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WO2024130119A9 - Compositions symbiotiques pour la production d'acides gras à chaîne courte - Google Patents

Compositions symbiotiques pour la production d'acides gras à chaîne courte Download PDF

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Publication number
WO2024130119A9
WO2024130119A9 PCT/US2023/084290 US2023084290W WO2024130119A9 WO 2024130119 A9 WO2024130119 A9 WO 2024130119A9 US 2023084290 W US2023084290 W US 2023084290W WO 2024130119 A9 WO2024130119 A9 WO 2024130119A9
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Prior art keywords
lacto
propionate
human milk
producing bacterium
strain
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WO2024130119A3 (fr
WO2024130119A2 (fr
Inventor
Gregory Mckenzie
Julie E. Button
Abigail REENS
Casey COSETTA
Aislinn ROWAN-NASH
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Prolacta Bioscience Inc
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Prolacta Bioscience Inc
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Publication of WO2024130119A9 publication Critical patent/WO2024130119A9/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • 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.
  • 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.
  • 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.
  • a prebiotic mixture e.g., of human milk oligosaccharides
  • Bifidobacterium e.g., B. longum subsp. inf antis
  • propionate producing bacterium e.g., a 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.
  • 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 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.
  • 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.
  • the prebiotic mixture comprises one or more human milk oligosaccharides.
  • 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.
  • 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 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.
  • the disease, condition, or disorder is associated with an infection.
  • the infection comprises a bacterial infection or gut domination.
  • 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, Plesi
  • the bacterial infection or gut domination comprises an infection or gut domination by drug-resistant bacteria.
  • 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.
  • the subject has undergone or will undergo an ileal pouch-anal anastomosis (IPAA) surgery, and wherein the disease, condition, or disorder comprises pouchitis.
  • AIPAA ileal pouch-anal anastomosis
  • the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella.
  • 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.
  • the at least one propionate producing bacterium comprises one or more strains of the genus Megasphaera.
  • 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).
  • the at least one propionate producing bacterium comprises one or more of M. elsdenii ndJo M. massiliensis.
  • 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.
  • 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.
  • 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.
  • the Bifidobacterium comprises B. breve, B. bifidum, or B. longum subsp. infantis. In certain embodiments, the Bifidobacterium comprises B. longum subsp. infantis.
  • the method further comprising at least one butyrate producing strain of bacteria.
  • the at least one butyrate producing strain comprises a strain of Clostridium Cluster IV or Clostridium Cluster XlVa bacteria.
  • 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.
  • the at least one butyrate producing strain comprises one or more of Anaerostipes caccae, Clostridium innocuum, Roseburia hominis, or Roseburia intestinalis.
  • 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.
  • 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.
  • the prebiotic mixture comprises one or more of 2'-fucosyllactose, 3- fucosyllactose, lacto-N-tetraose, or lacto-N-neotetraose.
  • 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.
  • 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.
  • 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.
  • the human skim milk is obtained from human milk pooled from at least 25, 50, or 100 individual donors.
  • kits 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.
  • 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.
  • the kit further comprises at least one butyrate producing strain of bacteria.
  • a method for increasing propionate concentration and/or propionate production in the gut of a subject in need thereof 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.
  • 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.
  • 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 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.
  • the at least one Bifidobacterium comprises B. longum subsp. infantis.
  • the method further comprises administering to the subject in need thereof iii) at least one propionate producing bacterium.
  • the at least one propionate producing bacterium comprises one or more strains of the genus Veillonella.
  • the one or more symptoms of acute radiation syndrome comprise gastrointestinal damage.
  • 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.
  • FIG. 2C 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 10 3 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.
  • 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. 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
  • 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.
  • 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.
  • 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.
  • 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. 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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],
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • V sp. 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.
  • 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.
  • V sp. 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.
  • 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.
  • V sp. 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.
  • 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.
  • HMO human milk oligosaccharide composition
  • 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.
  • compositions, kits, and articles of manufacture as well as methods of use thereof.
  • the provided compositions, kits, and articles of manufacture are or include prebiotics and at least two probiotic strains of bacteria.
  • the prebiotics are or include a mixture, e.g., of oligosaccharides such as human milk oligosaccharides.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions, methods, kits, and articles of manufacture address these needs.
  • 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.
  • prebiotics such as human milk oligosaccharides
  • probiotics such as Bifidobacterium including B. longum subspecies (subsp.) infantis and bacteria capable of producing propionate, e.g., Veillonella sp.
  • 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.
  • the prebiotic mixture is or includes human milk oligosaccharides.
  • the Bifidobacteria is or includes B. longum subsp. infantis.
  • the one or more strains of bacteria capable of producing propionate are or include strains of bacteria from the genus Veillonella.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the prebiotic mixture is free or essentially free of oligosaccharides that are not human milk oligosaccharides.
  • 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-t
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the prebiotic mixture is a liquid formulation.
  • the prebiotic mixture is in powdered form, e.g., a lyophilized or spray dried composition.
  • the prebiotic mixture is incorporated into a dosage form that is a separate composition from the Bifidobacterium strain.
  • the prebiotic mixture is incorporated into a dosage form that is a separate composition from the propionate producing strain.
  • the prebiotic mixture is or includes human milk oligosaccharides (HMOs) obtained or purified from ultra-filtered permeate from donated human milk.
  • the donated human milk is pooled to provide a pool of human milk.
  • a pool of human milk comprises milk from two or more (e.g., ten or more) donors.
  • the pooled human milk contains milk from at least 50, 75, 100, 150, or 200 individual donors.
  • the pooled human milk contains human milk from at least 100 individual donors or between 100 and 300 individual donors.
  • 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.
  • 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.
  • synthetic 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.
  • one or more synthetically derived human milk oligosaccharides are added to a concentrated human milk permeate to arrive at a prebiotic mixture.
  • the mixture of oligosaccharides described herein are produced from human milk permeate, e.g., concentrated ultra-filtered permeate from pooled human milk.
  • the mixture of oligosaccharides described herein contain or are formulated with human milk permeate, e.g., concentrated ultra-filtered permeate from pooled human milk.
  • the concentrated ultra-filtered permeate may be made according to any suitable method or technique known in the art.
  • 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.
  • the prebiotic mixture is or includes human milk permeate that has been generated or produced by a method described herein.
  • 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.
  • 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.
  • the pooled milk is filtered, e.g., through about a 200- micron filter.
  • the pooled milk is heated, e.g., at about 63°C or greater for about 30 minutes or more.
  • the milk is transferred to a separator, e.g., a centrifuge, to separate the cream from the skim.
  • the cream may go through separation once again to yield more skim.
  • a desired amount of cream is added to the skim prior to ultra-filtration.
  • 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.
  • the skim fraction undergoes ultra-filtration.
  • 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.
  • 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.
  • 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.
  • the ultra-filtered permeate undergoes a process for reducing lactose.
  • a process for producing a concentrated human milk permeate composition with substantially reduced levels of lactose is provided.
  • 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.
  • the permeate is free or essentially free of lactose following the enzymatic digestion.
  • 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.
  • the order of when steps (a)-(c) are performed may be varied.
  • 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.
  • 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.
  • 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.
  • lactase enzyme is added to the pH-adjusted, heated permeate to create a permeate/lactase mixture.
  • lactose within the permeate/lactase mixture is broken down into monosaccharides.
  • lactase enzyme is added at about 0.1% w/w to about 0.5% w/w concentration.
  • lactase enzyme is added at about 0.1% w/w, or 0.2% or 0.3% or 0.4% or 0.5% w/w.
  • lactase enzyme may be derived from any origin (e.g., fungal or bacterial in origin).
  • the pH-adjusted, heated permeate is incubated with the lactase enzyme for about 5 to about 225 minutes.
  • the incubation time is about 15 min to about 90 min.
  • the incubation time is about 30 minutes to about 90 minutes.
  • the incubation time is about 60 minutes.
  • pH, temperature, and enzyme incubation conditions 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.
  • 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.
  • the permeate/lactase mixture is clarified to remove insoluble constituents.
  • insoluble material may form throughout the change in pH and temperature.
  • 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.
  • 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.
  • the clarification step is important in the reusability of the filtration membranes and thus to the scalability of the process.
  • 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.
  • the spent and excess lactase enzyme is removed from the clarified permeate/lactase mixture.
  • the inactivated foreign protein will carry no biological risk and therefore the added steps of lactase removal or even inactivation may not be necessary.
  • 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.
  • lactase enzyme removal may be accomplished by ultrafiltration.
  • 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.
  • 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.
  • filtration can be accomplished using a nanofiltration membrane.
  • 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.
  • the additional nanofiltration removes monosaccharides, minerals, particularly calcium, and smaller molecules to produce the final purified HMO composition.
  • 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.
  • the nanofiltration comprises filtration through a membrane with a molecular cut-off of ⁇ 600 Dalton.
  • these additional steps may be performed at any stage of the process, including but not limited to prior to or after pasteurization.
  • the permeate is treated to reduce bioburden, such as by any means known in the art.
  • 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.
  • 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
  • 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.
  • the at least one butyrate producing strain is capable of consuming lactate and/or acetate.
  • the at least one butyrate producing strain is capable of producing, generating, and/or creating butyrate in the presence of lactate and/or acetate.
  • 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.
  • 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.
  • 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.
  • the butyrate producing strain is or includes at least three strains of bacteria capable of producing butyrate.
  • the butyrate producing strain is or includes between one and five species, subspecies, or strains of bacteria capable of producing butyrate.
  • the at least one butyrate producing strain has one or more genes that contribute to the production, generation, or making of butyrate.
  • the at least one butyrate producing strain has a functional butyryl-Co A: acetate CoA-transferase (but) gene.
  • the at least one butyrate producing strain has a functional butyrate kinase (buk) gene.
  • the at least one butyrate producing strain has a functional butyryl-CoA:4-hydroxybutyrate CoA transferase (4Hbf) gene.
  • the at least one butyrate producing strain has a functional butyryl-Co A: acetoacetate CoA transferase (Ato) gene.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • compositions, kits, and articles of manufacture 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.
  • prebiotics e.g., prebiotic mixtures of non- digestible carbohydrates such as human milk oligosaccharides
  • 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.
  • the prebiotic mixture and probiotics may be formulated as a pharmaceutical composition or a nutritional composition.
  • the provided composition includes or incorporates the prebiotic mixture and the probiotic strains.
  • the probiotic strains may be formulated as a pharmaceutical composition or a nutritional composition.
  • the prebiotic mixture and the probiotic are formulated or manufactured as separate compositions.
  • kits or articles of manufacture that are or include separate prebiotic and probiotic compositions.
  • 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.
  • the prebiotic mixture is or includes any of the prebiotic mixtures described herein, e.g., in Section I-C.
  • 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.
  • the one or more strains of Bifidobacteria are or include one or more strains of Bifidobacteria described herein, e.g., in Section I-B.
  • the one or more strains of Bifidobacteria are capable of consuming, internalizing, and/or hydrolyzing human milk oligosaccharides.
  • 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.
  • the one or more strains of propionate producing bacteria are or include at least one strain from the genus Veillonella or Megasphaera.
  • 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.
  • 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 B
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • provided herein are methods for treating, preventing, or ameliorating one or more diseases, disorders, or conditions in a subject in need thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • targeting the microbiome with the provided prebiotic and probiotic compositions may successfully treat, alleviate, or prevent a wide range of conditions, diseases, and disorders.
  • a method for treating, reducing, ameliorating, or preventing dysbiosis 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.
  • 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
  • a prebiotic mixture such as any described herein, e.g., in Section I-C.
  • the one or more diseases, disorders, or conditions is, includes, or is associated with dysbiosis, e.g., of the intestinal microbiome.
  • the microbiome is an intestinal microbiome of a human.
  • the microbiome is the intestinal microbiome of an infant or young child.
  • the microbiome is an intestinal microbiome of an adult human.
  • 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.
  • the one or more conditions, diseases, or disorders is, includes, or is associated with dysbiosis.
  • the one or more conditions, diseases, or disorders is, includes, or is associated with inflammation.
  • the one or more condition, disease, or disorder is, includes, or is associated with an autoimmune disease.
  • the one or more conditions, diseases, or disorders is or is associated with an allergy.
  • 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.
  • the prebiotic mixture and the probiotic strain prevent a condition described herein, e.g, in Section II-B.
  • 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.
  • 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.
  • the subject is a human.
  • the subject is an infant, a child, a juvenile, or an adult.
  • the subject is at least 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months of age.
  • the subject is at least 1 year, 2 years, 5 years, 10 years, 12 years, 16 years, or at least 18 years of age.
  • the subject is at least 12 years old.
  • the subject is at least 18 years old.
  • the subject is an adult.
  • the subject is elderly, e.g, at least 65 or 75 years of age.
  • 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.
  • the method is or includes a treatment where all three of the propionate producing bacterium, the Bifidobacterium, and the prebiotic mixture are administered.
  • 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.
  • the prebiotic mixture is administered daily for at least 2, 3, 4, 5, 7, 10, 14, 21, or 28 days, e.g., consecutive days.
  • 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.
  • 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.
  • 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.
  • doses of 9 g of total human milk oligosaccharides are administered twice daily for a total of 18 g/day.
  • 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 10 1 , 5 x 10 1 ,! x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 colony forming units (CFU) per day.
  • CFU colony forming units
  • the at least one Bifidobacterium is administered in an amount of at least 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , l x 10 8 , or 5 x 10 8 colony forming units (CFU) per dose.
  • the at least one Bifidobacterium is administered in an amount of between 1 x 10 6 and 1 x 10 12 , 5 x 10 6 and 1 x 10 10 , 1 x 10 7 and 1 x 10 9 , or 1 x 10 7 and 1 x 10 8 CFU per day.
  • the Bifidobacterium strain is administered in an amount of, of about, or at least 5 x 10 6 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 10 7 colony forming units (CFU) per dose or per day.
  • 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 10 1 , 5 x 10 1 ,! x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 ,l x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 colony forming units (CFU) per day.
  • CFU colony forming units
  • the at least one propionate producing bacterium is administered in an amount of at least 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , or 5 x 10 8 colony forming units (CFU) per dose.
  • the at least one propionate producing bacterium is administered in an amount of between 1 x 10 6 and 1 x 10 12 , 5 x 10 6 and 1 x IO 10 , 1 x 10 7 and 1 x 10 9 , or 1 x 10 7 and 1 x 10 8 CFU per day.
  • the at least one propionate producing bacterium is administered in an amount of, of about, or at least 5 x 10 6 colony forming units (CFU) per dose or per day.
  • the probiotic strain is administered in an amount of, of about, or at least 8 x 10 7 colony forming units (CFU) per dose or per day.
  • 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.
  • 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.
  • 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.
  • 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.
  • the prebiotic mixture is administered prior to and after the at least one Bifidobacterium.
  • 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.
  • 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.
  • 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.
  • 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.
  • a treatment phase is or is at least seven days.
  • a treatment phase is or is at least fourteen days.
  • the treatment regimen includes more than one treatment phase.
  • one treatment phase e.g., a first treatment phase
  • the prebiotic mixture and the at least one Bifidobacterium strain are administered in one treatment phase, e.g., a first treatment phase.
  • 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.
  • 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.
  • 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.
  • 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.
  • the at least one propionate producing strain is also administered during the treatment phase, e.g., the first treatment phase.
  • 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.
  • 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.
  • the treatment phase e.g., the first treatment phase
  • 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.
  • 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.
  • the at least one propionate producing strain is also administered during the second treatment phase.
  • 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.
  • 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.
  • the first and second treatment phases occur once during a treatment regimen.
  • 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.
  • 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.
  • the administered oligosaccharides 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • colon cancer diabetes, liver disorders, cystic fibrosis, and allergies.
  • 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.
  • the prebiotic mixture e.g., of human milk oligosaccharides
  • 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.
  • 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.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel disease
  • SIBO small intestinal bacterial overgrowth
  • gastroenteritis small intestinal bacterial overgrowth
  • leaky gut syndrome and gastric lymphoma.
  • the gastrointestinal condition, disease, or disorder is associated with a bacterial, viral or parasitic infection or overgrowth.
  • the disease or disorder is associated with infection by drug-resistant bacteria, e.g., vancomycin-resistant enterococcus (VRE).
  • VRE vancomycin-resistant enterococcus
  • 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.
  • 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.
  • the subject is immunocompromised.
  • the administration prevents, reduces, treats, or ameliorates an infection in the immunocompromised subject.
  • the administration prevents, reduces, treats, or ameliorates overgrowth or domination of pathogenic bacteria.
  • the immunocompromised subject has undergone one or more treatments for cancer.
  • the treatments are or include chemotherapy.
  • the treatment is or includes an allogenic transplant, e.g, a hematopoietic stem cell transplant or bone marrow transplant.
  • 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).
  • 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.
  • the probiotic strains e.g., the Bifidobacterium and the propionate producing bacterium
  • 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).
  • 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.
  • overgrowth or domination may be determined by routine techniques in the art, such as including but not limited to PCR or high throughput sequencing.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • pathogenic taxa e.g., Enterob acteriaceae, Enterococcus, Staphylococcus
  • 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.
  • the prebiotic mixture e.g., of human milk oligosaccharides
  • the growth of the Bifidobaclerium ivVm e.g., B. longum subsp. infantis
  • 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.
  • the subject has an increased risk of infection or gut domination, e.g., as compared to the general population.
  • 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.
  • 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
  • ICU intensive care unit
  • 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.
  • 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.
  • 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.
  • the prebiotic mixture and the probiotic strains are administered at least once during the medical procedure, e.g., surgery or chemotherapy.
  • 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.
  • Pathogenic bacteria may include known microbes with pathogenicity for the gastrointestinal tract, e.g., from esophagus down to rectum.
  • pathogenic bacteria are or include one or more species, subspecies, or strains of Proteobacteria.
  • 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 .
  • administration of the prebiotic mixture and the probiotic strains reduces or decreases the presence, growth, or abundance of pathogenic bacteria within the gut.
  • administration of the prebiotic mixture the propionate producing strain, and the Bifidobacterium strain impairs the growth of one or more pathogens.
  • 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.
  • 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
  • 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).
  • ARB antibioticresistant bacterium
  • VRE Vancomycin Resistant Enterococcus
  • CRE Carbapenem Resistant Enterobacteriaceae
  • ESBL-E Extended Spectrum Beta-Lactamase producing Enterobacterales
  • 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.
  • ARB antibiotic-resistant bacterium
  • CRE Carbapenem-resistant Enterobacteriaceae
  • ESBL-E Extended Spectrum Beta-Lactamase producing Enterobacterales
  • VRE vancomycin-resistant Enterococci
  • multi-drug resistant A. coli or multi-drug resistant Klebsiella.
  • 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 cristat
  • 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, Streptoc
  • the condition, disease, or disorder is an immune dysfunction that is an autoimmune disorder.
  • 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
  • ADAM acute disseminated
  • 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.
  • acute bloody diarrhea e.g., dysentery
  • acute watery diarrhea e.g., cholera
  • checkpoint inhibitor-associated colitis e.g., cholera
  • diarrhea due to food poisoning e.g., chronic diarrhea, and traveler's diarrhea.
  • 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.
  • administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain reduces GI immunoactivation and/or inflammation.
  • GI immunoactivation and inflammation may be assessed by known methods that are routine in the art.
  • 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.
  • IBD inflammatory bowel disease
  • 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.
  • administration of the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain treats or prevents various bloodstream infections (BSI).
  • BAI bloodstream infections
  • administration of the probiotic strains and the prebiotic mixture treats or prevents catheter or intravascular-line infections (e.g., central-line infections).
  • administration of the probiotic strains and the prebiotic mixture treats or prevents chronic inflammatory diseases.
  • 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.
  • pneumonia e.g., ventilator-associated pneumonia
  • skin and soft tissue infections e.g., surgical- site infections
  • urinary tract infections e.g., antibiotic-resistant urinary tract infections and catheter-associated urinary tract infections
  • wound infections e.g., antibiotic-resistant infections and antibiotic-sensitive infections.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the immunotherapy comprises administration of Keytruda (Pembrolizumab), Opdivo (Nivolumab), Yervoy (Ipilimumab), Tecentriq (atezolizumab), Bavencio (avelumab), and Imfinzi (durvalumab).
  • the subject is refractory and/or non-responsive to an anti-cancer therapy.
  • 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.
  • 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.
  • 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.
  • a treatment directed to a checkpoint molecule comprises administration of Keytruda (Pembrolizumab), Opdivo (Nivolumab), Yervoy (Ipilimumab), Tecentriq (atezolizumab), Bavencio (avelumab), or Imfinzi (durvalumab).
  • 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.
  • the administration prevents, reduces, treats, or ameliorates an infection in the immunocompromised subject.
  • the administration prevents, reduces, treats, or ameliorates overgrowth or domination of pathogenic bacteria.
  • the immunocompromised subject has undergone one or more treatments for cancer.
  • the treatments are or include chemotherapy.
  • the treatment is or includes an allogenic transplant, e.g., a hematopoietic stem cell transplant or bone marrow transplant.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the prebiotic mixture e.g., of human milk oligosaccharides
  • the propionate producing strain e.g., Veilloinella sp.
  • the at least one Bifidobacterium strain e.g, B. longum subsp. infantis
  • 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.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel disease
  • SBS short bowel syndrome
  • SIBO small intestinal bacterial overgrowth
  • gastroenteritis small intestinal bacterial overgrowth
  • leaky gut syndrome and gastric lymphoma
  • gastric lymphoma irritable bowel syndrome
  • the disease or disorder is associated with a bacterial, viral, or parasitic infection or overgrowth, e.g., by drug-resistant bacteria.
  • 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.
  • 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.
  • the prebiotic mixture, the propionate producing strain, and the Bifidobacterium strain are administered to a subject to treat, prevent, or ameliorate an allergy.
  • the allergy is a food allergy.
  • 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.
  • the prebiotic mixture the propionate producing strain, and the Bifidobacterium strain are administered to treat or ameliorate an allergy, e.g., a food allergy.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • pouchitis is inflammation that occurs in the lining of a pouch created during surgery to treat ulcerative colitis or certain other diseases.
  • the surgery is or includes removal of a diseased colon or portion thereof.
  • the surgery is a J pouch surgery (ileoanal anastomosis — IPAA).
  • 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.
  • administration of the prebiotic mixture and the probiotic strains prevents, reduces, decreases, remedies, or ameliorates one or more symptoms associated with pouchitis.
  • 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.
  • symptoms associated with more severe pouchitis include, but are not limited to, fever, dehydration, malnutrition, fatigue, iron-deficiency anemia, or joint pain.
  • 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.
  • 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.
  • the prebiotic mixture e.g., of human milk oligosaccharides
  • the propionate producing strain e.g., Veilloinella sp.
  • the at least one Bifidobacterium strain e.g., B. longum subsp. infantis
  • 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.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel disease
  • SBS short bowel syndrome
  • SIBO small intestinal bacterial overgrowth
  • gastroenteritis small intestinal bacterial overgrowth
  • leaky gut syndrome and gastric lymphoma.
  • the disease or disorder is associated with a bacterial, viral, or parasitic infection or overgrowth, e.g., by drug-resistant bacteria.
  • 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.
  • 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
  • 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
  • pouchitis is inflammation that occurs in the lining of a pouch created during surgery to treat ulcerative colitis or certain other diseases.
  • the surgery is or includes removal of a diseased colon or portion thereof.
  • the surgery is a J pouch surgery (ileoanal anastomosis — IPAA).
  • 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.
  • symptoms associated with more severe pouchitis include, but are not limited to, fever, dehydration, malnutrition, fatigue, iron-deficiency anemia, or joint pain.
  • the subject is a patient in an intensive care unit (ICU).
  • the subject is an organ transplant recipient.
  • the subject is a geriatric patient (e.g., at least 65, 70, 75, 80, or 85 years old).
  • 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).
  • the subject is a recipient of a broad-spectrum antibiotic treatment.
  • 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).
  • parenteral nutrition e.g., total parenteral nutrition or partial parenteral nutrition.
  • the subject is a recipient of enteral nutrition.
  • provided herein are methods of preventing or reducing the incidence or severity of graft versus host disease (GVHD) in a subject in need thereof.
  • 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.
  • 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.
  • 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.
  • 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
  • the prebiotic mixture such as any described herein, e.g, in Section I-C
  • the subject is a human infant, child, adolescent, or adult.
  • the subject is at risk or suspected of being at risk of having GVHD.
  • 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).
  • BMT bone marrow transplant
  • HSCT allogenic hematopoietic stem cell transplant
  • 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.
  • a prebiotic mixture e.g., of human milk oligosaccharides
  • a prebiotic mixture e.g., of human milk oligosaccharides
  • the allogenic transplant is a bone marrow transplant (BMT).
  • the allogenic transplant is a hematopoietic stem cell transplantation (HSCT).
  • 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.
  • 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.
  • 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.
  • administering reduces or decreases the probability or likelihood of experiencing GVHD.
  • 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.
  • 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.
  • the Bifidobacterium e.g., B. longum subsp. infantis
  • a propionate producing strain e.g., a Veillonella sp.
  • a prebiotic mixture e.g., of human milk oligosaccharides
  • the Bifidobacterium, e.g., B. longum subsp. infantis, the propionate producing strain e.g., a Veillonella sp.
  • 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.
  • an allogenic transplant e.g., BMT or HSCT
  • the Bifidobacterium e.g., B. longum subsp. infantis
  • a propionate producing strain e.g., the Veillonella sp.
  • the prebiotic mixture e.g., of human milk oligosaccharides
  • the GVHD is acute GVHD.
  • the GVHD is chronic GVHD.
  • 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.
  • 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.
  • administration of the Bifidobacterium e.g., B. longum subsp.
  • the propionate producing strain e.g., a Veillonella sp.
  • 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.
  • 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.
  • the Bifidobacterium e.g., B. longum subsp. infantis
  • the prebiotic mixture e.g., of human milk oligosaccharides
  • ARS acute radiation syndrome
  • a propionate producing strain e.g., the Veillonella sp.
  • the subject has or is suspected of having ARS.
  • 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.
  • ARS resulting from such exposure may present as hematopoietic dysfunction, gastrointestinal damage, and/or neurovascular injury.
  • the Bifidobacterium e.g., B. longum subsp. infantis
  • the prebiotic mixture e.g., of human milk oligosaccharides
  • the at least one propionate producing bacterium e.g., Veillonella sp.
  • 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.
  • CNS cardiovascular/central nervous system
  • 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).
  • 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.
  • 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.
  • the administration prevents the onset of one or more symptoms associated with ARS.
  • 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.
  • 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.
  • 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.
  • the subject has been exposed to a high dose of ionizing radiation.
  • 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.
  • 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.
  • 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.
  • the dose is associated with an external source of radiation, e.g., a source outside of the subject’s body.
  • 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.
  • 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.
  • 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.
  • the at least one propionate producing bacterium e.g., Veillonella sp.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • the at least one Bifidobacteria, the provided propionate producing bacterium, and the provided prebiotic mixture are formulated together in the same pharmaceutical composition.
  • the at least one Bifidobacterium and the prebiotic mixture are formulated together into the same pharmaceutical composition.
  • the at least one Bifidobacterium and the at least one propionate producing bacterium are formulated together into the same pharmaceutical composition.
  • the at least one Bifidobacteria, the provided propionate producing bacterium, and the provided prebiotic mixture are formulated separately into separate pharmaceutical compositions.
  • compositions described herein 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.
  • physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use.
  • 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.
  • the probiotic strains e.g., the provided Bifidobacterium and/or the provided propionate producing bacterium
  • 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).
  • 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
  • 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 10 5 to 10 12 bacteria, e.g., at, at about, or at least 10 5 bacteria, 10 6 bacteria, 10 7 bacteria, 10 8 bacteria, 10 9 bacteria, IO 10 bacteria, IO 11 bacteria, or 10 12 bacteria, or more.
  • the prebiotic mixture e.g., prebiotic mixture of human milk oligosaccharides
  • total prebiotic weight such as weight of total human milk oligosaccharides
  • 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.
  • 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.
  • the prebiotic mixture is administered to the subject in an amount of or about 5 g per day.
  • 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.
  • 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
  • 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.
  • 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.
  • the pharmaceutical compositions containing the provided at least one Bifidobacterium, the at least one propionate producing bacterium, and the prebiotic mixture 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).
  • 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
  • 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.
  • 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).
  • binding agents e.g, pregelatinized maize starch, hydroxypropyl methylcellulose, carboxymethyl cellulose, polyethylene
  • 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.
  • membrane selected from, but not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, hydroymethylacrylate-methyl methacrylate (
  • 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).
  • the pH profile may be modified.
  • the coating is degraded in specific pH environments in order to specify the site of release.
  • at least two coatings are used.
  • the outside coating and the inside coating are degraded at different pH levels.
  • 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.
  • 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.
  • a 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.
  • a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.
  • 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.
  • the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life.
  • the gummy candy may also comprise sweeteners or flavors.
  • the pharmaceutical composition may include a flavor.
  • 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.
  • 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.
  • 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.
  • HPMC hydroxypropylmethyl cellulose
  • 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.
  • 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.
  • the composition is a nutritional composition such as food product.
  • 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.
  • the nutritional composition or food product is a fermented food, such as a fermented dairy product.
  • the fermented dairy product is yogurt.
  • the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir.
  • the probiotic strain of the invention e.g., B. longum subsp. infantis strain
  • the food product is a beverage.
  • the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts.
  • 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.
  • the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.
  • 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.
  • compositions containing the at least one Bifidobacterium, the at least one propionate producing bacterium, and/or the prebiotic mixture in 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.
  • 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.
  • the composition can be delivered in a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent.
  • a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical compositions e.g., containing one or both of the probiotic strain and prebiotic mixtures are administered with food.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • human milk oligosaccharides also encompass 3'-sialyllactose (3'- SL) and 6'-sialyllactose (6'-SL) oligosaccharides that are found in human milk.
  • 3'- SL 3'-sialyllactose
  • 6'-SL 6'-sialyllactose
  • 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.
  • 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.
  • compositions containing particular recited components refers to compositions containing particular recited components while excluding other major bioactive factors.
  • Probiotic 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.
  • a host organism e.g., a mammal such as a human
  • those of skill in the art may readily identify species, strains, and/or subtypes of non-pathogenic bacteria that are recognized as probiotic bacteria.
  • 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).
  • 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.
  • 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.
  • 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.
  • gut microbiome and “intestinal microbiome” are used interchangeably unless otherwise noted.
  • 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.
  • 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.
  • unavoidable impurities e.g., no more impurities than what might be unavoidable with standard techniques for manufacture, formulation, transporting, and storage.
  • essentially free of a given substance may mean no more of the given substance than is unavoidable, e.g., as an impurity.
  • 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.
  • 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
  • HMO mixture human milk oligosaccharide mixture
  • 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 8xl0 9 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.
  • EXAMPLE 3 B. LONGUM SUBSP. INFANTIS DETECTION BY QPCR IN HEALTHY SUBJECTS AFTER VANCOMYCIN AND METRONIDAZOLE EXPOSURE
  • 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.
  • Results are displayed in FIGS. 2A-2C.
  • subjects from Cohort 1 antibiotics alone
  • 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.
  • B. longum subsp. infantis levels were significantly higher as early as day 11 (p 0.0004; Sidak’s post-hoc test; Figure 7A).
  • 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).
  • B. longum subsp. infantis levels in these subjects were maintained at -10 3 copies/ng DNA through day 35, which is -100-fold lower than for engrafted subjects who also received HMO.
  • 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.
  • 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
  • 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.
  • OFT Operational Taxonomic Unit
  • an OTU table was calculated with the read counts per genome per sample.
  • Alpha diversity metrics observed reads and Shannon entropy
  • 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.
  • 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.
  • EXAMPLE 5 HUMAN MILK OLIGOSACCHARIDE-MEDIATED ENGRAFTMENT WITH B. LONGUM SUBSP. INFANTIS ALTERS MlCROBIOME COMMUNITIES AFTER TRANSIENT, ANTIBIOTIC-INDUCED DYSBIOSIS
  • 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).
  • 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.
  • 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.
  • 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.
  • EXAMPLE 6 QUANTIFICATION OF SHORT-CHAIN FATTY ACIDS AND LACTATE IN HUMAN STOOL
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HESI-II heated electrospray ionization
  • 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 MS n scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z.
  • 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.
  • RI retention time/index
  • m/z mass to charge ratio
  • chromatographic data including MS/MS spectral data
  • 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.
  • 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
  • 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).
  • pCS uremic toxin p-cresol sulfate
  • 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.
  • FIGS. 9M and 9N uremic toxins phenol sulfate
  • pCG p- cresol-glucuronide
  • 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.
  • 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.
  • EXAMPLE 10 ISOLATION OF VEILLONELLA SP. FROM STOOL SAMPLES
  • 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).
  • EXAMPLE 11 ENHANCED IN VITRO GROWTH OF AND PRODUCTION OF PROPIONATE BY VEILLONELLA SP. CO-CULTURED WITH B. LONGUM SUBSP. INFANTIS AND HMO
  • 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.
  • EXAMPLE 12 ENHANCED IN VIVO GROWTH OF AND PRODUCTION OF PROPIONATE BY VEILLONELLA SP. CO-CULTURED WITH B. LONGUM SUBSP. INFANTIS AND HMO
  • mice 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.
  • mice 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.
  • EXAMPLE 13 EVALUATING ADMINISTRATION OF B. LONGUM SUBSP. INFANTIS AND HMO IN A MOUSE MODEL OF ACUTE RADIATION SYNDROME (ARS)
  • 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.
  • 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.
  • 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.
  • 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.
  • tissue regeneration e.g., intestinal crypt regeneration
  • 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 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.
  • 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.
  • mice administered human milk oligosaccharides 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.
  • Similar experiments may be performed in other animal models, such as in rats, cats, dogs, or non-human primates.
  • 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
  • EXAMPLE 15 ENHANCED IN VIVO PRODUCTION OF PROPIONATE BY VEILLONELLA SP. COCULTURED WITH B. LONGUM SUBSP. INFANTIS AND HMO
  • 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.
  • 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.
  • stool levels of B. longum subsp. infantis increased from the level of detection at day 8 to detectable levels at days 10 and 11.
  • mice 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.
  • EXAMPLE 16 ENHANCED IN VIVO PRODUCTION OF PROPIONATE BY MEGASPHAERA SP. COCULTURED WITH B. LONGUM SUBSP. INFANTIS AND HMO
  • mice 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.
  • PBS phosphate buffered saline
  • HMO human milk oligosaccharides
  • 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.
  • 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.
  • stool levels of B. longum subsp. infantis increased from the level of detection at day 8 to detectable levels at days 10 and 11.
  • mice 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.

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Abstract

La présente invention concerne des compositions, des méthodes, des stratégies, des kits et des articles manufacturés qui sont utiles, entre autres, dans le traitement ou la prévention de maladies, de désordres ou d'affections qui peuvent être associés à une inflammation, une infection, une allergie, un dysfonctionnement immunitaire ou une dysbiose du microbiome intestinal.<i /> Selon certains aspects, l'invention concerne une combinaison synergique d'un prébiotique, par exemple, un mélange d'oligosaccharides de lait humain et de souches probiotiques de bactéries telles qu'une ou plusieurs souches capables d'internaliser et de consommer le prébiotique, par exemple, Bifidobacterium longum subsp.infantis et une ou plusieurs souches capables de produire des acides gras à chaîne courte tels que le propionate, par exemple Veillonella sp. <i /> <i />
PCT/US2023/084290 2022-12-16 2023-12-15 Compositions symbiotiques pour la production d'acides gras à chaîne courte Ceased WO2024130119A2 (fr)

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