WO2020227689A1 - Oligosaccharide compositions and methods of use thereof - Google Patents
Oligosaccharide compositions and methods of use thereof Download PDFInfo
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- WO2020227689A1 WO2020227689A1 PCT/US2020/032240 US2020032240W WO2020227689A1 WO 2020227689 A1 WO2020227689 A1 WO 2020227689A1 US 2020032240 W US2020032240 W US 2020032240W WO 2020227689 A1 WO2020227689 A1 WO 2020227689A1
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- 0 *OC[C@]([C@](C(C1O*)O*)O[C@]1O*)O* Chemical compound *OC[C@]([C@](C(C1O*)O*)O[C@]1O*)O* 0.000 description 9
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- 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
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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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
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present disclosure relates to oligosaccharide compositions and uses thereof.
- CRE carbapenem-resistant Enterobacteriaceae
- VRE vancomycin-resistant Enterococci
- Colonization may be associated with transmission as well as increased risk of infection.
- CRE and vancomycin-resistant enterococcus (VRE) colonization are associated with increased risk of infection.
- the relative risk of infection is higher in VRE and CRE colonized patients compared to non-colonized patients.
- BI bloodstream infection
- ESBL extended- spectrum beta-lactamase
- colonization is highly associated with spread of hospital acquired infections (HAI).
- CRE, VRE and C. difficile infections are associated with being acquired after receiving healthcare (e.g ., central line-associated bloodstream infections (CLABSI), catheter-associated urinary tract Infection (CAUTI), and C. difficile infections (CDI)).
- CLABSI central line-associated bloodstream infections
- CAUTI catheter-associated urinary tract Infection
- CDI C. difficile infections
- microbiome metabolic therapies utilizing oligosaccharide compositions that are useful for driving functional outputs of the gut microbiome organ, e.g., to treat disease.
- Some aspects of the disclosure relate to a recognition that commensal microbes offer protection from pathogen infection and that in immunocompromised hosts or with antibiotic treatment, the protective properties of the microbial community are compromised, leaving the gut susceptible to pathogen colonization.
- microbiome metabolic therapies utilizing oligosaccharide compositions are particularly effective for reducing the acquisition of, colonization of, or reducing the reservoir of a pathogen (e.g., a drug or antibiotic resistant pathogen, or an MDR pathogen) in a subject, e.g., by modulating the abundance (e.g., relative abundance or absolute abundance) of commensal microbial populations.
- a pathogen e.g., a drug or antibiotic resistant pathogen, or an MDR pathogen
- An exemplary mechanism for the reduction of pathogens and increase of commensal bacteria is shown in FIG. 1.
- microbiome metabolic therapies utilizing oligosaccharide compositions disclosed herein are useful for treating a subject having or at risk of developing an infection (e.g., a gastrointestinal infection, or another infection, e.g.
- a pathogen e.g., a bacterial or fungal pathogen or pathobiont (e.g., an opportunistic pathogen that is a symbiotic organism capable of causing disease only when certain genetic and/or environmental conditions are present), including a pathogen resistant to most or even all available antibiotics) such as carbapenem-resistant
- CRE Enterobacteriaceae
- VRE vancomycin-resistant Enterococcus
- ESDLE extended- spectrum beta lactamase-producing Enterobacteriaceae
- selected synthetic oligosaccharide compositions that affect the structure (e.g., composition) and/or function (e.g. metabolic activity) of the gut microbiota.
- the selected oligosaccharide compositions confer beneficial health effects on a subject.
- the selected oligosaccharide compositions described herein reduce the abundance (e.g., relative abundance or absolute abundance) of pathogens or pathobionts (e.g., in the gastrointestinal tract), e.g., when compared to a baseline (e.g., untreated (population of) subject(s), or a subject prior to treatment).
- the selected oligosaccharide compositions described herein promote growth of commensal bacteria over growth of pathogens or pathobionts (e.g., in the gastrointestinal tract, e.g., the intestines, e.g., the large intestine or colon).
- pathogens or pathobionts e.g., in the gastrointestinal tract, e.g., the intestines, e.g., the large intestine or colon.
- subjects achieve decolonization with MDR pathogens (e.g ., vancomycin-resistant Enterococcus (VRE), extended-spectrum beta lactamase- producing Enterobacteriaceae (ESBLE), and carbapenem-resistant Enterobacteriaceae (CRE), e.g., levels of these bacteria are near to or fall below detectable levels.
- VRE vancomycin-resistant Enterococcus
- EMBLE extended-spectrum beta lactamase- producing Enterobacteriaceae
- the reduction in the abundance (e.g., relative abundance or absolute abundance) of a pathogen or pathobiont may be determined, e.g., by subjecting a sample (e.g., a stool sample) from a subject to nucleic acid sequencing (e.g., whole genome sequencing) and other assays (e.g., colony-forming units (cfu)/g feces by culture).
- nucleic acid sequencing e.g., whole genome sequencing
- other assays e.g., colony-forming units (cfu)/g feces by culture.
- the selected oligosaccharide compositions described herein promote an increase in alpha-diversity (e.g. an increase in bacterial taxa diversity, e.g., as determined by measuring Shannon diversity, e.g. by nucleic acid sequencing).
- the selected oligosaccharide compositions described herein promote richness of the bacterial community.
- the selected oligosaccharide compositions described herein reduce inflammation, e.g. inflammation associated with pathogens or pathobionts or other bacteria. The reduction may be determined by measuring one or more markers of inflammation, e.g. IFN-g, IL-Ib, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-a.
- the markers can be determined, e.g., from stool or blood samples.
- the selected oligosaccharide compositions described herein reduce infections (e.g., the rate of infections), including secondary or opportunistic infections (e.g., hospital acquired infections (HAI)), including, e.g., central line-associated bloodstream infections (CLABSI), catheter-associated urinary tract Infection (CAUTI), and C. difficile infections (CDI)).
- infections e.g., the rate of infections
- HAI hospital acquired infections
- CLABSI central line-associated bloodstream infections
- CAUTI catheter-associated urinary tract Infection
- CDI C. difficile infections
- the selected oligosaccharide compositions described herein reduce the rate of hospitalizations, e.g., due to or caused by infections.
- the selected oligosaccharide compositions described herein shorten the time period of hospitalization required, e.g., to treat or resolve the infections.
- the disclosure provides an oligosaccharide composition comprising a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III):
- each R independently is selected from hydrogen, and Formulae (la), (lb), (Ic), (Id), (Ila), (lib), (lie), (lid), (Ilia), (Illb), (IIIc), (Hid):
- the oligosaccharide composition is produced by a process comprising: (a) forming a reaction mixture comprising dextrose monomer, galactose monomer, and mannose monomer wherein the molar ratio of dextrose to galactose is about 1:1 and the molar ratio of dextrose to mannose is about 4.5:1 with an acid catalyst comprising positively charged hydrogen ions; and
- step (b) comprises promoting acid catalyzed oligosaccharide formation in the reaction mixture by transferring sufficient heat to the reaction mixture to maintain the reaction mixture at its boiling point until the weight percent of total monomer content in the oligosaccharide composition is in a range of 2% to 20%, wherein the total monomer content comprises dextrose monomer, galactose monomer, and/or mannose monomer.
- the disclosure provides an oligosaccharide composition comprising a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III):
- each R independently is selected from hydrogen, and Formulae (Ila), (lib), (lie), (lid), (Ilia), (mb), ( me ), (Hid):
- oligosaccharide composition is produced by a process comprising:
- step (b) comprises loading the preparation with an acid catalyst comprising positively charged hydrogen ions, in an amount such that the molar ratio of positively charged hydrogen ions to total dextrose monomer, galactose monomer, and mannose monomer content is in an appropriate range.
- steps (a) and (b) occur simultaneously.
- step (a) comprises heating the reaction mixture under agitation conditions to a temperature in a range of 100°C to 160 °C.
- step (a) comprises heating the reaction mixture under agitation conditions to a temperature in a range of 135 °C to 145 °C.
- step (a) comprises heating the reaction mixture under agitation conditions at a temperature in a range of 100°C to 160 °C.
- step (a) comprises heating the reacion mixture under agitation conditions at a temperature in a range of 135 °C to 145 °C.
- step (a) comprises gradually increasing the temperature (e.g., from room temperature) to about 140 °C, under suitable conditions to achieve homogeneity and uniform heat transfer.
- step (b) comprises maintaining the reaction mixture at
- step (b) comprises gradually increasing the temperature (e.g., from room temperature) to about 140 °C, under suitable conditions to achieve homogeneity and uniform heat transfer.
- said heating comprises melting the preparation and/or heating the preparation under suitable conditions to achieve homogeneity and uniform heat transfer.
- the acid catalyst is a soluble catalyst.
- the soluble catalyst is an organic acid, optionally a weak organic acid.
- the acid catalyst is citric acid, acetic acid, or propionic acid.
- the acid catalyst is a strong acid cation exchange resin having one or more physical and chemical properties according to Table 1 and/or wherein the catalyst comprises > 3.0 mmol/g sulfonic acid moieties and ⁇ 1.0 mmol/gram cationic moieties.
- the catalyst has a nominal moisture content of 45-50 weight percent.
- the catalyst has some or all of the properties shown in Table 1.
- the oligosaccharide composition further comprises water at a level below that which is necessary for microbial growth upon storage at room temperature.
- step (b) further comprises removing water from the reaction mixture by evaporation. In some embodiments, step (b) further comprises maintaining the reaction mixture at 93-94 weight percent dissolved solids.
- the process further comprises: (c) quenching the reaction mixture, for example, using water, while bringing the temperature of the reaction mixture to a temperature in the range of 55 °C to 95 °C (e.g., 85 °C, 90 °C); and optionally, (d) separating
- step (c) the water is deionized water. In some embodiments, in (c) the water has a temperature of about 95 °C. In some embodiments, in (c) the water is added to the reaction mixture under conditions sufficient to avoid solidifying the mixture.
- step (d) said separating comprises removing the catalyst by filtration. In some embodiments, (d) comprises cooling the reaction mixture to below about 85 °C before filtering. [0020] In some embodiments, the process further comprises: (e) diluting the oligosaccharide composition of (d) with water to a concentration of about 45-55 weight percent; (f) passing the diluted composition through a cationic exchange resin; (g) passing the diluted composition through a decolorizing polymer resin; and/or (h) passing the diluted composition through an anionic exchange resin; wherein each of (f), (g), and (h) can be performed one or more times in any order.
- the process further comprises diluting the oligosaccharide composition of (d) with water to a concentration of about 35-55 weight percent and passing the diluted composition through a 45 pm filter.
- composition comprises water at a level below that which is necessary for microbial growth upon storage at room temperature.
- the composition comprises water in a range of 45-55 weight percent.
- the composition has a MWw (g/mol) in a range of 1905-2290. In some embodiments, the composition has a MWw (g/mol) in a range of 1740-2407. In some embodiments, the composition has a MWw (g/mol) in a range of 1863-2268. In some embodiments, the composition has a MWw (g/mol) in a range of 1700-2295. In some embodiments, the composition has a MWn (g/mol) in a range of 1033-1184. In some
- the composition has a MWn (g/mol) in a range of 975-1155.
- the composition has a MWn (g/mol) in a range of 984-1106.
- the composition has a MWn (g/mol) in a range of 938-1120.
- a solution comprising the oligosaccharide composition has a pH in a range of 2.50-7.00, optionally 2.50-3.50.
- the composition comprises oligomers having two or more repeat units (DP2+) in a range of 86-96 weight percent. In some embodiments, the composition comprises oligomers having two or more repeat units (DP2+) in a range of 81-100 weight percent. In some embodiments, the composition comprises oligomers having at least three linked monomer units (DP3+) in a range of 85-90 weight percent.
- the composition further comprises: 0.18-0.51% w/w
- levoglucosan 0.01-0.05% w/w lactic acid, and/or 0.04-0.07% w/w formic acid.
- the composition further comprises: 0.40-0.53% w/w levoglucosan, 0.01-0.02% w/w lactic acid, 0.01-0.04% w/w formic acid, and/or 0.01-0.04% w/w citric acid.
- the disclosure provides an oligosaccharide composition
- oligosaccharide composition comprising a plurality of oligosaccharides that are minimally digestible in humans, the composition being characterized by a multiplicity-edited gradient-enhanced 3 ⁇ 4-3 ⁇ 4 heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 5, 6, 7, and 15 of the following table, wherein the spectrum is generated using a sample of the oligosaccharide composition having less than 2% monomer:
- HSQC heteronuclear single quantum correlation
- the oligosaccharide composition is characterized by a multiplicity- edited gradient-enhanced 1 H- 13 C heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 5, 6, 7, 10, 14, and 15 of the following table, wherein the spectrum is generated using a sample of the oligosaccharide composition having less than 2% monomer:
- HSQC heteronuclear single quantum correlation
- the oligosaccharide composition is characterized by a multiplicity- edited gradient-enhanced 1 H- 13 C heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 5, 6, 7, and 10-15 of the following table, wherein the spectrum is generated using a sample of the oligosaccharide composition having less than 2% monomer:
- HSQC heteronuclear single quantum correlation
- the oligosaccharide composition is characterized by a multiplicity- edited gradient-enhanced 1 H- 13 C heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 1-15 of the following table, wherein the spectrum is generated using a sample of the oligosaccharide composition having less than 2% monomer:
- HSQC heteronuclear single quantum correlation
- the disclosure provides an oligosaccharide composition comprising a plurality of oligosaccharides that are minimally digestible in humans, the composition being characterized by a multiplicity-edited gradient-enhanced heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 5, 6, 7, and 15 of the following table, wherein the spectrum is generated using a sample of the oligosaccharide composition having less than 2% monomer:
- HSQC multiplicity-edited gradient-enhanced heteronuclear single quantum correlation
- the oligosaccharide composition is characterized by a multiplicity- edited gradient-enhanced 1 H- 13 C heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 5, 6, 7, 10, 14, and 15 of the following table, wherein the spectrum is generated using a sample of the oligosaccharide composition having less than 2% monomer:
- HSQC heteronuclear single quantum correlation
- the oligosaccharide composition is characterized by a multiplicity- edited gradient-enhanced 1 H- 13 C heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 5, 6, 7, and 10-15 of the following table, wherein the spectrum is generated using a sample of the oligosaccharide composition having less than 2% monomer:
- HSQC heteronuclear single quantum correlation
- the oligosaccharide composition is characterized by a multiplicity- edited gradient-enhanced 1 H- 13 C heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 1-15 of the following table, wherein the spectrum is generated using a sample of the oligosaccharide composition having less than 2% monomer:
- HSQC heteronuclear single quantum correlation
- any one of signals 1-15 are further characterized by an ' H integral region and a 13 C integral region, defined as follows:
- the disclosure provides an oligosaccharide composition comprising a plurality of oligosaccharides that are minimally digestible in humans, each oligosaccharide comprising a plurality of monomer radicals;
- the plurality of oligosaccharides comprising two or more types of monomer radicals selected from radicals (l)-(40):
- t-mannopyranose monoradicals representing 3.0-4.1 mol% of monomer radicals in the plurality of oligosaccharides
- t-galactopyranose monoradicals representing 8.3-12.5 mol% of monomer radicals in the plurality of oligosaccharides
- 3-glucopyranose monoradicals representing 3.0-4.9 mol% of monomer radicals in the plurality of oligosaccharides
- 2-mannopyranose and/or 3-mannopyranose monoradicals representing 1.2- 1.9 mol% of monomer radicals in the plurality of oligosaccharides
- 6-mannopyranose monoradicals representing 2.0-2.9 mol% of monomer radicals in the plurality of oligosaccharides
- 6-galactofuranose monoradicals representing 1.4-5.0 mol% of monomer radicals in the plurality of oligosaccharides
- (21) 3, 4-glucopyranose and/or 3, 5-glucofuranose diradicals, representing 0.1- 1.1 mol% of monomer radicals in the plurality of oligosaccharides;
- (22) 2,4-glucopyranose and/or 2,5-glucofuranose and/or 2,4-galactopyranose and/or 2,5-galactofuranose diradicals, representing 0.9- 1.4 mol% of monomer radicals in the plurality of oligo s accharides ;
- the oligosaccharide composition comprising at least one glucofuranose or glucopyranose radical, at least one mannofuranose or mannopyranose radical, and at least one galactofuranose or galactopyranose radical.
- the disclosure provides an oligosaccharide composition comprising a plurality of oligosaccharides that are minimally digestible in humans, each oligosaccharide comprising a plurality of monomer radicals;
- the plurality of oligosaccharides comprising two or more types of monomer radicals selected from radicals (l)-(43):
- t-mannopyranose monoradicals representing 3.0-4.1 mol% of monomer radicals in the plurality of oligosaccharides
- t-galactopyranose monoradicals representing 9.7-11.7 mol% of monomer radicals in the plurality of oligosaccharides
- 2-mannopyranose and/or 3-mannopyranose monoradicals representing 0.8-2.0 mol% of monomer radicals in the plurality of oligosaccharides
- 2-glucopyranose monoradicals representing 2.7-3.0 mol% of monomer radicals in the plurality of oligosaccharides
- 6-mannopyranose monoradicals representing 2.1-2.5 mol% of monomer radicals in the plurality of oligosaccharides
- 6-galactofuranose monoradicals representing 2.3-2.7 mol% of monomer radicals in the plurality of oligosaccharides
- (21) 3, 4-galactopyranose and/or 3, 5-galactofuranose and/or 2, 3-galactopyranose diradicals, representing 0.9- 1.1 mol% of monomer radicals in the plurality of oligosaccharides;
- (22) 3, 4-glucopyranose and/or 3, 5-glucofuranose diradicals, representing 0.5-0.8 mol% of monomer radicals in the plurality of oligosaccharides;
- the oligosaccharide composition comprising at least one glucofuranose or glucopyranose radical, at least one mannofuranose or mannopyranose radical, and at least one galactofuranose or galactopyranose radical.
- the molar percentages of the monomer radicals are determined using a permethylation assay.
- the composition is substantially non-absorbable in a human.
- the disclosure provides a method of reducing a ratio of pathogenic bacteria to commensal bacteria in the gastrointestinal tract of a human subject.
- a method of reducing a ratio of pathogenic bacteria to commensal bacteria in the gastrointestinal tract of a human subject comprises administering to the gastrointestinal tract of the subject an effective amount of an oligosaccharide composition as described herein.
- the disclosure provides a method of reducing the relative or absolute abundance of pathogens in the gastrointestinal tract of a human subject.
- a method of reducing the relative or absolute abundance of pathogens in the gastrointestinal tract of a human subject comprises administering to the gastrointestinal tract of the subject an effective amount of an oligosaccharide composition as described herein.
- the oligosaccharide composition is administered in an amount effective to modulate (e.g. reduce or inhibit) colonization or to modulate (e.g. increase) decolonization of the pathogen in the gut (e.g., small intestine, large intestine and/or colon) of the human subject.
- the oligosaccharide composition is administered in an amount effective to reduce or inhibit colonization (e.g., colonization by VRE, CRE, and/or ESBLE).
- the oligosaccharide composition is administered in an amount effective to increase decolonization (e.g ., decolonization by VRE, CRE, and/or ESBLE).
- a method reduces the abundance of pathogenic bacteria in the gastrointestinal tract, relative to a control (e.g., a control subject or baseline measurement).
- a method increases the abundance of commensal bacteria in the gastrointestinal tract, relative to a control (e.g., a control subject or baseline measurement).
- the reduction of the relative or absolute abundance of pathogens is determined by performing nucleic acid sequencing (e.g., 16S metagenomic sequencing) of a fecal sample collected from the subject.
- nucleic acid sequencing e.g., 16S metagenomic sequencing
- the reduction of the relative or absolute abundance of pathogens is determined by: (i) performing 16S metagenomic sequencing of a fecal sample collected from the subject prior to administration of the oligosaccharide composition; (ii) performing 16S metagenomic sequencing of a fecal sample collected from the subject following administration of the oligosaccharide composition; and (iii) comparing the relative or absolute abundance of pathogens determined using the sequencing data provided in (ii) relative to the relative or absolute abundance of pathogens determined using the sequencing data provided in (i).
- the disclosure provides a method of treating a subject for a pathogen infection.
- a method of treating a subject for a pathogen infection comprises administering to the gastrointestinal tract of the subject an effective amount of an oligosaccharide composition as described herein, thereby treating the subject.
- a method of treating a subject for a pathogen infection comprises administering to the gastrointestinal tract of the subject an effective amount of an
- oligosaccharide composition wherein the oligosaccharide composition has an average degree of polymerization of 5-20 and comprises a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III):
- each R independently is selected from hydrogen, and Formulae (la), (lb), (Ic), (Id), (Ila), (lib), (lie), (lid), (Ilia), (Illb), (IIIc), (Hid):
- a method reduces the rate of infection. In some embodiments, a method reduces the abundance of pathogen. In some embodiments, a method reduces the abundance of pathogen of infection by at least 5%, 10%, 20%, or 30%, relative to a baseline measurement ( e.g ., wherein the baseline measurement is determined prior to treatment). In some embodiments, a method treats the infection. In some embodiments, a method prevents the onset of an infection.
- a pathogen infection is an infection of the gastrointestinal tract, lungs, bloodstream, central nervous system, lymphatic system, and/or soft tissues of the subject.
- the oligosaccharide composition is administered in an amount sufficient, to reduce or prevent dysbiosis in the gut (e.g., small intestine, large intestine and/or colon) of the human subject.
- the oligosaccharide composition reduces the risk of an adverse effect of the pathogen on the human subject.
- the oligosaccharide composition is administered in an amount effective to: (a) reduce pathogen biomass (e.g., the number of pathogens and/or the number of drug- or antibiotic-resistance gene or MDR element carriers); (b) modulate (e.g., increase) the level of anti-microbial compounds produced by the subject (e.g., by the resident gut microbiota and/or the host (e.g., human cells)); (c) modulate the environment of the GI tract (e.g., small intestine, large intestine or colon), e.g. reducing the pH (e.g., by increasing production or levels of lactic acid, e.g.
- pathogen biomass e.g., the number of pathogens and/or the number of drug- or antibiotic-resistance gene or MDR element carriers
- modulate e.g., increase
- the level of anti-microbial compounds produced by the subject e.g., by the resident gut microbiota and/or the host
- the oligosaccharide composition is administered in an amount effective to: (a) decrease the relative abundance or absolute abundance of pathogens and/or drug- or antibiotic -resistance gene or MDR element carriers; and/or (b) increase the relative abundance or absolute abundance of commensal or beneficial bacteria.
- the pathogen is a bacterial microorganism or a fungal
- the pathogen is a drug or antibiotic resistant pathogen, optionally a multi-drug resistant (MDR) pathogen.
- MDR multi-drug resistant
- the pathogen is vancomycin resistant Enterococcus (VRE) or carbapenem resistant Enterobacteriaceae (CRE).
- the pathogen is VRE Enterococcus faecium.
- the pathogen is CRE Escherichia coli or CRE Klebsiella pneumoniae.
- the pathogen is Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, or Candida lusitaniae.
- the pathogen is Clostridium difficile.
- the pathogen is gram-positive bacteria or gram-negative bacteria.
- the pathogen is a fungus.
- the pathogen is Candida.
- a human subject has received cancer treatment.
- a human subject is a transplant recipient.
- a human subject has received immunosuppression.
- a human subject has an auto-immune disease (e.g ., systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, or Crohn's disease).
- a human subject has a hematological malignancy.
- a human subject has cirrhosis.
- a human subject has or is at risk of having end-stage liver disease (ESLD).
- ESLD end-stage liver disease
- a human subject is preparing for or recovering from a gastrointestinal surgery.
- a human subject is a patient in an intensive care unit (ICU).
- ICU intensive care unit
- a human subject has had multiple courses of antibiotics, and/or chronic use of antibiotics.
- a human subject has a positive stool culture for Carbapenem-resistant Enterobacteriaciae (CRE), extended spectrum beta lactamase (ESBL) producing Enterobacteriaciae (ESBLE), and
- VRE Vancomycin-resistant Enterococcus
- a human subject has a positive stool culture for Carbapenem-resistant Enterobacteriaciae (CRE).
- CRE Carbapenem-resistant Enterobacteriaciae
- a human subject has a positive stool culture for extended spectrum beta lactamase (ESBL) producing Enterobacteriaciae (ESBLE). In some embodiments, a human subject has a positive stool culture for Vancomycin-resistant Enterococcus (VRE). In some embodiments, a human subject has low diversity of bacterial communities in the gastrointestinal tract. In some embodiments, a human subject is a hematopoietic stem cell transplant (HSCT) recipient. In some embodiments, a human subject is a solid organ transplant recipient. In some embodiments, a human subject has recently had a central line-associated bloodstream infection (CLABSI). In some embodiments, a human subject has recently had a catheter-associated urinary tract infection (CAUTI). In some embodiments, a human subject has recently had a C. difficile infections). [0059] In some aspects, the disclosure provides a method comprising
- a method further comprises administering to the human subject a population of commensal or probiotic bacteria.
- a human subject is a patient having a gut microbiome devoid of any detectable levels of commensal bacteria.
- a method further comprises administering to the human subject antibiotics (e.g., broad spectrum antibiotics) or other standard-of-care treatment concurrent with the oligosaccharide composition.
- antibiotics e.g., broad spectrum antibiotics
- other standard-of-care treatment concurrent with the oligosaccharide composition.
- the subject has been treated with antibiotics (e.g., broad spectrum antibiotics) or other standard-of-care treatment prior to administration with the oligosaccharide composition.
- antibiotics e.g., broad spectrum antibiotics
- other standard-of-care treatment prior to administration with the oligosaccharide composition.
- the oligosaccharide composition is administered to the subject one to twenty-eight days before a cancer treatment, surgery (e.g., transplant, e.g., hematopoietic stem cell), or admision to an intensive care unit.
- the oligosaccharide composition is administered to the subject one to twenty-eight days after a cancer treatment, surgery (e.g., transplant, e.g., hematopoietic stem cell), or admision to an intensive care unit.
- the oligosaccharide composition is administered to the subject at least one to twenty-eight days after onset of a pathogen infection.
- the oligosaccharide composition is administered to the intestines ( e.g ., the large intestine).
- the oligosaccharide composition is self-administered to the subject. In some embodiments, the oligosaccharide composition is formulated as a
- the oligosaccharide composition is orally administered to the subject. In some embodiments, the oligosaccharide composition is formulated as a pharmaceutical composition for delivery by a feeding tube. In some embodiments, the oligosaccharide composition is administered to the subject by a feeding tube.
- the oligosaccharide composition is administered to the subject once per day or twice per day.
- the disclosure provides a method of reducing the relative or absolute abundance of pathogens in the gastrointestinal tract of a human subject, the method comprising administering to the gastrointestinal tract of the subject an effective amount of an oligosaccharide composition, wherein the oligosaccharide composition comprises a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III):
- each R independently is selected from hydrogen, and Formulae (la), (lb), (Ic), (Id), (Ila), (lib), (lie), (lid), (Ilia), (Illb), (IIIc), (Hid):
- oligosaccharide composition is produced by a process comprising:
- step (b) comprises promoting acid catalyzed oligosaccharide formation in the reaction mixture by transferring sufficient heat to the reaction mixture to maintain the reaction mixture at its boiling point until the weight percent of total monomer content in the oligosaccharide composition is in a range of 2% to 20%, wherein the total monomer content comprises dextrose monomer, galactose monomer, and/or mannose monomer.
- the disclosure relates to a oligosaccharide composition (which may be useful as a microbiome metabolic therapy) that comprises a plurality of oligosaccharides selected from Formula (I) Formula (II) and Formula (III):
- each R independently is selected from hydrogen, and Formulae (la), (lb), (Ic), (Id), (Ila), (lib), (lie), (lid), (Ilia), (Illb), (IIIc), (Hid), :
- the oligosaccharide composition is produced by a process comprising:
- oligosaccharide composition (which may be useful as a microbiome metabolic therapy) that comprises a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III): (I) (II) (III) wherein each R independently is selected from hydrogen, and Formulae (Ila), (lib), (lie), (lid), (Ilia), (mb), (IIIc), (Hid):
- oligosaccharide composition is produced by a process comprising:
- FIG. 1 depicts exemplary uses of oligosaccharide compositions to reduce the
- Bacteria depictions in dark color (red) symbolize pathogens or pathobionts.
- Bacteria depictions in light color (blue) symbolize commensal bacteria.
- FIG. 2 provides graphs showing reduction in pathogen growth in cultures of single pathogen strains ( Clostridium difficile strains) incubated in the presence of samples of a selected oligosaccharide composition.
- FIG. 3 provides graphs showing reduction in pathogen growth in cultures of single pathogen strains (VRE Enterococcus faecium) incubated in the presence of samples of a selected oligosaccharide composition.
- FIG. 4 provides graphs showing reduction in pathogen growth in cultures of single pathogen strains (CRE Escherichia coli, CRE Klebsiella pneumoniae ) incubated in the presence of samples of a selected oligosaccharide composition.
- FIG. 5 provides graphs showing reduction in pathogen growth in cultures of single pathogen strains ( Candida albicans , Candida glabrata, Candida krusei, Candida tropicalis) incubated in the presence of samples of a selected oligosaccharide composition.
- FIGs. 6A-6B provide graphs showing reduction in pathogen growth in cultures of single pathogen strains ( Candida lusitaniae ) incubated in the presence of samples of a selected oligosaccharide composition.
- FIG. 6A provides graphs specific for ATCC 66035 strain.
- FIG. 6B provides graphs specific for ATCC 42720 strain.
- FIGs. 7A-7B provide graphs showing reduction in pathogen growth (normalized to water controls) in an ex vivo pathogen reduction assay where fecal samples from 11 ICU patients were incubated with the selected oligosaccharide composition.
- FIG. 7A is a graph showing reduction of pathogens in fecal samples spiked with carbapenem-resistant Enterobacteriaceae .
- FIG. 7B is a graph showing reduction of pathogens in fecal samples spiked with vancomycin-resistant Enterococcaceae.
- FIGs. 8A-8B provide graphs showing reduction in pathogen growth (normalized to water controls) in an ex vivo pathogen reduction assay where fecal samples from hepatic
- FIG. 8A is a graph showing reduction of pathogens in fecal samples spiked with carbapenem- resistant Enterobacteriaceae.
- FIG. 8B is a graph showing reduction of pathogens in fecal samples spiked with vancomycin -resistant Enterococcaceae .
- FIG. 9 depicts a SEC-HPLC chromatogram of a selected oligosaccharide composition using the method provided in Example 15.
- FIG. 10 depicts an overlay of SEC-HPLC chromatograms of standard saccharides for use in Example 17.
- FIG. 11 is a HSQC NMR spectra of the selected oligosaccharide composition.
- FIG. 12 provides a graph showing the microbial compositions of fecal samples collected from ICU patients and fecal samples collected from healthy subjects. Presented are relative proportions of discrete bacterial taxa (genus-level) in each fecal sample.
- FIGs. 13A-13B provide graphs showing reduction in relative proportions of pathogenic microbes (e.g ., VRE E. faecium, Enterobacteriales ) and increase in relative proportions of commensal microbes in an ex vivo pathogen reduction assay.
- pathogenic microbes e.g ., VRE E. faecium, Enterobacteriales
- Fecal samples that had been spiked with vancomycin-resistant Enterococcaceae or carbapenem-resistant Enterobacteriaceae were incubated with the selected oligosaccharide composition, FOS, or water.
- aspects of the disclosure relate to oligosaccharide compositions that are effective for reducing pathogen levels, abundance and/or colonization and colonization in a subject.
- Some aspects of the disclosure are based on the results of an extensive screening effort that was performed to identify oligosaccharide compositions that are capable of modulating, e.g., reducing, levels of pathogens in a subject. Hundreds of unique oligosaccharide compositions were assayed for their effect on pathogen levels. The oligosaccharide compositions examined in the screen were produced using different saccharide monomers, e.g., dextrose monomers, xylose monomers, etc., and under conditions involving differing reaction temperatures, for varying periods of time, and/or in the presence of different catalyst conditions.
- saccharide monomers e.g., dextrose monomers, xylose monomers, etc.
- this oligosaccharide composition is particularly useful for treating subjects having high levels of pathogen colonization in their GI tract (e.g., subjects colonized with pathogens in their intestines) and/or receiving broad spectrum antibiotics. Further aspects of the disclosure, including a description of defined terms, are provided below.
- Agitation conditions refers to conditions that promote or maintain a substantially uniform or homogeneous state of a mixture (e.g., a reaction mixture comprising dextrose monomer, galactose monomer, and mannose monomer) with respect to dispersal of solids (e.g., solid catalysts), uniformity of heat transfer, or other similar parameters.
- Agitation conditions generally include stirring, shaking, and/or mixing of a reaction mixture.
- agitation conditions may include the addition of gases or other liquids into a solution.
- agitation conditions are used to maintain substantially uniform or homogenous distribution of a catalyst, e.g., an acid catalyst.
- a monosaccharide preparation is heated in the presence of an acid catalyst under suitable conditions to achieve homogeneity and uniform heat transfer in order to synthesize an oligosaccharide composition.
- an acid catalyst under suitable conditions to achieve homogeneity and uniform heat transfer in order to synthesize an oligosaccharide composition.
- the term“approximately” or“about” refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
- Dextrose monomer refers to a D-isomer of a glucose monomer, known as D -glucose.
- a dextrose monomer is dextrose monohydrate or 70DS com syrup.
- Effective amount refers to an administered amount or concentration of an oligosaccharide composition that is necessary and sufficient to elicit a biological response, e.g., in a subject or patient.
- an effective amount of an oligosaccharide composition is capable of modulating, e.g., increasing or decreasing, the activity or levels of an enzyme in a subject.
- an effective amount of an oligosaccharide composition is capable of modulating, e.g., increasing or decreasing, the processing of a metabolite.
- an effective amount of an oligosaccharide composition is capable of modulating, e.g., increasing or decreasing, the concentration or number of at least one microbial species. In some embodiments, an effective amount of an oligosaccharide composition is capable of modulating, e.g., decreasing, the symptoms of a disease associated with elevated pathogen colonization in a subject (e.g., the severity or number of symptoms). In some embodiments, an effective amount of an oligosaccharide composition is capable of modulating, e.g., increasing or decreasing, the concentration or number of at least one microbial species. In some embodiments, an effective amount of an oligosaccharide composition is capable of modulating, e.g., decreasing, the symptoms of a disease associated with elevated pathogen colonization in a subject (e.g., the severity or number of symptoms). In some embodiments, an effective amount of an oligosaccharide composition is capable of modulating, e.g., increasing or decreasing, the concentration or number of at least
- oligosaccharide composition is capable of reducing the acquisition of, colonization of, or reducing the reservoir of a pathogen (e.g., a drug or antibiotic resistant pathogen, or an MDR pathogen) in a subject.
- a pathogen e.g., a drug or antibiotic resistant pathogen, or an MDR pathogen
- an effective amount of an oligosaccharide composition is capable of treating a subject having intestinal colonization with a pathogen, e.g., CRE or VRE.
- Galactose monomer As used herein, the term“galactose monomer” generally refers to a D-isomer of a galactose monomer, known as D -galactose.
- Mannose monomer As used herein, the term“mannose monomer” generally refers to a D-isomer of a mannose monomer, known as D -mannose.
- Monosaccharide Preparation As used herein, the term“monosaccharide preparation” refers to a preparation that comprises two or more monosaccharides (e.g ., dextrose monomer, galactose monomer, and mannose monomer). In some embodiments, a monosaccharide preparation comprises dextrose monomers, galactose monomers, and mannose monomers.
- Oligosaccharide refers to a saccharide molecule comprising at least two monosaccharides (e.g., dextrose monomers, galactose monomers, mannose monomers) linked together via a glycosidic bond (having a degree of polymerization (DP) of at least 2 (e.g., DP2+)).
- monosaccharides e.g., dextrose monomers, galactose monomers, mannose monomers
- DP degree of polymerization
- an oligosaccharide comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten monosaccharides subunits linked by glycosidic bonds.
- an oligosaccharide is in the range of 3-20, 4-16, 5-15, 8-12, 5-25, 10-25, 20-50, 40-80, or 75-100 monosaccharides linked by glycosidic bonds.
- an oligosaccharide comprises at least one 1,2; 1,3; 1,4; and/or 1,6 glycosidic bond.
- Oligosaccharides may be linear or branched. Oligosaccharides may have one or more glycosidic bonds that are in alpha-configurations and/or one or more glycosidic bonds that are in beta- configurations.
- composition refers to a composition having pharmacological activity or other direct effect in the mitigation, treatment, or prevention of disease, and/or a finished dosage form or formulation thereof and is for human use.
- a pharmaceutical composition or pharmaceutical preparation is typically produced under good manufacturing practices (GMP) conditions.
- GMP good manufacturing practices
- Pharmaceutical compositions or preparations may be sterile or non-sterile. If non-sterile, such pharmaceutical compositions or preparations typically meet the microbiological specifications and criteria for non-sterile pharmaceutical products as described in the U.S. Pharmacopeia (USP) or European Pharmacopoeia (EP). Any oligosaccharide composition described herein may be formulated as a pharmaceutical composition.
- Subject refers to a human subject or patient. Subjects may include a newborn (a preterm newborn, a full-term newborn), an infant up to one year of age, young children (e.g., 1 yr to 12 yrs), teenagers, (e.g., 13-19 yrs), adults (e.g., 20-64 yrs), and elderly adults (65 yrs and older). In some embodiments, a subject is of a pediatric population, or a subpopulation thereof, including neonates (birth to 1 month), infants (1 month to 2 years), developing children (2-12 years), and adolescents (12-16 years). In some embodiments, a subject is a healthy subject.
- a subject is a patient having higher abundance of pathogen relative to a healthy subject, e.g., a subject colonized with a pathogen (e.g., CRE and/or VRE pathogens) in their gastrointestinal tract (e.g., their colon or intestines.).
- a pathogen e.g., CRE and/or VRE pathogens
- their gastrointestinal tract e.g., their colon or intestines.
- a subject is a patient receiving broad spectrum antibiotics.
- the subject is particularly susceptible to pathogen infection, e.g., the subject is critically-ill and/or immunocompromised.
- the subject is a patient having a lower abundance of commensal bacteria relative to a healthy subject in their gastrointestinal tract (e.g., their colon or intestines).
- treatment and Treating refer to the administration of a composition to a subject (e.g., a symptomatic subject afflicted with an adverse condition, disorder, or disease) so as to affect a reduction in severity and/or frequency of a symptom, eliminate a symptom and/or its underlying cause, and/or facilitate improvement or remediation of damage, and/or preventing an adverse condition, disorder, or disease in an asymptomatic subject who is susceptible to a particular adverse condition, disorder, or disease, or who is suspected of developing or at risk of developing the condition, disorder, or disease.
- a subject e.g., a symptomatic subject afflicted with an adverse condition, disorder, or disease
- treating a subject with an oligosaccharide composition reduces the relative or absolute abundance of pathogens in the gastrointestinal tract of the subject. In some embodiments, treating a subject with an oligosaccharide composition slows or reduces the rate of a pathogen infection (e.g., in the gastrointestinal tract). In some embodiments, treating a subject with an oligosaccharide composition prevents the onset of a pathogen infection (e.g., in the gastrointestinal tract). In some embodiments, the oligosaccharide composition treats an infection (e.g., bacterial infection). In some embodiments, the oligosaccharide composition treats a localized infection.
- an infection e.g., bacterial infection
- the oligosaccharide composition treats a localized infection.
- the oligosaccharide composition treats a nascent infection. In some embodiments, the oligosaccharide composition treats a systemic infection, e.g., a systemic C. diff infection. However, in some embodiments, the selected oligosaccharide composition prevents an infection, e.g., a localized, nascent, or systemic infection. In some embodiments, treating a subject with an oligosaccharide composition eliminates a pathogen infection and/or reduces pathogenic load (e.g., in the gastrointestinal tract). In some
- a subject has or is at risk of a pathogenic (e.g., bacterial or fungal) infection (e.g., an infection of the gastrointestinal tract).
- a subject has recently had and/or recovered pathogenic (e.g., bacterial or fungal) infection (e.g., an infection of the gastrointestinal tract).
- a subject is an immunocompromised subject (e.g ., a hematopoietic stem cell transplantation (HSCT) patient).
- a subject is a healthy subject.
- oligosaccharide compositions and their methods of use for modulating levels of pathogens in a human subject.
- oligosaccharide compositions comprise a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III):
- each R independently is selected from hydrogen, and Formulae (la), (lb), (Ic), (Id), (Ila), (lib), (lie), (lid), (Ilia), (Illb), (IIIc), (Hid):
- oligosaccharide compositions are produced by a process that initially involves heating a preparation comprising dextrose monomers, galactose monomers, and mannose monomers to a temperature in a range of 100 °C to 160 °C, 100 °C to 120 °C, 110 °C to 130 °C, 120 °C to 140 °C, 130 °C to 150 °C, or about 140 °C.
- the ratio of dextrose monomers to galactose monomers may be 1:1.
- the ratio of dextrose monomers to mannose monomers may be 4.5:1.
- the ratio of galactose monomers to mannose monomers may be 4.5:1.
- Heating may be performed under agitation conditions.
- Heating may comprises gradually increasing the temperature (e.g ., from room temperature) to about 130 °C, about 135 °C about 140 °C about 145 °C, or about 150 °C under suitable conditions to achieve homogeneity and uniform heat transfer.
- An acid catalyst comprising positively charged hydrogen ions is added to the preparation (e.g., following heating).
- the acid catalyst is a solid catalyst.
- the catalyst is a strong acid cation exchange resin having one or more physical and chemical properties according to Table 1.
- the catalyst comprises > 3.0 mmol/g sulfonic acid moieties and ⁇ 1.0 mmol/gram cationic moieties.
- the catalyst has a nominal moisture content of 45-50 weight percent.
- the catalyst is added at the same time as the dextrose monomers, galactose monomers, and mannose monomers.
- the resultant reaction mixture is held at atmospheric pressure and at a temperature in a range of 100 °C to 160 °C, 100 °C to 120 °C, 110 °C to 130 °C, 120 °C to 140 °C, 130 °C to 150 °C, or about 140 °C under conditions that promote acid catalyzed oligosaccharide formation.
- total monomer content comprises the amount of dextrose monomer, galactose monomer, and/or mannose monomer
- total monomer content comprises the amount of dextrose monomer, galactose monomer, and/or mannose monomer
- total monomer content comprises the amount of dextrose monomer, galactose monomer, and/or mannose monomer
- total monomer content comprises the amount of dextrose monomer, galactose monomer, and/or mannose monomer
- the reaction mixture is quenched. Quenching typically involves using water (e.g ., deionized water) to dilute the reaction mixture, and gradually decrease the temperature of the reaction mixture to 55 °C to 95 °C. In some embodiments, the water used for quenching is about 95 °C.
- the water may be added to the reaction mixture under conditions sufficient to avoid solidifying the mixture.
- water may be removed from the reaction mixture by evaporation.
- the reaction mixture may contain 93-94 weight percent dissolved solids.
- the composition is generally separated from the acid catalyst, typically by diluting the quenched reaction mixture with water to a concentration of about 45-55 weight percent and a temperature of below about 85 °C and then passing the mixture through a filter or a series of chromatographic resins.
- the filter used is a 0.45 pm filter.
- a series of chromatographic resins may be used and generally involves a cationic exchange resin, an anionic exchange resin, and/or a decolorizing polymer resin.
- the oligosaccharide composition comprises water at a level below that which is necessary for microbial growth upon storage at room temperature.
- the mean degree of polymerization of all oligosaccharides is in a range of 7-15.5, optionally 11-15.
- the oligosaccharide composition comprises water in a range of 45-55 weight percent.
- the oligosaccharide composition comprises oligosaccharides that have a MWw (weight- average molecular weight) (g/mol) in a range of 1905-2290.
- the oligosaccharide composition comprises oligosaccharides that have a MWn (number-average molecular weight) (g/mol) in a range of 1030-1095.
- a solution comprising the oligosaccharide composition has a pH in a range of 2.50-3.50.
- the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 86-96 weight percent.
- oligosaccharide compositions may be de- monomerized.
- de-monomerization involves the removal of residual saccharide monomers.
- de-monomerization is performed using
- compositions can be prepared depending upon the percent of monomer present.
- percent of monomer present in some embodiments,
- oligosaccharide compositions are de-monomerized to a monomer content of about 1%, about 3%, about 5%, about 10%, or about 15%. In some embodiments, oligosaccharide compositions are de-monomerized to a monomer content of about 1-3%, about 3-6%, about 5-8%, about 7- 10%, or about 10-15%. In one embodiment, the oligosaccharide compositions is de- monomerized to a monomer content of less than 1%. In one embodiment, the oligosaccharide composition is de-monomerized to a monomer content between about 7% and 10%. In one embodiment, the oligosaccharide compositions is de-monomerized to a monomer content between about 1% and 3%. In one embodiment, de-monomerization is achieved by osmotic separation. In a second embodiment de-monomerization is achieved by tangential flow filtration (TFF). In a third embodiment de-monomerization is achieved by ethanol precipitation
- oligosaccharide compositions with different monomer contents may also have different measurements for total dietary fiber, moisture, total dietary fiber (dry basis), or percent Dextrose Equivalent (DE).
- total dietary fiber is measured according to the methods of AO AC 2011.25.
- moisture is measured by using a vacuum oven at 60°C.
- total dietary fiber is (dry basis) is calculated.
- percent DE is measured according to the Food Chemicals Codex (FCC).
- the oligosaccharide compositions have a total dietary fiber content of 87.4 percent (on dry basis). In some embodiments, the oligosaccharide compositions have a total dietary fiber content of 81.9-93.0, 82-85, 85-88, 88-90, or 90-93 percent (on dry basis). In some embodiments, the oligosaccharide compositions have a total dietary fiber content of about 82, about 85, about 87, about 90, or about 93 percent (on dry basis). In some embodiments, the oligosaccharide compositions have a total dietary fiber content of 78-97 percent (on dry basis).
- the oligosaccharide compositions have a total dietary fiber content of 82-93 percent (on dry basis). In some embodiments, the oligosaccharide compositions have a total dietary fiber content of 14.5-100 percent (on dry basis). In some embodiments, the oligosaccharide compositions have a total dietary fiber content of 34-94 percent (on dry basis).
- the oligosaccharide compositions have a total reducing sugar content (Dextrose Equivalence (DE) (dry solids)) of 6.5-35 percent.
- DE Dextrose Equivalence
- the oligosaccharide compositions have a total reducing sugar content (Dextrose Equivalence (DE) (dry solids)) of 12-29 percent. In some embodiments, the oligosaccharide compositions have a total reducing sugar content (Dextrose Equivalence (DE) (dry solids)) of 5- 40, 5-30, 5-25, 10-30, 10-25, 10-20, 15-30, 15-25, or 15-20 percent.
- DE Dextrose Equivalence
- oligosaccharides compositions can be performed in a batch process or a continuous process.
- oligosaccharide compositions are produced in a batch process, where the contents of the reactor are subjected to agitation conditions (e.g ., continuously mixed or blended), and all or a substantial amount of the products of the reaction are removed (e.g., isolated and/or recovered).
- the methods of using the catalyst are carried out in an aqueous environment.
- aqueous solvent is water, which may be obtained from various sources. Generally, water sources with lower concentrations of ionic species (e.g., salts of sodium, phosphorous, ammonium, or magnesium) may be used, in some embodiments, as such ionic species may reduce effectiveness of the catalyst. In some embodiments where the aqueous solvent is water, the water has less than 10% of ionic species (e.g., salts of sodium, phosphorous, ammonium, magnesium).
- the water has a resistivity of at least 0.1 megaohm-centimeters, of at least 1 megaohm- centimeters, of at least 2 megaohm-centimeters, of at least 5 megaohm-centimeters, or of at least 10 megaohm-centimeters.
- the methods described herein may further include monitoring the amount of water present in the reaction mixture and/or the ratio of water to monomer or catalyst over a period of time.
- the water content of the reaction mixture may be altered over the course of the reaction, for example, removing evolved water produced.
- Appropriate methods may be used to remove water (e.g., evolved water) in the reaction mixture, including, for example, by evaporation, such as via distillation.
- the method comprises including water in the reaction mixture.
- the method comprises removing water from the reaction mixture through evaporation.
- the ratio of dextrose monomer to galactose monomer is about 1:2, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, or 2:1. In some embodiments, the ratio of dextrose monomer to galactose monomer is about 1:1.
- the ratio of dextrose monomer to mannose monomer is about 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, or 5.5:1. In some embodiments, the ratio of dextrose monomer to mannose monomer is about 4.5:1.
- the ratio of galactose monomer to mannose monomer is about 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, or 5.5:1. In some embodiments, the ratio of galactose monomer to mannose monomer is about 4.5:1.
- the monosaccharide preparation comprises about 30-60% dextrose monomer, about 30-60% galactose monomer, and 1-25% mannose monomer. In some embodiments, the monosaccharide preparation comprises about 30-60% dextrose monomer, about 30-60% galactose monomer, and about 5-15% mannose monomer. In some embodiments, the monosaccharide preparation comprises about 40-50% dextrose monomer, about 40-50% galactose monomer, and about 5-15% mannose monomer. In some embodiments, the monosaccharide preparation comprises about 45% dextrose monomer, about 45% galactose monomer, and about 10% mannose monomer.
- the preparation is loaded with an acid catalyst comprising positively charged hydrogen ions.
- an acid catalyst is a solid catalyst (e.g ., Dowex Marathon C).
- an acid catalyst is a soluble catalyst (e.g., citric acid).
- the molar ratio of positively charged hydrogen ions to total dextrose monomer, galactose monomer, and mannose monomer content is in an appropriate range. In some embodiments, the molar ratio of positively charged hydrogen ions to total dextrose monomer, galactose monomer, and mannose monomer content is in a range of 0.01 to 0.1, 0.02 to 0.08, 0.03 to 0.06, or 0.05 to 0.06.
- the molar ratio of positively charged hydrogen ions to total dextrose monomer, galactose monomer, and mannose monomer content is in a range of 0.003 to 0.01, 0.005 to 0.02, 0.01 to 0.02, 0.01 to 0.03, 0.02 to 0.03, 0.02 to 0.04, 0.03 to 0.05, 0.03 to 0.08, 0.04 to 0.07, 0.05 to 0.1, 0.05 to 0.2, 0.1 to 0.2, 0.1 to 0.3, or 0.2 to 0.3.
- the molar ratio of positively charged hydrogen ions to total dextrose monomer, galactose monomer, and mannose monomer content is in a range of 0.050 to 0.052.
- the molar ratio of positively charged hydrogen ions to total dextrose monomer, galactose monomer, and mannose monomer content is in a range of 0.020 to 0.035. In some embodiments, the molar ratio of positively charged hydrogen ions to total dextrose monomer, galactose monomer, and mannose monomer content is 0.028.
- the molar ratio of soluble acid catalyst (e.g., citric acid catalyst) to total dextrose monomer, galactose monomer, and mannose monomer content is in an appropriate range. In some embodiments, the molar ratio of soluble acid catalyst (e.g., citric acid catalyst) to total dextrose monomer, galactose monomer, and mannose monomer content is in a range of 0.01 to 0.1, 0.02 to 0.08, 0.03 to 0.06, or 0.05 to 0.06.
- the molar ratio of soluble acid catalyst (e.g., citric acid catalyst) to total dextrose monomer, galactose monomer, and mannose monomer content is in a range of 0.003 to 0.01, 0.005 to 0.02, 0.01 to 0.02, 0.01 to 0.03, 0.02 to 0.03, 0.02 to 0.04, 0.03 to 0.05, 0.03 to 0.08, 0.04 to 0.07, 0.05 to 0.1, 0.05 to 0.2, 0.1 to 0.2, 0.1 to 0.3, or 0.2 to 0.3.
- soluble acid catalyst e.g., citric acid catalyst
- the molar ratio of soluble acid catalyst (e.g., citric acid catalyst) to total dextrose monomer, galactose monomer, and mannose monomer content is in a range of 0.050 to 0.052. In some embodiments, the molar ratio of soluble acid catalyst (e.g., citric acid catalyst) to total dextrose monomer, galactose monomer, and mannose monomer content is in a range of 0.020 to 0.035. In some embodiments, the molar ratio of soluble acid catalyst (e.g., citric acid catalyst) to total dextrose monomer, galactose monomer, and mannose monomer content is 0.028.
- soluble acid catalyst e.g., citric acid catalyst
- water is added to the reaction mixture to quench the reaction by bringing the temperature of the reaction mixture to 100 °C or below.
- the water used for quenching is deionized water.
- the water used for quenching is USP water.
- the water has a temperature of about 60 °C to about 100 °C.
- the water used for quenching is about 95 °C.
- the water is added to the reaction mixture under conditions sufficient to avoid solidifying the mixture.
- the viscosity of the reaction mixture may be measured and/or altered over the course of the reaction.
- viscosity refers to a measurement of a fluid’s internal resistance to flow (e.g .,“thickness”) and is expressed in centipoise (cP) or pascal- seconds.
- the viscosity of the reaction mixture is between about 100 cP and about 95,000 cP, about 5,000 cP and about 75,000 cP, about 5,000 and about 50,000 cP, or about 10,000 and about 50,000 cP.
- the viscosity of the reaction mixture is between about 50 cP and about 200 cP.
- oligosaccharide compositions provided herein may be subjected to one or more additional processing steps.
- Additional processing steps may include, for example, purification steps.
- Purification steps may include, for example, separation, demonomerization, dilution, concentration, filtration, desalting or ion-exchange,
- the methods described herein further include a dilution step.
- deionized water is used for dilution.
- USP water is used for dilution.
- composition comprises water in a range of about 5-75, 25-65, 35-65, 45-55, or 47-53 weight percent. In certain embodiments, after dilution, the oligosaccharide composition comprises water in a range of about 45-55 weight percent.
- the methods described herein further include a
- the one or more oligosaccharide compositions produced may undergo a decolorization step using appropriate methods, including, for example, treatment with an absorbent, activated carbon, chromatography (e.g. , using ion exchange resin), and/or filtration (e.g., microfiltration).
- appropriate methods including, for example, treatment with an absorbent, activated carbon, chromatography (e.g. , using ion exchange resin), and/or filtration (e.g., microfiltration).
- the one or more oligosaccharide compositions produced are contacted with a material to remove salts, minerals, and/or other ionic species.
- a material to remove salts, minerals, and/or other ionic species For example, in certain embodiments, the one or more oligosaccharide compositions produced are flowed through an anionic exchange column. In other embodiments, oligosaccharide compositions produced are flowed through an anionic/cationic exchange column pair.
- the methods described herein may further include a concentration step.
- the oligosaccharide compositions may be subjected to evaporation (e.g., vacuum evaporation) to produce a concentrated
- the oligosaccharide compositions may be subjected to a spray drying step to produce an oligosaccharide powder.
- the oligosaccharide compositions may be subjected to both an evaporation step and a spray drying step.
- the oligosaccharide compositions be subjected to a lyophilization (e.g ., freeze drying) step to remove water and produce powdered product.
- the methods described herein further include a
- Oligosaccharide compositions prepared and purified may be subsequently separated by molecular weight using any method known in the art, including, for example, high- performance liquid chromatography, adsorption/desorption (e.g. low-pressure activated carbon chromatography), or filtration (for example, ultrafiltration or diafiltration).
- high- performance liquid chromatography e.g. high- performance liquid chromatography
- adsorption/desorption e.g. low-pressure activated carbon chromatography
- filtration for example, ultrafiltration or diafiltration.
- oligosaccharide compositions are separated into pools representing 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or greater than 98% short (about DP1-2), medium (about DP3-10), long (about DPI 1-18), or very long (about DP>18) species.
- prepared oligosaccharide compositions are fractionated by adsorption onto a carbonaceous material and subsequent desorption of fractions by washing the material with mixtures of an organic solvent in water at a concentration of 1%, 5%, 10%, 20%, 50%, or 100%.
- the adsorption material is activated charcoal.
- the adsorption material is a mixture of activated charcoal and a bulking agent such as diatomaceous earth or Celite 545 in 5%, 10%, 20%, 30%, 40%, or 50% portion by volume or weight.
- prepared oligosaccharide compositions are separated by passage through a high-performance liquid chromatography system.
- prepared oligosaccharide compositions are separated by ion-affinity chromatography, hydrophilic interaction chromatography, or size-exclusion chromatography including gel- permeation and gel-filtration.
- catalyst is removed by filtration.
- a 0.45 pm filter is used to remove catalyst during filtration.
- low molecular weight materials are removed by filtration methods.
- low molecular weight materials may be removed by dialysis, ultrafiltration, diafiltration, or tangential flow filtration.
- the filtration is performed in static dialysis tube apparatus. In other embodiments, the filtration is performed in a dynamic flow filtration system. In other embodiments, the filtration is performed in centrifugal force-driven filtration cartridges. In certain embodiments, the reaction mixture is cooled to below about 85 0 C before filtration.
- oligosaccharides is in a range of 6-16. In certain embodiments, the mean degree of polymerization of all oligosaccharides is in a range of 10-15. In certain embodiments, the mean degree of polymerization of ah oligosaccharides is in a range of 9-16. In certain embodiments, the mean degree of polymerization of ah oligosaccharides is in a range of 10.5-15. In certain embodiments, the mean degree of polymerization of ah oligosaccharides is in a range of 9-15. In certain embodiments, the mean degree of polymerization of ah oligosaccharides is in a range of 10.5-14.
- the mean degree of polymerization of ah oligosaccharides is in a range of 7-15, 7-12, 7-10, 7-8, 9-10, 10-11, 11-12, 11-15, 12-13, 12-14 13-14, 14-15, 15-16, 17- 18, 15-20, 3-8, 4-7, or 5-6.
- the weight percent of dextrose monomer, galactose monomer, and mannose monomer in the oligosaccharide composition is in a range of 10-18. In certain embodiments, the weight percent of dextrose monomer, galactose monomer, and mannose monomer in the oligosaccharide composition is in a range of 11-17. In certain embodiments, the weight percent of dextrose monomer, galactose monomer, and mannose monomer in the oligosaccharide composition is in a range of 12-16. In certain embodiments, the weight percent of dextrose monomer, galactose monomer, and mannose monomer in the oligosaccharide composition is in a range of 13-15.
- the oligosaccharide composition is a mixture of polymers of dextrose, galactose, and mannose in proportions of approximately 45%, 45%, and 10%, by weight respectively.
- Each monomer unit may be unsubstituted, singly, doubly, or triply substituted with another dextrose, galactose, or mannose unit by any glycosidic isomer.
- the oligosaccharide composition comprises water in a range of 5-75 weight percent. In some embodiments, the oligosaccharide composition comprises water in a range of 25-65 weight percent. In some embodiments, the oligosaccharide
- composition comprises water in a range of 35-65 weight percent. In some embodiments, the oligosaccharide composition comprises water in a range of 45-55 weight percent.
- the oligosaccharide composition comprises
- the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1905-2290. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1753-2395. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1750-2400. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1500-2500.
- the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1800-2000. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 2000-2300. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1515-2630. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1500-2500.
- the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1740-2400. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1700-2300. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWw (g/mol) in a range of 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, or 2400-2500.
- the oligosaccharide composition comprises
- the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 1030-1095. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 981-1214. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 980-1220 In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 1000-1050.
- the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 1050-1100. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 890-1300. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 975-1155. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 875-1180.
- the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 940-1120. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a MWn (g/mol) in a range of 900-950, 950-1000, 1000- 1050, 1050-1100, 1100-1150, 1150-1200, or 1200-1250.
- a solution comprising the oligosaccharide composition has a pH in a range of 1.50-6.00. In some embodiments, a solution comprising the oligosaccharide composition has a pH in a range of 1.50-5.00. In some embodiments, a solution comprising the oligosaccharide composition has a pH in a range of 2.00-4.00. In some embodiments, a solution comprising the oligosaccharide composition has a pH in a range of 2.50-3.50.
- the oligosaccharide composition comprises
- the oligosaccharide composition comprises oligosaccharides that have a degree of branching in a range of about 8.5% to about 32%. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a degree of branching in a range of about 10% to about 35%. In some embodiments, the oligosaccharide composition comprises oligosaccharides that have a degree of branching in a range of about 13% to about 29%. In some embodiments, the oligosaccharide composition comprises
- oligosaccharides that have a degree of branching in a range of 5-50%, 5-40%, 5-30%, 5-20%, 5- 15%, 10-50%, 10-40%, 10-30%, 10-25%, 15-30%, or 15-20%.
- the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 80-100 weight percent.
- DP2+ repeat units
- the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 86-96 weight percent. In some embodiments, the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 86-91 weight percent. In some embodiments, the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 91-96 weight percent. In some
- the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 81-100 weight percent. In some embodiments, the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 80-94 weight percent. In some embodiments, the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 91-96 weight percent. In some
- the oligosaccharide composition comprises oligomers having two or more repeat units (DP2+) in a range of 80-85, 85-87, 86-88, 87-90, 88-91, 89-92, 90-93, 91-94, 92-95, 93-96, or 95-98 weight percent.
- the oligosaccharide composition has a polydispersity index (PD I) of 1.8-2.0. In some embodiments, the oligosaccharide composition has a polydispersity index (PD I) of 1.8-2.1. In some embodiments, the oligosaccharide composition has a PDI of 1.0- 1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.7-1.8, 1.8-2.0, 2.0-2.2, 2.2-2.4, or 2.4-2.6. In some embodiments, the oligosaccharide composition has a PDI of about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, or about 2.2.
- the MWw, MWn, PDI, monomer content (DPI) and/or DP2+ values of oligosaccharides in an oligosaccharide composition are determined using the size exclusion chromatography method described in Example 15.
- oligosaccharides in an oligosaccharide composition are determined using the size exclusion chromatography method described in Example 17.
- the oligosaccharide composition comprises oligomers having at least three linked monomer units (DP3+) in a range of 80-95 weight percent. In some embodiments, the oligosaccharide composition comprises oligomers having at least three linked monomer units (DP3+) in a range of 85-90 weight percent. In some embodiments, the oligosaccharide composition comprises oligomers having at least three linked monomer units (DP3+) in a range of 80-85, 85-87, 86-88, 87-90, 88-91, 89-92, 90-93, 91-94, or 92-95 weight percent.
- the oligosaccharide composition comprises 4.20% to 6.28% monomer (DPI). In some embodiments, the oligosaccharide composition comprises 4% to 5%, 5% to 6%, or 6% to 7% monomer (DPI). In some embodiments, the oligosaccharide composition comprises 6.20% to 8.83% disaccharide (DP2). In some embodiments, the oligosaccharide composition comprises 6% to 6.5%, 6.5% to 7%, 7.5% to 8%, 8% to 8.5%, or 8.5% to 9% disaccharide (DP2). In some embodiments, the oligosaccharide composition comprises 84.91% to 89.58% oligomers having at least three linked monomer units (DP3+). In some embodiments, the oligosaccharide composition comprises 84% to 85%, 85% to 86%, 86% to 87%, 87% to 88%, or 88% to 90% oligomers having at least three linked monomer units (DP3+).
- the oligosaccharide composition comprises less than 0.10% total impurities (excluding monomer). In some embodiments, the oligosaccharide composition comprises less than 0.05% total impurities (excluding monomer). In some embodiments, the oligosaccharide composition comprises less than 0.20%, 0.15%, 0.10%, or 0.05% total impurities (excluding monomer).
- the oligosaccharide composition comprises less than 0.10% w/w levoglucosan, less than 0.10% w/w glucuronic acid, less than 0.10% w/w lactic acid, less than 0.10% w/w formic acid, less than 0.10% w/w acetic acid, and less than 0.10% w/w hydroxymethylfurfural (HMF).
- the oligosaccharide composition comprises 0.35% w/w levoglucosan, 0.03% w/w lactic acid, and/or 0.06% w/w formic acid.
- the oligosaccharide composition comprises 0.28-0.43% w/w levoglucosan, 0.00-0.03% w/w lactic acid, and/or 0.05-0.07% w/w formic acid.
- the oligosaccharide composition comprises a MWw of 1753-2395, a MWn of 981-1214, and/or a PDI of 1.8-2.0.
- oligosaccharide compositions described herein, and prepared according to the methods described herein can be characterized and distinguished from prior art compositions using permethylation analysis. See, e.g., Zhao, Y., et al.‘Rapid, sensitive structure analysis of oligosaccharides,’ PNAS March 4, 1997 94 (5) 1629-1633; Kailemia, M.J., et al.
- oligosaccharide compositions are provided herein that comprise a plurality of oligosaccharides that are minimally digestible in humans, the plurality of oligosaccharides comprising monomer radicals.
- the molar percentages of different types of monomer radicals in the plurality of oligosaccharides can be quantified using a permethylation assay as described in Example 13. The permethylation assay is performed on a de-monomerized sample of the composition.
- the plurality of oligosaccharides comprises two or more monomer radicals selected from radicals (l)-(40):
- t-mannopyranose monoradicals representing 3.0-4.1 mol% of monomer radicals in the plurality of oligosaccharides
- t-galactopyranose monoradicals representing 8.3-12.5 mol% of monomer radicals in the plurality of oligosaccharides
- 2-mannopyranose and/or 3-mannopyranose monoradicals representing 1.2- 1.9 mol% of monomer radicals in the plurality of oligosaccharides
- 2-glucopyranose monoradicals representing 2.4-3.2 mol% of monomer radicals in the plurality of oligosaccharides
- 6-mannopyranose monoradicals representing 2.0-2.9 mol% of monomer radicals in the plurality of oligosaccharides
- 6-glucofuranose monoradicals representing 0-1.6 mol% of monomer radicals in the plurality of oligosaccharides
- 6-galactofuranose monoradicals representing 1.4-5.0 mol% of monomer radicals in the plurality of oligosaccharides
- (21) 3, 4-glucopyranose and/or 3, 5-glucofuranose diradicals, representing 0-1.1 mol% of monomer radicals in the plurality of oligosaccharides;
- (22) 2, 4-glucopyranose and/or 2, 5-glucofuranose and/or 2, 4-galactopyranose and/or 2,5- galactofuranose diradicals, representing 0.9- 1.4 mol% of monomer radicals in the plurality of oligo s accharides ;
- (23) 4,6-mannopyranose and/or 5,6-mannofuranose diradicals, representing 0.5-0.7 mol% of monomer radicals in the plurality of oligosaccharides;
- oligosaccharide composition are 1,2 glycosidic bonds. In some embodiments, about 10.5-25% of the total glycosidic bonds in an oligosaccharide composition are 1,2 glycosidic bonds. In some embodiments, about 9.5-32% of the total glycosidic bonds in an oligosaccharide composition are
- 1.2 glycosidic bonds In some embodiments, about 13-27% of the total glycosidic bonds in an oligosaccharide composition are 1,2 glycosidic bonds. In some embodiments, 5-50%, 5-40%, 5- 30%, 5-20%, 5-15%, 10-50%, 10-40%, 10-30%, 10-25%, 15-30%, or 15-20% of the total glycosidic bonds in an oligosaccharide composition are 1,2 glycosidic bonds.
- about 14.5-34% of the total glycosidic bonds in an oligosaccharide composition are 1,3 glycosidic bonds. In some embodiments, about 17-30% of the total glycosidic bonds in an oligosaccharide composition are 1,3 glycosidic bonds. In some embodiments, about 9.5-27% of the total glycosidic bonds in an oligosaccharide composition are
- glycosidic bonds In some embodiments, about 12.5-23.5% of the total glycosidic bonds in an oligosaccharide composition are 1,3 glycosidic bonds. In some embodiments, 5-50%, 10- 40%, 10-30%, 10-20%, 5-15%, 10-50%, 10-40%, 10-30%, 10-25%, 15-30%, or 15-20% of the total glycosidic bonds in an oligosaccharide composition are 1,3 glycosidic bonds.
- about 10-26% of the total glycosidic bonds in an oligosaccharide composition are 1,4 glycosidic bonds.
- about 12-22% of the total glycosidic bonds in an oligosaccharide composition are 1,4 glycosidic bonds.
- about 10-29.5% of the total glycosidic bonds in an oligosaccharide composition are 1,4 glycosidic bonds.
- about 13-25% of the total glycosidic bonds in an oligosaccharide composition are 1,4 glycosidic bonds.
- 5-50%, 10- 40%, 10-30%, 10-20%, 5-15%, 10-50%, 10-40%, 10-30%, 10-25%, 15-30%, or 15-20% of the total glycosidic bonds in an oligosaccharide composition are 1,4 glycosidic bonds.
- about 32-57% of the total glycosidic bonds in an oligosaccharide composition are 1,6 glycosidic bonds. In some embodiments, about 35-52% of the total glycosidic bonds in an oligosaccharide composition are 1,6 glycosidic bonds. In some embodiments, about 23-65% of the total glycosidic bonds in an oligosaccharide composition are 1,6 glycosidic bonds. In some embodiments, about 30-56% of the total glycosidic bonds in an oligosaccharide composition are 1,6 glycosidic bonds. In some embodiments, 15-70%, 20-60%, 20-40%, 25-50%, 30-50%, 30-40%, or 30-60% of the total glycosidic bonds in an
- oligosaccharide composition are 1,6 glycosidic bonds.
- an oligosaccharide composition comprises 17.5-43% total furanose. In some embodiments, an oligosaccharide composition comprises 20.5-37% total furanose. In some embodiments, an oligosaccharide composition comprises 14-60% total furanose. In some embodiments, an oligosaccharide composition comprises 20.5-50% total furanose. In some embodiments, an oligosaccharide composition comprises 10-60%, 10-50%, 15-40%, 20-40%, 20-30%, or 30-50% total furanose.
- the oligosaccharide composition comprises at least one glucofuranose or glucopyranose radical, at least one mannofuranose or mannopyranose radical, and at least one galactofuranose or galactopyranose radical.
- an oligosaccharide composition comprising a plurality of oligosaccharides comprising monomer radicals (l)-(40) in the molar percentages shown in Table 2.
- the oligosaccharide compositions are free from monomer. In other embodiments, the oligosaccharide compositions comprise monomer. [00153]
- the oligosaccharide compositions described herein, and prepared according to the methods described herein, can be characterized and distinguished from prior art compositions using two-dimensional heteronuclear NMR. Accordingly, in another aspect, oligosaccharide compositions are provided that comprise a plurality of oligosaccharides that are minimally digestible in humans, the compositions being characterized by a heteronuclear single quantum correlation (HSQC) NMR spectrum comprising signals 5, 6, 7, and 15, each signal having a center position and an area:
- HSQC heteronuclear single quantum correlation
- the spectrum further comprises 1-2 (e.g ., one or two) signals selected from signals 10 and 14, and defined as follows:
- the spectrum further comprises 1-3 (e.g., one, two, or three) signals selected from signals 11, 12, and 13, and defined as follows:
- the spectrum comprises 1-3 (e.g ., one, two, or three) signals selected from signals 11, 12, and 13, and defined as follows:
- the spectrum comprises 1-15 (e.g ., one, two, or three) signals selected from signals 1-15, and defined as follows:
- the spectrum comprises 1-15 (e.g., one, two, or three) signals selected from signals 1-15, and defined as follows:
- the spectrum comprises 1-15 (e.g ., one, two, or three) signals selected from signals 1-15, and defined as follows:
- signals 5, 6, 7, 15, 10, 14, 11, 12, and 13 are each further characterized by an ' H integral region and a 13 C integral region, defined as follows:
- signals 1-15 are each characterized by an ' H integral region and a 13 C integral region, defined as follows:
- the NMR spectrum is obtained by subjecting a sample of the composition to HSQC NMR, wherein the sample is a solution in a deuterated solvent.
- Suitable deuterated solvents in include deuterated acetonitrile, deuterated acetone, deuterated methanol, D2O, and mixtures thereof.
- the deuterated solvent is D2O.
- the NMR spectrum is obtained using the conditions described in Example 14.
- Exemplary oligosaccharide compositions may be prepared according to the procedures described herein.
- oligosaccharide compositions may be used to reduce pathogen (e.g., CRE or VRE) levels and/or pathogen colonization in subjects with elevated pathogen levels.
- pathogen e.g., CRE or VRE
- oligosaccharide compositions may be used to increase levels of commensal bacteria in subjects.
- the selected oligosaccharide composition is useful for controlling ( e.g . reducing) pathogen levels. In some embodiments, the selected oligosaccharide composition is useful for controlling (e.g. reducing) pathogen levels relative to commensals (e.g., non-pathogenic commensals). In some embodiments, the selected oligosaccharide composition is useful for controlling (e.g. reducing) absolute pathogen levels in a subject.
- commensal bacteria refer to bacteria commonly associated with a healthy state of a microbiome in a particular niche, e.g., the gastrointestinal tract (e.g., the intestines), and/or are generally considered non-pathogenic.
- the selected oligosaccharide composition is useful for controlling (e.g. reducing) pathobiont levels. In some embodiments, the selected oligosaccharide composition is useful for controlling (e.g. reducing) pathobiont levels relative to commensals (e.g., non-pathogenic commensals). In some embodiments, the selected oligosaccharide composition is useful for controlling (e.g. reducing) absolute pathobiont levels in a subject.
- pathobionts include bacteria (and fungi) that are potentially pathological (disease-causing), though, under normal circumstances, non-pathologicahy co-exist with the subject, e.g., as a non-harming symbiont.
- dysbiosis causes a non- pathological bacterium to become pathological.
- pathobionts are as described in Hornef, M.“Pathogens, Commensal Symbionts, and Pathobionts: Discovery and Functional Effects on the Host”, ILAR Journal, Volume 56, Issue 2, 2015, Pages 159-162.
- the selected oligosaccharide composition is useful for controlling relative levels of pathogens and commensals. In some embodiments, the selected oligosaccharide composition is useful for controlling relative levels of pathobionts and commensals. In some embodiments, the selected oligosaccharide composition is useful for controlling relative levels of pathogenic commensals and non-pathogenic commensals.
- oligosaccharide compositions may be used to affect the structure (e.g., composition) and/or function (e.g. metabolic activity) of the gut microbiota.
- the selected oligosaccharide compositions confer beneficial health effects on a subject.
- Subjects that may benefit from the methods and uses described herein e.g., uses of the oligosaccharide compositions to treat subjects (e.g. , treat infections) and uses of the
- oligosaccharide compositions to reduce the abundance of pathogens or pathobionts include immunocompromised or immunosuppressed subjects.
- Subjects that may benefit from the methods and uses described herein include subjects undergoing transplant procedures, e.g., hematopoietic stem cell transplantation (HSCT) or solid organ transplant, or other medical procedures, e.g., surgery, e.g. of the gastrointestinal tract.
- Subjects that may benefit from the methods and uses described herein include other immunocompromised subjects, e.g., subjects with hematological malignancies or cirrhosis (e.g., liver cirrhosis).
- Subjects that may benefit from the methods and uses described herein include subjects admitted to intensive care units.
- the selected oligosaccharide compositions described herein reduce the abundance (e.g., relative abundance or absolute abundance) of pathogens or pathobionts (e.g., in the gastrointestinal tract), e.g., when compared to a baseline (e.g., untreated (population of) subject(s), or a subject prior to treatment).
- the selected oligosaccharide compositions described herein promote growth of commensal bacteria over growth of pathogens or pathobionts (e.g., in the gastrointestinal tract, e.g., the intestines, e.g., the large intestine or colon).
- subjects achieve decolonization with MDR pathogens (e.g., vancomycin-resistant Enterococcus (VRE), extended-spectrum beta lactamase- producing Enterobacteriaceae (ESBLE), and carbapenem-resistant Enterobacteriaceae (CRE), e.g., levels of these bacteria are near to or fall below detectable levels.
- MDR pathogens e.g., vancomycin-resistant Enterococcus (VRE), extended-spectrum beta lactamase- producing Enterobacteriaceae (ESBLE), and carbapenem-resistant Enterobacteriaceae (CRE)
- the reduction in the abundance (e.g., relative abundance or absolute abundance) of a pathogen or pathobiont may be determined, e.g., by subjecting a sample (e.g., a stool sample) from a subject to nucleic acid sequencing (e.g., whole genome sequencing) and other assays (e.g., colony-forming units (cfu)/g feces by culture).
- nucleic acid sequencing e.g., whole genome sequencing
- other assays e.g., colony-forming units (cfu)/g feces by culture.
- the selected oligosaccharide compositions described herein promote an increase in alpha-diversity (e.g. an increase in bacterial taxa diversity, e.g., as determined by measuring Shannon diversity, e.g. by nucleic acid sequencing).
- the selected oligosaccharide compositions described herein promote richness of the bacterial community.
- the selected oligosaccharide compositions described herein reduce inflammation, e.g. inflammation associated with pathogens or pathobionts or other bacteria. The reduction may be determined by measuring one or more markers of inflammation, e.g. IFN-g, IL-Ib, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-a.
- the markers can be determined, e.g., from stool or blood samples.
- the selected oligosaccharide compositions described herein treat infections, e.g., bacterial infections or fungal infections. In some embodiments, the selected oligosaccharide compositions described herein reduce infections (e.g., the rate of infections), including secondary or opportunistic infections (e.g ., hospital acquired infections (HAI)), including, e.g., central line- associated bloodstream infections (CLABSI), catheter-associated urinary tract Infection
- infections e.g., the rate of infections
- secondary or opportunistic infections e.g ., hospital acquired infections (HAI)
- CLABSI central line- associated bloodstream infections
- the selected oligosaccharide compositions described herein reduce the rate of hospitalizations, e.g., due to or caused by infections. In some embodiments, the selected oligosaccharide compositions described herein shorten the time period of hospitalization required, e.g., to treat or resolve the infections.
- the selected oligosaccharide compositions described herein is administered to a subject having a high likelihood of developing an infection, e.g., to prevent the infection or slow the progression of an infection.
- treatment with the selected oligosaccharide compositions described herein is provided until the subject’s infection is resolved or the subject is at a low risk of acquiring an infection, or is at a low risk of acquiring a re-infection.
- the selected oligosaccharide compositions described herein is administered to a subject having a high likelihood of developing a rejection of a transplant, e.g., graft-versus-host disease (GvHD), e.g., to prevent the rejection or slow the progression of the rejection, e.g., at a time after the transplant is received by the subject.
- GvHD graft-versus-host disease
- treatment with the selected oligosaccharide compositions described herein is provided until the subject’s transplant rejection reaction is resolved or the subject is at a low risk of rejecting the transplant.
- the selected oligosaccharide compositions described herein can be provided with standard-of-care treatment (e.g., administration of antibiotics). In some embodiments, the selected oligosaccharide compositions described herein can be provided without standard-of-care treatment (e.g., administration of antibiotics).
- an oligosaccharide composition described herein can be used to benefit (e.g., treat) patients having detectable commensal bacteria in the gut, e.g., patients with gut microbiota that are not devoid of detectable commensal bacteria.
- an oligosaccharide composition described herein is administered to a patient with low levels of commensal bacteria, e.g., a patient with gut microbiota that is not devoid of commensal bacteria or a patient with a gut microbiota that has not been completely depleted (e.g., resulting from use of antibiotics or chemotherapy), e.g., for treatment purposes.
- a patient with low levels of commensal bacteria e.g., a patient with gut microbiota that is not devoid of commensal bacteria or a patient with a gut microbiota that has not been completely depleted (e.g., resulting from use of antibiotics or chemotherapy), e.g., for treatment purposes.
- the oligosaccharide composition is formulated as powder, e.g., for reconstitution (e.g., in water) for oral administration.
- the oligo saccharide composition is formulated as a pharmaceutical composition for delivery by a feeding tube.
- the oligosaccharide composition is formulated as a pharmaceutical composition for delivery by total parenteral nutrition (TPN).
- TPN total parenteral nutrition
- the oligosaccharide composition may be administered to the subject on a daily, weekly, biweekly, or monthly basis. In some embodiments, the composition is administered to the subject more than once per day (e.g., 2, 3, or 4 times per day). In some embodiments, the composition is administered to the subject once or twice per day for one, two, three, or four weeks in a row.
- the composition is administered to the subject according to the following schedule: 18 grams total on each of days 1 and 2 of a treatment protocol; 36 grams total on each of days 3 and 4 of a treatment protocol; and 72 grams total on each of days 5-14 of a treatment protocol.
- the composition is administered to the subject according to the following schedule: 18 grams total on each of days 1-7 of a treatment protocol; 36 grams total on each of days 8-14 of a treatment protocol; 54 grams total on each of days 15-21 of a treatment protocol; and 72 grams total on each of days 22-28 of a treatment protocol.
- an effective amount of an oligosaccharide is a total of 5- 200 grams, 5-150 grams, 5-100 grams, 5-75 grams, 5-50 grams, 5-25 grams, 10-50 grams, 25-50 grams, 30-60 grams, 50-75 grams, 50-100 grams, 18-72 grams, or 36-72 grams administered daily.
- oligosaccharide composition of the disclosure is well tolerated by a subject
- oligosaccharide compositions do not cause or cause minimal discomfort, e.g., production of gas or gastrointestinal discomfort, in subjects.
- 5-200 grams, 5-150 grams, 5-100 grams, 5-75 grams, 5-50 grams, 5-25 grams, 10-50 grams, 25-50 grams, 30-60 grams, 50-75 grams, 50-100 grams, 18-72 grams, or 36-72 grams of total daily dose are well tolerated by a subject.
- the amount of an oligosaccharide composition that is administered to the subject at a single time or in a single dose is well tolerated by the subject.
- the amount of the oligosaccharide composition that is administered to the subject at a single time or in a single dose is more tolerated by the subject than a similar amount of commercial low-digestible sugars such as fructooligosaccharides (FOS).
- Commercial low-digestible sugars are known in the art to be poorly tolerated in subjects (See, e.g., Grabitske, H.A., Critical Reviews in Food Science and Nutrition, 49:327-360 (2009)), e.g., at high doses.
- tolerability studies of FOS indicate that 20 grams FOS per day causes mild gastrointestinal symptoms and that 30 grams FOS per day causes major discomfort and gastrointestinal symptoms.
- oligosaccharide compositions provided herein effectively reduce colonization with, prevent colonization with, or reduce the risk of an adverse effect of a pathogen or pathobiont to a subject.
- the method comprises shifting the microbial community in the gastrointestinal tract toward a commensal population, e.g., thereby replacing (e.g. outcompeting) a pathogen or an antibiotic resistance gene carrier.
- the oligosaccharide compositions provided herein are administered to a subject reduce the spread of pathogen to other untreated subjects.
- the oligosaccharide composition is administered in an effective amount and/or to a sufficient number of subjects that the spread of the pathogen, e.g., from a first subject to a second subject, is reduced. Such reduction might be measured by any of the methods described herein or any other conceivable method.
- a method of reducing a pathogen or pathobiont reservoir in a subject by administering an oligosaccharide composition to the subject, e.g., in an effective amount and/or to a sufficient number of subjects that the pathogen or pathobiont reservoir is reduced.
- the pathogen or pathobiont reservoir is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%, e.g., relative to a reference standard.
- a pathogen or pathobiont reservoir may represent about 5%, about 10%, about 15%, about 20%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85% of the total bacterial reservoir of a subject (e.g., about 5%, about 10%, about 15%, about 20%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85% of the total bacterial population in the gut or intestines of a subject).
- the pathogen or pathobiont reservoir comprises the pathogen or pathobiont biomass.
- the bacterial reservoir comprises the total bacterial biomass.
- methods described herein comprise administering an oligosaccharide composition to a subject in an effective amount to reduce the total pathogen or pathobiont reservoir from about 80% to about 5% of the total bacterial reservoir, from about 80% to about 10% of the total bacterial reservoir, from about 80% to about 20% of the total bacterial reservoir, from about 80% to about 30% of the total bacterial reservoir, from about 80% to about 40% of the total bacterial reservoir, or from about 80% to about 50% of the total bacterial reservoir.
- methods described herein comprise administering an oligosaccharide composition to a subject in an effective amount to reduce the total pathogen or pathobiont reservoir from about 50-80% to about 5% of the total bacterial reservoir, from about 50-80% to about 10% of the total bacterial reservoir, from about 50-80% to about 20% of the total bacterial reservoir, from about 50-80% to about 30% of the total bacterial reservoir, or from about 50-80% to about 40% of the total bacterial reservoir.
- a method of modulating the biomass of a pathogen or pathobiont or an antibiotic resistance gene carrier comprises increasing or decreasing, e.g., the biomass of a pathogen or pathobiont or an antibiotic resistance gene carrier.
- the oligosaccharide composition is administered in an effective amount and/or to a sufficient number of subjects, that the reservoir or biomass of a pathogen or pathobiont is reduced.
- the oligosaccharide composition is administered in an effective amount that pathogen or pathobiont biomass is modulated, e.g., reduced (e.g., the number of pathogen or pathobionts and/or the number of drug- or antibiotic -resistance gene or MDR element carriers is modulated).
- pathogen or pathobiont biomass is modulated, e.g., reduced (e.g., the number of pathogen or pathobionts and/or the number of drug- or antibiotic -resistance gene or MDR element carriers is modulated).
- a method of modulating the number of pathogen or pathobionts or antibiotic resistance gene carriers e.g., in a population, e.g., a microbial population).
- Exemplary pathogens include Enterobacteriaciae (e.g., a family comprising
- Plesiomonas, Shigella, or Salmonella Clostridium (e.g., a genus comprising Clostridium difficile), Enterococcus, Staphylococcus (e.g., a genus comprising Staphylococcus aureus), Campylobacter, Vibrio, Aeromonas, Norovims, Astrovirus, Adenovirus, Sapovims, or Rotavirus.
- Clostridium e.g., a genus comprising Clostridium difficile
- Enterococcus e.g., a genus comprising Clostridium difficile
- Staphylococcus e.g., a genus comprising Staphylococcus aureus
- Campylobacter Vibrio, Aeromonas, Norovims, Astrovirus, Adenovirus, Sapovims, or Rotavirus.
- the pathogen is a carbapenem-resistant Enterobacteriaceae
- the pathogen is a vancomycin-resistant Enterococci (VRE). In some embodiments, the pathogen is an extended- spectrum beta-lactamase (ESBL) producing organism.
- VRE vancomycin-resistant Enterococci
- ESBL extended- spectrum beta-lactamase
- the pathogen includes Enterobacteriaciae (e.g ., a family comprising Plesiomonas, Shigella, or Salmonella ).
- the pathogen includes Clostridium (e.g., a genus comprising Clostridium difficile').
- the pathogen includes Enterococcus.
- the pathogen includes Staphylococcus.
- the method comprises reducing the spread of a pathogen by administering to a subject a oligosaccharide composition, e.g., in an effective amount and/or to a sufficient number of subjects that the spread of the pathogen is reduced.
- the spread of a pathogen is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%, e.g., relative to a reference standard.
- the spread of a pathogen comprises the spread from a first subject to a second subject.
- the spread of a pathogen comprises the spread from a first subject or a second subject to an entity which can harbor the pathogen (e.g., another individual or an inanimate object, e.g., facility built surface (e.g. sink, door handle, toilet, faucet) or medical supply (e.g., a package comprising a dressing or device, or a dressing or device itself).
- entity which can harbor the pathogen e.g., another individual or an inanimate object, e.g., facility built surface (e.g. sink, door handle, toilet, faucet) or medical supply (e.g., a package comprising a dressing or device, or a dressing or device itself).
- the oligosaccharide composition is administered in an effective amount and/or to a sufficient number of subjects, that the spread of drug- or antibiotic-resistance gene, or a MDR element, e.g., from a first subject to a second subject, is reduced. This reduction might be measured by any of the methods described herein.
- a method of reducing a drug-resistance gene reservoir e.g., an antibiotic resistance gene reservoir or MDR gene reservoir
- a drug-resistance gene reservoir e.g., an antibiotic resistance gene reservoir or MDR gene reservoir
- administering a oligosaccharide composition e.g., in an effective amount and/or to a sufficient number of subjects that the drug-resistance gene reservoir (e.g., antibiotic resistance gene reservoir or MDR gene reservoir) is reduced.
- a drug-resistance gene reservoir (e.g., an antibiotic resistance gene reservoir or MDR gene reservoir) is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%, e.g., relative to a reference standard.
- antibiotic resistance genes include penicillin resistance genes, MecA (conferring methicillin, penicillin and other penicillin-like antibiotic resistance) and other genes that encode the protein PBP2A (penicillin binding protein 2A), carbapenemase resistance genes (e.g., Klebsiella pneumonia carbapenemase (KPC)),
- betalactamase resistance genes e.g., New Delhi betalactamase (NDM), OXA, SHV, TIM, CTX- M, VIM
- vancomycin resistance genes e.g., VanA, VanB, vancomycin resistance genes in Enterococcus
- AmpC carbapenem and beta lactam resistance genes in Enter obacteriaceae
- fluoroquinoline resistance genes e.g., Qnr
- trimethoprim resistance genes e.g.
- dihydrofolate reductase sulfamethoxazole resistance genes (e.g., dihydropteroate synthetase), ciprofloxacin resistance genes, and aminoglycoside resistance genes (e.g., ribosomal methyl transferase).
- a drug-resistance gene reservoir e.g., an antibiotic resistance gene reservoir or MDR gene reservoir
- any technique described herein e.g., a technique described for the assessment of a pathogen reservoir.
- a method of reducing the spread of a drug- resistance gene comprising administering a oligosaccharide composition to a subject, e.g., in an effective amount and/or to a sufficient number of subjects that the spread of the drug-resistance gene (e.g., antibiotic resistance gene or MDR gene) is reduced.
- a drug- resistance gene e.g., an antibiotic resistance gene or MDR gene
- the spread of an antibiotic resistance gene is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%, e.g., relative to a reference standard.
- the spread of a drug- resistance gene e.g., an antibiotic resistance gene or MDR gene
- a first subject e.g., a first subject
- a second subject e.g., a second subject
- the oligosaccharide composition is administered in an effective amount and/or to a sufficient number of subject(s), that the rate at which a drug- or antibiotic-resistance gene, or an MDR element, is transferred from a first pathogen to a second pathogen is reduced.
- This transfer might be measured by showing the presence of a similar gene or toxin, identified by any of the methods described herein, present in a second pathogen distinct from the first pathogen.
- This distinction can be at the level of organism identification (e.g., metabolite production, species identity, or susceptibility to antibiotics), or by molecular methods to show other differences, such as any of those described herein.
- a method of reducing the rate at which a pathogen causes infection or colonization e.g ., in a subject
- a oligosaccharide composition to the subject, e.g., in an effective amount and/or to a sufficient number of subjects that the rate of infection is reduced.
- the oligosaccharide composition is administered in an effective amount and/or to a sufficient number of subject(s), that the rate at which a pathogen causes infection, or the severity of pathogen infection, as indicated by assessment of symptoms associated with infection, is reduced.
- the rate of infection is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%, e.g., relative to a reference standard.
- oligosaccharide compositions provided herein effectively prevent onset of a pathogenic infection (e.g., in immunocompromised (e.g., HSCT) patients or ICU patients).
- oligosaccharide compositions may be provided prior to, during, or after a certain medical procedure or medical event to prevent onset of a pathogenic infection.
- oligosaccharide compositions provided herein effectively prevent onset of a pathogenic infection in at least 1 out of every 100 subjects, at least 5 out of every 100 subjects, at least 10 out of every 100 subjects, at least 20 out of every 100 subjects, at least 30 out of every 100 subjects, at least 40 out of every 100 subjects, at least 50 out of every 100 subjects, at least 60 out of every 100 subjects, at least 70 out of every 100 subjects, at least 80 out of every 100 subjects, at least 90 out of every 100 subjects, or at least 95 out of every 100 subjects.
- oligosaccharide compositions provided herein effectively prevent onset of a pathogenic infection in 10-100%, 10-20%, 15-25%, 20-50%, 40-60%, 50-75%, 60-80%, 75- 90%, 80-100%, or 90-100% of subjects.
- oligosaccharide compositions provided herein effectively minimizes or prevents progression of an infection (e.g., progression from mild or moderate symptoms to severe symptoms).
- oligosaccharide compositions may be provided prior to, during, or after the onset of infection to prevent progression of the infection.
- oligosaccharide compositions provided herein effectively minimizes or prevents progression of an infection in at least 1 out of every 100 subjects, at least 5 out of every 100 subjects, at least 10 out of every 100 subjects, at least 20 out of every 100 subjects, at least 30 out of every 100 subjects, at least 40 out of every 100 subjects, at least 50 out of every 100 subjects, at least 60 out of every 100 subjects, at least 70 out of every 100 subjects, at least 80 out of every 100 subjects, at least 90 out of every 100 subjects, or at least 95 out of every 100 subjects.
- oligosaccharide compositions provided herein effectively minimizes or prevents progression of an infection in 10-100%, 10-20%, 15-25%, 20-50%, 40- 60%, 50-75%, 60-80%, 75-90%, 80-100%, or 90-100% of subjects.
- Reduction in the rate of infection or colonization using a method described herein may be prospective or retrospective, e.g., relative to an infection.
- the method described herein comprises monitoring a subject or a population of subjects for a similar infection, e.g., through observation of similar symptoms or similar features to those known to be caused by or identified with a pathogen of interest.
- any of the methods described herein might be used to more specifically determine the type of the pathogen involved, and its relationship -if any- to spread or a reservoir.
- a method of modulating the gastrointestinal tract e.g., ah of the GI tract or a part thereof, e.g., the small intestine, the large intestine, the colon, and the like
- the method comprises modulating the environment (e.g., chemical or physical environment) of the gastrointestinal tract of a subject to make the gastrointestinal tract (and the microbial community therein) less selective or less receptive for a pathogen or an antibiotic resistance gene carrier.
- the method further comprises administering a second agent in combination with a oligosaccharide composition, e.g., charcoal or an antibiotic-degrading enzyme (e.g., beta-lactamase), or a synbiotic (e.g., an engineered beta-lactamase (e.g., a non-inf ectious beta-lactamase).
- a oligosaccharide composition e.g., charcoal or an antibiotic-degrading enzyme (e.g., beta-lactamase), or a synbiotic (e.g., an engineered beta-lactamase (e.g., a non-inf ectious beta-lactamase).
- a method of reducing the transfer of a drug- resistance gene e.g., an antibiotic resistance gene or an MDR gene
- a drug- resistance gene e.g., an antibiotic resistance gene or an MDR gene
- one organism e.g., bacterial taxa, e.g., taxa containing the antibiotic resistance gene or an MDR gene
- another organism e.g., taxa that do not contain the antibiotic resistance gene or an MDR gene
- a oligosaccharide composition to a subject , e.g., in an effective amount and/or to a sufficient number of subjects that the transfer of the drug-resistance gene (e.g., an antibiotic resistance gene or MDR gene) is reduced.
- the method comprises reducing the transfer of a drug-resistance gene (e.g., an antibiotic resistance gene or an MDR gene) to an organism with increased pathogenic potential.
- a drug-resistance gene e.g., an antibiotic resistance gene or an MDR gene
- the method comprises reducing the number of recipient bacteria, (e.g., commensal bacterial strains), capable of taking up an antibiotic resistance gene, in a subject .
- the method comprises reducing the probability of a pathogen or an antibiotic resistance gene carrier to spread or transfer an antibiotic resistance gene.
- the method comprises reducing the ability of a pathogen or an antibiotic resistance gene carrier to reach a state of competency.
- Competency refers to the bacteria’s ability to take up genes (e.g ., antibiotic resistance genes) from the environment (von Wintersdorff et al. Front. Microbiol. (2016); 7: 173).
- the method comprises reducing exchange of gene material (e.g., conjugation- based) in a pathogen or an antibiotic resistance gene carrier.
- the method comprises reducing the level of free nucleic acid (e.g. microbial DNA, e.g., comprising an antibiotic resistance gene cassette), in a pathogen or antibiotic resistance gene carrier, e.g., after the pathogen or antibiotic resistance gene carrier reaches competency.
- the method comprises increasing microbial metabolism of a nucleic acid (e.g.
- the method comprises use of a nucleic acid binding molecule as a scavenger, e.g., for binding to a pathogen- derived or antibiotic resistance gene carrier-derived nucleic acid.
- a method of reducing pathogen infectivity as determined by the incidence of the number of pathogens in a population.
- the oligosaccharide composition is administered in an effective amount to reduce the number of pathogen cells that can transmit a drug or antibiotic resistance gene, or MDR element, to another organism.
- the oligosaccharide composition is administered in an effective amount to reduce the number of organisms (e.g. bacteria) that can receive a drug or antibiotic resistance gene, or MDR element.
- the oligosaccharide composition is administered in an effective amount to reduce the ability of a pathogen cell to enter the state in which it can donate a drug or antibiotic resistance gene, or MDR element, to another organism. In some embodiments, the oligosaccharide composition is administered in an effective amount to reduce the ability of a pathogen cell to enter the state in which it can receive a drug or antibiotic resistance gene, or MDR element, from another organism.
- a method of reducing the presence of a drug or antibiotic resistance gene, or MDR element in a microbe e.g., a pathogen, e.g. a bacterial pathogen
- a microbe e.g., a pathogen, e.g. a bacterial pathogen
- the oligosaccharide composition is administered in an effective amount to reduce the copy number of a drug or antibiotic resistance gene, or MDR element, in a microbe (e.g. a bacterial pathogen, on a cell-by-cell basis) or reduce the total number in a microbial population.
- the oligosaccharide composition is administered in an effective amount to increase the population of a gut microbe that is not a (potential) host for a drug or antibiotic resistance gene, or MDR element. In some embodiments, the oligosaccharide composition is administered in an effective amount to reduces the competence of a pathogen, e.g., Streptococcus, to take up a drug or antibiotic resistance gene, or MDR element. In some embodiments, the oligosaccharide composition is administered in an effective amount to reduces the ability of a bacterial cell (e.g., a pathogen), e.g., a gram-negative organism, e.g., E. coli or Klebsiella, to take up a drug or antibiotic resistance gene, or MDR element.
- a bacterial cell e.g., a pathogen
- a gram-negative organism e.g., E. coli or Klebsiella
- the oligosaccharide composition is administered in an effective amount to shift the microbial community of a subject to displace or inhibit a pathogen, an organism that can donate a drug or antibiotic resistance gene, or MDR element (donor microbes), or an organism that can receive a drug or antibiotic resistance gene, or MDR element (recipient microbes).
- the oligosaccharide composition is administered in an effective amount to reduce the probability of donor microbes to spread a drug or antibiotic resistance gene, or MDR element.
- a method of managing an infection by a pathogen comprises treating, preventing, and/or reducing the risk of developing an infection by a pathogen.
- treating an infection by a pathogen comprises administering a oligosaccharide composition to a subject or population upon detection of a pathogen.
- preventing an infection by a pathogen comprises administering a oligosaccharide composition to a subject or population at risk of developing an infection.
- the subject or population may include those who may have been exposed to the pathogen directly and/or infected individuals.
- reducing the risk of developing an infection by a pathogen comprises administering a oligosaccharide composition to a subject or population that may become exposed to a pathogen.
- a method to reduce the expression or release (e.g., by a pathogen) of a factor having an adverse effect on a subject such as a virulence factor or toxin.
- the factor causes a disease.
- a pathogen e.g., a virulence factor or toxin.
- oligosaccharide composition is administered in an effective amount and/or to a sufficient number of subject(s), that the expression or release by a microbe (e.g., a pathogen) of a factor having an adverse effect on a subject, e.g., a virulence factor or a toxin, e.g., that causes disease, is reduced.
- a microbe e.g., a pathogen
- a factor having an adverse effect on a subject e.g., a virulence factor or a toxin, e.g., that causes disease
- the expression of a factor is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%, e.g., relative to a reference standard.
- An adverse effect in a subject includes causing a disease, delaying diagnosis of a disease, or reducing the effectiveness of a disease treatment.
- a method of modulating the number of gene donors (donor microbes) in a population e.g., the state of competency/conjugation).
- a method of modulating the number of gene recipients (recipient microbes) in a population e.g., a oligosaccharide composition is administered in an effective amount to reduce the number of donor microbes (e.g., microbes that carry drug- or antibiotic -resistance genes or MDR elements).
- a oligosaccharide composition is administered in an effective amount to reduce the number of recipient microbes.
- a oligosaccharide composition may affect bacterial activity such that it reduces the frequency of transfer of toxins or determinants of antibiotic resistance between strains or species. This may be accomplished, for instance by transformation, conjugation, phage production or transduction, plasmid release or plasmid replication, such that fewer pathogens are able to access these toxins or resistance determinants. This in turn may reduce the availability of those markers to new pathogens. Such a reduction might be accomplished by modulating a state of competency or conjugation property.
- a method of modulating the copy number of a resistance gene in a population is administered in an effective amount that the copy number of a drug- or antibiotic-resistance gene, toxin or virulence factor is reduced.
- the copy number of a drug- or antibiotic-resistance gene, toxin or virulence factor is reduced.
- a oligosaccharide composition resulting in decreased copy number of a drug or antibiotic resistance gene, toxin or virulence factor might be expected to increase susceptibility to an antibiotic, reduce adverse effects of a pathogen, or reduce the availability of the gene, toxin or virulence factor to other microbial recipients.
- a method of modulating the fitness deficit e.g ., increase the burden of carrying a drug- or antibiotic -resistance gene or MDR element
- the modulating comprises increasing the burden of carrying a resistance gene.
- the oligosaccharide composition is administered in an effective amount that the fitness deficit is increased.
- a oligosaccharide composition that increases the fitness deficit reduces the number of microbes (e.g., bacterial pathogens) carrying it, or their ability to persist in particular subjects (e.g., subjects).
- the oligosaccharide composition alters the ecology of the GI tract (or a subset thereof, e.g., small intestine, large intestine, or colon) such that nitrogen sources is in short supply. This in turn can increase the cost of maintaining additional genetic elements by nucleic acid synthesis.
- the fitness deficit results from enhanced recognition or response by the host (e.g., a human subject). For example, some factors, such as bacterial lipopolysaccharide (LPS) are directly recognized by human cells, resulting in immune responses.
- LPS bacterial lipopolysaccharide
- pathogens e.g., viruses and bacteria, e.g., Vibrio cholerae and Norovirus
- pathogens have been shown to have glycan receptors, or glycan moieties that are necessary to infect gut cells (Holmer, et al. FEBS Letters 584 (2010) 2548-2555).
- a method of decreasing the binding of a pathogen to a cell decreasing the activity of a pathogen on or in a cell, decreasing the entry of a pathogen into or onto a cell, or decreasing the effect of a pathogen on a cell, wherein the method comprises administering of a oligosaccharide
- the cell is a human cell.
- the oligosaccharide composition binding to a pathogen prevents said pathogen or another pathogen from reaching and entering a cell.
- a oligosaccharide composition may directly or indirectly induce a modification of the gut lining or the mucous membrane, or affect another property such that pathogen entry into a cell, pathogen effect on a cell, the ability of a pathogen to persist within a cell or avoid antibody or immune recognition.
- a method of modulating the anti-microbial output (e.g., immune response) of a subject for example, a oligosaccharide composition is administered to increase mucus production, or antibody production or secretion, or the production of antimicrobial peptides (e.g., such as Regllfy) thereby increasing resistance to pathogens.
- Regllly is an antimicrobial protein that binds intestinal bacteria via interactions with peptidoglycan carbohydrate (Cash et ah, Science. (2006) August 25; 313(5790): 1126-1130).
- the modulation of the immune response of the subject may be determined by measuring one or more markers of inflammation, e.g. IFN-g, IL-Ib, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-a.
- the markers can be determined, e.g., from stool or blood samples.
- a ratio of pathogenic bacteria to commensal bacteria is reduced by decreasing the abundance of pathogenic bacteria. In some embodiments, a ratio of pathogenic bacteria to commensal bacteria is reduced by increasing the abundance of commensal bacteria.
- a oligosaccharide composition is administered in an effective amount to modulate the microbial community and alter the environment of the GI tract, (e.g., altering pH, altering lactic acid, altering microbial density, etc.).
- the method comprises outcompeting a pathogen, pathobiont or an antibiotic resistance gene carrier for space or nutrients in the gastrointestinal tract.
- a oligosaccharide composition is administered in an effective amount to reduce the“space” for a pathogen or pathobiont to colonize, e.g., physical space.
- the method comprises making non- pathogenic bacteria fitter (e.g., providing a more selective food source or encouraging growth of fitter (e.g., faster) growing species/strains).
- the method comprises outcompeting a pathogen, pathobiont or an antibiotic resistance gene carrier by increasing the population of a commensal bacterial strain, or by increasing an anti-microbial defense mechanism in a commensal bacterial strain, e.g., production of a bacteriocin, anti-microbial peptide, hydrogen peroxide, or low pH (e.g., through increased level of an acid (e.g., acetate, butyrate, and the like).
- an acid e.g., acetate, butyrate, and the like.
- a ratio of pathogenic bacteria to commensal bacteria is determined by performing nucleic acid sequencing (e.g., 16S metagenomic sequencing) of a fecal sample (e.g., collected from a subject prior to, or following treatment with the oligosaccharide composition).
- 16S metagenomic sequencing of a fecal sample may be accomplished, for example, by extracting genomic DNA from the fecal sample and performing standard 16S sequencing.
- variable region 4 of the 16S rRNA gene is amplified and sequenced (e.g., in accordance with the Earth Microbiome Project protocol www.earthmicrobiome.org/emp-standard-protocols/16s/ and/or Caporaso JG et al. Ultra-high-throughput microbial community analysis on the Ihumina HiSeq and MiSeq platforms. ISME J. (2012) Aug;6(8): 1621-4)).
- Raw sequences may be demultiplexed, and each sample may be processed separately with UN0ISE2 (Robert Edgar UN0ISE2: improved error- correction for Ihumina 16S and ITS amplicon sequencing. bioRxiv (2016) Oct. 15).
- Reads from 16S rRNA amplicon sequencing data may be rarefied to 5000 reads, without replacement, and the resulting OTU table used in downstream calculations.
- the analyzed sequencing data may allow for calculations of the total abundance of various bacterial species (e.g., pathogenic and commensal bacterial species), from which the relative abundance or absolute abundance of pathogenic and commensal bacteria may be determined.
- the reduction of a ratio of pathogenic bacteria to commensal bacteria is determined by (i) performing 16S metagenomic sequencing of a fecal sample collected from the subject prior to administration of the oligosaccharide composition; (ii) performing 16S metagenomic sequencing of a fecal sample collected from the subject following administration of the oligosaccharide composition; and (hi) comparing the relative or absolute abundance of pathogens determined using the sequencing data provided in (ii) relative to the relative or absolute abundance of pathogens determined using the sequencing data provided in (i).
- pathogens include bacterial pathogens (e.g., Abiotrophia spp., (e.g., A. defective), Achromobacter spp., Acinetobacter spp., (e.g., A. baumanii), Actinobaculum spp., (e.g., A. schallii), Actinomyces spp., (e.g., A. israelii), Aerococcus spp., (e.g., A. urinae), Aeromonas spp., (e.g., A., A.
- Abiotrophia spp. e.g., A. defective
- Achromobacter spp. e.g., A. baumanii
- Actinobaculum spp. e.g., A. schallii
- Actinomyces spp. e.g., A. israelii
- Aggregatibacter spp. e.g. A. aphrophilus, Bacillus anthracis, Bacillus cereus group, Bordetella spp., Brucella spp., e.g. B. henselae, Burkholderia spp., e.g., B. cepaciae, Campylobacter spp., e.g., C. jejuni, Chlamydia spp., Chlamydophila spp., Citrobacter spp., e.g., C.
- Enterobacteriaceae including many of the genera below, Ehrlichia spp., Enterobacter spp., e.g., E. cloacae, Enterococcus spp., e.g. E. faecium, Escherichia spp., including enteropathogenic, uropathogenic, and enterohemorrhagic strains of E. coli, Francisella spp., e.g. F. tularensis, Fusobacterium spp., e.g. F. necrophorum, Gemella spp., e.g. G. mobillorum, Granulicatella spp., e.g. G.
- Haemophilus spp. e.g. H. influenza
- Helicobacter spp. e.g. H. pylori
- Kingella spp. e.g. K. kingae
- Klebsiella spp. e.g. K. pneumoniae
- Fegionella spp. e.g. F.
- pneumophila Feptospira spp., Fisteria spp., e.g. F. monocytogenes, Morganella spp., e.g. M. morganii, Mycobacterium spp., e.g. M. abcessus, Neisseria spp., e.g. N. gonorrheae, Nocardia spp., e.g. N. asteroids, Ochrobactrum spp., e.g. O. anthropic, Pantoea spp., e.g. P. agglomerans, Pasteurella spp., e.g. P.
- multocida multocida
- Pediococcus spp. Plesiomonas spp., e.g. P. shigelloides
- Proteus spp. e.g. P. vulgaris
- Providencia spp. e.g. P. stuartii
- Pseudomonas spp. e.g. P.
- Raoultella spp. e.g. R. ornithinolytica
- Rothia spp. e.g. R. mucilaginosa
- Salmonella spp. e.g. S. enterica
- Serratia spp. e.g. S. marcesens
- Shigella spp. e.g. S. flexneri
- Staphylococcus aureus e.g. S. enterica
- Staphylococcus lugdunensis e.g. S. flexneri
- Staphylococcus saprophyticus Stenotrophomonas spp., e.g. S. maltophilia, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus constellatus, Streptococcus dysgalactiae, Streptococcus intermedius, Streptococcus milleri, Streptococcus pseudopneumoniae,
- Streptococcus pyogenes Streptooccus pneumoniae, Treponema spp., Ureaplasma ureolyticum, Vibrio spp., e.g. V. cholerae, and Yersinia spp., (e.g., Y.
- enterocolitica enterocolitica
- viral pathogens e.g., Adenovirus, Astrovims, Cytomegalovirus, Enterovirus, Norovirus, Rotavirus, and Sapovims
- gastrointestinal pathogens e.g., Cyclospora spp., Cryptosporidium spp., Entamoeba histolytica, Giardia lamblia, and Microsporidia, (e.g., Encephalitozoon canaliculi)
- the method comprises reducing the spread of antibiotic resistant organisms.
- Antibiotic resistant organisms include: Beta-lactamase producing
- Enterobacteriaceae including extended spectrum beta lactamase and carbapenemase producers, possessing genes such as TIM, OXA, VIM, SHV, CTX-M, KPC. NDM or AmpC); Vancomycin- resistant Enterococcus (e.g., possessing genes such as VanA or VanB) ⁇ , Fluoroquinolone-resistant Enterobacteriaceae (e.g., with genes such as Qnr ); Carbapenem-resistant and multidmg resistant Pseudomonas; Methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae (e.g., possessing the MecA gene); Multidmg resistant Acinetobacter (often containing beta lactamase); Trimethoprim resistant organisms (e.g., dihydrofolate reductase); Sulfamethoxazole resistant organisms (e.g., dihydropteroate synthetase); and Amino
- a method to manage an infection by a pathogen comprising, administering to a first and/or second subject, a second treatment.
- the second treatment comprises administering charcoal, or other adsorbing agent.
- the adsorbing agent might serve to reduce the presence of antibiotic within the GI tract (e.g., small intestine, large intestine, colon), so as to reduce the selective pressure of maintaining a resistance determinant, thereby allowing its reservoir, level, spread or adverse effect to be reduced.
- the adsorbing agent might increase the beneficial effect of the oligosaccharide composition.
- the second treatment comprises administering a nonabsorbable antibiotic such as a beta lactam, or a beta lactamase inhibitor to a subject .
- the second treatment comprises administering an antibiotic degrading enzyme, e.g., beta-lactamase enzyme.
- the subject is critically ill and/or a transplant patient.
- Critically ill subjects and/or transplant patients are prone to infections (e.g. have a high rate of infections), such as bloodstream infections.
- infectious microbes are carried in the gut (e.g., can be acquired through colonization) and include E. coli, Klebsiella, other Enterobacteriaceae, and Enterococcus.
- the microbes e.g., pathogens
- are drug resistant e.g. carbapenem-resistant Enterobacteriaceae, vancomycin- resistant Enterococcus).
- assessment of colonization is used to predict the risk of infection (e.g., bloodstream infection, urinary tract infection (UTI), or respiratory infection, bacteremia), e.g., by correlating levels of colonization (e.g., by assessing a suitable sample for presence or absence of predetermined bacterial taxa and/or assessing pathogen load) with risk of infection, wherein evidence of colonization is correlated with an increased risk of infection, wherein culture-negative subjects are at lower risk of infection.
- higher levels of bacteria lead to higher rates of infection.
- intestinal colonization e.g. by a pathogen, e.g.
- gastrointestinal tract-colonizing pathogens may include: Enterobacteriaceae (e.g. E. coli, Klebsiella, Enterobacter, Proteus) and Enterococcus. In some embodiments, gastrointestinal tract-colonizing pathogens further include multidmg resistant bacteria (e.g., Carbapenem resistant Enterobacteriaceae, Vancomycin resistant Enterococcus).
- the outcome of screening subject populations for pathogen status determines the course of bloodstream infection management.
- screening methods comprise stool sampling ( e.g . by rectal swab) of subjects.
- the method comprises assessing the presence/absence (abundance) of
- dmg/antibiotic resistant pathogens e.g., VRE
- the level of pathogens within the gut is correlated with infection risk.
- intensive care unit (ICU) subjects, transplant subjects, chemotherapy-receiving subjects, and antibiotic receiving subjects have a higher risk of having pathogen colonization from antibiotic resistant bacteria such as carbapenem resistant Enterobacteriaciae and Vancomycin-resistant
- reducing the level of pathogens within the gut reduces risk (e.g., by administering a oligosaccharide composition if desired in combination with an antibiotic).
- the subject is administered a oligosaccharide composition to prevent infection (e.g., bloodstream infection) or bacteremia.
- the subject is administered a oligosaccharide composition to reduce infection (e.g., bloodstream infection) or bacteremia.
- a method to reduce the colonization level or prevalence of antibiotic resistant pathogens carried in the GI tract of high-risk subjects e.g. subjects.
- antibiotic resistant pathogens include Carbapenem-resistant
- Enterobacteriaciae e.g., extended spectrum beta lactamase (ESBL) producing
- ESDLE Enterobacteriaciae
- Vancomycin-resistant Enterococcus Vancomycin-resistant Enterococcus
- a method to reduce the rate of infections e.g., from pathogens that colonize the GI tract
- the method comprises reducing the rate of urinary tract infections.
- the method comprises reducing the rate of bloodstream infections.
- the method comprises reducing the rate of respiratory tract infections.
- the method comprises managing infections in subjects.
- subject groups with infections include: subjects with urinary infections (e.g., infected with Enterococcus , Enterobacteriaciae), subjects with bloodstream infections (e.g., infected with Enterococcus, Enterobacteriaciae), transplant subjects (e.g., bone marrow (e.g., undergoing hematopoietic stem cell transplantation), solid organ (e.g., liver)), intensive care patients (e.g., infected with Carbapenem resistant Enterobacteriaciae and ESBL producing pathogens), pre-transplant liver failure patients (e.g., infected with Vancomycin resistant Enterococcus), post-transplant liver failure patients (e.g., infected with Vancomycin resistant Enterococcus).
- subjects with urinary infections e.g., infected with Enterococcus , Enterobacteriaciae
- subjects with bloodstream infections e.g., infected with
- antibiotic-treated subjects comprise higher pathogen loads, including antibiotic resistant pathogens.
- subjects undergoing or about to undergo a transplant subjects with cancer, subjects with liver disease (e.g., end-stage renal disease), or subjects with suppressed immune system (e.g., immunocompromised subjects) may have high risk of developing infections, e.g., gut-derived infections.
- the method comprises prophylactic treatment, e.g., with a oligosaccharide composition, of a subject, e.g., a subject with a high risk of developing an infection.
- subjects who are undergoing chemotherapy or antibiotic treatment have reduced diversity of commensal bacteria.
- the method comprises treatment of a subject to reduce the colonization of pathogens, e.g., multidrug resistant pathogens, in a subject, e.g., subjects in a facility, e.g., a hospital or long-term care facility.
- the method comprises treatment of a subject to reduce the transmission of pathogens, e.g., multidrug resistant pathogens, from a first subject to a second subject, e.g., subjects in a facility, e.g., a hospital or long-term care facility.
- pathogens e.g., multidrug resistant pathogens
- bacteria that pose a risk of colonization in subjects comprise resistant subpopulations of Enterobacteriaceae (e.g., E. cloacae), Enterococcus, C. difficile (including Napl (pandemic hypervirulent) C. difficile strain), and bacteria that cause infectious diarrhea (e.g., Campylobacter, Salmonella, Shigella,
- EHEC enterohemorrhagic E. coli
- ETEC enterotoxigenic E. coli
- EPEC enteropathogenic E. coli
- EIEC enteroinvasive E. coli
- EAEC enteroaggregative E. coli
- DAEC diffusely adherent E. coli
- the method comprises managing infections in subjects who are in need of an organ transplant, e.g., a liver or bone marrow transplant.
- an organ transplant e.g., a liver or bone marrow transplant.
- the method comprises managing infections in subjects immediately, or shortly, before said subject receives an organ transplant, e.g., a liver or bone marrow transplant. In some embodiments, the method comprises managing infections in subjects immediately, or shortly, after said subject receives an organ transplant, e.g., a liver or bone marrow transplant. In some embodiments, the method comprises managing infections in subjects who have, are suspected of having, or at risk of having end-stage liver disease (ESLD). [00223] In some embodiments, the method reduces a ratio of pathogenic bacteria to commensal bacteria, e.g., in a subject, e.g., in the gastrointestinal tract of the subject (e.g, the colon).
- ESLD end-stage liver disease
- a ratio of pathogenic bacteria to commensal bacteria is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200%. In some embodiments, a ratio of pathogenic bacteria to commensal bacteria is reduced by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90- 110%, 100-125%, 110-150%, 125-175%, or 150-200%.
- the method reduces the abundance of pathogens and/or increases the abundance of commensal bacteria, e.g., in a subject, e.g., in the gastrointestinal tract of the subject (e.g, the colon). In some embodiments, the method increases the alpha- diversity (e.g., a high degree of diversity) of a microbial community (e.g., a community of commensal bacteria), e.g., of the gut of a subject.
- a microbial community e.g., a community of commensal bacteria
- the method reduces the abundance of pathogens by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200%. In some embodiments, the method reduces the abundance of pathogens by 1-10%, 5-20%, 10-25%, 20- 40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200%.
- the method increases the abundance of commensal bacteria by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200%. In some embodiments, the method increases the abundance of commensal bacteria by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90- 110%, 100-125%, 110-150%, 125-175%, or 150-200%.
- the method increases the alpha-diversity (e.g., a high degree of diversity) of the microbiome in the gastrointenstinal tracts.
- Alpha diversity may be measured using the Shannon index in combination with nucleic acid sequencing.
- the Shannon Index indicates that subjects in need of treatment (e.g., ICU patients) have communities that are considerably less diverse in their representation of bacterial taxa than healthy subjects.
- community richness, or the number of unique taxa within a sample is considerably lower in subjects in need of treatment (e.g., ICU patients) relative to healthy subjects.
- oligosaccharide compositions are substantially fermented or consumed by commensal bacteria and are not fermented or consumed by pathogens. In some embodiments, oligosaccharide compositions are substantially fermented or consumed by commensal bacteria and are fermented or consumed by pathogens at low levels. In some embodiments, a oligosaccharide composition that is substantially consumed by commensal bacteria may increase the diversity and biomass of the commensal microbiota and lead to a reduction in the relative abundance or absolute abundance of a pathogen(s), such as a bacterial pathogen (e.g., a pathogenic taxa). In some embodiments, a oligosaccharide composition is substantially non- fermented or not consumed by VRE or CRE species. In some embodiments, a oligosaccharide composition is substantially non-fermented or not consumed by C. difficile.
- a oligosaccharide composition supports the growth of commensal or probiotic bacteria, e.g., in a gut microbiome.
- a oligosaccharide composition supports the growth of commensal or probiotic bacteria, e.g., in a gut microbiome.
- oligosaccharide composition does not support the growth of at least one pathogen, e.g., does not support the growth of a CRE, VRE, and/or C. difficile species.
- administration of a oligosaccharide composition may increase the concentration, amount or abundance (e.g., relative abundance or absolute abundance) of commensal bacteria relative to pathogenic bacteria in the microbiome of a subject (e.g., a human patient).
- administration of a oligosaccharide composition and a population of viable commensal or probiotic bacteria may increase the concentration, amount, or abundance (e.g., relative abundance or absolute abundance) of commensal bacteria relative to pathogenic bacteria in the microbiome of a subject (e.g., a human patient).
- administration of a oligosaccharide composition that supports the growth of commensal or probiotic bacteria, e.g., in a gut microbiome may increase the concentration, amount or abundance (e.g., relative abundance or absolute abundance) of commensal bacteria relative to pathogenic bacteria in the microbiome of a subject (e.g., a human patient).
- administration of a oligosaccharide composition that does not support the growth of at least one pathogen e.g., does not support the growth of a CRE, VRE, and/or C.
- a oligosaccharide composition that supports the growth of commensal or probiotic bacteria and does not support the growth of at least one pathogen, e.g., does not support the growth of a CRE, VRE, and/or C.
- difficile species e.g., in a gut microbiome, may increase the concentration, amount or abundance (e.g., relative abundance or absolute abundance) of commensal bacteria relative to pathogenic bacteria in the microbiome of a subject (e.g., a human patient).
- a subject e.g., a human patient
- administration of an oligosaccharide composition may increase the concentration, amount or abundance (e.g., relative abundance or absolute abundance) of Bacteroidetes (e.g., Bacteroidales ) relative to pathogenic bacteria in the microbiome of a subject (e.g., a human patient).
- Bacteroidetes e.g., Bacteroidales
- an oligosaccharide composition described herein is co administered with commensal or probiotic bacterial taxa and bacteria that are generally recognized as safe (GRAS) or known commensal or probiotic microbes.
- GRAS commensal or probiotic bacterial taxa
- probiotic or commensal bacterial taxa (or preparations thereof) may be administered to a subject before or after administration of an oligosaccharide composition to the subject.
- probiotic or commensal bacterial taxa (or preparations thereof) may be administered to a subject before or after administration of an oligosaccharide composition to the subject.
- probiotic or commensal bacterial taxa (or preparations thereof) may be administered to a subject before or after administration of an oligosaccharide composition to the subject.
- probiotic or commensal bacterial taxa (or preparations thereof) may be
- oligosaccharide composition administered to a subject simultaneously with administration of an oligosaccharide composition to the subject.
- an oligosaccharide composition described herein is administered with a population of Bacteroidetes. In embodiments, an oligosaccharide composition described herein is administered with a population of Bacteroidales.
- a commensal or probiotic bacteria is also referred to a probiotic.
- Probiotics can include the metabolites generated by the probiotic bacteria during fermentation. These metabolites may be released to the medium of fermentation, e.g., into a host organism (e.g., subject), or they may be stored within the bacteria.
- Probiotic bacteria includes bacteria, bacterial homogenates, bacterial proteins, bacterial extracts, bacterial ferment supernatants and
- Useful probiotics include at least one lactic acid and/or acetic acid and/or propionic acid producing bacteria, e.g., microbes that produce lactic acid and/or acetic acid and/or propionic acid by decomposing carbohydrates such as glucose and lactose.
- the probiotic bacteria is a lactic acid bacterium.
- lactic acid bacteria include
- Suitable probiotic bacteria can also include other bacterias which beneficially affect a host by improving the hosts intestinal microbial balance, such as, but not limited to yeasts such as Saccharomyces,
- Debaromyces Candida, Pichia and Torulopsis, molds such as Aspergillus, Rhizopus, Mucor, and Penicillium and Torulopsis, and other bacteria such as but not limited to the genera Bacteriodes, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Enterococcus, Lactococcus, Staphylococcus, Peptostreptococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, and Oenococcus, and combinations thereof.
- Non-limiting examples of lactic acid bacteria useful in the disclosure herein include strains of Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetylactis, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bifidus, Lactobacillus casei, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus delbruekii, Lactobacillus thermophilus, Lactobacillus fermentii, Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus brevis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobcterium animalis, Bifidob
- Pediococcus cerevisiae and combinations thereof in particular Lactobacillus, Bifidobacterium, and combinations thereof.
- Commensal or probiotic bacteria which are particularly useful in the present disclosure include those which (for human administration) are of human origin (or of the origin of the mammal to which the probiotic bacteria is being administered), are non-pathogenic to the host, resist technological processes ( i.e . can remain viable and active during processing and in delivery vehicles), are resistant to gastric acidity and bile toxicity, adhere to gut epithelial tissue, have the ability to colonize the gastrointestinal tract, produce antimicrobial substances, modulate immune response in the host, and influence metabolic activity (e.g . cholesterol assimilation, lactase activity, vitamin production).
- metabolic activity e.g . cholesterol assimilation, lactase activity, vitamin production.
- the commensal or probiotic bacteria can be used as a single strain or a combination of multiple strains, wherein the total number of bacteria in a dose of probiotic bacteria is from about 1 x 10 3 to about 1 x 10 14 , or from about 1 x 10 to about 1 x 10 12 , or from about 1 x 10 7 to about 1 x 10 11 CFU per dose.
- the commensal or probiotic bacteria can be formulated with the oligosaccharide compositions while the probiotic bacteria are alive but in a state of“suspended animation” or somnolence.
- the viable cultures(s) of probiotic bacteria are handled so as to minimize exposure to moisture that would reanimate the cultures because, once reanimated, the cultures can experience high rates of morbidity unless soon cultured in a high moisture environment or medium. Additionally, the cultures are handled to reduce possible exposure to high temperatures (particularly in the presence of moisture) to reduce morbidity.
- the probiotic bacterias can be used in a powdered, dry form.
- the probiotic bacterias can also be administered in the oligosaccharide composition or in a separate oligosaccharide composition, administered at the same time or different time as the
- probiotic bacteria suitable include Bifidobacterium lactis, B. animalis, B. bifidum, B. longum, B. adolescentis, and B. inf antis.
- a commensal bacterial taxa that can be used in and/or in combination with an oligosaccharide composition described herein comprises Akkermansia, Anaerococcus, Bacteroides, Bifidobacterium (including Bifidobacterium lactis, B. animalis, B. bifidum, B. longum, B. adolescentis, B. breve, and B. infantis ), Blautia, Clostridium,
- Corynebacterium Dialister, Eubacterium, Faecalibacterium, Finegoldia, Fusobacterium, Factobacillus (including, F. acidophilus, F. helveticus, F. bifidus, F. lactis, F. fermentii, F.
- Peptococcus Peptostreptococcus, Peptoniphilus, Prevotella, Roseburia, Ruminococcus, Staphylococcus, and/or Streptococcus (including S. lactis, S. cremoris, S. diacety lactis, S.
- thermophiles are thermophiles.
- a commensal bacterial taxa e.g., GRAS strain
- an oligosaccharide composition described herein comprises Bacillus coagulans GBI-30, 6086; Bifidobacterium animalis subsp. Lactis BB-12;
- Factobacillus acidophilus NCFM Factobacillus casei DN 114-001 ( Factobacillus casei Immunitas(s)/Defensis); Factobacillus casei CRL431; Factobacillus casei F19; Factobacillus paracasei Stl 1 (or NCC2461); Factobacillus johnsonii Lai ( Factobacillus LCI, Factobacillus johnsonii NCC533); Factococcus lactis LI A; Factobacillus plantarum 299V; Factobacillus reuteri ATTC 55730 ( Factobacillus reuteri SD2112); Factobacillus rhamnosus ATCC 53013; Factobacillus rhamnosus LB21; Saccharomyces cerevisiae ( boulardii ) lyo; mixture of
- Factobacillus rhamnosus GR-1 and Factobacillus reuteri RC-14 mixture of Factobacillus acidophilus NCFM and Bifidobacterium lactis BB-12 or BL-04; mixture of Lactobacillus acidophilus CL1285 and Lactobacillus cased, and a mixture of Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011, and/ or Lactobacillus rhamnosus GG (LGG).
- the method comprises the administration of a
- oligosaccharide composition and the administration of a commensal or probiotic bacterial species may be used to benefit patients with depleted microbiomes (e.g ., patients with few or no detectable commensal bacteria), e.g., patients who are undergoing chemotherapy or receiving antibiotics.
- the combined administration of oligosaccharide compositions and commensal bacteria may be used to benefit a subject or patient having a gut microbiome devoid of any detectable commensal bacteria.
- the method comprises combined administration of oligosaccharide compositions and commensal bacteria to a subject or patient who has a gut microbiome devoid of any detectable commensal bacteria.
- an oligosaccharide composition described herein can be used for treating an urea cycle disorder (UCD) in a human subject.
- the subject has hepatic encephalopathy (HE).
- composition described herein can be used for treating a subject having end-stage liver disease (ESLD).
- ESLD end-stage liver disease
- the subject is of a pediatric population (e.g., 2-24 months or 2- 18 years old).
- an oligosaccharide composition described herein can be used for treating an infection, e.g., a bacterial infection, e.g. associated with a bacterial pathogen or pathobiont.
- an oligosaccharide composition described herein can be used for treating an infection, e.g., a bacterial infection, e.g. associated with a antibiotic-resistant (e.g., multi-dmg-resistant) bacterial pathogen
- an oligosaccharide composition described herein can be used for treating an infection, e.g., a fungal infection.
- an oligosaccharide composition described herein can be used for reducing the relative or absolute abundance of pathogens or pathobionts in the human subject (e.g., in the gastrointestinal tract, the UTI tract, the bloodstream, or a different site, e.g. within the cardiovascular system or the respiratory system).
- the oligosaccharide composition is administered in an amount effective to modulate (e.g . reduce or inhibit) colonization or to modulate (e.g. increase) decolonization by the pathogen, e.g., in the gut (e.g., small intestine, large intestine and/or colon) of the human subject.
- treatment includes one or both of (i) reducing the abundance of pathogenic bacteria, e.g., in the gastrointestinal tract, relative to a control (e.g., a control subject or baseline measurement), and (ii) increasing the abundance of commensal bacteria, e.g., in the gastrointestinal tract, relative to a control (e.g., a control subject or baseline measurement).
- a subject for the methods described herein e.g., treatment of infection, or methods reducing the relative or absolute abundance of pathogens
- the methods relate to a subject who is a patient receiving broad spectrum antibiotics.
- the methods relate to a subject who is particularly susceptible to pathogen infection, e.g., the subject is critically-ill and/or immunocompromised. In some embodiments, the methods relate to a subject who is a patient having a lower abundance of commensal bacteria relative to a healthy subject in their gastrointestinal tract (e.g., their colon or intestines).
- the methods relate to a subject who has received or is currently receiving cancer treatment. In some embodiments, the methods relate to a subject who has received or is currently receiving immunosuppression. In some embodiments, the methods relate to a subject who is preparing for or recovering from a gastrointestinal surgery. In some embodiments, the methods relate to a subject who is a patient in an intensive care unit (ICU). In some embodiments, the methods relate to a subject who is a healthy subject. In some
- the methods relate to a subject who is asymptomatic, yet is detectably colonized by pathogens. In some embodiments, the methods relate to a subject who is at risk of developing a pathogenic infection (e.g., and treatment with the oligosaccharide composition reduces the likelihood of infection). In some embodiments, the methods relate to a subject who is a transplant recipient or is preparing to receive a transplant. In some embodiments, the methods relate to a subject who is a hematopoietic stem cell treatment (HSCT) recipient or is preparing to receive a hematopoietic stem cell treatment. In some embodiments, the methods relate to a subject who is a solid organ transplant recipient or is preparing to receive a solid organ transplant. A subject undergoing any treatment or surgery (e.g., cancer treatment,
- gastrointestinal surgery, transplantation surgery may be treated with an oligosaccharide composition before, during, and/or after the treatment or surgery.
- treatment with an oligosaccharide composition before, during, and/or after the other treatment or surgery e.g ., in an ICU facility
- prevents a pathogenic infection e.g., treatment with an oligosaccharide composition before, during, and/or after the other treatment or surgery (e.g., in an ICU facility) prevents graft-vs-host disease (GvHD).
- GvHD graft-vs-host disease
- the methods relate to a subject who has an auto-immune disease (e.g., systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, or Crohn's disease).
- the methods relate to a subject who has a hematological malignancy.
- the methods relate to a subject who has cirrhosis.
- the methods relate to a subject who has a positive stool culture for Carbapenem- resistant Enterobacteriaciae (CRE), extended spectrum beta lactamase (ESBL) producing Enterobacteriaciae (ESBLE), and/or Vancomycin-resistant Enterococcus (VRE).
- CRE Carbapenem- resistant Enterobacteriaciae
- ESBL extended spectrum beta lactamase
- EMBLE Vancomycin-resistant Enterococcus
- the methods relate to a subject who has low diversity of bacterial communities in the gastrointestinal tract.
- the methods relate to a subject who has or is at risk of developing end-stage liver disease (ESLD).
- the methods relate to a subject who has had multiple courses of antibiotics, and/or chronic use of antibiotics and/or has been overprescribed antibiotics.
- the methods relate to a subject who is experiencing or is at risk of an over-aggressive immune response such as, for example, a cytokine storm.
- a cytokine storm (also known as hypercytokinemia), in some embodiments, involves an immune reaction in which the body releases too many cytokines into the blood too quickly (e.g., at the same time). The release of a large amount of cytokines at one time can be harmful.
- a cytokine storm is characterized by high fever, inflammation (e.g., redness and swelling), severe fatigue and/or nausea.
- a cytokine storm is severe or life threatening and/or can lead to multiple organ failure.
- the methods comprise the administration of an oligosaccharide composition concurrent with administration of antibiotics or any other standard-of-care.
- the methods provided herein comprise the administration of an oligosaccharide composition subsequent to administration of antibiotics or any other standard-of-care.
- an oligosaccharide composition provides an additive benefit to subjects being administered antibiotics or any other standard-of- care.
- an oligosaccharide composition provides an synergistic benefit (e.g., by reducing the ratio of pathogenic bacteria to commensal bacteria) to subjects being administered antibiotics or any other standard-of-care.
- the antibiotics are broad spectrum antibiotics.
- the antibiotics are intravenous Gram positive (e.g., vancomycin) or Gram-negative (e.g., ceftriaxone, cefepime, or piperacillin-tazobactam) antibiotics, or both Gram positive and Gram-negative agents together.
- Kits also are contemplated.
- a kit can comprise unit dosage forms of the oligosaccharide composition, and a package insert containing instructions for use of the composition in treatment.
- the composition is provided in a dry powder format.
- the composition is provided in solution.
- the kits include an oligosaccharide composition in suitable packaging for use by a subject in need thereof. Any of the compositions described herein can be packaged in the form of a kit.
- a kit can contain an amount of an oligosaccharide composition sufficient for an entire course of treatment, or for a portion of a course of treatment.
- oligosaccharide composition can be individually packaged, or the oligosaccharide composition can be provided in bulk, or combinations thereof.
- a kit provides, in suitable packaging, individual doses of an oligosaccharide composition that correspond to dosing points in a treatment regimen, wherein the doses are packaged in one or more packets.
- Kits can further include written materials, such as instructions, expected results, testimonials, explanations, warnings, clinical data, information for health professionals, and the like.
- the kits contain a label or other information indicating that the kit is only for use under the direction of a health professional.
- the container can further include scoops, syringes, bottles, cups, applicators or other measuring or serving devices.
- Example 1 Reduction of pathogen abundance in a defined microbial community in the presence of oligosaccharide compositions
- oligosaccharide compositions were tested for their ability to modulate (e.g., reduce) the abundance (e.g., relative abundance or absolute abundance) of pathogens and to support the growth of commensal bacteria in a defined microbial community (comprising 46 different commensal bacterial strains).
- This screening was conducted by spiking the defined microbial community with three drug-resistant bacteria (CRE Klebsiella pneumoniae, CRE Escherichia coli, and VRE Enterococcus faceium) and
- the defined microbial community was constructed by combining 46 strains that belonged to phyla Actinobacteria, Firmicutes, and Bacteroidetes: Blautia producta, Blautia hansenii, Clostridium celatum, Bacteroiodes cellulosilyticus, Odoribacter splanchnicus, Bifidobacterium catenulatum, Eubacterium hallii, Bacteroides dorei, Bifidobacterium
- pseudocatenulatum Bifidobacterium adolescentis , Bacteroides coprophilus, Lactobacillus casei, Coprococcus catus, Bifidobacterium angulatum, Eubacterium ventriosum, Lachnospira multipara, Parabacteroides merdae, Bacteroides finegoldii, Parabacteroides distasonis, Bacteroides thetaiotaomicron, Blautia hydro genotrophica, Blautia coccoides, Clostridium bolteae, Clostridium scindens, Holdemanella biformis, Bifidobacterium longum sub.
- each of the 46 different commensal bacterial strains were independently grown in standard chopped meat glucose medium (CMG) for 18-48 hours, depending on the strain. After growth, the optical density (ODeoo) of each bacterial strain was adjusted to 0.2 and equal volumes of each of the 46 strains were combined into one bottle at a final glycerol concentration of 15%. 1.5-mL aliquots of the defined microbial community were frozen at -80 °.
- CMG chopped meat glucose medium
- the CRE Klebsiella pneumoniae, CRE Escherichia coli, and VRE Enterococcus faecium strains for use in this Example were obtained from the Centers for Disease Control (CDC) and were grown aerobically in BHI medium for 12 hours at 37 °C prior to their addition to the defined microbial community.
- CDC Centers for Disease Control
- Each spiked aliquot was provided one of the four hundred and fifteen different oligosaccharide compositions (as the sole carbon source present in the aliquot) at a final concentration of 0.5% w/v or 0.05% w/v and incubated for 24 hours in the anaerobic chamber at 37 °C. Water was used as a negative control ( . ⁇ ? ., no carbon source). Each oligosaccharide composition was replicated up to 3 times. After 24 hours of incubation, the O ⁇ ⁇ oo of each spiked microbial community was measured to provide an approximation of total anaerobic growth.
- oligosaccharide compositions that reduced the abundance of pathogens and supported commensal growth in the spiked microbial community of Example 1 were further assessed for their abilities to similarly function in ex vivo fecal suspensions from humans that were spiked with single pathogen strains (VRE E. faecium, CRE K. pneumoniae , or CRE E. coli). Oligosaccharide compositions were prepared at 5% w/v in water, filter-sterilized and added to 96-well deep well microplates assay plates for a final concentration of 0.5% or 0.05% w/v in the assay, with water supplied as a negative control.
- a human fecal sample donation was stored at -80 °C.
- the fecal sample was transferred into the anaerobic chamber and allowed to thaw.
- the fecal sample was then prepared in 20% w/v in phosphate buffered saline (PBS) pH 7.4 (P0261, Teknova Inc., Hollister, CA), 15% glycerol.
- PBS phosphate buffered saline
- the 20% w/v fecal suspension + 15% glycerol was centrifuged at 2,000xg, supernatant was removed, and the pellet was suspended in 1% PBS prior to dilution in a CM medium consisting of 900 mg/L sodium chloride, 26 mg/L calcium chloride dihydrate, 20 mg/L magnesium chloride hexahydrate, 10 mg/L manganese chloride tetrahydrate, 40 mg/L ammonium sulfate, 4 mg/L iron sulfate heptahydrate, 1 mg/L cobalt chloride hexahydrate, 300 mg/L potassium phosphate dibasic, 1.5 g/L sodium phosphate dibasic, 5 g/L sodium bicarbonate, 0.125 mg/L biotin, 1 mg/L pyridoxine, 1 m/L pantothenate,
- CM medium with 0.5% D-glucose in an anaerobic chamber.
- aliquots of the pathogenic cultures were washed with PBS and the optical density (ODeoo) of each pathogenic culture and the 1% fecal suspension were adjusted to OD 0.1 in CM media.
- Each of the three pathogen cultures was then separately added to three aliquots of the fecal suspension such that the pathogen cultures comprised 8% of the final volume of the fecal suspension/pathogen mixture.
- Each of the three fecal suspension/pathogen mixtures were exposed to the 96-well plates of oligosaccharide compositions at a final concentration of 0.05% w/v or 0.5% w/v, 350 pL final volume per well, at 37 0 C for 45 hours, anaerobically.
- Example 3 Testing of ability oligosaccharide compositions to support the growth of single pathogens
- oligosaccharide compositions can support the growth of single pathogens.
- Mega Medium contains lOg/L tryptone peptone, 5g/L yeast extract, 4.1mM L-cysteine, lOOmM potassium phosphate buffer (pH 7.2), 0.008mM magnesium sulfate, 4.8mM sodium bicarbonate, 1.37mM sodium chloride, 5.8mM vitamin K, 0.8% calcium chloride, 1.44mM iron (II) sulfate heptahydrate, 4mM resazurin, 0.1% histidine-hematin, 1% ATCC trace mineral supplement, 1% ATCC vitamin supplement, 29.7mM acetic acid, 0.9mM isovaleric acid, 8.1mM propionic acid, 4.4mM N-butyric acid with the pH adjusted to 7 using sodium hydroxide.
- This medium was filter sterilized using a 0.2um filter and stored in an anaerobic chamber prior to use to allow any dissolved oxygen to dissipate.
- the single strains of E. coli BAA-2340, BAA-97, 4 strains isolated from patients, and ECO.139), K.
- oligosaccharide compositions as the sole carbon source in each well. Water added to medium (e.g., CM or MM) without any carbon source functioned as a control. These microplates were then incubated at 37 °C in the COY anaerobic chamber for a total of 45 hours and the ODeoo was measured every 15 minutes to generate a growth curve for each experimental well. Each oligosaccharide composition was tested in three replicates against each bacterial pathogen.
- medium e.g., CM or MM
- AUC area under the curve
- a selected oligosaccharide composition did not support the growth (or supported very low growth) of CRE E. coli, CRE K. pneumoniae, VRE E. faecium, or C. difficile. These results further demonstrated that the selected oligosaccharide composition does not support the growth of pathogens, and thereby disadvantages pathogen growth and abundance in microbial communities by selectively favoring the growth of commensal bacteria.
- a selected oligosaccharide composition comprised of a plurality of
- oligosaccharides selected from Formula (I), Formula (II), and Formula (III) and produced by a process as described in Examples 7-9 was further tested for its ability to reduce growth and abudance of single strains of pathogens that frequently encountered in critically ill and immunocompromised patients.
- Three Napl strains of C. difficile and one C. difficile strain from ribotype 012 were obtained from the ATCC® (ATCC® BAA- 1870TM, ATCC® BAA- 1803TM, ATCC® BAA- 1805TM, and ATCC® BAA-1382TM). Each strain was grown anaerobically in CM medium at 37 °C for 24 hours until each strain achieved an optical density (ODeoo) of about 1. Each culture was adjusted to an ODeoo of 0.01 and then incubated with glucose or a sample of the selected oligosacchride composition. Water was added to media without any added carbon source as a negative control.
- the final concentration of glucose or the selected oligosacchride composition in each assay was 0.5% w/v and each assay was replicated 3 times within each growth plate. Plates were incubated at 37 °C in an anaerobic chamber for a total of 48 hours. Optical density was determined for each strain every 15 minutes for 48 hours.
- the selected oligosaccharide composition was further tested for its ability to reduce the growth and abundance of individual strains of CRE Escherichia coli, CRE Klebsiella pneumoniae , and VRE E. faecium.
- Single strains of E. coli one strain obtained from the CDC’s Enterobacteriaceae-carbapenem-breakpoint panel, the other isolated from a patient
- K K.
- Each culture was adjusted to an ODeoo of 0.01 and then incubated with glucose, fructooligosaccharide (FOS), or a sample of the selected oligosacchride composition. Water was added to media without any added carbon source as a negative control. The final concentration of glucose, FOS, or the selected oligosacchride composition in each assay was 0.5% w/v and each assay was replicated 3 times within each growth plate. Plates were incubated at 37 °C in an anaerobic chamber for a total of 45 hours. Optical density was determined for each strain every 15 minutes for 48 hours.
- FOS fructooligosaccharide
- the CRE and VRE pathogens exhibited little-to-no growth in the presence of samples of the selected oligosaccharide composition, similar to the growth of pathogens in the presence of the water control (FIG. 3 and FIG. 4).
- the selected oligosaccharide composition was tested for its ability to reduce the growth and abundance of individual strains of fungal pathogens ( Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis). Each of four strains of Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis were obtained from ATCC (ATCC MYA-2950, ATCC 14243, ATCC 201380 and ATCC MYA-2876).
- Candida lusitaniae ATCC 66035 and ATCC 42720 were also tested. All Candida strains were grown aerobically in modified Sabouraud broth (10 g/L peptone solution) with glucose at 2% final concentration at 37 °C for 24 hours until each strain achieved optical density (ODeoo) of about 1. 200 pL of each culture was diluted in 3 mL of modified Sabouraud broth and 120 pL was added to each well of a 96 well plate containing 80 pL of one of the following 5% w/v solutions per well: glucose, FOS, or a sample of the selected oligosaccharide composition.
- modified Sabouraud broth 10 g/L peptone solution
- ODeoo optical density
- Water was used as a negative control.
- the final concentration of glucose, FOS, or the selected oligosaccharide composition in each assay to test Candida albicans, Candida glabrata, Candida krusei, or Candida tropicalis was 2%, each assay was replicated 3 times, and plates were incubated at 37 °C for a total of 65 hours.
- the final concentration of glucose, FOS, or the selected oligosaccharide composition in each assay to test Candida lusitaniae strains was 0.5%, each assay was replicated 3 times, and plates were incubated at 37 °C for a total of 48 hours.
- Optical density data was collected for each of the Candida albicans, Candida glabrata, Candida krusei, or Candida tropicalis strains every 15 minutes; optical density data was collected for the Candida lusitaniae strains at the end of the experiment.
- each of the Candida albicans, Candida glabrata, Candida krusei, or Candida tropicalis strains grew minimally in the presence of the samples of selected oligosaccharide composition (FIG. 5). Meanwhile, each of these strains grew to high ODeoo in the presence of glucose. Further, growth of each Candida strain in the presence of the selected oligosaccharide composition was similar to the amount of growth in the presence of water (negative control, no carbon source).
- Example 5 Assessment of selected oligosaccharide compositions in fecal suspensions from hospitalized patients
- oligosaccharide composition comprised of a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III) as produced by a process similar to as described in Examples 7-9 to reduce pathogen growth in microbiome samples from fecal suspensions of thirteen hospitalized patients receiving antibiotic treatment from an Intensive Care Unit (ICU) facility was assessed.
- ICU Intensive Care Unit
- CRE Carbapenem-resistant Enterobacteriaceae
- VRE vancomycin-resistant Enterococcaceae
- the fecal suspensions from ICU patients and healthy subjects were mixed with either of the Carbapenem-resistant Enterobacteriaceae (CRE) culture or the vancomycin- resistant Enterococcaceae (VRE) culture such these pathogens comprised 8% (v/v) of the final mixture.
- CRE Carbapenem-resistant Enterobacteriaceae
- VRE vancomycin- resistant Enterococcaceae
- the fecal suspensions were then subjected to 16S metagenomic sequencing to determine the initial abundance (e.g ., relative abundance or absolute abundance) of pathogen and commensal bacteria.
- the cultures were then added to 96-well microplates with one of the following carbon sources (final concentration of 0.5% w/v) in each well: maltodextrin, fructooligosaccharide, a sample of the selected oligosaccharide composition, or water (negative control, i.e., no carbon source). These microplates were then incubated at 37 °C in the COY chamber for a total of 45 hours, with each experimental condition being tested in three replicates on each plate.
- carbon sources final concentration of 0.5% w/v
- the fecal suspensions from healthy subjects contained a greater diversity in commensal taxa compared to the fecal suspensions from the ICU patients (FIG. 12).
- the fecal suspensions of three of the thirteen ICU patients contained low levels of commensal bacteria (FIG. 12).
- the selected oligosaccharide composition reduced the abundance of Carbapenem- resistant Enterobacteriaceae (FIG. 7A) and vancomycin-resistant Enterococcaceae (FIG. 7B) in spiked fecal suspensions from ICU patients, as assessed by 16S sequencing.
- a reduction in the abundance (e.g., relative abundance or absolute abundance) of Carbapenem-resistant Enterobacteriaceae FIG. 7A
- vancomycin-resistant Enterococcaceae FIG. 7B
- Enterobacteriaceae was observed in the fecal suspensions from ten of the thirteen ICU patients. There was a smaller reduction in the abundance of Carbapenem-resistant Enterobacteriaceae in the three ICU patients that had few commensal bacteria, indicating that the abundance of commensal bacteria in the gut microbiome can influence the degree of pathogen reduction by the selected oligosaccharide composition. The abundance of each of these pathogens (carbapenem- resistant Enterobacteriaceae and vancomycin-resistant Enterococcaceae ) was greater in those spiked fecal suspensions that were incubated in the presence of FOS (a commercial fiber) or maltodextrin.
- FOS a commercial fiber
- the selected oligosaccharide composition comprised of a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III) as produced by a process similar to as described in Examples 7-9 is capable of reducing or preventing the growth of pathogens such as Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococcaceae (VRE) in a medically relevant model.
- CRE Carbapenem-resistant Enterobacteriaceae
- VRE vancomycin-resistant Enterococcaceae
- Example 6 Assessment of selected oligosaccharide compositions in fecal suspensions from hepatic encephalopathy (HE) patients
- pathogen infection could potentially be a precipitating factor of hepatic encephalopathy (HE) in certain patients.
- HE patients can be
- oligosaccharide composition comprised of a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III) as produced by a process similar to as described in Examples 7-9 to reduce pathogen abundance in patients with HE was testes.
- Microbiome samples from 44 HE patients were spiked with a single pathogen strain (CRE E. coli or VRE E. faecium) and then grown in the presence of selected oligosaccharide composition, FOS, or water (negative control, i.e., no carbon source).
- Fecal samples from HE patients and a healthy subject were collected and stored at -80 °.
- PBS phosphate buffered saline
- the cell pellet was resuspended in PBS and was then further diluted into a 1% solution in Mega Medium (MM). This medium was filter sterilized using a 0.2 pm filter and stored in an anaerobic chamber prior to use to allow any dissolved oxygen to dissipate.
- MM Mega Medium
- CRE Carbapenem-resistant Enterobacteriaceae
- VRE vancomycin-resistant Enterococcaceae
- the CRE strain or VRE strain was added to the fecal suspensions at 8% (v/v) of the total culture.
- Each batch of CRE strain and VRE strain that was normalized to an ODeoo of 0.01 was added to 12 of the fecal suspensions.
- the remaining 32 fecal suspensions were supplemented with cultures of these pathogens that were normalized to an O ⁇ ⁇ oo of 0.1.
- a sample of each pathogen-supplemented fecal suspension was then subjected to shallow shotgun sequencing (16S sequencing) to determine the initial abundance (e.g ., relative abundance or absolute abundance) of pathogen and commensal bacteria.
- the mixed culture was then added to 96-well microplates with one of the following carbon sources (final concentration of 0.5% w/v) in each well: selected oligosaccharide composition, FOS, or water. These microplates were then incubated at 37 °C in the COY chamber for a total of 45 hours. Each oligosaccharide composition was tested in three replicates on each plate, with each experimental condition being tested in three replicates on each plate.
- Enterococcaceae was accompanied by an increase in the abundance of commensal bacteria in the patient fecal suspensions (FIG. 13).
- FOG. 13 For example, there was a decrease in Firmicutes (such as VRE E. faecium and Clostridiales ) and an increase in commensal bacteria such as
- Bacteroidetes e.g., Bacteroidales
- VRE E. faecium a decrease in Proteobacteria (such as Enterobacteriales ) and an increase in commensal bacteria such as Bacteroidetes (e.g., Bacteroidales ) in fecal samples that had been spiked with CRE E. coli.
- composition comprised of a plurality of oligosaccharides selected from Formula (I), Formula (II), and Formula (III) as produced by a process similar to as described in Examples 7-9 is capable of reducing or preventing the growth of pathogens such as Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococcaceae (VRE) in a relevant model of hepatic encephalopathy (HE). Further, the selected oligosaccharide composition is capable of inducing the growth and increasing the abundance of commensal microbial species in this relevant model of hepatic encephalopathy (HE).
- CRE Carbapenem-resistant Enterobacteriaceae
- VRE vancomycin-resistant Enterococcaceae
- HE hepatic encephalopathy
- Example 7 Production of a selected oligosaccharide composition at 10 kg scale from dextrose monohydrate, galactose and mannose using a solid polymeric catalyst
- the temperature controller was set to 140 °C, and stirring (agitation) of the contents of the vessel at 30 RPM was initiated to promote uniform heat transfer and melting of the sugar solids, as the temperature of the syrup was brought to approximately 140 °C, under ambient (atmospheric) pressure gradually over a 2.5 hour period.
- the reaction mixture was maintained at temperature of approximately 140 °C for 1.5 hours (90 min), after which the heating was stopped and pre -heated water was gradually added to the reaction mixture at a rate of 60 mL/min until the temperature of the reactor contents decreased to 120 °C, then at a rate of 150 mL/min until the temperature of the reactor contents decreased to 110 °C, and then at a rate of 480 mL/min until the temperature of the reactor contents decreased below 100 °C and a total of 6 kg of water was added. An additional 1.75 kg of water was added to the reactor for further dilution.
- reaction mixture was drained from the vessel and the solids were removed by filtration, resulting in 15 kg of crude oligosaccharide composition product material as an aqueous solution (approximately 45 wt%).
- the oligosaccharide composition composition was purified by flowing it through a cationic exchange resin (Dowex® Monosphere® 88H) column, two columns of decolorizing polymer resin (Dowex® OptiPore® SD-2), and an anionic exchange resin (Dowex®
- oligosaccharide composition had a concentration of about 35 wt% and was then concentrated to a final concentration of about 75 wt% solids by vacuum rotary evaporation.
- Example 8 Production of oligosaccharide composition at 100 g scale from dextrose monohydrate, galactose and mannose using a solid polymeric catalyst
- reaction vessel 1 L three-neck round-bottom flask
- the reaction vessel was equipped with a heating mantle configured with an overhead stirrer.
- a probe thermocouple was disposed in the vessel through a septum, such that the probe tip sat above the stir blade and not in contact with the walls of the reaction vessel.
- the reaction vessel Prior to addition of catalyst, the reaction vessel was equipped with a condenser in a reflux position.
- the procedure also used an acidic oligomerization catalyst (Dowex Marathon C) (3-5% w/w) and de-ionized water for quenching.
- the catalyst was handled in wet form, e.g., at a nominal moisture content of 45 - 50 wt% H2O.
- the exact catalyst moisture content was generally determined on a per-experiment basis using, for example, using a moisture analyzing balance (e.g., Mettler-Toledo MJ-33).
- the reaction vessel was equipped in a distillation position to remove excess water throughout the course of the reaction.
- the temperature controller was set to a target temperature (100 to 160 °C), and stirring of the contents of the vessel was initiated to promote uniform heat transfer and melting of the sugar solids, as the temperature of the syrup was brought to the target temperature, under ambient (atmospheric) pressure.
- the reaction was then quenched by slowly adding approximately 60 mL of deionized (DI) water (room temperature) to dilute and cool the product mixture, to target a final concentration of 60-70 wt % dissolved solids. Generally, the water addition rate was performed to control the mixture viscosity as the oligosaccharide composition was cooled and diluted.
- DI deionized
- the oligosaccharide composition was cooled to approximately 60°C.
- the catalyst was then removed by vacuum filtration through a 100 micron mesh screen or fritted-glass filter, to obtain the final oligosaccharide composition at around 40 °Bx.
- Example 9 Production of the selected oligosaccharide composition at 10 kg scale from dextrose monohydrate, galactose and mannose using a citric acid catalyst
- the contents were agitated at approximately 30 RPM and the vessel temperature was gradually increased over a 2.5 hour period to about 139 °C at atmospheric pressure.
- the mixture was maintained at temperature for one and half hours.
- the heating was subsequently stopped and pre-heated water was gradually added to the reaction mixture at a rate of 60 mL/min until the temperature of the reactor contents decreased to 120 °C, then at 150 mL/min until the temperature of the reactor contents decreased to 110 °C, then at 480 mL/min until a total of 6 kg of water was added, and the temperature of the reactor contents decreased below 100 °C.
- An additional 1.75 kg water was added to the reactor for further dilution.
- the reaction mixture was drained from the vessel, resulting in 15 kg of crude oligosaccharide composition product as an aqueous solution (approximately 53 wt%).
- the sample was purified on a Biotage Isolera equipped with an ELSD detector using a 20/80 to 50/50 (v/v) deionized water/ ACN mobile phase gradient over 55 column volumes.
- Other flash chromatography systems such as the Teledyne ISCO Rf may also be used.
- the flow rate was set in accordance with the
- the mobile phase was set to 100% water until the remainder of the oligosaccharide composition eluted and was collected.
- the non-monomer containing fractions were concentrated by rotary evaporation to afford the de-monomerized product.
- samples may be collected into the Faeces Tube 54x28mm (Sarstedt AG, 25ml SC Feces Container w/Scoop), Globe Scientific Screw Cap Container with Spoon (Fisher Scientific) or the OMNIgene-GUT collection system (DNA Genotek, Inc.), which stabilizes microbial DNA for downstream nucleic acid extraction and analysis. Aliquots of fecal samples were stored at -20 °C and -80 °C following standard protocols known to one skilled in the art.
- Example 12 Determining the level of pathogens in subjects.
- fecal samples or rectal swabs are collected by a suitable method.
- Sample material is cultured on, e.g., i) Cycloserine-Cefoxitin Fructose Agar (available for instance from Anaerobe Systems) cultured anaerobically to selectively and differentially grow Clostridium difficile ⁇ , ii) Eosin Methylene Blue Agar (available for instance from Teknova) cultured aerobically to titer Escherichia coli and other Gram- negative enteric bacteria, most of which are opportunistic pathogens; hi) Bile Esculin Agar (BD) cultured aerobically to titer Enterococcus species; iv) phenyl-ethylalcohol blood agar (Becton Dickinson), or Colistin-Nalidixic Acid (CNA) blood agar (for instance, from Hardy Diagnostics) cultured
- Cycloserine-Cefoxitin Fructose Agar available for
- coli and other Gram-negative enteric bacteria Additional antibiotics can be used as appropriate to select drug-resistant subsets of these bacteria, for instance vancomycin (e.g., for vancomycin-resistant Enterococcus), cefoxitin (e.g., for extended spectrum beta lactamases or Enterococcus), ciprofloxacin (e.g., for fluoroquinolone resistance), ampicihin (e.g., for ampicihin resistant bacteria), and ceftazidime (e.g., for cephalosporin resistant bacteria).
- chromogenic substrates may be added to facilitate the differentiation of pathogens from commensal strains, such as with ChromID plates (Biomerieux) or ChromAgar (Becton Dickinson). Plates are incubated at 35-37 °C under aerobic, anaerobic or microaerophilic conditions as appropriate for the pathogen. After 16-48 hours, colonies are counted and used to back-calculate the concentration of viable cells in the original sample.
- the quantity of a pathogen is measured by quantitative PCR.
- primers specific to one or more of the pathogens (including bacterial pathogens, viral pathogens and pathogenic protozoa) described herein are designed and used in a real-time quantitative PCR (for instance, using a PCR reaction to which a double- stranded- specific fluorescent dye such as Sybr Green, or a sequence-specific Taqman probe (Applied to the PCR).
- Genomic DNA is extracted from each sample using the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) according to the manufacturer's instructions or by bead beating, e.g., performed for 2 minutes using a BioSpec Mini-Beadbeater-96 (BioSpec Products, Bartlesville, Okla.).
- the genomic DNA is isolated using the Mo Bio Powersoil® DNA Isolation Kit (Mo Bio
- analyte-specific reagents are available for many of the pathogens, for instance from Luminex, Inc (www.luminexcorp.com).
- universal ribosomal primers are used to quantitatively measure the total copy number of genomes from pathogens to determine relative instead of absolute abundance of pathogens. If desired, the ratio of pathogen to total copies is calculated.
- the colony counts can be normalized (e.g., a ratio is calculated) to the total DNA content of the sample, or to the quantitative measure, e.g., determined by a qPCR using universal ribosomal primers. [00311] Alternatively, the colony count of a pathogen, or all pathogens combined, is compared to the total colony count of the sample cultured under non-selective conditions.
- Samples are cultured on rich media or agar such as Brucella Blood Agar (Anaerobe Systems), Brain Heart Infusion Broth (Teknova), or chocolate agar (Anaerobe Systems).
- agar such as Brucella Blood Agar (Anaerobe Systems), Brain Heart Infusion Broth (Teknova), or chocolate agar (Anaerobe Systems).
- the maximum number of colonies on these media, grown anaerobically are used as the denominator in a normalized ratio of pathogens to commensals as a relative measure.
- the amount of pathogen may also be estimated by 16s ribosomal DNA profiling.
- Genomic DNA is extracted from subject samples (e.g . fecal samples, rectal swabs, skin or mucosal swabs, biopsies or tissue samples), and variable region 4 of the 16S rRNA gene is amplified and sequenced (Earth Microbiome Project protocol www.earthmicrobiome.org/emp- standard-protocols/16s/ and Caporaso JG et al. 2012. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J.).
- Operational Taxonomic Units are generated by aligning 16S rRNA sequences at 97% identity, or lower as appropriate. Then the OTUs potentially representing pathogenic species are assessed by aligning the OTUs to known taxonomic structures such as those maintained by NCBI
- Reagents used were methanol, acetic acid, sodium borodeuteride, sodium carbonate, dichioromethane, isopropanol, trifluoro acetic acid (TFA), and acetic anhydride.
- Equipment included a heating block, drying apparatus, gas chromatograph equipped for capillary columns and with a RID/MSD detector, and a 30 meter RTX®-2330 (RESTEK). All derivation procedures were done in a hood.
- A. Standard preparation [00315] 1 mg/mL solutions of the following standard analytes were prepared: arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose, and inositol. The standard was prepared by mixing 50 pL of each of arabinose, xylose, fucose, glucose, mannose, and galactose with 20 pL of inositol in a vial. The standard was subsequently lyophilized.
- Each sample was prepared by mixing 100-500 pg of of the selected
- oligosaccharide composition (as weighed on an analytical balance) with 20 pg (20 pL) of inositol in a vial.
- the GC temperature program SP2330 was utilized for GC-MS analysis.
- the initial temperature was 80 °C and the initial time was 2.0 minutes.
- the first ramp was at a rate of 30 °C/min with a final temperature of 170 °C and a final time of 0.0 minutes.
- the second ramp was at a rate of 4 °C/min with a final temperature of 240 °C and a final time of 20.0 minutes.
- Each sample was prepared by mixing 600-1000 pg of the selected oligosaccharide composition (as weighed on an analytical balance) with 200 pL DMSO. The sample was stirred overnight until the oligosaccharide composition dissolved.
- the GC temperature program SP2330 was utilized for GC-MS analysis.
- the initial temperature was 80 °C and the initial time was 2.0 minutes.
- the first ramp was at a rate of 30 °C/min with a final temperature of 170 °C and a final time of 0.0 minutes.
- the second ramp was at a rate of 4 °C/min with a final temperature of 240 °C and a final time of 20.0 minutes.
- oligosaccharide composition as produced by the processes in Examples 7 and 9, were performed using a Varian Unity Inova NMR, according to the protocol described below.
- the sample was analyzed in a Varian Unity Inova operating at 499.83 MHz (125.69 MHz 13C) equipped with a XDB broadband probe with Z-axis gradient, tuned to 13C, and operating at 25 °C.
- the sample was subjected to a heteroatomic single quantum coherence (HSQC), echo-antiecho, with gradient selection HSQCETGP pulse sequence experiment using the following acquisition and processing parameters in Table 3:
- HSQC heteroatomic single quantum coherence
- Echo-antiecho with gradient selection HSQCETGP pulse sequence experiment using the following acquisition and processing parameters in Table 3:
- a representative HSQC NMR spectra of the selected oligosaccharide composition is provided in FIG. 11.
- the mobile phase (0.1 M NaNO,) was prepared by weighing 17 g of NaN0 3 (ACS grade reagent) and dissolving in 2000 mL of deionized (DI) water (from MiliQ water filter). The solution was filtered through a 0.2 pm filter.
- DI deionized
- a polymer solution mixture #1 was prepared by weighing 10 mg of each standard of glucose, maltose, maltooctaose and Mp 9600 into an HPLC vial, adding 1.0 mL of diluent and mixing well.
- a polymer solution mixture #1 was prepared by weighing 10 mg of each standard of maltotetraose, Mp 6100 and Mp 21100 into an HPLC vial, adding 1.0 mL of diluent and mixing well.
- Sample A was prepared in duplicate. Approximately 300 mg of oligosaccharide sample was weighed into a 20 mL scintillation vial and 10 mL of DI water was added. The solution was mixed and filtered through a PES syringe filter with a 0.2 pm polyethersulfone membrane.
- Sample B was prepared in duplicate. Approximately 220 mg of oligosaccharide sample was weighed into a 20 mL scintillation vial and 10 mL of Di-water was added. The solution was mixed and filtered a PES syringe filter with a 0.2 pm polyethersulfone membrane.
- the flow rate was set to 0.7 mL/min at least 2 hours before running samples with the column temperature set to 65 °C and the RI detector temperature set to 50 °C with the RI detector purge turned on.
- a blank sample consisting of DI water was run. Samples of standard mixtures #1 and #2 were run. Sample A was run. Sample B was run.
- oligosaccharide composition produced using the process in Example 7 (Marathon C catalyst) were analyzed using the above SEC methods.
- the batches of oligosaccharide composition comprised oligosaccharides with an average MWw of 2074 g/mol (ranging from 1905-2286 g/mol), an average MWn of 1097 g/mol (ranging from 1033-1184 g/mol), and an average PDI of 1.9 (ranging from 1.84-1.97).
- Assayed batches comprised a DP2+ of 91.1% (DP2+ ranging from 86.3-95.9%) and about 8.9% monomer on average (ranging from 4.1-13.8% monomer).
- Assayed batches had an average degree of polymerization (DP) of 12.7 (ranging from 11.6-14.0).
- oligosaccharide composition produced using the process in Example 9 (citric acid catalyst) were analyzed using the above SEC methods.
- the batches of oligosaccharide composition comprised oligosaccharides with an average MWw of 1998 g/mol (ranging from 1863-2268 g/mol), an average MWn of 1030 g/mol (ranging from 984-1106.00 g/mol), and an average PDI of 1.94 (ranging from 1.88-2.05).
- Assayed batches comprised a DP2+ of 87.3% (DP2+ ranging from 83.6-91.0%) and about 12.7% monomer on average (ranging from 9.0-16.4% monomer).
- Assayed batches had an average degree of polymerization (DP) of 12.2 (ranging from 11.4-13.9).
- the mobile phase (25 mM H2SO4 in water) was prepared by filling a bottle with 2000 mL Di-water and slowly adding 2.7 mL of H2SO4. The solution was filtered through a 0.2 pm filter.
- a standard solution was prepared by measuring 50 + 2 mg of reference standard into a 100-mL volumetric flask, adding mobile phase to 100-mL mark and mixing well..
- sample A A sample of a selected oligosaccharide composition (Sample A) was prepared in duplicate. Approximately 1000 mg of oligosaccharide sample was weighed into a 10 mL volumetric flask and mobile phase was added up to the mark. The solution was mixed and filtered through a PES syringe filter with a 0.2 pm polyethersulfone membrane.
- sample B A sample of a selected oligosaccharide composition (Sample B) was prepared in duplicate. Approximately 700 mg of oligosaccharide sample was weighed into a 10 mL volumetric flask and mobile phase was added up to the mark. The solution was mixed and filtered through a PES syringe filter with a 0.2 pm polyethersulfone membrane.
- the flow rate was set to 0.65 mL/min at least 2 hours before running samples with the column temperature set to 50 °C and the RI detector temperature set to 50 °C with the RI detector purge turned on. [00359] Before running samples wherein the injection volume for all samples was 50 pL and run time was 40 minutes, the detector purge was turned off and the pump was run at 0.65 mL/min until an acceptable baseline was obtained.
- a blank sample consisting of DI water was run.
- the standard, sample A, and sample B were each independently run.
- oligosaccharide composition comprised 0.35% w/w ( ⁇ 0.05%) levoglucosan, 0.03% w/w ( ⁇ 0.01%) lactic acid, and 0.06% w/w ( ⁇ 0.01%) formic acid.
- oligosaccharide composition comprised 0.28-0.43% w/w levoglucosan, 0.00-0.03% w/w lactic acid, and 0.05-0.07% w/w formic acid.
- the selected oligosaccharide composition comprised 0.47% w/w ( ⁇ 0.02%) levoglucosan (23 batches of the selected oligosaccharide), 0.01% w/w lactic acid (11 batches of the selected oligosaccharide), 0.02% w/w formic acid (12 batches of the selected oligosaccharide), and 0.02% w/w citric acid (23 batches of the selected oligosaccharide).
- Samples of the selected oligosaccharide composition comprised 0.43-0.51% w/w levoglucosan, 0.01-0.02% w/w lactic acid, 0.00-0.03% w/w formic acid, and 0.00-0.03% w/w citric acid.
- the mobile phase (0.1 M NaNOs) was prepared by weighing 42.5 g of NaNC>3 (ACS grade reagent) and dissolving in 5000 mL of deionized (DI) water (from MiliQ water filter). The solution was filtered through a 0.2 pm filter.
- DI deionized
- Samples of the selected oligosaccharide composition were prepared as lOmg/mL concentrated samples or dilute aqueous samples to 2.5-3.5 Brix.
- the flow rate was set to 1.0 mL/min at least 2 hours before running samples with the column temperature set to 70 °C and the RI detector temperature set to 40 °C with the RI detector purge turned on.
- a blank sample consisting of DI water, individual standards, and sample were independently run.
- oligosaccharide composition comprised 5.24% ( ⁇ 0.35%) monomer (DPI), 7.52% ( ⁇ 0.44%) disaccharide (DP2), and 87.25% ( ⁇ 0.78%) oligomers having at least three linked monomer units (DP3+).
- compositions, as produced by the process in Example 7 were measured according to the methods of AO AC 2011.25 (AO AC International, AO AC Official Method 2011.25).
- the average amount of total dietary fiber was 87.44% (on dry basis) across 10 batches (ranging from 84.9-90.5%).
- the percent Dextrose Equivalent (DE) (dry basis) of these oligosaccharide batches was also measured according to the Food Chemicals Codex (FCC).
- the average amount of dextrose equivalent (on dry basis) was 16.60% across two batches (one at 15.10% DE and the other at 18.10% DE).
- the percent Dextrose Equivalent (DE) (dry basis) of these oligosaccharide batches was also measured according to the Food Chemicals Codex (FCC).
- the average amount of total dietary fiber was 64.14% (on dry basis) across fourteen batches (ranging from 47.10-73.10%).
- the average amount of dextrose equivalent (on dry basis) was 20.60% across two batches (one at 18.60% DE and the other at 22.60% DE).
- Example 19 Clinical trial to assess ability of the selected oligosaccharide composition to reduce the relative or absolute abundance of pathogens
- the study is a randomized, controlled, multi-site, open label clinical study to assess the selected oligosaccharide composition on safety as well as the proportion of subjects with a clinically significant reduction from baseline in abundance of taxa of interest
- a reasonable number of patients are randomized 3:2 to a treatment group (i.e ., treatment with the selected oligosaccharide composition) or to an observational control group.
- Patients are at least 18 years of age and must have a positive fecal sample for VRE, ESBLE, or CRE, based on a 16S metagenomic analysis of a fecal sample of the patient.
- Patients in the treatment group are administered (e.g ., orally self-administered) a dosage of the oligosaccharide composition (e.g., once or twice daily) for the length of the study (e.g., 28 days).
- Patients in the treatment group and the observational group undergo regular physical checkups throughout the length of the study, which may include observation of vital signs, fecal stool sample collection for microbiologic and/or 16S metagenomic testing, recordation of stool frequency and BSS evaluation, and/or recordation of adverse effects.
- the endpoints of the study may include the number of patients experiencing study product-related treatment emergent adverse events (TEAEs); serious adverse events (SAEs), change from baseline in vital signs, electrocardiograms (ECGs), physical examinations, safety laboratory analyses; and change from baseline in stool frequency, Bristol Stool Score (BSS); and the proportion of subjects with a reduction from baseline (e.g., a >30% reduction) at the end of the study (e.g., on Day 28) in the abundance (e.g., relative abundance or absolute abundance) of taxa of interest (Enterobacteriaceae, Enterococcus, and C. difficile, combined and/or
- Additional endpoints may include (i) proportion of subjects with a reduction in level of MDR organisms (cfu/g feces by culture) including VRE, ESBLE, and CRE, combined and/or individually; (ii) alpha diversity (Shannon index) by nucleic acid sequencing over the intake phase versus baseline; (iii) abundance of bacteria as measured by nucleic acid sequencing over the intake phase versus baseline; (iv) change in level of stool inflammatory biomarkers (e.g., lipocalin) over the intake phase versus baseline; (v) Change in blood inflammatory markers (e.g., IFN-g, IL-Ib, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-a) over the intake phase versus baseline; (vi) change in microbial metabolite concentration (for example, p-cresol sulfate, trimethylamine oxide) in stool, urine or blood samples over the
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MX2021013612A MX2021013612A (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide compositions and methods of use. |
KR1020217039532A KR20220007090A (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide compositions and methods of use thereof |
CA3138903A CA3138903A1 (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide compositions and methods of use thereof |
EP20802108.9A EP3965776A4 (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide compositions and methods of use |
US17/609,234 US20220233560A1 (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide compositions and methods of use |
JP2021565821A JP2022532066A (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide composition and its usage |
SG11202112314XA SG11202112314XA (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide compositions and methods of use |
CN202080048988.2A CN114126625A (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide compositions and methods of use thereof |
BR112021022293A BR112021022293A2 (en) | 2019-05-08 | 2020-05-08 | Oligosaccharide compositions and methods of using them |
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US20100284972A1 (en) * | 2009-05-07 | 2010-11-11 | Tate & Lyle Ingredients France SAS | Compositions and methods for making alpha-(1,2)-branched alpha-(1,6) oligodextrans |
US20160007642A1 (en) * | 2014-07-09 | 2016-01-14 | Midori Usa, Inc. | Oligosaccharide compositions and methods for producing thereof |
WO2018106845A1 (en) * | 2016-12-06 | 2018-06-14 | Kaleido Biosciences, Inc. | Glycan polymers and related methods thereof |
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EP4142742A4 (en) * | 2020-04-30 | 2024-05-29 | DSM Nutritional Products, LLC | Oligosaccharide compositions and methods of use thereof for treating viral infections |
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US20100284972A1 (en) * | 2009-05-07 | 2010-11-11 | Tate & Lyle Ingredients France SAS | Compositions and methods for making alpha-(1,2)-branched alpha-(1,6) oligodextrans |
US20160007642A1 (en) * | 2014-07-09 | 2016-01-14 | Midori Usa, Inc. | Oligosaccharide compositions and methods for producing thereof |
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