times, at least once per week, at least twice per week, at least three time per week, every other day, and/or every day during the treatment phase, e.g., the second treatment phase. In particular embodiments, the at least one propionate producing strain is also administered during the second treatment phase. In some embodiments, the at least one propionate producing strain is administered at least once, at least twice, at least three times, at least once per week, at least twice per week, at least three time per week, every other day, and/or every day during the second treatment phase. In certain embodiments, the at least one propionate producing strain is not administered during the second treatment phase. In particular embodiments, the second treatment phase has a duration of at least one, two, three, four, five, six, seven, ten, fourteen days, or at least one, two, three, four, five, or six weeks, or from one day to fourteen days or from three days to seven days.

[0105] In some embodiments, the first and second treatment phases occur once during a treatment regimen. In particular embodiments, the first and second treatment phases occur more than once during a treatment regimen, such as cycling or repeating throughout the duration of the treatment regimen. In some embodiments, there may be a gap or duration, e.g., for at least one, two, three, five, seven, ten, or fourteen, days, with no treatments between cycles, e.g., after the end of the second treatment phase and before the beginning of a subsequent first treatment phase.

[0106] In some embodiments, the prebiotic mixture and the at least one Bifidobacterium strain are administered together or separately for a period of time, such as in a treatment regimen. In some embodiments, the administration of the prebiotic mixture, e.g., of HMOs, allows for the engraftment and expansion of the Bifidobacterium strain, e.g., B. longum subsp. infantis. In certain embodiments, the Bifidobacterium strain is exogenous to the subject’s microbiome, e.g., intestinal microbiome. In particular embodiments, the Bifidobacterium strain is not present within the subject’s microbiome prior to administration. In certain embodiments, the prebiotic mixture is administered concurrently with and/or subsequently to administration of the at least one Bifidobacterium strain. In some embodiments, the at least one Bifidobacterium strain is present and/or expands within the subject’s microbiome during a time period in which the prebiotic mixture is administered. In certain embodiments, the presence or amount of the at least one Bifidobacterium strain within the microbiome is reduced when administration of the prebiotic mixture ends, is ceased or is terminated. In particular embodiments, the Bifidobacterium strain is absent and/or undetectable following the termination or end of administration of the prebiotic mixture. In certain embodiments, the presence of the Bifidobacterium strain, e.g., B. infantis, is transient and is regulated by administration of the prebiotic mixture.

[0107] In certain embodiments, at least one Bifidobacterium strain is capable of consuming or metabolizing some or all of the oligosaccharides, e.g., HMOs, of the prebiotic mixture. In some embodiments, the timing or dosing for administering the at least one Bifidobacterium strain and the prebiotic mixture achieves a growth or expansion of the Bifidobacterium strain in vivo, e.g., within the microbiome of the subject. In certain embodiments, the administered oligosaccharides, e.g., HMOs, selectively or exclusively serve as a carbon source for the at least one Bifidobacterium strain, e.g., as opposed to other bacterial strains present in the gut or microbiome. In some embodiments, the oligosaccharides of the mixture selectively or exclusively serve as an energy source for the at least one Bifidobacterium strain e.g., as opposed to other bacterial strains present in the gut or microbiome.

[0108] In certain embodiments, subjects are administered (e.g., at least once daily) all of the at least one Bifidobacterium strain, e.g., B. longum subsp. infantis, the at least one propionate producer, and the prebiotic mixture, e.g., of human milk oligosaccharides, during a first or initial treatment phase, e.g., for at least 1, 3, 7, or 14 days, and then are administered the prebiotic mixture alone during a subsequent treatment phase, e.g., such that occurs immediately after the first or initial treatment phase. In some embodiments, administration of the prebiotic mixture extends the duration of the colonization of the Bifidobacterium strain within the subject’s gut and/or microbiome.

[0109] In certain embodiments, administering the prebiotic mixture regulates the expansion, level, or amount of the Bifidobacterium strain, e.g., B. longum subsp. infantis, and/or the production or generation of metabolites, e.g., lactate and/or acetate, by the Bifidobacterium strain. In some aspects, expansion of the Bifidobacterium strain and/or generation or production of metabolites by the Bifidobacterium strain promotes the engraftment and/or the expansion of the propionate producing strain and/or promotes the production or generation of propionate by the propionate producing strain. Thus, in some aspects, the Bifidobacterium and propionate producing strains are administered to the subject, and the concurrent or subsequent administration of the prebiotic mixture may be adjusted to provide a therapeutic response, e.g., to promote growth or expansion of beneficial microbiota and/or to promote the generation or production of propionate within the subject’s gut or microbiome. In some embodiments, the dosage and/or duration of treatment with the prebiotic mixture, e.g., of HMOs, can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease.

[0110] As used herein, “subject” and “subject in need thereof’ are used interchangeably. In some embodiments, the subject is an infant, a child, a juvenile, or an adult. In certain embodiments, the subject is at least 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months of age. In certain embodiments, the subject is at least 1 year, 2 years, 5 years, 10 years, 12 years, 16 years, or at least 18 years of age. In some embodiments, the subject is at least 12 years old. In certain embodiments, the subject is at least 18 years old. In some embodiments, the subject is an adult.

[OHl] In some embodiments, provided herein is a method for treating, reducing, ameliorating, or preventing dysbiosis. In particular embodiments, a method is or includes steps for administering to the subject a prebiotic mixture, such as any described herein e.g., in Section I-C, at least one Bifidobacterium strain, such as a Bifidobacterium strain described herein, e.g., in Section I-B or listed in Table 1, and a propionate producing bacterium, e.g., as described in such as in Section I-A. In some embodiments, the method treats or prevents one or more diseases or conditions that include or are associated with dysbiosis, e.g., of the intestinal microbiome. In certain embodiments, the microbiome is an intestinal microbiome of a human.

B. Conditions, Diseases, and Disorders

[0061] In certain embodiments, provided herein are methods for treating, preventing, or ameliorating one or more diseases, disorders, or conditions that are or may be associated with dysbiosis, e.g., of the intestinal microbiome, in a subject in need thereof. In certain embodiments, administration of the at least one strain of Bifidobacterium, e.g., such as any described herein, e.g., in Section I-B, the strain of bacterium capable of producing propionate, such as any of the propionate producing bacterium described herein, e.g., in Section I- A, and the prebiotic mixture, such as any described herein, e.g., in Section I-C, are useful to treat, ameliorate, remedy, or prevent diseases, disorders, or conditions such as obesity, inflammatory bowel disease (IBD), celiac disease, irritable bowel syndrome (IBS), colon cancer, diabetes, liver disorders, cystic fibrosis, and allergies.

[0062] In certain embodiments, the at least one strain of Bifidobacterium, e.g., B. longum subsp. infantis, the strain of bacterium capable of producing propionate, e.g., Veillonella sp., and the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to a subject to treat, ameliorate, remedy, or prevent a gastrointestinal condition, disease, or disorder associated with, related to, or caused by dysbiosis, e.g., of the intestinal microbiome. In certain embodiments, the gastrointestinal condition, disease, or disorder is or includes one or more of a chronic inflammatory disease, an autoimmune disease, an infection, bowel resection, and/or a condition associated with chronic diarrhea. In certain embodiments, the gastrointestinal condition, disease, or disorder is or includes one or more of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) including Crohn's Disease and colitis, short bowel syndrome (SBS), celiac disease, small intestinal bacterial overgrowth (SIBO), gastroenteritis, leaky gut syndrome, and gastric lymphoma. In certain embodiments, the gastrointestinal condition, disease, or disorder is associated with a bacterial, viral or parasitic infection or overgrowth. In a particular embodiment, the disease or disorder is associated with infection by drug-resistant bacteria, e.g., vancomycin-resistant enterococcus (VRE). In particular embodiments, administration of the at least one prebiotic mixture, e.g., of human milk oligosaccharides, the strain of bacterium capable of producing propionate, e.g., Veillonella sp., and the at least one probiotic strain, e.g., B. longum subsp. infantis, prevents, reduces, or ameliorates one or more symptoms of the gastrointestinal condition.

[0063] In some embodiments, the at least one strain of Bifidobacterium, e.g., B. longum subsp. infantis, the strain of bacterium capable of producing propionate, e.g., Veillonella sp., and the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to a subject with an immune dysfunction. In some embodiments, the subject is immunocompromised. In certain embodiments, the administration prevents, reduces, treats, or ameliorates an infection in the immunocompromised subject. In some embodiments, the administration prevents, reduces, treats, or ameliorates overgrowth or domination of pathogenic bacteria. In some embodiments, the immunocompromised subject has undergone one or more treatments for cancer. In some embodiments, the treatments are or include chemotherapy. In certain embodiments, the treatment is or includes an allogenic transplant, e.g, a hematopoietic stem cell transplant or bone marrow transplant. In certain embodiments, the immunocompromised subject is in an ICU, has received an organ transplant, is elderly (e.g., at least 65 or 75 years old) and/or has been on prolonged antibiotic treatment (e.g., for at least 2, 3, 4, 6, 8, 10, or 12 weeks, or at least 1, 2, 3, 6, 12, 18, or 24 months). In certain embodiments, the administration prevents or reduces the probability or likelihood of a systemic infection by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%, e.g., as compared to a subject administered an alternative treatment and/or not administered the probiotic strains (e.g., the Bifidobacterium and the propionate producing bacterium) and/or the prebiotic mixture.

[0064] In particular embodiments, the at least one strain of Bifidobacterium, e.g., B. longum subsp. infantis, the strain of bacterium capable of producing propionate, e.g., Veillonella sp., and the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to treat or prevent overgrowth or domination of pathogenic bacteria (also referred to herein as gut domination). In some aspects, domination of pathogenic bacteria refers to the presence of a species of bacteria (e.g, a pathogenic species), of at least 1%, 5%, 10%, 20%, or 30%, relative to the bacteria present in the subject’s gut or intestinal microbiome. Particular embodiments contemplate that overgrowth or domination may be determined by routine techniques in the art, such as including but not limited to PCR or high throughput sequencing.

[0065] In certain embodiments, the at least one strain of Bifidobacterium, e.g., B. longum subsp. infantis, the strain of bacterium capable of producing propionate, e.g., Veillonella sp., and the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to a subject having, suspected of having, or at risk of having dysbiosis, e.g., of the intestinal microbiome. In certain embodiments, the transient presence, engraftment, or expansion of the Bifidobacterium strain, e.g., B. longum subsp. infantis, and the propionate producing strain, e.g., Veilloinella sp. reduces, decreases, or ameliorates the dysbiosis. Particular embodiments contemplate that the presence, engraftment, or expansion of the Bifidobacterium strain, e.g., B. longum subsp. infantis, and the propionate producing strain, e.g., Veilloinella sp., creates, promotes, or generates an environment and/or one or more conditions that (i) promotes the presence, growth, or expansion of beneficial microbiota; (ii) decreases the presence, growth, or expansion of pathogenic microbiota; (iii) promotes diversity of microbiota present within the microbiome; or (iv) any or all of (i) through (iii).

[0066] In certain embodiments, administration of the Bifidobacterium strain, e.g., B. longum subsp. infantis, and the propionate producing strain, e.g., Veilloinella sp., reduces the presence or abundance of pathogenic bacteria in the subject’s gut. In certain embodiments, administration of the Bifidobacterium strain, e.g., B. longum subsp. infantis, and the propionate producing strain, e.g., Veilloinella sp., reduces gut domination by pathogenic taxa (e.g., Enterob acteriaceae, Enterococcus, Staphylococcus). In particular embodiments, the growth of the Bifidobacterium strain, e.g., B. longum subsp. infantis, and the propionate producing strain, e.g., Veilloinella sp., within the gut or microbiome reduces the abundance, level, activity, or presence of pathogenic taxa. In certain embodiments, administration of the Bifidobacterium strain, e.g., B. longum subsp. infantis, and the propionate producing strain, e.g., Veilloinella sp. and the prebiotic mixture, e.g., of human milk oligosaccharides, reduces the abundance, level, activity, or presence of pathogenic bacteria and/or taxa by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 100%, e.g., as compared to prior to the administration or as compared to the gut or microbiome of a subject not administered the Bifidobacterium strain, the propionate producing strain, and/or the prebiotic mixture. In particular embodiments, the growth of the Bifidobaclerium ivVm, e.g., B. longum subsp. infantis, within the gut or microbiome increases the amount, level, presence, or concentration of at least one short chain fatty acid, e.g., acetate, propionate, or butyrate, within the gut.

[0067] In certain embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., and the Bifidobacterium strain, e.g., B. longum subsp. infantis, are administered to a subject who is at risk of an infection or gut domination, e.g., by pathogenic bacteria. In some embodiments, the subject has an increased risk of infection or gut domination, e.g., as compared to the general population. In certain embodiments the subject is immunocompromised, undergoing an extended antibiotic treatment regimen (e.g., lasting at least 2, 3, 4, 5, 6, 8 10, or 12 weeks or 2, 3, 6, 12, 18, or 24 months), is elderly, is hospitalized e.g., in an intensive care unit (ICU), has received an organ transplant, and/or is immunosuppressed. In certain aspects, the subject will undergo or has received a medical procedure such as a surgery or a chemotherapy that may increase the risk, likelihood, or probability of infection. [0068] In certain embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain reduces the risk, likelihood, or probability of infection, e.g, by pathogenic bacteria, is reduced by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 99%, or 99.9% as compared to alternative treatments or no treatments, or as compared to administration of the probiotic strains or prebiotic mixture alone. In some embodiments, the prebiotic mixture and the probiotic strains are administered at least once at least 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 1 week, 2 weeks, 4 weeks, 6 weeks, one month, or two months prior to the medical procedure, e.g, surgery or chemotherapy. In particular embodiments, the prebiotic mixture and the probiotic strains are administered at least once during the medical procedure, e.g., surgery or chemotherapy. In certain embodiments, the prebiotic mixture and the probiotic strains are administered at least once at least 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 1 week, 2 weeks, 4 weeks, 6 weeks, one month, or two months after to the medical procedure, e.g., surgery or chemotherapy.

[0069] Pathogenic bacteria may include known microbes with pathogenicity for the gastrointestinal tract, e.g., from esophagus down to rectum. In some embodiments, pathogenic bacteria are or include one or more species, subspecies, or strains of Proteobacteria. In certain embodiments, the pathogenic bacteria may include, but are not limited to strains, species, subspecies, or strains of one or more of Firmicutes, Clostridium, Enter obacteriaceae, Enterococcus, Staphylococcus, Corynebacteria, Salmonella, Shigella, Staphylococcus, Campylobacter (e.g., Campylobacter jejuni), Clostridia, Escherichia coli, Yersinia, Vibrio cholerae, Mycobacterium avium subspecies paratuberculosis, Brachyspira hyodysenteriae, or Law sonia intracellularis . In particular embodiments, administration of the prebiotic mixture and the probiotic strains reduces or decreases the presence, growth, or abundance of pathogenic bacteria within the gut.

[0070] In some embodiments, administration of the prebiotic mixture the propionate producing strain, and the Bifidobacterium strain impairs the growth of one or more pathogens. Such pathogens treated by the provided methods include, but are not limited to, Aeromonas hydr ophila, Bacillus, e.g., Bacillus cereus, Bifidobacterium, Bordetella, Borrelia, Brucella, Burkholderia, C. difficile, Campylobacter, e.g., Campylobacter fetus and Campylobacter jejuni, Chlamydia, Chlamydophila, Clostridium, e.g., Clostridium botulinum, Clostridium difficile, and Clostridium perfringens, Corynebacterium, Coxiella, Ehrlichia, Enterob acteriaceae, e.g., Carbapenem-resistant Enterobacteriaceae (CRE) and Extended Spectrum Beta-Lactamase producing Enterobacterales (ESBL-E), fluoroquinolone-resistant Enterobacteriaceae, Enterococcus, e.g., vancomycin-resistant enterococcus spp., extended spectrum beta-lactam resistant Enterococci (ESBL), and vancomycin-resistant Enterococci (VRE), Escherichia, e.g., enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enterop athogenic E. coli, enterotoxigenic Escherichia coli (such as but not limited to LT and/or ST), Escherichia coli 0157:H7, and multi-drug resistant bacteria E. coli, Francisella, Haemophilus, Helicobacter, e.g., Helicobacter pylori, Klebsiella, e.g., Klebsiellia pneumonia and multi-drug resistant bacteria Klebsiella, Legionella, Leptospira, Listeria, e.g., Lysteria monocytogenes, Morganella, Mycobacterium, Mycoplasma, Neisseria, Orientia, Plesiomonas shigelloides, Antibiotic-resistant Proteobacteria, Proteus, Pseudomonas, Rickettsia, Salmonella, e.g., Salmonella paratyphi, Salmonella spp., and Salmonella typhi, Shigella, e.g., Shigella spp., Staphylococcus, e.g., Staphylococcus aureus and Staphylococcus spp., Streptococcus, Treponema, Vibrio, e.g., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio spp., and Vibrio vulnificus, and Yersinia, e.g., Yersinia enter ocolitica. At least one of the one or more pathogens can be an antibioticresistant bacterium (ARB), e.g., Antibiotic-resistant Proteobacteria, Vancomycin Resistant Enterococcus (VRE), Carbapenem Resistant Enterobacteriaceae (CRE), fluoroquinoloneresistant Enterobacteriaceae, or Extended Spectrum Beta-Lactamase producing Enterobacterales (ESBL-E).

[0071] In some embodiments, administration of the prebiotic mixture the propionate producing strain, and the Bifidobacterium strain impairs the growth of of antibiotic-resistant bacterium (ARB), Antibiotic-resistant Proteobacteria, Carbapenem-resistant Enterobacteriaceae (CRE), Extended Spectrum Beta-Lactamase producing Enterobacterales (ESBL-E), fluoroquinolone-resistant Enterobacteriaceae, vancomycin-resistant Enterococci (VRE), multi-drug resistant A. coli, or multi-drug resistant Klebsiella.

[0072] In some embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treat, prevent, or ameliorate an infection or gut domination by one or more of Caproiciproducens, Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Papillibacter cinnamivorans, Papillibacter, Proteus mirabilis, Serratia marcescens, Sporobacter, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus anginosus, Streptococcus australis, Streptococcus constellatus, Streptococcus cristatus, Streptococcus gordonii, Streptococcus infantis, Streptococcus intermedius, Streptococcus mitis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus tigurinus, Streptococcus vestibularis, or Syntrophococcus sp. In certain embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treat, prevent, or ameliorate an infection or gut domination by one or more of Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus anginosus, Streptococcus australis, Streptococcus constellatus, Streptococcus cristatus, Streptococcus gordonii, Streptococcus infantis, Streptococcus intermedius, Streptococcus mitis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus tigurinus, or Streptococcus vestibularis.

[0073] In some embodiments, the condition, disease, or disorder is an immune dysfunction that is an autoimmune disorder. In some embodiments, the autoimmune disorder includes, but is not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressier's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Tumer syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, & Ill autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis.

[0074] In some embodiments, the condition, disease, or disorder is a diarrheal disease including, but not limited to, acute bloody diarrhea (e.g., dysentery), acute watery diarrhea (e.g., cholera), checkpoint inhibitor-associated colitis, diarrhea due to food poisoning, persistent diarrhea, and traveler's diarrhea.

[0075] In some embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treats or prevents various GI disorders known to result from or be associated or accompanied with dysbiosis of the intestinal microbiome. In certain embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain reduces GI immunoactivation and/or inflammation. In some embodiments, GI immunoactivation and inflammation may be assessed by known methods that are routine in the art. In some embodiments, the condition, disease, or disorder is an inflammatory bowel disease (IBD) or related disease including, but not limited to, Behcet's disease, collagenous colitis, Crohn's disease, diversion colitis, fulminant colitis, intermediate colitis, left-sided colitis, lymphocytic colitis, pancolitis, pouchitis, proctosigmoiditis, short bowel syndrome, ulcerative colitis, and ulcerative proctitis.

[0076] In various embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treats or prevents various bloodstream infections (BSI). In certain embodiments, administration of the probiotic strains and the prebiotic mixture treats or prevents catheter or intravascular-line infections (e.g., central-line infections). In some embodiments, administration of the probiotic strains and the prebiotic mixture treats or prevents chronic inflammatory diseases.

[0077] In particular embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treats or prevents meningitis; pneumonia, e.g., ventilator-associated pneumonia; skin and soft tissue infections; surgical- site infections; urinary tract infections (e.g., antibiotic-resistant urinary tract infections and catheter-associated urinary tract infections); wound infections; and/or antibiotic-resistant infections and antibiotic-sensitive infections.

[0078] In certain embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treats or prevents diseases or disorders relating to the "gut-brain axis", including neurodegenerative, neurodevelopmental, and neurocognitive disorders, such as anorexia, anxiety, autism-spectrum disorder, depression, Parkinson's, and Schizophrenia. In certain embodiments, administration of the probiotic strains and the prebiotic mixture reduces one or more symptoms associated with anorexia, anxiety, autism-spectrum disorder, depression, Parkinson's, and/or Schizophrenia.

[0079] In some embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treats or prevents a side effect of an anticancer therapy and/or increases efficacy of an anti -cancer therapeutic agent and/or anticancer therapy. In some embodiments, the anti -cancer therapy is surgery, radiation therapy, chemotherapy (including hormonal therapy) and/or targeted therapy (including an immunotherapy). Illustrative chemotherapeutics agents are provided elsewhere herein. In particular embodiments, the immunotherapy binds to and/or recognizes a tumor-cell antigen and/or a cancer-cell antigen, e.g., CTLA-4, PD-1, PD-L1, or PD-L2. In some embodiments, the immunotherapy comprises administration of Keytruda (Pembrolizumab), Opdivo (Nivolumab), Yervoy (Ipilimumab), Tecentriq (atezolizumab), Bavencio (avelumab), and Imfinzi (durvalumab).

[0080] In some embodiments, the subject is refractory and/or non-responsive to an anti-cancer therapy. In certain embodiments, the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treats a subject that presents a non-curative response, a limited response, or no response to the anti-cancer therapy, or even progress, after 12 weeks or so of receiving the anti -cancer therapy. Thus, in some aspects, the provided probiotic strains and prebiotic mixture of the present invention can rescue subjects that are refractory and/or non-responsive to the anti-cancer therapy. In certain embodiments, the subject is refractory and/or non-responsive to a treatment directed to a checkpoint molecule, e.g., CTLA-4, PD-1, PD-L1, and/or PD-L2. In particular embodiments, the treatment directed to a checkpoint molecule comprises administration of Keytruda (Pembrolizumab), Opdivo (Nivolumab), Yervoy (Ipilimumab), Tecentriq (atezolizumab), Bavencio (avelumab), or Imfinzi (durvalumab).

[0081] In some embodiments, the prebiotic mixture, c.g, of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., and the at least one Bifidobacterium strain, e.g, B. longum subsp. infantis, are administered to an immunocompromised subject. In certain embodiments, the administration prevents, reduces, treats, or ameliorates an infection in the immunocompromised subject. In some embodiments, the administration prevents, reduces, treats, or ameliorates overgrowth or domination of pathogenic bacteria. In some embodiments, the immunocompromised subject has undergone one or more treatments for cancer. In some embodiments, the treatments are or include chemotherapy. In certain embodiments, the treatment is or includes an allogenic transplant, e.g., a hematopoietic stem cell transplant or bone marrow transplant. In certain embodiments, the administration prevents or reduces the probability or likelihood of a systemic infection by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%, e.g., as compared to an alternative treatment or treatment with either the probiotic strains or prebiotic mixture alone.

[0082] In certain embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., and the at least one Bifidobacterium strain, e.g, B. longum subsp. infantis, are administered to a subject who has or is at risk of sepsis. In some embodiments, the probability or likelihood of sepsis is reduced or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, e.g, as compared to a subject (e.g., who has or is at risk for sepsis) not administered the prebiotic mixture or the probiotics. In certain embodiments, the administration of the prebiotic mixture and the probiotics improves or increases the survival of the subject over 6 months, 12 months, 18 months, 1 year, 2 years, 5 years, 10 years, and/or 20 years or more by, by about, or by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, or 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold greater than in subjects (e.g., who have or are at risk for sepsis) not administered the prebiotic mixture and the probiotic strains.

[0083] In particular embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain prevents, reduces, decreases, remedies, or ameliorates one or more symptoms associated with a gastrointestinal condition, disease, or disorder. In certain embodiments, the one or more symptoms associated with gastrointestinal condition, disease, or disorder may include, but are not limited to, diarrhea, fever, fatigue, abdominal pain and cramping, blood in stool, mouth sores, weight loss, fistula, inflammation (of skin, eyes, or joints), inflamed liver or bile ducts, delayed growth (in children). In particular embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain reduces the risk or probability for the subject of experiencing one or more symptoms associated with the gastrointestinal condition, disease, or disorder by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%, e.g., as compared to a subject not administered the probiotic strains and/or the prebiotic mixture. In certain embodiments, administration of the prebiotic mixture and the probiotic strains increases probability or likelihood for remission by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, or 1-fold, 2-fold, 5-fold, 10- fold, 20-fold, 50-fold, or 100-fold e.g., as compared to a subject not administered the probiotic strains and/or the prebiotic mixture. In some embodiments, administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain increases probability or likelihood for remission within 12 weeks, 10 weeks, 8 weeks, 6 weeks, 4 weeks, or less than 4 weeks, e.g., from the initiation or termination of the administration.

[0084] In various embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., and the at least one Bifidobacterium strain, e.g, B. longum subsp. infantis, are administered to a subject to treat, ameliorate, remedy, or prevent a chronic inflammatory disease, an autoimmune disease, an infection, bowel resection, and/or a condition associated with chronic diarrhea. According to particular embodiments, the pathology is selected from the group consisting of: irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), short bowel syndrome (SBS), celiac disease, small intestinal bacterial overgrowth (SIBO), gastroenteritis, leaky gut syndrome, and gastric lymphoma. In another embodiment the disease or disorder is associated with a bacterial, viral, or parasitic infection or overgrowth, e.g., by drug-resistant bacteria. In some embodiments, administration of the prebiotic mixture the propionate producing strain, and the Bifidobacterium strain increases probability or likelihood for cure or remission of the chronic inflammatory disease, autoimmune disease, infection, bowel resection, and/or chronic diarrhea for by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, or 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold e.g, as compared to a subject not administered the probiotic strains and/or the prebiotic mixture. In some embodiments, administration of the prebiotic mixture the propionate producing strain, and the Bifidobacterium strain increases probability or likelihood for the cure or remission within 12 weeks, 10 weeks, 8 weeks, 6 weeks, 4 weeks, or less than 4 weeks, e.g., from the initiation or termination of the administration.

[0085] In certain embodiments, the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain are administered to a subject to treat, prevent, or ameliorate an allergy. In some embodiments, the allergy is a food allergy. In certain embodiments, the food allergy is or includes a chronic or acute immunological hypersensitivity reaction (e.g., a type I hypersensitivity reaction) elicited in a mammal in response to an ingested material or food antigen (also referred to in the art as a “food allergen”). Identification and diagnosis of food allergy is routine among persons of ordinary skill in the art. Food allergies may include, but are not limited to, allergies to nuts, peanuts, shellfish, fish, milk, eggs, wheat, or soybeans.

[0086] In some embodiments, the prebiotic mixture the propionate producing strain, and the Bifidobacterium strain are administered to treat or ameliorate an allergy, e.g., a food allergy. In certain embodiments, the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain reduce or decrease the severity of the allergic response to the allergen, e.g., as compared to the allergic response prior to any treatment with the probiotic strains and prebiotic mixture. In certain embodiments, the prebiotic mixture the propionate producing strain, and the Bifidobacterium strain attenuates or reduces the severity or intensity of one or more symptoms or clinical manifestations of the allergy, e.g., food allergy, to subsequent exposures to the allergen, e.g., as compared to symptoms or clinical manifestations observed prior to treatment with the probiotic strains and prebiotic mixture. In some embodiments, the symptoms or clinical manifestations of the allergy may include, but are not limited to rash, eczema, atopic dermatitis, hives, urticaria, angiodema, asthma, rhinitis, wheezing, sneezing, dyspnea, swelling of the airways, shortness of breath, other respiratory symptoms, abdominal pain, cramping, nausea, vomiting, diarrhea, melena, tachycardia, hypotension, syncope, seizures, and anaphylactic shock.

[0087] In particular embodiments, the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain are administered to a subject, e.g., a subject at risk of having or developing an allergy, to prevent or reduce or decrease the probability or likelihood experiencing an allergic response. In certain embodiments, administration of the probiotic strains and prebiotic mixture reduce the likelihood or probability of having an allergic response within the next month, 3 months, 6 months, 12 months, 18 months, year, 2 years, 3 years, 5 years, 10 years, or 20 years. In some embodiments, the probability or likelihood of developing the allergy is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 99% as compared to a subject with a similar risk profile who is not administered the probiotic strains and the prebiotic mixture. In some embodiments, administration of the probiotic strains and prebiotic mixture reduces the severity of one or more symptoms or clinical manifestations of an allergic response following exposure to the allergen over the next month, 3 months, 6 months, 12 months, 18 months, year, 2 years, 3 years, 5 years, 10 years, or 20 years, e.g., as compared to exposure of the allergen to a subject with the same or similar allergy who was not administered the probiotic strains and the prebiotic mixture. [0088] In some embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., and the at least one Bifidobacterium strain, e.g., B. longum subsp. infantis, are administered to a subject to treat, ameliorate, remedy, or prevent pouchitis. In certain aspects, pouchitis is inflammation that occurs in the lining of a pouch created during surgery to treat ulcerative colitis or certain other diseases. In some embodiments, the surgery is or includes removal of a diseased colon or portion thereof. In certain embodiments, the surgery is a J pouch surgery (ileoanal anastomosis — IPAA).

[0089] In some embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., and the at least one Bifidobacterium strain, e.g., B. longum subsp. infantis, are administered to a subject to treat, ameliorate, remedy, or prevent pouchitis in a subject in need thereof, e.g., a subject who has undergone an IPAA surgery. In particular embodiments, administration of the prebiotic mixture and the probiotic strains prevents, reduces, decreases, remedies, or ameliorates one or more symptoms associated with pouchitis. In certain embodiments, the one or more symptoms associated with pouchitis may include, but are not limited to, increased stool frequency, tenesmus, straining during defecation, blood in the stool, incontinence, seepage of waste matter during sleep, abdominal cramps, pelvic or abdominal discomfort, or tail bone pain. In certain embodiments, symptoms associated with more severe pouchitis include, but are not limited to, fever, dehydration, malnutrition, fatigue, iron-deficiency anemia, or joint pain. In particular embodiments, administration of the prebiotic mixture and the at least one probiotic strain reduces the risk or probability for the subject of experiencing pouchitis by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%, e.g., as compared to a subject not administered the probiotic strains and/or the prebiotic mixture.

[0090] In various embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., and the at least one Bifidobacterium strain, e.g., B. longum subsp. infantis, are administered to a subject to treat, ameliorate, remedy, or prevent a chronic inflammatory disease, an autoimmune disease, an infection, bowel resection, and/or a condition associated with chronic diarrhea. Such pathology includes, but is not limited to: irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), short bowel syndrome (SBS), celiac disease, small intestinal bacterial overgrowth (SIBO), gastroenteritis, leaky gut syndrome, and gastric lymphoma. In some embodiments the disease or disorder is associated with a bacterial, viral, or parasitic infection or overgrowth, e.g., by drug-resistant bacteria. In some embodiments, administration of the prebiotic mixture and the probiotic strains increases probability or likelihood for cure or remission of the chronic inflammatory disease, autoimmune disease, infection, bowel resection, and/or chronic diarrhea for by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, or 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold e.g., as compared to a subject not administered the probiotic strains and prebiotic mixture and/or a subject administered an alternative therapy. In some embodiments, administration of the prebiotic mixture and the probiotic strains increases probability or likelihood for the cure or remission within 12 weeks, 10 weeks, 8 weeks, 6 weeks, 4 weeks, or less than 4 weeks, e.g., from the initiation or termination of the administration e.g., as compared to a subject not administered the probiotic strains and prebiotic mixture and/or a subject administered an alternative therapy.

[0091] In some embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., the Bifidobacterium strain, e.g., B. longum subsp. infantis, are administered to a subject to treat, ameliorate, remedy, or prevent pouchitis. In certain aspects, pouchitis is inflammation that occurs in the lining of a pouch created during surgery to treat ulcerative colitis or certain other diseases. In some embodiments, the surgery is or includes removal of a diseased colon or portion thereof. In certain embodiments, the surgery is a J pouch surgery (ileoanal anastomosis — IPAA).

[0092] In some embodiments, the prebiotic mixture, e.g., of human milk oligosaccharides, the propionate producing strain, e.g., Veilloinella sp., and the Bifidobacterium strain, e.g., B. longum subsp., are administered to a subject to treat, ameliorate, remedy, or prevent pouchitis in a subject in need thereof, e.g., a subject who has undergone an IPAA surgery. In particular embodiments, administration of the prebiotic mixture and the probiotic strains prevents, reduces, decreases, remedies, or ameliorates one or more symptoms associated with pouchitis. In certain embodiments, the one or more symptoms associated with pouchitis may include, but are not limited to, increased stool frequency, tenesmus, straining during defecation, blood in the stool, incontinence, seepage of waste matter during sleep, abdominal cramps, pelvic or abdominal discomfort, or tail bone pain. In certain embodiments, symptoms associated with more severe pouchitis include, but are not limited to, fever, dehydration, malnutrition, fatigue, iron-deficiency anemia, or joint pain. In particular embodiments, administration of the prebiotic mixture, the Bifidobacterium strain, and the propionate producing strain reduces the risk or probability for the subject of experiencing pouchitis by, by about, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%, e.g, as compared to a subject not administered the probiotic strains and/or the prebiotic mixture.

[0093] In some embodiments, the subject is a patient in an intensive care unit (ICU). In some embodiments, the subject is an organ transplant recipient. In some embodiments, the subject is a geriatric patient (e.g., at least 65, 70, 75, 80, or 85 years old). In some embodiments, the subject has received prolonged antibiotic treatment (e.g., at least 2, 3, 4, 5, 6, 8, 10, or 12 weeks, or at least 1, 2, 3, 6, 12, 18, or 24 months). In some embodiments, the subject is a recipient of a broad-spectrum antibiotic treatment. In some embodiments, the subject is a recipient, or recent recipient (e.g., within at least 1, 2, 3, 4, 5, 6, or 7 days, or within at least 1, 2, 3, or 4 weeks), of parenteral nutrition (e.g., total parenteral nutrition or partial parenteral nutrition). In some embodiments, the subject is a recipient of enteral nutrition.

[0094] In particular embodiments, provided herein are methods of preventing or reducing the incidence or severity of graft versus host disease (GVHD) in a subject in need thereof. In certain embodiments, the provided methods prevent or reduce incidence or severity of GVHD in a subject that has received or will receive an allogenic stem cell transplant. In some embodiments, the provided mixtures of HMOs are formulated to be administered to subjects who have, are, or will undergo an allogenic transplant, e.g., BMT or HSCT. In some embodiments, the at least one strain of Bifidobacterium and the at least one propionate producing strain are formulated to be administered to subject who has underwent, is undergoing, or will undergo an allogenic transplant. In particular embodiments, at least one strain of Bifidobacterium, e.g., such as any described herein, e.g., in Section I-B, the strain of bacterium capable of producing propionate, such as any of the propionate producing bacterium described herein, e.g., in Section I-A, and the prebiotic mixture, such as any described herein, e.g, in Section I-C, are administered to a subject who will undergo or who has undergone an allogenic transplant.

[0095] In certain embodiments, the subject is a human infant, child, adolescent, or adult. In particular embodiments, the subject is at risk or suspected of being at risk of having GVHD. In some embodiments, the GHVD is associated with, or accompanied by, an allogenic transplant, such as an allogenic bone marrow transplant (BMT) or an allogenic hematopoietic stem cell transplant (HSCT). [0096] In some embodiments, at least one strain of the Bifidobacterium, e.g., B. longum subsp. infantis, a strain of bacterium capable of producing propionate, e.g., a Veillonella sp. and a prebiotic mixture, e.g., of human milk oligosaccharides, is administered to a subject has undergone or will undergo an allogenic stem cell transplant. In certain embodiments, the allogenic transplant is a bone marrow transplant (BMT). In particular embodiments, the allogenic transplant is a hematopoietic stem cell transplantation (HSCT). In particular embodiments, the subject has undergone the allogenic stem cell transplant within 12 weeks, 8 weeks, 6 weeks, 4 weeks, 3 weeks, 2 weeks, 14 days, 12 days 10 days, 7 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration of a first dose of the prebiotic mixture or the at least one probiotic strain. In certain embodiments, the first dose of the prebiotic mixture or the at least one probiotic strain is administered within 12 weeks, 8 weeks, 6 weeks, 4 weeks, 3 weeks, 2 weeks, 14 days, 12 days 10 days, 7 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to receiving the allogenic stem cell transplant.

[0097] In some embodiments, provided herein are methods for treating, preventing, or ameliorating GVHD in a subject in need thereof. In certain embodiments, provided herein are methods for treating, preventing, or ameliorating a condition or disease associated or accompanied with GVHD in a subject in need thereof. In certain embodiments, provided herein are methods for treating, preventing, reducing, decreasing, or ameliorating the severity or presence of one or more symptoms associated with GVHD or a disease or condition associated or accompanied with GVHD in a subject in need thereof.

[0098] In particular embodiments, administration of the Bifidobacterium, e.g., B. longum subsp. infantis, a propionate producing strain, e.g., a Veillonella sp., and a prebiotic mixture, e.g., of human milk oligosaccharides, reduces or decreases the probability or likelihood of experiencing GVHD. In certain embodiments, the probability or likelihood is reduced or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, e.g., as compared to a subject not administered the prebiotic mixture or the probiotics. In certain embodiments, the probability or likelihood of experiencing GVHD within 20 years, 10 years, 7 years, 5 years, 2 years or 1 year, or within the subject’s lifetime, is reduced or decreased, e.g., as compared to a subject not administered the prebiotic mixture or the probiotics.

[0099] In certain embodiments, the Bifidobacterium, e.g., B. longum subsp. infantis, a propionate producing strain, e.g., a Veillonella sp. and a prebiotic mixture, e.g., of human milk oligosaccharides, are administered to decrease or reduce mortality associated with an allogenic transplant, e.g., BMT or HSCT, or with GVHD. In some embodiments, the Bifidobacterium, e.g., B. longum subsp. infantis, the propionate producing strain, e.g., a Veillonella sp. and the prebiotic mixture, e.g., of human milk oligosaccharides are administered to increase survival of subjects who undergo an allogenic transplant, e.g., BMT or HSCT. In particular embodiments, administration improves or increases the survival of the subject over 6 months, 12 months, 18 months, 1 year, 2 years, 5 years, 10 years, and/or 20 years or more by, by about, or by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, or 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold greater than in subjects (e.g., subjects who received an allogenic transplant, e.g., BMT or HSCT) not administered the prebiotic mixture and the probiotic strains.

[0100] In some embodiments, the Bifidobacterium, e.g., B. longum subsp. infantis, a propionate producing strain, e.g., the Veillonella sp. And the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to treat, prevent, ameliorate, reduce, or decrease the severity, occurrence, or likelihood of experiencing one or more symptoms, e.g., symptoms associated with or accompanying GHVD. In particular embodiments, the GVHD is acute GVHD. In some embodiments, the GVHD is chronic GVHD. In particular embodiments, the symptoms of GVHD are or include, but are not limited to, a rash, such as with burning or itching sensation; blistering, e.g., of the skin; flaking of the skin; nausea; vomiting; abdominal cramps; loss of appetite; diarrhea; and jaundice. In some embodiments, the symptoms of GVHD are or include, but are not limited to dry mouth, mouth ulcers, difficulty eating, gum disease, tooth decay, rash, itchy sensation, thickening and tightening of the skin, jaundice, changes in skin coloration, hair loss, premature gray hair, loss of body hair, loss of appetite, unexplained weight loss, nausea, vomiting diarrhea, stomach pain, shortness of breath, difficulty breathing, persistent or chronic cough, wheezing, impaired liver function, abdominal swelling, muscle weakness, muscle cramps, and joint stiffness. In particular embodiments, administration of the Bifidobacterium, e.g., B. longum subsp. infantis, the propionate producing strain, e.g., a Veillonella sp., and the prebiotic mixture, e.g., of human milk oligosaccharides treats, prevents, ameliorates, reduces, or decreases the severity, occurrence, or likelihood of the one or more symptoms as compared to what is observed in subjects (e.g., subjects who have had or will undergo an allogeneic transplant) that are not administered the Bifidobacterium, the propionate producing strain, and the prebiotic mixture. In some aspects, the presence, occurrence, and severity of a symptom may be recognized, identified, or scored by skilled person (e.g., a healthcare practitioner) as a matter of routine. [0101] In certain embodiments, the Bifidobacterium, e.g., B. longum subsp. infantis, and the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to treat, prevent, ameliorate, reduce, or decrease the severity, occurrence, or likelihood of experiencing one or more symptoms, e.g., symptoms associated with or accompanying acute radiation syndrome (ARS). In particular embodiments, a propionate producing strain, e.g., the Veillonella sp., is administered with or concurrent to the administration of the Bifidobacterium and the prebiotic mixture.

[0102] In some embodiments, the subject has or is suspected of having ARS. In certain embodiments, the subject has been exposed or is suspected of having been exposed to high doses of ionizing radiation. Subjects may be exposed to high doses of ionizing radiation under various circumstances that may include but are not limited to accidental, medical, or terrorist incidents. In humans, ARS resulting from such exposure may present as hematopoietic dysfunction, gastrointestinal damage, and/or neurovascular injury. Particular embodiments contemplate that while medical countermeasures exist to treat hematopoietic dysfunctions, there are currently no approved therapies to treat or prevent gastrointestinal damage associated with ARS.

[0103] In particular embodiments, the Bifidobacterium, e.g., B. longum subsp. infantis, and the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to prevent, treat, and/or reduce the severity of one or more symptoms associated with ARS. In some embodiments, the at least one propionate producing bacterium, e.g., Veillonella sp., is also administered. In certain embodiments, the symptoms are or include symptoms associated with hematopoietic or bone marrow syndrome and/or damage, gastrointestinal syndrome and/or damage, and/or cardiovascular/central nervous system (CNS) syndrome and/or damage. In some embodiments, the symptoms of hematopoietic syndrome and/or hematopoietic damage are or include anorexia, nausea and vomiting, fever, malaise, and/or drops in blood cell counts (e.g., red, white, or both). In certain embodiments, the symptoms of gastrointestinal syndrome and/or gastrointestinal damage are or include anorexia, severe nausea, vomiting, cramps, diarrhea, malaise, fever, dehydration, and/or electrolyte imbalance. In particular embodiments, the symptoms of cardiovasculature/CNS syndrome and/or cardiovasculature/CNS damage are or include extreme nervousness and confusion, severe nausea, vomiting, and diarrhea, loss of consciousness, burning sensations on the skin, convulsions, and/or coma. In certain embodiments, the administration prevents the onset of one or more symptoms associated with ARS. In particular embodiments, the administration reduces the severity or intensity of one or more symptoms associated with ARS, e.g., as compared to an unadministered subject exposed to a similar dose of ionizing radiation.

[0104] In certain embodiments, the Bifidobacterium, e.g., B. longum subsp. inf amis. and the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to prevent, treat, and/or reduce the severity of gastrointestinal damage associated with ARS and/or associated with exposure to a high dose of ionizing radiation. In some embodiments, the at least one propionate producing bacterium, e.g., Veillonella sp., is also administered. In certain embodiments, the administration preserves gut barrier integrity and development of the gastrointestinal tract resulting from or associated with ARS or a high dose of ionizing radiation, e.g., as compared to an untreated subject exposed to the same or similar ionizing radiation. In particular embodiments, administration prevents or reduces the likelihood of infection due to pathogen outgrowth and/or translocation associated with ARS or a high dose of ionizing radiation, e.g., as compared to an untreated subject exposed to the same or similar ionizing radiation.

[0105] In some embodiments, the subject has been exposed to a high dose of ionizing radiation. For example, exposure to ionizing radiation doses of 0.3 Gray (Gy) or 30 rads may be sufficient to cause or induce symptoms associated with ARS, and doses of at least 0.7 Gray (Gy) or 70 rads may generally be considered as sufficient to cause or induce ARS. In certain embodiments, the dose is at least 0.3 Gy, 0.5 Gy, 0.7 Gy, 1.0 Gy, 2.0 Gy, 3.0 Gy, 4.0 Gy, 5.0 Gy, 6.0 Gy, 7.0 Gy, 8.0 Gy, 9.0 Gy, 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 40 Gy, or at least 50 Gy. In particular embodiments, the dose is at least 30 rad, 50 rad, 70 rad, 100 rad, 150 rad, 200 rad, 250 rad, 300 rad, 500 rad, 600 rad, 700 rad, 750 rad, 800 rad, 900 rad, 1,000 rad, 2,000 rad, 3,000 rad, 4,000 rad, or at least 5,000 rad. In some embodiments, the dose is associated with an external source of radiation, e.g., a source outside of the subject’s body. In particular embodiments, the ionizing radiation dose may be penetrating and/or able to reach the subject’s internal organs, and may include, but are not limited to, high energy X-rays, gamma rays, and/or neutrons. In some embodiments, the subject may be exposed to the ionizing radiation dose over of relatively short amount of time, e.g., within minutes, such as over an amount of time less than one hundred eighty minutes, one hundred twenty minutes, ninety minutes, sixty minutes, thirty minutes, fifteen minutes, ten minutes, five minutes, three minutes, two minutes, or less than one minute.

[0106] In some embodiments, the Bifidobacterium, e.g., B. longum subsp. inf antis. and the prebiotic mixture, e.g., of human milk oligosaccharides, are administered to prevent or reduce mortality associated with and/or resulting from ARS. In certain embodiments, the at least one propionate producing bacterium, e.g., Veillonella sp., is also administered. In some embodiments, at least one butyrate producing bacterium, such as those described herein, e.g., in Section I-D such as Anaerostipes caccae, Clostridium innocuum, Roseburia hominis, o Roseburia inleslinalis. is also administered. In certain embodiments, B. longum subsp. infantis, the prebiotic mixture of human milk oligosaccharides, and the propionate producing bacterium are administered to prevent or reduce mortality associated with and/or resulting from ARS. In some embodiments, B. longum subsp. infantis, the prebiotic mixture of human milk oligosaccharides, and the butyrate producing bacterium are administered to prevent or reduce mortality associated with and/or resulting from ARS. In various embodiments, B. longum subsp. infanlis. the prebiotic mixture of human milk oligosaccharides, the propionate producing bacterium, and the butyrate producing bacterium are administered to prevent or reduce mortality associated with and/or resulting from ARS.

[0107] In some embodiments, the administration improves survival in subjects that have or are suspected of having ARS and/or that have been exposed or are suspected of having been exposed to a high dose of ionizing radiation. In particular embodiments, the subjects’ survival rate is about or at least 50%, 60%, 75%, 80%, 90%, or at least 95% or more after about, at least, or at least about two weeks, four weeks, six weeks, eight weeks, ten weeks, twelve weeks, two months, three months, four months, six months, nine months, twelve months, sixteen months, eighteen months, one year, two years, or three years from the exposure to the dose of ionizing radiation and/or the initial onset of one or more ARS symptoms. In certain embodiments, the subjects who are administered have a survival rate that at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 95%, or at least 1-fold, 1.5 fold, 2 fold, 3-fold, 4-fold, or 5-fold or more after about, at least, or at least about two weeks, four weeks, six weeks, eight weeks, ten weeks, twelve weeks, two months, three months, four months, six months, nine months, twelve months, sixteen months, eighteen months, one year, two years, or three years from the exposure to the dose of ionizing radiation and/or the initial onset of one or more ARS symptoms than is observed in unadmini stered subj ects . III. PHARMACEUTICAL FORMULATIONS

[0108] In certain embodiments, the provided at least one Bifidobacteria, e.g., B. longum subsp. infantis, the provided propionate producing bacterium, e.g., Veillonella sp., and the provided prebiotic mixture, e.g, of human milk oligosaccharides, are formulated together or separately, e.g, for administering to a human subject. In some embodiments, the at least one Bifidobacteria, the provided propionate producing bacterium, and the provided prebiotic mixture, are formulated together in the same pharmaceutical composition. In certain embodiments, the at least one Bifidobacterium and the prebiotic mixture are formulated together into the same pharmaceutical composition. In particular embodiments, the at least one Bifidobacterium and the at least one propionate producing bacterium are formulated together into the same pharmaceutical composition. In certain embodiments, the at least one Bifidobacteria, the provided propionate producing bacterium, and the provided prebiotic mixture are formulated separately into separate pharmaceutical compositions.

[0109] The compositions described herein, e.g., any or all of the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture, may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.). In some embodiments, the compositions described herein are subjected to tableting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

[0110] The probiotic strains (e.g., the provided Bifidobacterium and/or the provided propionate producing bacterium) and prebiotic mixture described herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, immediate-release, pulsatile-release, delay ed- release, or sustained release). Suitable dosage amounts for the provided probiotics may range from about 10⁵ to 10¹² bacteria, e.g., at, at about, or at least 10⁵ bacteria, 10⁶ bacteria, 10⁷ bacteria, 10⁸ bacteria, 10⁹ bacteria, IO¹⁰ bacteria, IO¹¹ bacteria, or 10¹² bacteria, or more.

[OHl] In some embodiments, the prebiotic mixture, e.g., prebiotic mixture of human milk oligosaccharides, is administered to the subject generally in the range of about 20 mg to about 20 g, e.g., total prebiotic weight (such as weight of total human milk oligosaccharides) per dose. In certain embodiments, between or between about 50 mg and 10 g, 100 mg and 7.5 g, 500 mg to 5 g, 1 g and 2.5 g, 50 mg and 20 g, 100 mg and 15 g, 500 mg and 10 g, or 1 g and 7.5 g prebiotic weight per dose. In some embodiments, during an initial treatment phase, the dosing can be higher; for example, 100 mg to 20 g, 100 mg to 30 g, 500 mg to 15 g, 1 g to 10 g, or 2.5 g to 7.5 g per dose. During a secondary treatment phase, the dosing can be reduced; for example, in certain embodiments, to 20 mg to 10 g per dose, 100 mg to 7.5 g per dose, 500 mg to 2.5 g per dose, 750 mg to 1.5 g per dose, 20 mg to 20 g per dose, 100 mg to 10 g per dose, 500 mg to 7.5 g per dose, or 750 mg to 5 g per dose. In certain embodiments, the prebiotic mixture is administered to the subject in an amount of or about 5 g per day. In some embodiments, a dose of the prebiotic mixture is administered at least once per month, once per week, or once per day. In some embodiments, a dose of the prebiotic mixture is administered at least once, twice, three times, four times, five times, six times, eight times, ten times, or twelve times daily.

[0112] In some embodiments, the pharmaceutical compositions may be administered once or more daily, weekly, or monthly. The probiotics (e.g., the at least one Bifidobacterium and/or the at least one propionate producing bacteria) may be formulated, together or separately, into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the probiotic strain may be formulated in a solution of sodium bicarbonate, e.g., 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The prebiotic mixture may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, tri ethyl amine, 2- ethylamino ethanol, histidine, procaine, etc.

[0113] In some embodiments, the pharmaceutical compositions containing the provided at least one Bifidobacterium, the at least one propionate producing bacterium, and the prebiotic mixture, e.g., together or as separate compositions, may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as) polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked agar, alginic acid or a salt thereof such as sodium alginate.

[0114] Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g, pregelatinized maize starch, hydroxypropyl methylcellulose, carboxymethyl cellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, and tragacanth); fillers (e.g, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, such as with membrane selected from, but not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyethylene glycol/poly pentamethylcyclopentasiloxane/ polydimethylsiloxane (PEG/PD5/PDMS), siliceous encapsulates, cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads,, poly(lactide-co-glycolides), carrageenan, starch polyanhydrides, starch polymethacrylates, and enteric coating polymers. [0115] In some embodiments, the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture are enterically coated, such as in order to remain viable during transit through the stomach, reduce contact with bile acids in the small intestine, or for release into the gut or a particular region of the gut, for example, the large intestine. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.

[0116] In certain embodiments, the pharmaceutical compositions are formulated as liquid preparations. Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the bacteria described herein.

[0117] In some embodiments, the at least one Bifidobacterium, the at least one propionate producing bacterium, and prebiotic mixture may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, taste blockers, or suitable to be mixed in a foodstuff, e.g., applesauce. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults. [0118] In certain embodiments, the pharmaceutical composition that is suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors.

[0119] In some embodiments, the pharmaceutical composition, e.g., composition suitable for administration to pediatric subjects, may include a flavor. As used herein, “flavor” is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, cherry, and chocolate.

[0120] In particular embodiments, the at least one Bifidobacterium, the at least one propionate producing bacterium, and the prebiotic mixture may, together or separately, be orally administered, such as with an inert diluent or an assimilable edible carrier. In some aspects, the pharmaceutical composition may also be enclosed in a hard or soft-shell gelatin capsule, a hydroxypropylmethyl cellulose (HPMC) capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the probiotic strain and prebiotic mixture may, together or separately, be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In some aspects, it may be necessary to coat or co-administer the pharmaceutical composition with a material to prevent inactivation of the probiotic strain and/or the prebiotic mixture.

[0121] In some embodiments, the composition containing the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture may be a nutritional or a comestible product, e.g., a food product or nutritional composition. In some embodiments, the composition is a nutritional composition such as food product. In certain embodiments, the food product or nutritional composition is or includes milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In some embodiments, the nutritional composition or food product is a fermented food, such as a fermented dairy product. In particular embodiments, the fermented dairy product is yogurt. In certain embodiments, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In some embodiments, the probiotic strain of the invention, e.g., B. longum subsp. infantis strain, is combined in a preparation containing other live bacterial cells intended to serve as probiotics. In some embodiments, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In certain embodiments, the food product or nutritional composition is a jelly or a pudding. Other food products suitable for administration of the probiotic strain and prebiotic mixtures provided herein are known, such as those described in U.S. Application Nos. 2015/0359894 and 2015/0238545. In yet another embodiment, the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.

[0122] In some embodiments, the composition, e.g, pharmaceutical composition, that includes the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[0123] In some embodiments, disclosed herein are pharmaceutically acceptable compositions containing the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a subject without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion. [0124] Single dosage forms of the pharmaceutical composition containing the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a subject.

[0125] In certain embodiments, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see, e.g., U.S. Pat. No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly((2 -hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[0126] Dosage regimens of the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture may be adjusted to provide a therapeutic response, e.g., to improve or maintain propionate production. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus of one or both of the mixture and the probiotic strain may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

[0127] In some embodiments, ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent.

[0128] The pharmaceutical compositions, e.g., containing the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture, may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C. and 8° C. and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0- 10% sucrose (optimally 0.5- 1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include poly dextrose, dextrins (e.g., maltodextrin (e.g., a native maltodextrin or a resistant maltodextrin)), inulin, P-glucan, resistant starches (e.g., resistant maltodextrin), hydrocolloids (e.g., one or more of gum Arabic, pectin, guar gum, alginate, carrageenan, xanthan gum and cellulose gum), corn syrup solids and the like and polysorbate 80.-. Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

[0129] In some embodiments, the pharmaceutical compositions, e.g., containing one or both of the probiotic strain and prebiotic mixtures are administered with food. In alternate embodiments, the pharmaceutical composition is administered before or after eating food. The pharmaceutical compositions may be administered in combination with one or more dietary modifications, e.g., low-protein diet and amino acid supplementation. The dosage of the pharmaceutical compositions and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.

IV. DEFINITIONS

[0112] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0113] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that aspects and variations described herein include "consisting" and/or "consisting essentially of’ aspects and variations.

[0114] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described or claimed subject matter. This applies regardless of the breadth of the range.

[0115] Throughout this disclosure, ranges that are presented or expressed as “between” two endpoints, e.g., “between A and B” are understood to include the endpoints, e.g. “A” and “B”, unless otherwise indicated.

[0116] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X". In some embodiments, “about” a value means within a range of ±25%, ±10%, ±5%, ±1%, ±0.1%, or ±0.01% of the value.

[0117] As used herein the term "pharmaceutical composition" means, for example, a mixture or formulation containing a specified amount, e.g., a therapeutically effective amount, of an active ingredient such as a human milk fraction, in a pharmaceutically acceptable carrier to be administered to a mammal, e.g., a human.

[0118] As used herein the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms, which are, within the scope of sound medical judgment, suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit/risk ratio. Such reasonable benefit/risk ratios may be determined by one of skill as a matter of routine.

[0119] By “human milk oligosaccharide(s)” (also referred to herein as “HMO(s)”) is meant a family of structurally diverse unconjugated glycans that are found in human breast milk. As used herein human milk oligosaccharides include oligosaccharides found in human milk that contain lactose at the reducing end and, typically, fucose, sialic acid or N- acetylglucosamine at the non-reducing end (Morrow et al., J. Nutri. 2005 135: 1304-1307). Unless otherwise indicated, human milk oligosaccharides also encompass 3'-sialyllactose (3'- SL) and 6'-sialyllactose (6'-SL) oligosaccharides that are found in human milk. [0120] Unless otherwise noted, a number of human milk oligosaccharides, e.g., “at least 5 human milk oligosaccharides,” refers to the number of unique species of human milk oligosaccharides, e.g., human milk oligosaccharides having different chemical structures or formulas.

[0121] Glycans in milk are found as oligosaccharides or conjugated to milk proteins as glycoproteins, or lipid as glycolipids etc. HMO are free glycans that constitute the third most abundant component of human milk, after lactose and lipid (Morrow, 2005). The majority of HMO, however, are not metabolized by the infant and can be found in infant feces largely intact.

[0122] By “consisting essentially” of, as used herein refers to compositions containing particular recited components while excluding other major bioactive factors.

[0123] “Probiotic” as used herein, refers to any live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism, e.g., a mammal such as a human, that contains an appropriate amount of the microorganism. In some aspects, those of skill in the art may readily identify species, strains, and/or subtypes of non-pathogenic bacteria that are recognized as probiotic bacteria. Examples of probiotic bacteria may include, but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Pat. Nos. 5,589,168; 6,203,797; 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).

[0124] “Bifidobacterium” or “Bifidobacteria” as used herein, refers to a genus of gram-positive, nonmotile, anaerobic bacteria. In some aspects, Bifidobacterium are ubiquitous inhabitants of the gastrointestinal tract, vagina, and mouth of mammals, including humans. In certain aspects, Bifidobacteria are one of the major genera of bacteria that make up the gastrointestinal tract microbiota in mammals. In certain aspects, some or all species, subspecies, or strains of Bifidobacterium are probiotics.

[0125] The term "dysbiosis" as used herein refers to a state of the microbiota of the gut or other body area in a subject, in which the normal diversity and/or function of the microbial populations is disrupted. This unhealthy state can be due to a decrease in diversity, the overgrowth of one or more pathogens or pathobionts, symbiotic organisms able to cause disease only when certain genetic and/or environmental conditions are present in a subject, or the shift to an ecological microbial network that no longer provides an essential function to the host subject, and therefore no longer promotes health. According to non-limitative examples, essential functions may include enhancement of the gut mucosal barrier, direct or indirect reduction and elimination of invading pathogens, enhancement of the absorption of specific substances, and suppression of GI inflammation.

[0126] As used herein, the terms “gut microbiome” and “intestinal microbiome” are used interchangeably unless otherwise noted.

[0127] As used herein, “non-digestible” as used in the term “non-digestible carbohydrate” refers to the fact that the carbohydrate is not digested by the host or human subject.

[0128] The term “essentially” such as when used in the phrase “essentially all” of a given substance may be used to infer that the substance, e.g., oligosaccharides, includes unavoidable impurities, e.g., no more impurities than what might be unavoidable with standard techniques for manufacture, formulation, transporting, and storage. Likewise, when used in the phrase “essentially free” of a given substance (or “essentially no” or “essentially none of’ a given substance) may mean no more of the given substance than is unavoidable, e.g., as an impurity.

[0129] The term "internalization" such as in reference to an internalization of an oligosaccharide by a bacterial cell refers to the transfer of the oligosaccharide from the outside of the bacterial cell to the inside of the bacterial cell. Unless otherwise indicated, “internalization of an oligosaccharide” refers to the internalization of the intact oligosaccharide.

EXAMPLES

[0130] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE 1: MANUFACTURE OF A CONCENTRATED HMO MIXTURE

[0131] Human milk from previously screened and approved donors was tested to verify donor identity and then mixed together to generate a pool of donor milk. In a clean room environment, the pool of donor milk was further tested, including for specific pathogens and bovine proteins. After testing, the pooled donor milk was filtered through a 200 pm filter, heated to a temperature of approximately 63 °C for 30 minutes, and then cooled to between 2°C and 8°C. The pooled human milk was then transferred to a centrifuge to separate the cream from the skim. The resulting skim milk was ultra-filtered with a 10 kDa membrane, and the material that passed through the filter was collected as the permeate fraction. The permeate was frozen and stored at approximately -20°C.

[0132] Multiple samples of the permeate fractions were thawed and pooled. The pH of the pooled permeate was adjusted to a target pH of 4.5 ± 0.2. The permeate was then heated to approximately 50°C. Lactase enzyme was added to the permeate at a 0.1% weight/weight (w/w) concentration and incubated at approximately 50°C for 60 minutes. The permeate and lactase enzyme mixture then was cooled to between 20°C and 30°C and clarified by depth filtration (Filtrox CHI 13P). The resulting depth filter filtrate was then ultra-filtered (Biomax- 1 OK membrane) to remove the lactase. The ultra-filtered permeate was then concentrated by nanofiltration using membranes with estimated 400 to 500 Dalton molecular weight cut-off (GE G-5 UF). The concentrated HMO composition was then pasteurized and clarified though 0.2 pm sterile filters. This final HMO composition was then filled into containers and stored at < -20°C. The final concentrations of HMO were quantified using high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD) with commercially available standards.

EXAMPLE 2: ADMINISTRATION OF B. LONGUM SUBSP. INFANTIS AND A CONCENTRATED HUMAN MILK OLIGOSACCHARIDE MIXTURE TO SUBJECTS TREATED WITH ANTIBIOTICS [0133] A clinical study assessing the administration of B. longum subsp. infantis and human milk oligosaccharides in healthy adult subjects who were administered antibiotics was performed as summarized in FIG. 1 and Table El. A total of 56 subjects were enrolled in three different cohorts: those who received antibiotics alone for days 1-5 (Cohort 1); those who received antibiotics on days 1-5 and B. longum subsp. infantis on days 1-14 (Cohort 2); and those who received antibiotics on days 1-5, B. longum subsp. infantis on days 1-14, and a human milk oligosaccharide mixture (HMO mixture) generated as described in Example 1 on days 1-28 (Cohort 3). For antibiotic treatment, subjects were administered 250 mg vancomycin three times daily, which preferentially affects gram positive bacteria, and 500 mg metronidazole three times daily, which affects anaerobes including gram negative bacteria. Used together, these antibiotics broadly impact the gut microbial community. Subjects administered B. longum subsp. infantis received 8xl0⁹ CFU per oral dose of a commercially available probiotic once a day from days 1-14. Subjects receiving the HMO mixture were administered HMO orally in liquid form twice a day from days 1-28 for a total of 18 g/day of total human milk oligosaccharides.

Table El. Study Design Summary

included in analyses. [0134] All subjects were followed to day 35, which was one week after completion of HMO dosing in the corresponding cohort. Stool samples were collected during eligibility screening and on days 1, 3, 4, 5, 7, 9, 11, 14, 17, 21, 28, and 35, where day 1 was the first day of the protocol. Some subjects were asked to return to the study site and provide a stool sample on or after day 90. Blood samples were also drawn on days 1, 5, 14, 28 and 35, processed to serum, and frozen. On study days 1, 5, 9, 14, 28, and 35, stool was refrigerated at 4°C after production and frozen at -80°C within 24 hours. On days 3, 4, 7, 11, 17, and 21 of the study, a portion of each stool was preserved in ethanol immediately after production and frozen at -80°C within one week.

[0135] No serious adverse effects were observed for any of the subjects.

EXAMPLE 3: B. LONGUM SUBSP. INFANTIS DETECTION BY QPCR IN HEALTHY SUBJECTS AFTER VANCOMYCIN AND METRONIDAZOLE EXPOSURE

[0136] Genomic DNA was extracted from stool samples collected from subjects as described in Example 2. Extracted DNA was assessed for B. longum subsp. infantis by qPCR performed similar to as described in Lawley et al., PeerJ. 2017 May 25;5:e3375 with forward and reverse primers to the sialidase gene identical to SEQ ID NOS: 17 and 18 and a probe sequence identical to SEQ ID NO: 19.

[0137] Results are displayed in FIGS. 2A-2C. As shown in FIG. 2A, subjects from Cohort 1 (antibiotics alone) had B. longum subsp. infantis levels that remained at or below the limit of detection (27 copies/ng DNA) or barely detectable (< 2% of samples at or below 93 copies/ng DNA). During the antibiotic dosing period (Days 1-5), low levels of B. longum subsp. infantis were detected in the DNA from stool of subjects in both cohorts that received B. longum subsp. infantis. These levels were consistent with a “pass through” reflecting the daily consumption of the bacteria, as B. longum subsp. infantis is sensitive to both antibiotics used in this study and was unlikely to remain viable in the gut. With continued dosing beyond the antibiotic treatment, B. longum subsp. infantis levels rose in both A longum subsp. infantis-treated cohorts above the “pass-through” levels by day 11. On days 21 and 28, a significant difference was observed between cohorts. Subjects from Cohort 2 (B. longum subsp. infantis alone) exhibited a steady decline in B. longum subsp. infantis levels after the end of dosing and with presumed recovery of the microbiome, while most Cohort 3 subjects (B. longum subsp. infantis and HMO) displayed stable, high levels of B. longum subsp. infantis through day 28. After HMO dosing ceased, levels of B. longum subsp. infantis declined rapidly. Together, these data demonstrate that while antibiotic treatment may temporarily reduce colonization resistance to enable transient expansion of B. longum subsp. infantis, co-dosing with HMO is essential for durable, high-level engraftment.

[0138] In both cohorts dosed with B. longum subsp. infantis (Cohorts 2 and 3), there was evidence of two subpopulations: an “engrafted” population for which B. longum subsp. infantis was consistently maintained during the HMO dosing period, defined as exhibiting B. longum subsp. infantis signal more than two standard deviations above the “pass through” level during antibiotic treatment for at least two consecutive time points, and a “not engrafted” population, where B. longum subsp. infantis was not consistently maintained (FIGS 2B and 2C). Of the Cohort 3 subjects (B. longum subsp. infantis and HMO), 76% (13/17) were deemed engrafted (FIG. 2B). Among the four subjects defined as not engrafted, each produced at least one stool sample without detectable levels of B. longum subsp. infantis during the 14-day dosing period, which may suggest issues with compliance that could have influenced engraftment success. The mean level of B. longum subsp. infantis at day 14 in engrafted subjects was approximately 5 -fold higher than the overall mean when all subjects were included (1.1 x 10⁵ vs 2.4 x 10⁴ copies/ng DNA). Moreover, when only engrafted subjects within the Cohort 3 were compared to Cohort 2, B. longum subsp. infantis levels were significantly higher as early as day 11 (p = 0.0004; Sidak’s post-hoc test; Figure 7A).

[0139] Engraftment was also observed in 4 of the Cohort 2 subjects (B. longum subsp. infantis alone), representing -24% (4/17) of the cohort (FIG. 2C). However, B. longum subsp. infantis levels in these subjects were maintained at -10³ copies/ng DNA through day 35, which is -100-fold lower than for engrafted subjects who also received HMO. The rate of engraftment was also significantly different between the two cohorts (13/17 engrafted vs 4/17 engrafted; p = 0.0053 by Fisher’s exact test). Here, engraftment in the absence of HMO treatment may be due to loss of colonization resistance to B. longum subsp. infantis following antibiotic exposure, although this was observed in only a subset of subjects.

[0140] Some engrafted subjects from both cohorts retained B. longum subsp. infantis signal at day 35. Additional stool samples were from a subset of engrafted subjects on or after day 90 (Table E2). B. longum subsp. infantis was below the limit of detection for the nine subjects analyzed from Cohort 3 (B. longum subsp. infantis and HMO). Of three engrafted subjects evaluated from Cohort 2 (B. longum subsp. infantis only), one displayed levels of B. longum subsp. infantis below the limit of detection and the other two had detectable but low levels (7.7 x 10¹ and 5.79 x 10² copies/ng DNA; Table E2). These data suggest that for the majority of subjects, administration of B. longum subsp. infantis did not lead to durable engraftment after antibiotic treatment in the absence of treatment with HMO.

Table E2: B. longum subsp. infantis levels of a subset of subjects that returned post study

EXAMPLE 4: B. LONGUM SUBSP. INFANTIS DETECTION BY WHOLE META GENOMIC SEQUENCING IN HEALTHY SUBJECTS AFTER VANCOMYCIN AND METRONIDAZOLE EXPOSURE

[0141] Whole metagenomic sequencing (WMS) on stool samples was used with a strain-specific tracking algorithm to quantify the relative abundance of B. longum subsp. infantis in the microbiome for each cohort. [0142] DNA extracted from human stool samples collected at days 1, 5, 9, 14, 28, and 35 as described in Example 2 was used to prepare libraries for shotgun metagenomic sequencing. Paired-end sequencing (2 x 150 bp) was performed on an Illumina NovaSeq instrument to generate a target of ~2 million reads per sample (BoosterShot, Diversigen Inc.).

[0143] Sequence analyses followed an established pipeline (Diversigen Inc.). Briefly, sequences were aligned to a curated database containing all representative genomes in RefSeq for bacteria with additional manually curated strains. Alignments were made at 97% identity against all reference genomes. Every input sequence was compared to every reference sequence in the Diversigen Venti database using fully gapped alignment with BURST 123. Ties were broken by minimizing the overall number of unique Operational Taxonomic Units (OTUs). For taxonomy assignment, each input sequence was assigned the lowest common ancestor that was consistent across at least 80% of all reference sequences tied for best hit. Samples with fewer than 10,000 sequences were discarded. OTUs accounting for less than one millionth of all strain-level markers and those with less than 0.01% of their unique genome regions covered (and < 0.1% of the whole genome) at the species level were discarded. The counts for each OTU in this filtered table were normalized to the OTU's genome length, and filtered tables for the normalized counts and relative abundance of each OTU were generated. Strain-level tracking, including for B. longum subsp. infantis, used the “capitalist” algorithm in BURST to assign reads to genomes in the Diversigen Venti database. Rather than applying a lowest common ancestor approach, the “capitalist” algorithm returns a minimal set of genomes that can explain all the reads in a sample. From that output, an OTU table was calculated with the read counts per genome per sample. Alpha diversity metrics (observed reads and Shannon entropy) were calculated using the filtered OTU table rarefied to a read depth of 76,000 using the R package phyloseq (v.1.41.0). Bray-Curtis dissimilarity was calculated on the same filtered data, aggregated at the genus taxonomic level and rarefied to 76,000 reads using the R package phyloseq (vl.41.0124). The filtered OTU table aggregated at the Family taxonomic level was used to calculate the top Families among cohorts. All taxa >1% abundance in any cohort were kept and all taxa <1% among taxa were grouped into an “Other” category. The count table of aggregated genera was then center log-ratio (CLR) transformed by taking the median CLR value for each OTU sampled 150 times from a Dirichlet model using the R package ALDEx2 (v.1.29.1125). [0144] As shown in FIG. 2D, engrafted Cohort 3 subjects (B. longum subsp. infantis and HMO) displayed a median B. longum subsp. infantis relative abundance of 45.9%, with a maximum of 81% observed in one subject at day 9. Abundance was much lower in engrafted Cohort 2 subjects (B. longum subsp. infantis alone), ranging from 0.7-7.8% with a median of 3.4%. Use of an orthogonal method with different sequence biases determined B. longum subsp. infantis relative abundance by calculating the ratio of B. longum subsp. infantis to total bacterial (16S) qPCR signals (FIG. 7B). This method similarly confirmed the high abundances observed by WMS with median relative abundance of engrafted subjects in the B. longum subsp. infantis and HMO cohort at 8.6% with a range of 0.05-48.4% during days 14- 28.

[0145] These data are consistent with promotion of B. longum subsp. infantis engraftment by human milk oligosaccharides in a higher frequency of subjects and at high abundance in subjects where dysbiosis has been induced by antibiotics. This durable, high- level engraftment is consistent with a high likelihood of positively impacting the gut microbiome.

EXAMPLE 5: HUMAN MILK OLIGOSACCHARIDE-MEDIATED ENGRAFTMENT WITH B. LONGUM SUBSP. INFANTIS ALTERS MlCROBIOME COMMUNITIES AFTER TRANSIENT, ANTIBIOTIC-INDUCED DYSBIOSIS

[0146] WMS data generated as described in Example 4 from genomic DNA collected from stool samples from Cohort 1 (antibiotics only control) and Cohort 3 (HMO and B. longum subsp. infantis) subjects described in Example 2 was assessed. Antibiotic treatment induced a transient, acute dysbiosis in all subjects of both Cohorts. Comparison of samples from days 1 (baseline) to samples from day 5 (end of antibiotic dosing) displayed robust decreases in microbial diversity and a consistent shift in the microbial community composition (FIGS. 3 and 8A). Shannon Entropy, which quantifies diversity and evenness in community structure, was significantly decreased in both cohorts at day 5 relative to day 1 (FIG. 3 A); similar results were observed when alpha diversity was quantified using observed reads (FIG.8A). As expected, there was also a significant decrease in total bacterial abundance, quantified using qPCR of the 16S rRNA gene (FIG. 8B). Overall microbiome community structure, as measured by Bray-Curtis dissimilarity, was also significantly different on day 5 relative to day 1 (FIG. 3B and Table E3). Levels of Lactobacillaceae and Enterob acteriaceae increased from <0.2% of the population to >76% and >16% respectively (FIG. 3C).

Table E3. PERMANOVA p-values per day of study, related to Figure 3.

Bray-Curtis dissimilarity was calculated for each sample with rarefied sequences aggregated to the genus taxonomic level, and PERMANOVA analysis was run with the pairwise Adonis package (v0.4).

[0147] From day 5 to 35, all measures of microbiome diversity rebounded in both cohorts. Shannon Entropy partially recovered, as values at day 35 increased from day 5, but also remained significantly different from day 1 baseline levels, suggesting recovery was incomplete at day 35 (FIG. 3 A). Incomplete recovery was also observed for measures of bacterial abundance (FIGS. 8A and 8B). Similarly, microbial community composition became progressively more similar to pre-treatment samples, although it remained distinct from baseline in all cohorts at all post-antibiotic timepoints by PERMANOVA analysis, consistent with incomplete recovery or recovery to an alternate stable state (FIG. 3B, Table E3). Levels of Lactobacillaceae and Enterobacteriaceae, which were at high abundance at the end of antibiotic treatment, decreased during the post-antibiotic period, and by day 28, bacterial families characteristic of a healthy human microbiome such as Lachnospiraceae and Bacteroidaceae were observed in both cohorts (FIG. 3C), suggesting that a high abundance of B. longum subsp. infantis does not preclude recovery of these taxa. [0148] Cohort 3 (B. longum subsp. infantis and HMO) was compared to Cohort 1 (antibiotics only control). Both Shannon Entropy and observed reads suggested a slower recovery toward baseline levels for Cohort 3 subjects (FIGS. 3A and 8A), although total bacterial abundance did not differ between cohorts (FIG. 8B). There were also significant differences in microbiome composition between the cohorts when days 14 and 28 were compared (p < 0.001, Table E4).

Table E4. PERMANOVA p-values between cohorts

Bray-Curtis dissimilarity was calculated for each sample with rarefied sequences aggregated to the genus taxonomic level, and PERMANOVA analysis was run with pairwise Adonis package (v0.4). “With Bifidobacterium" comparisons included all reads, while comparisons “Without Bifidobacterium" were run on samples with reads assigned to the genus Bifidobacterium removed prior to analysis.

[0149] Possible subtle changes in the underlying community structure of engrafted subjects were assessed by subtracting reads corresponding to the genus Bifidobacterium using an approach similar to the American Gut Project to exclude data resulting from microbial blooms during sample storage at room temperature (Amir et al., (2017). mSystems 2, e00199- 16). Even with Bifidobacterium excluded, there were statistically significant differences in microbial community structure between engrafted subjects in Cohort 3 compared to Cohort 1 at day 14 and day 28 (FIG. 3D, Table E4). Further, measuring the distance between each point and the centroid for its respective cohort indicated that there is significantly less Bray- Curtis dissimilarity among individual subjects within the B. longum subsp. infantis + HMO cohort compared to the antibiotic cohort at day 14, regardless of the inclusion of Bifidobacterium reads (FIG. 3D, p<0.05 by ANOVA). This result is consistent with the engraftment of B. longum subsp. infantis after antibiotic treatment may steer microbiome recovery toward a more consistent community structure. [0150] The abundance of specific taxa in engrafted Cohort 3 (B. longum subsp. infantis and HMO) subjects was compared to Cohort 1 subjects (antibiotics only control). Besides the expected increase in Bifidobacterium signal at various timepoints, the genera Lactobacillus and Veillonella were present at significantly higher levels in Cohort 3 at days 14 and 28, as was Pediococcus at day 14 (FIGS. 3E and 3F and 8E and 8F). Taxa that differed when Bifidobacterium reads were subtracted from the dataset displayed similarly significant increases of the same three genera. Lactobacillus became dominant in the microbiome of nearly all subjects during the antibiotic treatment period, and limited consumption of HMO has been reported for some strains, so it is possible that persistence of this genus was associated with either B. longum subsp. infantis abundance or the presence of HMO. Median Veillonella abundance increased from <0.05% at day 1 in both cohorts to a maximum of 9.8% and average of ~2% after antibiotic treatment at day 9 and appeared to be maintained at higher levels (average of ~2%) in the presence of B. longum subsp. infantis and HMO by day 14, whereas in the antibiotics only cohort had dropped below 1% by this point (FIG. 3F). Veillonella is known to consume lactate, one of the organic acids produced by B. longum subsp. infantis during fermentation of human milk oligosaccharides, so it may be enriched due to increased availability of lactate in the gastrointestinal environment when B. longum subsp. infantis is present. Enrichment of Lactobacillus could also lead to increased lactate availability, and this occurring during antibiotic treatment may explain early (day 9) increases in Veillonella in both cohorts, even though by day 14 it was significantly more abundant in subjects engrafted with B. longum subsp. infantis. Additionally, there was significantly less Clostridiales at the end of the antibiotic treatment period (day 5) in the B. longum subsp. infantis + HMO cohort (FIGS 3E and 8G), and Ruminococcus was also significantly lower but only at day 14 (FIGS. 3E and 8H)

EXAMPLE 6: QUANTIFICATION OF SHORT-CHAIN FATTY ACIDS AND LACTATE IN HUMAN STOOL

[0151] Stool samples collected as described in Example 2 were sent to Precion, Ltd. (Morrisville, NC) for extraction and analysis. A portion of a neat sample was transferred into a 2 mL cryotube containing three stainless steel 1/8” cone balls, and the exact weight of the sample was recorded. A solution of deuterium-labelled internal standards in water (50 pL) and 1.5 mL of methanol was added to the cryotube and the sample was homogenized by vortex-mixing for 2 minutes. Alternatively, ethanol-preserved stool samples were centrifuged and 50 pL of the supernatant was transferred to a 2 mL cryotube together with 50 pL standards and 1.0 mL methanol, and vortex-mixed for 2 minutes. The resulting suspensions were centrifuged at 2,000 x g at 20°C for 10 minutes, and 50 pL of the supernatant was transferred to a 96-well plate and derivatized using a modified version of the published protocol. An aliquot of the reaction mixture was analyzed by liquid-chromatography mass spectrometry (LC-MS/MS) using an Exion UHPLC (Sciex) coupled to a 5500+ Triple Quadrupole Mass Spectrometer (Sciex) in ESI negative mode using a C18 column (Zorbax Eclipse plus C18 1.8 micron, 2.1x50 mm, Agilent). The peak areas of the respective parent to product ion transitions were measured against the peak area of the parent to product ion transitions of the corresponding labelled internal standards for the quantitated metabolites. Quantitation was performed with Sciex OS-MQ software (Sciex) based on fortified calibration standards prepared immediately prior to each run.

[0152] For neat stool samples, raw data were weight corrected to account for the individual wet weight of each sample providing the analyte content in pg/g wet weight. Additionally, using a separate sample aliquot, the dry weight/wet weight ratio was determined for each sample and each dry weight/wet weight ratio was used to calculate the analyte content of each sample in pg/g dry weight. For ethanol-preserved samples, raw data were weight corrected to account for the individual dry weight of each sample. Using a separate sample aliquot (750 pL sample suspension), the dry weight/volume ratio was determined for each sample. Each dry weight/volume ratio was used to calculate the analyte content of each sample in pg/g dry weight. Data were then transformed to mmol/kg by dividing by the molecular weight of the analyte.

EXAMPLE 7: QUANTIFICATION OF METABOLITES IN HUMAN SERUM

[0153] Serum samples collected as described in Example 2 were quantitatively analyzed by LC-MS/MS for 70 analytes at Precion Ltd., Morrisville, NC, USA. This analysis consisted of two extractions of the serum samples. To the first extraction (100 pL), a solution of stable labelled internal standards was added followed by protein precipitation. After centrifugation, one portion of the supernatant was removed, evaporated to dryness, reconstituted and an aliquot analyzed on a Sciex Exion LC/Sciex 5500+ Triple Quadrupole Mass Spectrometer LC-MS/MS system in ESI negative mode using Cl 8 reversed phase chromatography. This assay covered organic acids, phenolic compounds, sulfates, and other analytes that are negatively charged under negative ESI mass spectrometer conditions. A second portion of the supernatant was removed, evaporated to dryness, reconstituted, and an aliquot analyzed on a Sciex Exion LC/Sciex 5500+ Triple Quadrupole Mass Spectrometer LC-MS/MS system in ESI positive mode using Cl 8 reversed phase chromatography. This analysis covered amino acids, amines, and other analytes that were positively charged under positive ESI mass spectrometer conditions.

[0154] A second extraction was performed from 50.0 pL of the serum sample. A solution of stable labelled internal standards was added to serum samples followed by protein precipitation. After centrifugation, a portion of the supernatant was removed and derivatized with a substituted hydrazine to form the corresponding acid hydrazides of short chain fatty acids. An aliquot of the resulting mixture was analyzed on a Sciex Exion LC/Sciex 5500+ Triple Quadrupole Mass Spectrometer LC-MS/MS system in ESI positive mode using Cl 8 reversed phase chromatography.

[0155] For all methods, the peak areas of the respective parent to product ion transitions were measured against the peak areas of the parent to product ion transitions of the corresponding labelled internal standards. Stable labelled versions of each of the 70 analytes were used as internal standards. Quantitation was performed using a weighted linear least squares regression analysis generated from fortified calibration standards (6 to 10 concentration levels, depending on analyte) prepared concurrently with study samples and quality control samples in each analytical run.

EXAMPLE 8: GLOBAL METABOLOMICS ANALYSIS OF HUMAN STOOL

[0156] Untargeted metabolomic profiling was performed on stool samples collected as described in Example 2 from days 1, 5, and 14 at Metabolon, Inc (Morrisville, NC, USA) using a combination of LC-MS methods (Evans et al., (2014). Metabolomics 4, 1000132). All methods utilized a Waters ACQUITY UPLC and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. Briefly, the sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. Based on Metabolon, Inc protocols and previously published methods, the first aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. The second aliquot was also analyzed using acidic positive ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. The third aliquot was analyzed using basic negative ion optimized conditions and a separate dedicated Cl 8 column. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1 x 150 mm, 1.7 pm) using a gradient consisting of water and acetonitrile with 10 mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSⁿ scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z.

[0157] Raw data were extracted, peak-identified, and QC processed using Metabolon’s hardware and software. These systems are built on a web-service platform utilizing Microsoft’s .NET technologies, which run on high-performance application servers and fiber-channel storage arrays in clusters to provide active failover and load-balancing. Compounds are identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a library based on authenticated standards that contains the retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Furthermore, biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library +/- 10 ppm, and the MS/MS forward and reverse scores. MS/MS scores are based on a comparison of the ions present in the experimental spectrum to ions present in the library entry spectrum. While there may be similarities between these molecules based on one of these factors, the use of all three data points can be utilized to distinguish and differentiate biochemicals. More than 4500 commercially available purified standard compounds have been acquired and registered into a database for analysis on all platforms for determination of their analytical characteristics. Additional mass spectral entries have been created for structurally unnamed biochemicals, which have been identified by virtue of their recurrent nature (both chromatographic and mass spectral). These compounds have the potential to be identified by future acquisition of a matching purified standard or by classical structural analysis.

[0158] Metabolites were identified by comparison to a referenced library of chemical standards, and area-under-the-curve analysis was performed for peak quantification and normalized to day median value. To ensure high quality of the dataset, control and curation processes were subsequently used to ensure true chemical assignment and remove artifacts and background noise. Metabolites were scaled by run-day medians and log-transformed before statistical analysis. Two-way repeated measures ANOVA contrasts used to analyze the data. For all analyses, missing values, if any, were imputed with the observed minimum for that compound. The statistical analyses were performed on natural log-transformed data using Array Studio.

EXAMPLE 9: ADMINISTRATION OF B. LONGUM SUBSP. INFANTIS AND HMO SIGNIFICANTLY ALTERS GUT METABOLITES IN SUBJECTS TREATED WITH ANTIBIOTICS

[0159] The impacts of B. longum subsp. infantis and HMO treatment on fecal and serum metabolite profiles were evaluated using a quantitative fecal assay for short-chain fatty acids and lactate as described in Examples 6, an untargeted global fecal metabolomics method as described in Example 7, and a targeted serum assay for microbiome-associated metabolites as described in Example 8. Only data from the engrafted subjects within Cohort 3 were included in these analyses.

[0160] Antibiotic treatment radically impacted metabolites detectable in both fecal and serum samples, with decreases between day 1 and day 5 in microbially-produced metabolites such as short-chain fatty acids, secondary bile acids, and certain tryptophan and tyrosine metabolites (FIGS. 4 and 9). A significant increase in lactate was observed in all subjects during this time frame as well (FIG. 4C), likely attributable to the blooms of Lactobacillaceae in all subjects (FIG. 3).

[0161] The high abundance of B. longum subsp. infantis in engrafted subjects, coupled with the availability of human milk oligosaccharides, suggested that the organic acids produced during the fermentation of human milk oligosaccharides should be detectable. Consistent with recovery of the microbiome after antibiotic treatment, levels of fecal acetate began to increase in all subjects in both cohorts after day 5 (FIG. 4 A). Although there were no significant differences in acetate at individual timepoints between cohorts, levels of acetate significantly differed between the cohorts when the data were analyzed over time (FIG. 4A). Recovery of fecal acetate levels was assessed across subjects in both cohorts using a Kaplan-Meier curve. All subjects in Cohort 3 (B. longum subsp. infantis + HMO) recovered back to the day 1 average and recovered significantly faster than those in Cohort 1 (antibiotics only control); moreover, 2 subjects in Cohort 1 had not returned to this baseline level by day 35 (FIG. 4B). This observation is consistent with promotion of acetate recovery by treatment with B. longum subsp. infantis and HMO. A correlation between acetate levels and B. longum subsp. infantis abundance was observed across days 5-28 in Cohort 3 (Pearson r=0.73; r2 0.53). Cohort 3 stool lactate levels were increased on days 11, 14, 17, and 28 (FIG 4C). Consistent with an increase in organic acids, stool from engrafted subjects within the B. longum subsp. infantis + HMO cohort had a significantly lower pH (5.8) on day 9 than stool from the control cohort (6.6) (FIG. 9C).

[0162] No difference in recovery of butyrate or propionate levels was observed between the two cohorts (FIGS. 9A and 9B). However, cross-sample analysis of SCFAs in stool samples may be confounded by differences in absorption through the distal gastrointestinal tract.

[0163] To look more broadly at the impact of B. longum subsp. infantis engraftment on the metabolome and the host, two complementary approaches were employed: global metabolomics analysis at days 1, 5, and 14 to identify differences in diverse gut metabolites, and a quantitative targeted panel for microbial metabolites in serum over time to highlight changes that translate to a systemic impact. Little difference was observed in fecal global metabolite profiles between cohorts on day 1 prior to antibiotic treatment (FIG. 9D). Within each cohort, significant changes were observed from days 1 to 5 and again from days 5 to 14, reflective of the changes in bacterial diversity caused by antibiotic treatment (FIGS. 9E-9I). However, on day 14, when B. longum subsp. infantis was at high levels in engrafted subjects, 231 metabolites were significantly different between the cohorts; 77 metabolites were decreased and 154 were increased (FIG. 4D). A subset of these (56/231) are unidentified compounds, and others could be linked directly to treatment with B. longum subsp. infantis and HMO (lactate, 3-fucosyllactose). Increased levels of indolelactate, a potentially beneficial immunomodulatory tryptophan metabolite known to be produced by B. longum subsp. infantis and other gut microbes, were observed in both serum and stool samples (FIG. 4E).

[0164] The uremic toxin p-cresol sulfate (pCS) was decreased in both fecal and serum samples of Cohort 3 subjects (FIG. 4F). pCS is a host-generated conjugate of p-cresol known to contribute to inflammatory conditions and is produced from tyrosine or phenylalanine by intestinal bacteria. Serum levels of other tyrosine and phenylalanine metabolites were also shifted, including decreases in the uremic toxins phenol sulfate (FIGS. 9M and 9N) and p- cresol-glucuronide (pCG), and in other pathway intermediates 4-hydroxyphenylpropionate, 3- hydroxyhippurate, and 3 -hydroxybenzoate. Conversely, there were increases in 4- hydroxyphenylacetate, the precursor of p-cresol, and phenyllactate, an alternative microbial metabolic pathway for tyrosine and phenylalanine. Together, these changes suggest /?. longum subsp. infantis + HMO treatment may redirect tyrosine and phenylalanine flux away from production of inflammatory toxins.

[0165] Several bile acids were differentially detected in fecal samples. Two hostgenerated glucuronide conjugates of the primary bile acid cholate and its associated secondary bile acid deoxycholate were increased in the B. longum subsp. infantis + HMO cohort (Figure 4D). Additionally, there were significant decreases in the secondary bile acids ursocholate, ursodeoxycholate, and isoursodeoxycholate, which are all 7-P-hydroxy epimers and associated derivatives of the primary bile acids cholate and chenodeoxycholate (FIG. 4D). Ursodeoxycholate was also included in the serum analysis, but no changes were observed between cohorts for that or two other bile acids: chenodeoxycholate and cholate. Interestingly, serum levels of the secondary bile acid deoxycholate were similar between the cohorts at day 14 but began to decrease in the B. longum subsp. infantis + HMO cohort at day 28.

[0166] Engraftment of B. longum subsp. infantis influenced the levels of a number of other metabolites indicative of changes in microbial metabolism (FIG. 4D). Over a quarter of the identified metabolites (47) are considered xenobiotics, or chemical compounds not naturally produced in the gut. Other identified metabolites are associated with carbohydrate metabolism (12), amino acids and derivatives (45), and lipid synthesis and metabolism (39), suggesting general shifts in microbial metabolism, gut mucosal properties, and/or enterocyte energetics. For example, increases in carbon sources, such as sugar alcohols and plant-based molecules like feruloylquinates, suggest either decreased degradation of these molecules or increased absorption, and increases in eight gamma-glutamyl amino acids suggest altered protein degradation, reflecting a change seen in infants that consume HMO that is linked to lower rates of respiratory illness.

EXAMPLE 10: ISOLATION OF VEILLONELLA SP. FROM STOOL SAMPLES

[0167] Bacterial strains of Veillonella sp. were isolated from fecal samples of Cohort 3 subjects (B. longum subsp. infantis and HMO) described in Example 2. The fecal samples had been previously preserved at -80°C, and an aliquot was thawed and serially diluted into anoxic filter-sterilized PBS (pH 7.0). Aliquots of serial dilutions were plated onto prereduced Brucella Blood Agar (Anaerobe Systems). Plates were incubated anaerobically at 37°C for 2-5 days. Individual colonies were picked, arrayed onto a fresh plate of the same agar, and regrown for 2-5 days. DNA was extracted from the arrayed isolates using an alkaline lysis buffer technique (Vyhlidalova et al. (2020). IJMS 21, 2614) and the bacterial 16S rRNA gene was amplified by PCR using the established universal 16S primers 27F and 1492R (Weisburg et al. (1991) J Bacteriol 173, 697-703). The PCR products were cleaned using Exo-SapIT (Applied Biosystems) and two-directional Sanger sequencing was performed using the same primers to assign taxonomic identity to each strain (Azenta Life Sciences).

[0168] Under anaerobic conditions, pure cultures of arrayed isolates identified as Veillonella were prepared by picking and streaking onto fresh media three times. A single colony was inoculated into 30 mL of liquid Reinforced Clostridial Media (RCM) with 60% sodium lactate, and the culture was grown anaerobically at 37°C for 1-3 days. The cell culture was mixed with pre-reduced glycerol and PBS to a final concentration of 15% glycerol, aliquots were distributed to cryovials, and the cryovials were frozen and stored at - 80°C. The same culturing conditions were used to propagate and bank V. parvula ATCC 10790, after initial revival from a lyophilized stock by streaking onto BRU.

[0169] Pure banks of isolates were subjected to PCR and Sanger sequencing using the 16S rRNA primers described above and primers specific to the Veillonella rpoB gene, with resulting sequences aligned to known Veillonella sequences to assign specific species identity. One strain was identified as highly similar to V. infantium and a second strain was identified as V. rogosae.

EXAMPLE 11: ENHANCED IN VITRO GROWTH OF AND PRODUCTION OF PROPIONATE BY VEILLONELLA SP. CO-CULTURED WITH B. LONGUM SUBSP. INFANTIS AND HMO

[0170] The two Veillonella strains isolated as described in Example 11 were evaluated for in vitro growth by optical density and species-specific qPCR. A commercially available strain of V. parvula sourced from the human gut was used for comparison. Levels of propionate, lactate, and acetate were measured during growth and, while kinetics varied between the strains, all Veillonella sp. grew and produced propionate and acetate in a lactatedependent manner (FIGS. 5A and 10A-10E). Veillonella sp. were cultured alone, with human milk oligosaccharides prepared from the mixture described in Example 1 (HMO), cocultured with B. longum subsp. infantis, or cocultured with B. longum subsp. infantis in the presence of human milk oligosaccharides. Neither human milk oligosaccharides alone nor B. longum subsp. infantis alone supported Veillonella growth or propionate production, however, when both B. longum subsp. infantis and HMO were included, all Veillonella sp. grew and produced more propionate than with an equivalent level of lactate alone (FIGS. 5B-5D). These data are consistent with B. longum subsp. infantis production of lactate during growth on HMO cross-feeding with Veillonella. resulting in promotion of Veillonella growth and propionate production.

EXAMPLE 12: ENHANCED IN VIVO GROWTH OF AND PRODUCTION OF PROPIONATE BY VEILLONELLA SP. CO-CULTURED WITH B. LONGUM SUBSP. INFANTIS AND HMO

[0171] Germ-free mice were inoculated with V. parvula or B. longum subsp. infantis as a control and, after a one-week stabilization, mice were gavaged with either PBS or B. longum subsp. infantis with human milk oligosaccharides prepared as described in Example 1 (HMO; FIG. 6A). Over the following three days, mice were gavaged once per day with either PBS or HMO. V. parvula levels increased over time only in mice treated with B. longum subsp. infantis + HMO but not in the control mice (FIGS. 6B and 11 A). B. longum subsp. infantis levels were predictably only detected after mice were dosed with B. longum subsp. infantis (FIG. 1 IB). Three hours after the final gavage, all animals were euthanized and levels of propionate, lactate, and acetate in different intestinal segments were quantified (FIGS. 6C-6D, 11C). Mice colonized with V. parvula and subsequently treated with B. longum subsp. infantis and HMO had an increase in Veillonella abundance and an increase in cecal propionate relative to PBS-treated mice (FIG. 6C), indicating that cross-feeding occurred. The differences in propionate levels were not significant in freshly collected rectal samples or cage-bottom fecal samples, suggesting absorption or other factors may introduce variability in samples from these compartments as has been previously observed in both rodents and humans. No propionate was detected in mice monocolonized with B. longum subsp. infantis (FIG. 6C). Mice monocolonized with A longum subsp. infantis and treated with HMO had high levels of lactate in cecal and rectal samples. In contrast, levels were lower in V. parvula-associated mice treated with B. longum subsp. infantis + HMO (FIG. 6D) even though similar levels of B. longum subsp. infantis were measured in both groups (FIG. 1 IB). Lower lactate levels are consistent with lactate consumption by Veillonella.

EXAMPLE 13: EVALUATING ADMINISTRATION OF B. LONGUM SUBSP. INFANTIS AND HMO IN A MOUSE MODEL OF ACUTE RADIATION SYNDROME (ARS)

[0172] Mice are exposed to a total body dose of ionizing radiation that is sufficient to induce gastrointestinal damage. The irradiated mice are orally administered human milk oligosaccharides and B. longum subsp. infantis or a control. Controls may include vehicle (e.g., saline or PBS) and/or B. longum subsp. infantis alone or human milk oligosaccharides alone. The mice are assessed for survival, clinical scores, and/or incidence of diarrhea.

[0173] Irradiated mice administered human milk oligosaccharides and B. longum subsp. infantis may have improved survival as compared to control mice. Irradiated mice administered human milk oligosaccharides and B. longum subsp. infantis may have clinical scores indicating reduced severity of gastrointestinal damage or disease in mice administered human milk oligosaccharides and B. longum subsp. infantis as compared to controls. These mice may also have less instances of diarrhea or runny stool than control mice.

[0174] At the study endpoint, mice are sacrificed and plasma, cecal contents, and tissue samples are harvested. Stool samples and/or cecal contents may be evaluated to quantify microbial compositions of intestinal microbiota. Samples from mice administered human milk oligosaccharides and B. longum subsp. infantis may have greater levels, amounts, or portions of B. longum subsp. infantis than samples from control mice. These samples may also have greater diversity or less levels, amounts, or portions or percentage (e.g., relative to total intestinal microbiota) of pathogenic bacteria.

[0175] Histological analysis of intestinal tissue samples may indicate a higher degree or more instances of tissue regeneration, e.g., intestinal crypt regeneration, in mice administered human milk oligosaccharides and B. longum subsp. infantis than are observed in control mice. Histological analyses may also reveal more cellular proliferation and/or more cells positive for proliferation markers (e.g., Ki-67) in intestinal tissues collected from mice administered human milk oligosaccharides and B. longum subsp. infantis than observed tissues from control mice. Tests may also indicate reduced_intestinal barrier permeability and/or reduced pathogen translocation in mice administered human milk oligosaccharides and B. longum subsp. infantis as compared to mice in control groups.

[0176] Tissues and/or plasma samples collected from irradiated mice may also be tested for markers of inflammation. Tissues and/or plasma samples collected from mice administered human milk oligosaccharides and B. longum subsp. infantis may have lower detectable levels or amounts of one or more inflammatory markers compared to plasma and/or tissues collected from mice in control groups.

[0177] Intestinal contents and plasma samples from irradiated mice may also be tested for the presence, amount, and/or concentration of metabolites, e.g., short chain fatty acids such as including acetate, lactate, butyrate, and/or propionate. Irradiated mice administered B. longum subsp. infantis may have altered levels of metabolites as compared to controls, including metabolites such as short chain fatty acids and/or metabolites with known or suspected anti-inflammatory properties.

[0178] Follow-on experiments may include experimental groups of irradiated mice administered human milk oligosaccharides, B. longum subsp. infantis, and Veillonella sp. Observations of increased survival, reduced gastrointestinal damage severity, increased regeneration, reduced intestinal permeability, and/or reduced inflammation with respect to irradiated mice administered with B. longum subsp. infantis and human milk oligosaccharides and/or mice treated with controls may be observed.

[0179] Similar experiments may be performed in other animal models, such as in rats, cats, dogs, or non-human primates. In these experiments, irradiated animals orally administered B. longum subsp. infantis and human milk oligosaccharides may have increased survival, reduced gastrointestinal damage severity, increased regeneration, reduced intestinal permeability, and/or reduced inflammation with respect to animals receiving control treatments.

EXAMPLE 14: VEILLONELLA SP. CULTURED IN MEDIA CONTAINING LACTATE PRODUCE PROPIONATE

[0180] Various strains of bacteria including strains from the genus Veillonella were cultured in a yeast casitone fatty acid (YCFA) broth similar to as described in Duncan et al., (International Journal of Systematic and Evolutionary Microbiology, 52(6): 2141-2146 (2002)), but lacking propionate and valerate, and with lactate added as the main fermentable carbohydrate source (1.25% weight/volume). After incubation for 48 hours, propionate and lactate levels were measured. As shown in FIG. 12, propionate was detected above background levels (approximately 0 pg/mL) in the incubated media accompanied by decreased levels of lactate as compared to initial levels (dotted vertical line) for all Veillonella sp. strains tested. Similar levels of propionate and lactate were observed in cultures of Anaerotignum lactatifermentans and Megasphera massiliensis . Detectable levels of propionate were also detected in media from Bacteroides fragilis (B. fragilis) cultures. While propionate detection was coupled with decreased lactate in media from some B. fragilis test strains, other media from other B. fragilis test strains had detectable propionate levels and increased lactate concentration relative to initial lactate levels. Notably, while lactate was provided as the main carbon source, the YCFA media also contained other potential carbon sources such as acetate and amino acids. Taken together, these results are consistent with the capability for Veillonella sp., as well as species of the genera Anaerotignum and Megasphaera and some strains of B. fragilis, to produce propionate in the presence of lactate.

EXAMPLE 15: ENHANCED IN VIVO PRODUCTION OF PROPIONATE BY VEILLONELLA SP. COCULTURED WITH B. LONGUM SUBSP. INFANTIS AND HMO

[0181] In vivo propionate production was assessed in various strains of Veillonella in mice initially inoculated with Veillonella sp. or B. longum subsp. infantis, and then subsequently treated with oral gavage of phosphate buffered saline (PBS) or B. longum subsp. infantis with human milk oligosaccharides (HMO) similar to as described in Example 12. The Veillonella test strains included V. parvula, V. atypica, and the two Veillonella strains isolated as described in Example 11.

[0182] As shown in FIGS. 13A-C, mice inoculated with V. parvula or the isolated Veillonella strains had detectable levels of Veillonella in stool at study days 8-11 regardless of whether they were treated with B. longum subsp. infantis and HMO. Similarly, mice inoculated with ZZ. longum subsp. infantis had detectable levels of B. longum subsp. infantis in stool at study days 8-11 regardless of whether they were treated with HMO. In mice initially inoculated with V. parvula or the Veillonella strains and later administered B. longum subsp. infantis and HMO, stool levels of B. longum subsp. infantis increased from the level of detection at day 8 to detectable levels at days 10 and 11. Of the eighteen mice inoculated with the strain of V. atypica prepared for these experiments, fifteen mice across experimental conditions did not display V. atypica engraftment (data not shown).

[0183] Three hours after the final gavage, mice were euthanized and levels of propionate, lactate, and acetate in different intestinal segments were quantified. Increased levels of propionate were observed in cecal samples collected from FeZZZcweZZa-inoculated test mice administered B. longum subsp. infantis and HMO with respect to cecal samples collected from control mice (FIG. 14A-14C). In contrast, lower levels of lactate were detected in cecal samples collected from Veillonella- -inoculated test mice administered B. longum subsp. infantis and HMO than in samples from control mice that received B. longum subsp. infantis and HMO (FIG. 15A-15C). Acetate levels did not appear to be reduced in cecal samples collected from Veillonella-inoculated test mice administered B. longum subsp.

I l l infantis and HMO as compared to samples from control mice that received B. longum subsp. infantis and HMO (FIG. 16A-16C). Taken together, these data are consistent with in vivo cross-feeding whereby Veillonella sp. consumes lactate produced by B. longum subsp. infantis through HMO consumption.

EXAMPLE 16: ENHANCED IN VIVO PRODUCTION OF PROPIONATE BY MEGASPHAERA SP. COCULTURED WITH B. LONGUM SUBSP. INFANTIS AND HMO

[0184] In vivo propionate production was assessed in Megasphaera elsdenii (M. elsdenii) in mice initially inoculated with M. elsdenii or B. longum subsp. infantis, and then subsequently treated with oral gavage of phosphate buffered saline (PBS) or B. longum subsp. infantis with human milk oligosaccharides (HMO) similar to as described in Example 12. Germ-free mice were initially inoculated with a A-/. elsdenii or B. longum subsp. infantis as a control. After a one-week stabilization, mice were gavaged with B. longum subsp. infantis and HMO followed by daily gavage with HMO for three days or with PBS.

[0185] As shown in FIG. 17A, mice inoculated with M. elsdenii had detectable levels of M. elsdenii in stool at study days 8-11 regardless of whether they were treated with PBS or B. longum subsp. infantis and HMO. Similarly, mice inoculated with B. longum subsp. infantis had detectable levels of B. longum subsp. infantis in stool at study days 8-11 regardless of whether they were treated with HMO. In mice initially inoculated with M. elsdenii and later administered B. longum subsp. infantis and HMO, stool levels of B. longum subsp. infantis increased from the level of detection at day 8 to detectable levels at days 10 and 11.

[0186] Three hours after the final gavage, mice were euthanized and levels of short chain fatty acids were assed in cecal samples collected from the mice. Levels of butyrate, propionate, valerate, lactate, and acetate in cecal contents were quantified (FIGS. 17B-17F). Increased levels of butyrate, propionate, and valerate were observed in samples from mice inoculated with

elsdenii that were administered B. longum subsp. infantis and HMO as compared to samples collected from control mice (FIGS. 17B-17D). In contrast, lower levels of lactate were detected in AL elsdenii inoculated mice administered B. longum subsp. infantis and HMO than in samples from control mice that received B. longum subsp. infantis and HMO (FIG. 17E). Acetate levels did not appear to be reduced in cecal samples collected from Veillonella-inoculated test mice administered B. longum subsp. infantis and HMO as compared to samples from control mice that received B. longum subsp. infantis and HMO (FIG. 17F).

SEQUENCES

Claims

1. A method of treating or preventing a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium.

2. A method of ameliorating a symptom of a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium.

3. The method of claim 1 or 2, wherein the subject has received or will receive an allogenic hematopoietic stem cell transplant, and wherein the disease, condition, or disorder comprises graft versus host disease.

4. The method of claim 1 or 2, wherein the disease, disorder, or condition comprises one or more of obesity, type II diabetes, a chronic inflammatory disease, an autoimmune disease, an infection, an infectious disease domination, bowel resection, or a condition associated with chronic diarrhea.

5. The method of claim 1 or 2, wherein the disease, disorder, or condition comprises one or more of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), short bowel syndrome (SBS), celiac disease, small intestinal bacterial overgrowth (SIBO), gastroenteritis, leaky gut syndrome, pouchitis, or gastric lymphoma.

6. The method of claim 1 or 2, wherein the disease, disorder, or condition comprises an atopic disease.

7. The method of claim 6, wherein the atopic disease comprises atopic dermatitis, food allergy, and/or asthma.

8. The method of any of claim 1 or 2, wherein the disease, condition, or disorder is associated with an infection.

9. The method of claim 8, wherein the infection comprises a bacterial infection or gut domination.

10. The method of claim 9, wherein the bacterial infection or gut domination comprises an infection or gut domination by one or more species, subspecies, or strains of Aeromonas, Bacillus, Bordetella, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterobacteriaceae, Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella, Legionella, Leptospira, Listeria, Morganella, Mycobacterium, Mycoplasma, Neisseria, Orientia, Plesiomonas, Proteus, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, or Yersinia, optionally one or more of Aeromonas hydrophila, Bacillus cereus, Campylobacter fetus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enteropathogenic E. coli, enterotoxigenic Escherichia coli, Escherichia coli 0157:H7, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Salmonella paratyphi, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, or Yersinia enterocolitica.

11. The method of claim 9 or 10, wherein the bacterial infection or gut domination comprises an infection or gut domination by one or more of Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus anginosus, Streptococcus australis, Streptococcus constellatus, Streptococcus cristatus, Streptococcus gordonii, Streptococcus infantis, Streptococcus intermedins, Streptococcus mitis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus tigurinus, or Streptococcus vestibularis.

12. The method of any of claims 9-11, wherein the bacterial infection or gut domination comprises an infection or gut domination by drug-resistant bacteria.

13. The method of claim 12, wherein the drug-resistant bacteria comprises one or more of antibiotic-resistant bacterium (ARB), Antibiotic-resistant Proteobacteria, Carbapenem-resistantEwtero zcterzaceae (CRE), Extended Spectrum Beta-Lactamase producing Enterobacterales (ESBL-E), fluoroquinolone-resistant Enterobacteriaceae, vancomycin-resistant Enterococci (VRE), multi-drug resistant A. coli, or multi-drug resistant Klebsiella.

14. The method of claim 1 or 2, wherein the subject has undergone or will undergo an ileal pouch-anal anastomosis (IPAA) surgery, and wherein the disease, condition, or disorder comprises pouchitis.

15. A method for increasing propionate concentration and/or propionate production in the gut of a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium.

16. The method of any of claims 1-15, wherein the at least one propionate producing bacterium comprises a strain that is isolated from a human intestinal microbiome and/or is capable of engrafting within the human intestinal microbiome.

17. The method of any of claims 1-16, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella.

18. The method of claim 1-17, wherein the at least one propionate producing bacterium comprises one or more of Veillonella atypica (V. atypica), Veillonella dispar (V. dispar), Veillonella infantium (V. infantium), Veillonella nakazawae (V. nakazawae), Veillonella parvula (V. parvula), Veillonella ratti (V. ratti), Veillonella rogosae (V. rogosae), Veillonella seminalis (V. seminalis), and/or Veillonella tobetsuensis (V. tobetsuensis).

19. The method of any of claims 1-17, wherein the at least one propionate producing bacterium comprises V atypica, V dispar, V. infantium, V. nakazawae, V. parvula, and/or V. rogosae.

20. The method of any of claims 1-17, wherein the at least one propionate producing bacterium comprises V. infantium, V. nakazawae, V. parvula, and/or V. rogosae.

21. The method of any of claims 1-17, wherein the at least one propionate producing bacterium comprises V. infantium, V. dispar, and/or V. nakazawae.

22. The method of any of claims 1-17, wherein the at least one propionate producing bacterium comprises V. parvula.

23. The method of any of claims 1-17, wherein the at least one propionate producing bacterium comprises V. rogosae.

24. The method of any of claims 1-23, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Megasphaera.

25. The method of claim 24, wherein the at least one propionate producing bacterium comprises one or more of Megasphaera elsdenii (M. elsdenii), Megasphaera hominis (M. hominis), Megasphaera indica (M. indica), Megasphaera massiliensis (M.massiliensis), and/ or Megasphaera micronuciformis (M. micronuciformis).

26. The method of claim 25, wherein the at least one propionate producing bacterium comprises one or more oiM. elsdenii ndJ M. massiliensis.

27. The method of any of claims 1-26, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Anaerotignum.

28. The method of claim 27, wherein the at least one propionate producing bacterium comprises Anaerotignum lactatifermentans (A. lactatifermentans).

29. The method of any of claims 1-28, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Bacteroides.

30. The method of claim 29, wherein the at least one propionate producing bacterium comprises Bacteroides fragilis (B. fragilis) o Bacteroides caccae (B. caccae).

31. The method of any of claims 1-30, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Coprococcus.

32. The method of claim 31, wherein the at least one propionate producing bacterium comprises Coprococcus catus (C. catus).

33. The method of any of claims 1-32, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Merdimmobilis.

34. The method of claim 33, wherein the at least one propionate producing bacterium comprises Merdimmobilis hominis.

35. The method of any of claims 1-34, wherein the propionate producing bacterium comprises a nucleotide sequence with at least 97%, 98%, or 99% sequence identity to any of SEQ ID NOS: 40-49 or 52-58 and/or an amino acid sequence with at least 97%, 98%, or 99% sequence identity to SEQ ID NO: 50 or 51.

36. The method of any of claims 1-35, wherein the Bifidobacterium comprises B. breve, B. bifidum, or B. longum subsp. infantis.

37. The method of any of claims 1-36, wherein the Bifidobacterium comprises B. longum subsp. infantis.

38. The method of any of claims 1-37, further comprising at least one butyrate producing strain of bacteria.

39. The method of claim 38, wherein the at least one butyrate producing strain comprises a strain of Clostridium Cluster IV or Clostridium Cluster XlVa bacteria.

40. The method of claim 38 or 39, wherein the at least one butyrate producing strain comprises one or more of Agathobacter rectalis, Anaerobutyricum hallii, Anaerostipes caccae, Blautia producta, Clostridium leptum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Roseburia hominis, or Roseburia intestinalis.

41. The method of any of claims 38-40, wherein the at least one butyrate producing strain comprises one or more of Anaerostipes caccae, Clostridium innocuum, Roseburia hominis, or Roseburia intestinalis.

42. The method of any of claims 1-41, wherein the prebiotic mixture comprises one or more of 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, 3’-sialyllactose, 6'- sialyllactose, lacto-N-tetraose, lacto-N-neo-tetraose, lacto-N-fucopentaose I, lacto-N- fucopentaose II, lacto-N-fucopentaose III, sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c, lacto-N-difuco-hexaose I, lacto-N-difuco-hexaose II, lacto-N- hexaose, para-1 acto-N-hexaose, disialyllacto-N-tetraose, Fucosyl-lacto-N-hexaose, difucosyl- lacto-N-hexaose a, difucosyl-lacto-N-hexaose b, lactodifucotetraose, 6’galactosyllactose,

3 ’galactosyllactose, 3-sialyl -3-fucosyllactose, sialylfucosyllacto-N-tetraose, sialyllacto-N- fucopentaose V, disialyl-lacto-n-fucopentaose II, disialyl-lacto-n-fucopentaose V, lacto-N- neo-difucohexaose II, 3-Fucosyl-sialylacto-N-tetraose c, para-lacto-N-neohexose, lacto-N- octaose, lacto-N-neooctaose, lacto-N-neohexaose, lacto-N-fucopentaose V, iso-lacto-N- octaose, para-lacto-N-octaose, lacto-decaose, or sialyl-lacto-N-fucopentaose I.

43. The method of any of claims 1-42, wherein the prebiotic mixture comprises one or more of 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N- tetraose, lacto-N-difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N- tetraose c, sialylacto-N-tetraose b, or disialyllacto-N-tetraose.

44. The method of any of claims 1-42, wherein the prebiotic mixture comprises one or more of 2'-fucosyl-lactose, 3-fucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N- tetraose, lacto-N-neotetraose, or difucosyllactose.

45. The method of any of claims 1-42, wherein the prebiotic mixture comprises one or more of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, or lacto-N-neotetraose.

46. The method of any of claims 1-42, wherein the prebiotic mixture comprises one or both of 2'-fucosyllactose and lacto-N-neotetraose.

47. The method of any of claims 1-46, wherein the prebiotic mixture comprises at least 10, at least 25, at least 50, at least 100, or at least 150 human milk oligosaccharides.

48. The method of any of claims 1-47, wherein the prebiotic mixture comprises 2'- fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N- difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, and disialyllacto-N-tetraose.

49. The method of any of claims 1-48, wherein the prebiotic mixture is, is derived from, or comprises a concentrated human milk permeate, wherein the concentrated human milk permeate is obtained by a process comprising the steps of ultra-filtering human skim milk to obtain human milk permeate and concentrating the human milk oligosaccharide content of the human milk permeate.

50. The method of claim 49, wherein the human skim milk is obtained from human milk pooled from at least 25, 50, or 100 individual donors.

51. A kit comprising i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium.

52. The kit of claim 51, wherein the at least one propionate producing bacterium comprises a strain that is isolated from a human intestinal microbiome and/or is capable of engrafting within the human intestinal microbiome.

53. The kit of claim 51 or 52, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella.

54. The kit of any of claims 51-53, wherein the at least one propionate producing bacterium comprises one or more of Veillonella atypica (V. atypica), Veillonella dispar (V. dispar), Veillonella infantium (V. infantium), Veillonella nakazawae (V. nakazawae), Veillonella parvula (V. parvula), Veillonella ratti (V. ratti), Veillonella rogosae (V. rogosae), Veillonella seminalis (V. seminalis), and/or Veillonella tobetsuensis (V. tobetsuensis).

55. The kit of any of claims 51-54, wherein the at least one propionate producing bacterium comprises V atypica, V dispar, V. infantium, V. nakazawae, V. parvula, and/or V. rogosae.

56. The kit of any of claims 51-54, wherein the at least one propionate producing bacterium comprises V. infantium, V. nakazawae, V. parvula, and/or V. rogosae.

57. The kit of any of claims 51-54, wherein the at least one propionate producing bacterium comprises V. infantium, V. dispar, and/or V. nakazawae.

58. The kit of any of claims 51-54, wherein the at least one propionate producing bacterium comprises V. parvula.

59. The kit of any of claims 51-54, wherein the at least one propionate producing bacterium comprises V. rogosae.

60. The kit of any of claims 51-59, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Megasphaera.

61. The kit of claim60, wherein the at least one propionate producing bacterium comprises one or more of Megasphaera elsdenii (M elsdenii), Megasphaera hominis (M hominis), Megasphaera indica (M indica), Megasphaera massiliensis (M massiliensis), and/ or Megasphaera micronuciformis (M micronuciformis).

62. The kit of claim 60 or 61, wherein the at least one propionate producing bacterium comprises one or more of A7. elsdenii a Mo M. massiliensis.

63. The kit of any of claims 51-62, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Anaerotignum.

64. The kit of claim 63, wherein the at least one propionate producing bacterium comprises Anaerotignum lactatifermentans (A. lactatifermentans).

65. The kit of any of claims 51-64, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Bacteroides.

66. The kit of claim 65, wherein the at least one propionate producing bacterium comprises Bacteroides fragilis (B. fragilis) o Bacteroides caccae (B. caccae).

67. The kit of any of claims 51-66, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Coprococcus.

68. The kit of claim 67, wherein the at least one propionate producing bacterium comprise Coprococcus catus (C. catus).

69. The kit of any of claims 51-68, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Merdimmobilis .

70. The kit of claim 69, wherein the at least one propionate producing bacterium comprises Merdimmobilis hominis.

71. The kit of any of claims 51-70, wherein the propionate producing bacterium comprises a nucleotide sequence with at least 97%, 98%, or 99% sequence identity to any of SEQ ID NOS: 40-49 or 52-58 and/or an amino acid sequence with at least 97%, 98%, or 99% sequence identity to SEQ ID NO: 50 or 51.

72. The kit of any of claims 51-71, wherein the Bifidobacterium comprises B. breve, B. bifidum, or B. longum subsp. infantis.

73. The kit of any of claims 51 -72, wherein the Bifidobacterium comprises B. longum subsp. infantis.

74. The kit of any of claims 51-73, further comprising at least one butyrate producing strain of bacteria.

75. The kit of claim 74, wherein the at least one butyrate producing strain comprises a strain of Clostridium Cluster IV or Clostridium Cluster XlVa bacteria.

76. The kit of claim 74 or 75, wherein the at least one butyrate producing strain comprises one or more of Agathobacter rectalis, Anaerobutyricum hallii, Anaerostipes caccae, Blautia producta, Clostridium leptum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Roseburia hominis, or Roseburia intestinalis.

77. The kit of claim 74 or 75, wherein the at least one butyrate producing strain comprises one or more of Anaerostipes caccae, Clostridium innocuum, Roseburia hominis, or Roseburia intestinalis.

78. The kit of any of claims 51-77, wherein the prebiotic mixture comprises one or more of 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neo-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, si alyl -lacto-N- tetraose c, lacto-N-difuco-hexaose I, lacto-N-difuco-hexaose II, lacto-N-hexaose, para-lacto- N-hexaose, disialyllacto-N-tetraose, Fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose a, difucosyl-lacto-N-hexaose b, lactodifucotetraose, 6’galactosyllactose, 3 ’galactosyllactose, 3- sialyl-3 -fucosyllactose, sialylfucosyllacto-N-tetraose, sialyllacto-N-fucopentaose V, disialyl- lacto-n-fucopentaose II, disialyl-lacto-n-fucopentaose V, lacto-N-neo-difucohexaose II, 3- Fucosyl-sialylacto-N-tetraose c, para-lacto-N-neohexose, lacto-N-octaose, lacto-N- neooctaose, lacto-N-neohexaose, lacto-N-fucopentaose V, iso-lacto-N-octaose, para-lacto-N- octaose, lacto-decaose, or sialyl-lacto-N-fucopentaose I.

79. The kit of any of claims 51-78, wherein the prebiotic mixture comprises one or more of 2'-fucosyllactose, 3 -fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, or disialyllacto-N-tetraose.

80. The kit of any of claims 51-78, wherein the prebiotic mixture comprises one or more of 2'-fucosyl-lactose, 3 -fucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N- tetraose, lacto-N-neotetraose, or difucosyllactose.

81. The kit of any of claims 51-78, wherein the prebiotic mixture comprises one or more of 2'-fucosyllactose, 3 -fucosyllactose, lacto-N-tetraose, or lacto-N-neotetraose.

82. The kit of any of claims 51-78, wherein the prebiotic mixture comprises one or both of 2'-fucosyllactose and lacto-N-neotetraose.

83. The kit of any of claims 51-82, wherein the prebiotic mixture comprises at least 10, at least 25, at least 50, at least 100, or at least 150 human milk oligosaccharides.

84. The kit of any of claims 51-83, wherein the prebiotic mixture comprises 2'- fucosyllactose, 3 -fucosyllactose, 3’-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N- difucohexaose I, lactodifucotetraose, lacto-N-fucopentaose I, sialylacto-N-tetraose c, sialylacto-N-tetraose b, and disialyllacto-N-tetraose.

85. The kit of any of claims 51-84, wherein the prebiotic mixture is, is derived from, or comprises a concentrated human milk permeate, wherein the concentrated human milk permeate is obtained by a process comprising the steps of ultra-filtering human skim milk to obtain human milk permeate and concentrating the human milk oligosaccharide content of the human milk permeate.

86. The kit of claim 85, wherein the human skim milk is obtained from human milk pooled from at least 25, 50, or 100 individual donors.

87. A method for increasing propionate concentration and/or propionate production in the gut of a subject in need thereof, the method comprising administering to the subject the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium of the kit of any of claims 51-86.

88. A method of treating or preventing a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium of the kit of any of claims 51-86.

89. A method of ameliorating a symptom of a disease, disorder, or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy, or dysbiosis of the intestinal microbiome in a subject in need thereof, the method comprising administering to the subject the prebiotic mixture, the at least one Bifidobacterium, and the at least one propionate producing bacterium of the kit of any of claims 51-86.

90. A pharmaceutical composition comprising i) a prebiotic mixture comprising one or more human milk oligosaccharides, ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides; and iii) at least one propionate producing bacterium.

91. The pharmaceutical composition of claim 90, wherein the at least one Bifidobacterium comprises B. longum subsp. inf antis and the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella.

92. The pharmaceutical composition of claim 90 or claim 91, further comprising at least one butyrate producing strain of bacteria, optionally wherein the at least one butyrate producing strain comprises one or more of Agathobacter rectalis, Anaerobutyricum hallii, Anaerostipes caccae, Blautia producta, Clostridium leptum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Roseburia hominis, or Roseburia intestinalis.

93. A method for treating one or more symptoms of acute radiation syndrome in a subject in need thereof, the method comprising administering to the subject i) a prebiotic mixture comprising one or more human milk oligosaccharides and ii) at least one Bifidobacterium capable of consuming the one or more human milk oligosaccharides, thereby treating the symptoms of acute radiation syndrome.

94. The method of claim 93, wherein the at least one Bifidobacterium comprises B. longum subsp. infantis.

95. The method of claim 93 or 94, further comprising administering to the subject in need thereof iii) at least one propionate producing bacterium.

96. The method of claim 95, wherein the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella.

97. The method of any of claims 93-96, wherein the one or more symptoms of acute radiation syndrome comprise gastrointestinal damage.

98. The method of any of claims 93-97, wherein the subject was exposed to an ionizing radiation dose of at least 0.3 Gray (Gy), optionally at least 0.7 Gy, 1 Gy, 2 Gy, 5 Gy, 6 Gy, or 10 Gy over a period of time lasting under 60 minutes, optionally under 30 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, 2 minutes, or 1 minute.

99. An article of manufacture, comprising the kit any one of claims 51-86 and instructions for use, wherein the instructions for use describe the method of any of claims 1- 50, 87-89, and 93-98.