WO2015074054A1 - Amélioration de la santé microbienne dans l'intestin de mammifères - Google Patents

Amélioration de la santé microbienne dans l'intestin de mammifères Download PDF

Info

Publication number
WO2015074054A1
WO2015074054A1 PCT/US2014/066173 US2014066173W WO2015074054A1 WO 2015074054 A1 WO2015074054 A1 WO 2015074054A1 US 2014066173 W US2014066173 W US 2014066173W WO 2015074054 A1 WO2015074054 A1 WO 2015074054A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
library
genes
gut
fold
Prior art date
Application number
PCT/US2014/066173
Other languages
English (en)
Inventor
Harris WANG
Original Assignee
The Trustees Of Columbia University In The City Of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of Columbia University In The City Of New York filed Critical The Trustees Of Columbia University In The City Of New York
Publication of WO2015074054A1 publication Critical patent/WO2015074054A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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 invention relates to methods and compositions for improving the health of a mammal by modulating the population of bacteria in the mammalian gastrointestinal (GI) tract.
  • the present invention relates to promoting the growth of beneficial gut bacteria.
  • the human body is colonized by trillions of microbes, with the highest concentration along the gastrointestinal (GI) tract.
  • GI gastrointestinal
  • the GI tract is a hostile environment for poorly adapted microbes. Nonetheless, diverse groups of microbes have evolved to prosper in the GI tract, in the setting of intense interspecies competition, physical and chemical stressors, and the host immune system (Ley et al, 2006; Dethlefsen et al, 2007).
  • microorganisms also support the normal homeostatic functions of the host by helping to extract nutrients, stimulate the immune system, and provide protection against colonization by pathogens (Ley et al, 2006; Gill et al, 2006; Backhed et al, 2005; Stappenbeck et al, 2002; Hooper, 2004). Only the most gut-adapted microbes are able to flourish, and thus the genes and pathways that they carry are enriched over time. It is now increasingly clear that the repertoires of genes encompassed in the microbiota, and not just simply the composition of the microbes themselves, drive the maintenance of microbiome health.
  • transposon mutagenesis may disrupt the expression of bystander genes that are near the relevant locus, thus causing confounding phenotypic effects.
  • the present invention provides for a method of identifying genes that enhance bacterial fitness in the gastrointestinal (GI) tract.
  • the method may comprise the steps of: (a) constructing a genomic or metagenomic library comprising fragments of the genome of at least one donor bacterium; (b) introducing the library to recipient bacteria; (c) introducing the recipient bacteria carrying the library into the GI tract of a mammal; (d) taking stool samples of the mammal at different time points, e.g., Ti through T n (n is an integer); (e) isolating DNA (e.g., plasmids and/or genomic DNAs) from the stool samples of step (d); and (f) sequencing the DNA of step (e).
  • the mammal may be a mouse, e.g., a mouse that is germ-free before introduction of the recipient bacteria.
  • the mouse may be healthy, or have inflamed GI tract.
  • a gene is identified to enhance bacterial fitness in the
  • time points may range from about 0 day to about 30 days after introduction of the recipient bacteria carrying the library into the GI tract of a mammal (e.g., step (c) of the method).
  • the DNA may be fragmented by sonication or digestion by at least one restriction enzyme.
  • the fragments of the donor bacterial genome can be under the control of a constitutive or inducible promoter in the recipient bacteria.
  • the DNA may be sequenced by deep sequencing, or Sanger sequencing.
  • the donor bacterium may belong to genera Bacteroides or Clostridium.
  • the donor bacterium can be Bacteroides thetaiotaomicron or Clostridium butyricum.
  • the donor bacterium can also be from a natural gut microbiota.
  • the natural gut microbiota may be from, e.g., the gut of a healthy mammal, or the gut of a mammal with inflamed GI tract.
  • the mammal with inflamed GI tract may have an inflammatory bowel disease (IBD, such as ulcerative colitis or Crohn's disease) or irritable bowel syndrome (IBS).
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • the healthy mammal, or the mammal with inflamed GI tract may be a human subject.
  • the recipient bacteria may belong to phyla Bacteroidetes, Firmicutes, Proteobacteria, or Actinobacteria.
  • the recipient bacteria may belong to genera Bacteroides, Clostridium,
  • Escherichia Bacillus, Lactobacillus, or Bifidobacterium.
  • recipient bacteria include Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Lactobacillus plantarum (L. plantarum), Lactobacillus reuteri (L. reuteri), Lactobacillus rhamnosus (L. rhamnosus), Bifidobacterium longum (B.
  • a probiotic composition comprising recombinant bacteria comprising a gene encoding a protein such as a glycoside hydrolase, a galactokinase or a glucose/galactose transporter.
  • the gene may be heterologous.
  • the gene may be endogenous which has been engineered to overexpress the encoded protein.
  • the gene may be under the control of a constitutive or inducible promoter.
  • the protein can be wild-type or a mutant.
  • the gene may be wild-type or mutated.
  • the gene may be truncated at the 5 ' end by from about 1 base pair (bp) to about 50 bp from the start codon.
  • the protein is Bacteroides thetaiotaomicron glycoside hydrolase whose encoding gene may be wild- type or may be truncated at the 5 ' end by about 4 bp from the start codon.
  • the recombinant bacteria may be capable of metabolizing sucrose, galactose, or both sucrose and galactose.
  • the gene may be integrated into the bacterial chromosome or may be episomal.
  • the probiotic composition may be a food composition, a beverage composition, a pharmaceutical composition, or a feedstuff composition.
  • the probiotic composition can be a dairy product.
  • Figure 8 Growth characterization of clones with genomic single nucleotide variants (SNVs). Growth curves over 42 hours at 37°C in M9 with 0.2% galactose and carbenicillin of (A) mouse- isolated clones from Day 28 and (B) cloned BT 0369, BT 0370, BT 0371, BT 0372, and BT 0370-BT 0372. The mean of four replicates is plotted in filled circles; error bars represent the standard deviation. (C) Endpoint optical density after 96 hours of growth. Two mouse- isolated strains with the BT 0370 insert were compared to isogenic strains transformed with those plasmids (4.0 or 4.3 kb insert). The strain with the galR SNV is shown in circles filled with dots. Lines represent the mean.
  • SNVs genomic single nucleotide variants
  • the present invention provides for a powerful and systematic method of identifying genes that enhance bacterial fitness in the mammalian gastrointestinal (GI) tract.
  • the identified genes can be used to generate recombinant bacteria to be included in a probiotic composition.
  • the composition may improve the health of a mammal, such as a human, or may be used to combat a gastrointestinal disorder.
  • the method of identifying a bacterial fitness gene may contain the following steps: (a) constructing a genomic or metagenomic library comprising fragments of the genome of at least one donor bacterium; (b) introducing the library to recipient bacteria; (c) introducing the recipient bacteria carrying the library into the GI tract of a mammal (to initiate the in vivo selection process); (d) taking stool samples of the mammal at different time points, e.g., Ti through T n ; (e) isolating DNA (e.g., plasmids and/or genomic DNAs) from the stool samples of step (d); and (f) sequencing the DNA of step (e).
  • the gene When the abundance of a gene or a DNA segment during the in vivo selection (e.g., at a time point, or multiple time points, of step (d)) is greater than its abundance before the in vivo selection (e.g., its abundance in the original library), the gene may be identified to be able to enhance bacterial fitness in the mammalian GI tract.
  • the abundance of a gene during the in vivo selection may be at least about 1.2 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.8 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least
  • a genomic library composed of 3-5 kb uniformly fragmented genomic DNA from donor Bacteroides thetaiotaomicron is first cloned and expressed in E. coli. Bacteroides thetaiotaomicron is an abundant Gram-negative commensal microbe with known important metabolic capabilities in the gut. A cell library containing ⁇ 10 5 unique clones will provide on average >50x coverage of the donor genome. This Gram-negative genomic library is then introduced by gavage to healthy, germ-free mice for passaging over a period of a month. Gnotobiotic murine experiments are conducted. In vivo selection of beneficial genes that improve gut colonization leads to a measureable enrichment of their relative abundance across the microbial population. The selected library from fecal pellets collected daily is extracted and deep-sequenced to identify and quantify genes that are enriched during the in vivo selection.
  • recombinant bacteria e.g., in a probiotic composition
  • genes that can enhance bacterial fitness in the mammalian GI tract.
  • the genes may encode a glycoside hydrolase, a galactokinase, a glucose/galactose transporter, or any other proteins involved in carbohydrate (e.g., sucrose, galactose, etc.) metabolism and/or transport.
  • the gene may be under the control of a constitutive or an inducible promoter.
  • the donor bacterium may be any suitable bacterium.
  • the donor bacterium may be a bacterium that exists naturally in the GI tract.
  • the donor bacterium is a commensal bacterium.
  • the donor bacterium is a probiotic bacterium.
  • the donor bacteria may be a mixture of bacteria, from, e.g., a metagenomic source.
  • the donor bacterium may be from a natural gut microbiota of the gut of a healthy mammal, or from the gut of an unhealthy mammal (e.g., with a gastrointestinal disorder).
  • the unhealthy mammal may have a condition influenced by the GI tract microbiota.
  • the unhealthy mammal may have inflamed GI tract, such as an inflammatory bowel disease (IBD), including ulcerative colitis or Crohn's disease.
  • IBD inflammatory bowel disease
  • the unhealthy mammal may have collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, indeterminate colitis, irritable bowel syndrome (IBS, or spastic colon), mucous colitis, microscopic colitis, antibiotic-associated colitis, constipation, diverticulosis, polyposis coli or colonic polyps.
  • the donor bacterium may be Gram-positive or Gram-negative.
  • the donor bacterium may be from any of the following phyla: Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, etc.
  • the donor bacterium may be from any of the following genera: Bacteroides, Clostridium, Bifidobacterium, Lactobacillales (lactic acid bacteria or LAB), Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Klebsiella, Escherichia, Enterobacter, Peptostreptococcus, Peptococcus, Bacillus, Propionibacteria, Ruminococcus, Gemmiger, Desulfomonas, Salmonella, etc.
  • the donor bacterium may be Bacteroides thetaiotaomicron, Clostridium butyricum, Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, Enterococcus faecalis, Escherichia coli, Bifidobacterium bifidum, Staphylococcus aureus, Clostridium perfringens, Proteus mirabilis, Clostridium tetani, Clostridium septicum, Pseudomonas aeruginosa,
  • Salmonella enteritidis Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus,
  • Lactobacillus casei Lactobacillus salivarius, Lactococcus lactis, Lactobacillus reuteri,
  • Lactobacillus rhamnosus Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, and Enterococcus faecium.
  • U.S. Patent No. 8,591,880 U.S. Patent No. 8,591,880.
  • Non limiting examples of the donor bacterium also include: Bacillus coagulans, B. lentus, Bacillus licheniformis, B. mesentericus, B. pumilus, Bacillus subtilis, B. natto, Bacteroides amylophilus, Bac. capillosus, Bac. ruminocola, Bac. suis, Bifidobacterium adolescentis, B.
  • the DNA library may be a genomic or metagenomic library.
  • a genomic library is a collection of the genomic DNA from a single organism.
  • a metagenomic library is a collection of the genomic DNAs of a mixture of organisms, such as a mixture of microbes (e.g., a mammalian GI tract microbiota).
  • DNA may be isolated from bacteria by any method well known in the art.
  • DNA extraction may include two or more of the following steps: cell lysis, addition of a detergent or surfactant, addition of protease, addition of RNase, alcohol precipitation (e.g., ethanol precipitation, or isopropanol precipitation), salt precipitation, organic extraction (e.g., phenol-chloroform extraction), solid phase extraction, silica gel membrane extraction, CsCl gradient purification.
  • Various commercial kits e.g., kits of Qiagen, Valencia, CA can be used to extract DNA.
  • Genomic or metagenomic DNA is fragmented prior to library construction.
  • DNA may be fragmented by methods including, but not limited to, sonication, needle shearing, nebulization, acoustic shearing, point-sink shearing and passage through a pressure cell.
  • DNA may also be fragmented by digestion with one or more restriction enzymes.
  • DNA fragments may or may not be separated by gel
  • the length of the DNA fragments to be used for library construction may range from about 50 base pairs to about 10 kb, from about 50 base pairs to about 2.5 kb, from about 200 base pairs to about 1 kb, from about 0.5 kb to about 10 kb, from about 10 kb to about 50 kb, from about 30 kb to about 40 kb, from about 50 kb to about 100 kb, from about 100 kb to about 200 kb, from about 1 kb to about 8 kb, from about 2 kb to about 6 kb, from about 2 kb to about 5 kb, from about 2 kb to about 4 kb, from about 2 kb to about 3 kb, from about 3 kb to about 5 kb, or longer.
  • DNA fragments are then inserted into vectors using, e.g., DNA ligase.
  • Each vector may contain a different insert of DNA.
  • fragmented DNA is end-repaired before being ligated to a vector.
  • Fragmented DNAs may be ligated to adapters before being inserted into vectors.
  • cloning vector and strategy largely reflects the desired library structure (i.e., insert size and number of clones) and target activities sought.
  • vectors include plasmids, phage lambda, cosmids, fosmids, bacteriophage PI, PI artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs).
  • Exemplary vectors include GMVlc, pCClFOS, pWE15, pFOSl, pIndigoBAC536, pWEB, pSMART, pUC18, pBR322 and its derivatives, Lambda ZAP, pHOS2, pUC and its derivatives, pBluescript and its derivatives, pTOPO-XL, and pCF430.
  • the DNA fragment may be under the control of a constitutive, inducible, and/or tissue-specific promoter, or promoters useful under the appropriate conditions to direct expression of the DNA fragment or a gene.
  • operatively positioned and “operatively linked” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence, a DNA fragment, or a gene, to control transcriptional initiation and/or expression of that sequence, DNA fragment or gene.
  • a constitutive promoter is an unregulated promoter that allows for continual transcription of the gene under the promoter's control.
  • constitutive promoters include constitutive E. coli ⁇ 70 promoters, constitutive E. coli o s promoters, constitutive E. coli
  • constitutive E. coli ⁇ promoters constitutive E. coli ⁇ promoters, constitutive E. coli ⁇ promoters, constitutive B. subtilis ⁇ ⁇ promoters, constitutive B. subtilis ⁇ ⁇ promoters, T7 promoters, and SP6 promoters.
  • a list of constitutive bacterial promoters may be found in the database of Registry of Standard Biological Parts. They are active in all circumstances in the cell.
  • the constitutive promoter is pL.
  • the DNA fragment or a gene may be under the control of an inducible promoter.
  • the transcriptional activity of these promoters is induced by either chemical or physical factors.
  • Chemically-regulated inducible promoters may include promoters whose transcriptional activity is regulated by the presence or absence of oxygen, a metabolite, alcohol, tetracycline, steroids, metal and other compounds. Physically-regulated inducible promoters, including promoters whose transcriptional activity is regulated by the presence or absence of heat, low or high temperatures, acid, base, or light. In one embodiment, the inducible promoter is pH-sensitive (pH inducible).
  • the inducer for the inducible promoter may be located in the biological tissue or environmental medium to which the composition is administered or targeted, or is to be administered or targeted.
  • the inducer for the inducible promoter may be located in the mammalian GI tract.
  • the pH level of a particular biological tissue can affect the inducibility of the pH inducible promoter. See, for example, Boron, et al, Medical Physiology: A Cellular and
  • acid inducible promoters include, but are not limited to, P170, PI , P3, baiAl , baiA3, lipF promoter, FiF 0 -ATPase promoter, gadC, gad D, glutamate decarboxylase promoter, etc. See, for example, Cotter and Hill, Microbiol, and Mol. Biol. Rev. vol. 67, no. 3, pp. 429-453 (2003); Hagenbeek, et al, Plant Phys., vol. 123, pp. 1553-1560 (2000); Madsen, et al, Abstract, Mol. Microbiol, vol. 56, no. 3, pp. 735-746 (2005); U.S. Pat. No.
  • promoters induced by a change in temperature include P2, P7, and PhS. See, for example, Taylor, et al, Cell, Abstract, vol. 38, no. 2, pp. 371 -381 (1984); U.S. Pat. No. 6,852,51 1 , Wang, et al, Biochem. and Biophys. Res. Commun. Abstract, vol. 358, no. 4, pp. 1 148-1 153 (2007), U.S. Pat. No. 7,462,708, each of which is incorporated herein by reference.
  • the acid inducible promoter is inducible at a pH of about 0.0, about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or any value therebetween or less.
  • the base inducible promoter is inducible at a pH of about 7.1 , about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 1 1.0, about 1 1.5, about 12.0, about 12.5, about 13.0, about 13.5, about 14.0, or any value therebetween or greater.
  • inducers that can induce the activity of the inducible promoters also include, but are not limited to, radiation, temperature change, alcohol, antibiotic, steroid, metal, salicylic acid, ethylene, benzothiadiazole, or other compound.
  • the at least one inducer includes at least one of arabinose, lactose, maltose, sucrose, glucose, xylose, galactose, rhamnose, fructose, melibiose, starch, inunlin, lipopolysaccharide, arsenic, cadmium, chromium,
  • inducers include, but are not limited to, at least a portion of one of an organic or inorganic small molecule, clathrate or caged compound, protocell, coacervate, microsphere, Janus particle, proteinoid, laminate, helical rod, liposome, macroscopic tube, niosome, sphingosome, vesicular tube, vesicle, unilamellar vesicle, multilamellar vesicle, multivesicular vesicle, lipid layer, lipid bilayer, micelle, organelle, nucleic acid, peptide, polypeptide, protein, glycopeptide, glycolipid, lipoprotein, lipopolysaccharide, sphingolipid, glycosphingolipid, glycoprotein, peptidoglycan, lipid, carbohydrate, metalloprotein, proteoglycan, chromosome, nucleus, acid, buffer, protic solvent, aprotic solvent, nitric oxide, vitamin
  • the recombinant DNAs are then introduced into recipient bacteria.
  • the recipient bacteria are transformed by any known method, including, but not limited to, electroporation, heat shock, biolistic transformation, and sonic transformation.
  • the vector is a viral vector
  • the DNA is introduced into recipient bacteria through transduction.
  • the recipient bacterium may be any suitable bacterium.
  • the recipient bacterium has low in vivo fitness or may be non-adapted in vivo.
  • the recipient bacterium has lower in vivo fitness than the donor bacterium, allowing for strong selection signals for clones harboring functional donor genes.
  • the recipient bacterium is a commensal bacterium.
  • the recipient bacterium is a probiotic bacterium.
  • the recipient bacterium may be Gram-positive or Gram-negative.
  • the recipient bacterium may be from any of the following phyla: Bacteroidetes,
  • the recipient bacterium may be from any of the following genera: Bacteroides,
  • Lactobacillales lactic acid bacteria or LAB
  • Lactobacillus Lactococcus
  • Enterococcus Streptococcus
  • Klebsiella Escherichia
  • Enterobacter Clostridium, Bifidobacterium, Lactobacillales (lactic acid bacteria or LAB), Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Klebsiella, Escherichia, Enterobacter,
  • Peptostreptococcus Peptococcus, Bacillus, Propionibacteria, Ruminococcus, Gemmiger, Desulfomonas, Salmonella, etc.
  • the recipient bacterium may be Bacteroides thetaiotaomicron, Clostridium butyricum, Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, Enterococcus faecalis, Escherichia coli, Bifidobacterium bifidum, Staphylococcus aureus, Clostridium perfringens, Proteus mirabilis, Clostridium tetani, Clostridium septicum, Pseudomonas aeruginosa,
  • Salmonella enteritidis Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus,
  • Lactobacillus casei Lactobacillus salivarius, Lactococcus lactis, Lactobacillus reuteri,
  • Lactobacillus rhamnosus Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, and Enterococcus faecium.
  • U.S. Patent No. 8,591,880 U.S. Patent No. 8,591,880.
  • the recipient bacteria are the strains E. coli K-12, E. coli MG1655, E. coli US, or E. coli Nissle 1917.
  • Non limiting examples of the recipient bacterium also include: Bacillus coagulans, B. lentus, Bacillus licheniformis, B. mesentericus, B. pumilus, Bacillus subtilis, B. natto,
  • gasseri L. helveticus, L. sakei, L. salivarius, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus acidilactici, P. pentosaceus, Propionibacterium freudenreichii, Prop, shermanii, Staphylococcus carnosus, Staph, xylosus, Streptococcus infantarius, Bacteroides uniformis, Streptococcus sanguinis, Streptococcus mutans, Salmonella enterica, Dorea formicigenerans, Strep, salivarius, Streptococcus thermophiles, Strep. Lactis, and E. mundtii.
  • the recipient bacteria carrying the genomic library are then introduced to the GI tract of one or more mammals.
  • the mammal may be gnotobiotic.
  • the mammal may be germ-free.
  • the mammal may have an already established microbiota.
  • the recipient bacteria carrying the genomic or metagenomic library may be enterally administered to a mammal.
  • the recipient bacteria can be introduced by gavage to a mouse.
  • Stool samples of the mammal are taken at different time points Ti through T n (n is an integer and indicates the number of time points for stool sampling).
  • stool samples can be taken at time points Ti through T n ranging from about 0 day to about 5 years, from about 0 day to about 3 years, from about 0 day to about 1 year, from about 0 day to about 6 months, from about 0 day to about 3 months, from about 0 day to about 30 days, from about 0 day to about 20 days, from about 0 day to about 15 days, from about 0 day to about 10 days, from about 0 day to about 5 days, or from about 0 day to about 3 days, after the recipient bacteria are introduced to the GI tract.
  • Stool samples can be taken at various numbers (i.e., the value of n) of time points, including, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more.
  • the time-series approach can allow discovery of the shifts in population dynamics of clones harboring different gene fragments.
  • the DNA from the stool samples is extracted and then studied by sequencing.
  • DNA may be amplified via polymerase chain reaction (PCR) before being sequenced.
  • PCR polymerase chain reaction
  • the DNA may be sequenced using vector-based primers; or a specific gene is sought by using specific primers.
  • PCR and sequencing techniques are well known in the art; reagents and equipment are readily available commercially.
  • Non-limiting examples of sequencing methods include Sanger sequencing or chain termination sequencing, Maxam-Gilbert sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al, Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass
  • spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al, Nat. BiotechnoL, 16:381-384 (1998)), and sequencing by
  • High-throughput sequencing technologies include, but are not limited to, Illumina/Solex sequencing technology (Bentley et al. 2008 Nature 456:53-59), Roche/454 (Margulies et al. 2005 Nature 437:376-380), Pacbio (Flusberg et al. 2010 Nature methods 7:461-465; Korlach et al. 2010 Methods in enzymology 472:431-455; Schadt et al. 2010 Nature reviews. Genetics 11 :647- 657; Schadt et al. 2010 Human molecular genetics 19:R227-240; Eid et al. 2009 Science
  • DNA from stool samples collected at different time points during in vivo selection is extracted and sequenced to identify genes that are enriched during the in vivo selection.
  • the term "read” refers to the sequence of a DNA fragment obtained after sequencing.
  • the reads are paired-end reads, where the DNA fragment is sequenced from both ends of the molecule.
  • Sequencing reads may be first subjected to quality control to identify overrepresented sequences and low-quality ends.
  • the start and/or end of a read may or may not be trimmed.
  • Sequences mapping to the recipient bacterium may be removed and excluded from further analysis.
  • Sequencing reads are mapped onto the reference genome of the donor bacterium (bacteria) using any alignment algorithms known in the art.
  • mapping algorithms include Bowtie; Bowtie2 (Langmead et al. 2009; Langmead et al, Fast gapped-read alignment with Bowtie 2. Nature methods 9(4), 357-9 (2012); Burrows- Wheeler Aligner (BWA, see, Li et al: Fast and accurate long-read alignment with Burrows- Wheeler transform. Bioinformatics, 26(5), 589-95 (2010)); SOAP2 (Li et al, SOAP2: an improved ultrafast tool for short read alignment.
  • Mathematical algorithms that can be used for alignment also include, the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine optimum alignment. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.
  • the BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra.
  • Gapped BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • GSNAP Thomas D. Wu, Serban Nacu "Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics. 2010 Apr. 1;
  • Algorithms and parameters for alignment can be adjusted depending on the type of bacteria selected, the type of target sequence being characterized, and the method of
  • Mapped reads may be post-processed by removing PCR duplicates (multiple, identical reads), etc.
  • the sequencing data after in vivo selection can be analyzed statistically.
  • the term “enrich” refers to an increase in abundance (or percentage or concentration) of a particular group of genomic DNA fragments. For example, after in vivo selection, the DNA library will contain a higher proportion of DNA fragments or genes than their proportion prior to the in vivo selection (enriching) process. A gene enriched at a time point may enhance bacterial fitness in the mammalian GI tract.
  • the gene When the abundance of a gene during the in vivo selection (e.g., at a time point, or multiple time points as discussed herein, of step (d)) is greater than its abundance before the in vivo selection (e.g., its abundance in the original library), the gene may be identified to be able to enhance bacterial fitness in the mammalian GI tract.
  • the abundance of a gene during the in vivo selection may be at least about 1.2 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.8 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 8 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 200 fold
  • enriched genes may be assessed by metabolic pathway analysis using the KEGG (Kyoto Encyclopedia of Genes and Genomes) and COG databases.
  • composition and abundance of the established microbiota after in vivo selection can also be studied by sequencing the 16S ribosomal R A (or 16S rRNA) gene.
  • 16S rRNA is a component of the 30S small subunit of prokaryotic ribosomes.
  • changes in a mammalian gut bacterial populations are assessed by fluorescent in situ hybridization (FISH) with 16S rRNA probes.
  • FISH fluorescent in situ hybridization
  • 16S rRNA probes specific for predominant classes of the gut microflora (bacteroides, bifidobacteria, Clostridia, and lactobacilli/enterococci), are tagged with fluorescent markers.
  • the probes can include Bifl64 (Langendijk et al, Appl. Environ. Microbiol, 61 : 3069-3075 (1995)), Bac303 (Manz, Microbiology, 142: 1097-1106 (1996)), Hisl50 (Franks, Appl. Environment.
  • DAPI nucleic acid stain 4'6-diamidino-2-phenylindole
  • the hybridization mix is vacuum filtered and the filter mounted on a microscope slide and examined using fluorescence microscopy, such that the bacterial groups could be enumerated (Ryecroft et al, J. Appl. Microbiol, 91 : 878 (2001)).
  • the present invention also involves recombinant bacteria engineered with genes that can enhance bacterial fitness in the mammalian GI tract ("fitness genes"), for example, any gene identified by the present methods.
  • the gene can be exogenous or endogenous. When the gene is endogenous, it can be overexpressed (i.e., having a generally higher expression than the gene in its natural form) and/or constitutively expressed.
  • the overexpressed gene can express its encoded protein at a level at least about 1.2 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.8 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 200 fold, of the protein expression level of the gene in its natural form.
  • the gene may be integrated into the bacterial chromosome or is episomal.
  • Expression of the gene requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as promoters that drive expression of the genes of interest in host cells.
  • the gene may be under the control of a constitutive, inducible, and/or tissue-specific promoter, or promoters useful under the appropriate conditions to direct expression of the introduced DNA segment or a gene.
  • the promoter may be exogenous (heterologous) or endogenous.
  • a promoter may be one naturally-associated with a gene or sequence, or may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as an endogenous promoter.
  • a recombinant or heterologous promoter refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • promoters may include promoters of other genes, and promoters isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not naturally-occurring, i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • constitutive promoters include constitutive E. coli ⁇ 70 promoters, constitutive E. coli o s promoters, constitutive E. coli ⁇ 32 promoters, constitutive E. coli ⁇ 54 promoters, constitutive E. coli ⁇ 70 promoters, constitutive B. subtilis ⁇ ⁇ promoters, constitutive B. subtilis ⁇ ⁇ promoters, T7 promoters, and SP6 promoters.
  • a list of constitutive bacterial promoters may be found in the database of Registry of Standard Biological Parts. They are active in all circumstances in the cell.
  • the constitutive promoter is pL.
  • the gene may be under the control of an inducible promoter.
  • the transcriptional activity of these promoters is induced by either chemical or physical factors.
  • Chemically-regulated inducible promoters may include promoters whose transcriptional activity is regulated by the presence or absence of oxygen, a metabolite, alcohol, tetracycline, steroids, metal and other compounds.
  • Physically-regulated inducible promoters including promoters whose
  • transcriptional activity is regulated by the presence or absence of heat, low or high temperatures, acid, base, or light.
  • the inducible promoter is pH-sensitive (pH inducible).
  • the inducer for the inducible promoter may be located in the biological tissue or environmental medium to which the composition is administered or targeted, or is to be administered or targeted.
  • the inducer for the inducible promoter may be located in the mammalian GI tract.
  • the pH level of a particular biological tissue can affect the inducibility of the pH inducible promoter. See, for example, Boron, et al, Medical Physiology: A Cellular and
  • acid inducible promoters include, but are not limited to, P170, PI , P3, baiAl , baiA3, lipF promoter, FiF 0 -ATPase promoter, gadC, gad D, glutamate decarboxylase promoter, etc. See, for example, Cotter and Hill, Microbiol, and Mol. Biol. Rev. vol. 67, no. 3, pp. 429-453 (2003); Hagenbeek, et al, Plant Phys., vol. 123, pp.
  • promoters induced by a change in temperature include P2, P7, and PhS. See, for example, Taylor, et al, Cell, Abstract, vol. 38, no. 2, pp. 371 -381 (1984); U.S. Pat. No. 6,852,51 1 , Wang, et al, Biochem. and Biophys. Res. Commun. Abstract, vol. 358, no. 4, pp. 1 148-1 153 (2007), U.S. Pat. No. 7,462,708, each of which is incorporated herein by reference.
  • the acid inducible promoter is inducible at a pH of about 0.0, about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or any value therebetween or less.
  • the base inducible promoter is inducible at a pH of about 7.1 , about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 1 1.0, about 1 1.5, about 12.0, about 12.5, about 13.0, about 13.5, about 14.0, or any value therebetween or greater.
  • inducers that can induce the activity of the inducible promoters also include, but are not limited to, radiation, temperature change, alcohol, antibiotic, steroid, metal, salicylic acid, ethylene, benzothiadiazole, or other compound.
  • the at least one inducer includes at least one of arabinose, lactose, maltose, sucrose, glucose, xylose, galactose, rhamnose, fructose, melibiose, starch, inunlin, lipopolysaccharide, arsenic, cadmium, chromium,
  • inducers include, but are not limited to, at least a portion of one of an organic or inorganic small molecule, clathrate or caged compound, protocell, coacervate, microsphere, Janus particle, proteinoid, laminate, helical rod, liposome, macroscopic tube, niosome, sphingosome, vesicular tube, vesicle, unilamellar vesicle, multilamellar vesicle, multivesicular vesicle, lipid layer, lipid bilayer, micelle, organelle, nucleic acid, peptide, polypeptide, protein, glycopeptide, glycolipid, lipoprotein, lipopolysaccharide, sphingolipid, glycosphingolipid, glycoprotein, peptidoglycan, lipid, carbohydrate, metalloprotein, proteoglycan, chromosome, nucleus, acid, buffer, protic solvent, aprotic solvent, nitric oxide, vitamin
  • a nucleic acid sequence or a gene can be endogenous, or "exogenous” or “heterologous” which means that it is foreign to the cell into which the vector is being introduced or that the sequence or gene is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • the genes that can enhance bacterial fitness in the mammalian GI tract may include the genes encoding a glycoside hydrolase, a galactokinase, a glucose/galactose transporter, or any other proteins involved in carbohydrate (e.g., sucrose, galactose, etc.) metabolism/transport.
  • the gene may be from any prokaryote or eukaryote, including the donor bacteria disclosed herein.
  • the recombinant bacteria are capable of metabolizing both sucrose and galactose. In another embodiment, the recombinant bacteria are capable of metabolizing sucrose. In yet another embodiment, the recombinant bacteria are capable of metabolizing galactose.
  • sucrose-utilizing probiotic strains to occupy the sucrose niche could also be an effective strategy to resist pathogen colonization.
  • the fitness gene engineered into the recombinant bacteria may be wild-type or be mutated. When the gene is a mutant, it may be truncated at the 5 ' end by from about 1 base pair (bp) to about 100 bp, from about 2 bp to about 50 bp, from about 3 bp to about 20 bp, or from about 4 bp to about 10 bp from the start codon.
  • the protein is Bacteroides thetaiotaomicron glycoside hydrolase and its gene is truncated at the 5 ' end by about 4 bp from the start codon.
  • a glycoside hydrolase also called glycosidase or glycosyl hydrolase, is an enzyme that catalyzes the hydrolysis of the glycosidic bond between two or more carbohydrates (e.g., in complex sugars), or between a carbohydrate and a non-carbohydrate moiety.
  • Glycoside hydrolases are typically classified into EC 3.2.1 as enzymes catalyzing the hydrolysis of O- or S- glycosides.
  • Glycoside hydrolases can also be classified according to the stereo-chemical outcome of the hydrolysis reaction: thus they can be classified as either retaining glycoside hydrolases or inverting glycoside hydrolases.
  • Glycoside hydrolases can also be classified as exo- or endo- acting, dependent upon whether they act at the end or in the middle, respectively, of a polysaccharide chain. Glycoside hydrolases may also be classified by sequence or structure based methods. Exemplary glycoside hydrolases include beta-galactosidase (also called beta-gal or ⁇ -gal), glucosidase, xylannase, lactase, amylase, chitinase, sucrase, maltase, neuraminidase, invertase, hyaluronidase and lysozyme. Samuel, PNAS, 2006, 103(26) 10011-10016.
  • beta-galactosidase also called beta-gal or ⁇ -gal
  • glucosidase also called beta-gal or ⁇ -gal
  • glucosidase glucosidase
  • xylannase lactas
  • the present invention encompasses both wild-type and mutant glycoside hydrolases.
  • the mutant glycoside hydrolase may have conservative amino acid substitutions or functional fragments that do not substantially alter its activity.
  • the gene of the glycoside hydrolase is truncated at the 5' end by from about 1 bp to about 50 bp from the start codon.
  • the glycoside hydrolase may be Bacteroides thetaiotaomicron glycoside hydrolase and its gene is truncated at the 5 ' end by about 4 bp from the start codon.
  • the present invention encompasses glycoside hydrolases from any of Glycoside
  • Galactokinase is an enzyme that facilitates the phosphorylation of a-D- galactose to galactose 1 -phosphate at the expense of one molecule of ATP.
  • Galactokinase may also phosphorylate 2-deoxy-D-galactose, 2-amino-deoxy-D-galactose, 3-deoxy-D-galactose and D-fucose.
  • the present invention also provides for a probiotic composition
  • a probiotic composition comprising the present recombinant bacteria.
  • the recombinant bacteria may constitutively express one or more of proteins involved in carbohydrate metabolism and/or transport.
  • the proteins can include a glycoside hydrolase, a galactokinase and a glucose/galactose transporter.
  • the gene of the protein may be under the control of a constitutive or inducible promoter.
  • Probiotics are microorganisms, or processed compositions of microorganisms which beneficially affect a host. Salminen et al., Probiotics: how should they be defined, Trends Food Sci. Technol. 1999: 10 107-10. U.S. Patent No. 8,216,563.
  • the present probiotic composition is to be administered enterally, such as oral, sublingual and rectal administrations.
  • the present probiotic composition can be a food composition, a beverage composition, a pharmaceutical composition, or a feedstuff composition.
  • the present probiotic composition may comprise a liquid culture.
  • the probiotic composition may be lyophilized, pulverized and powdered. As a powder it can be provided in a palatable form for reconstitution for drinking or for reconstitution as a food additive.
  • the composition can be provided as a powder for sale in combination with a food or drink.
  • the food or drink may be a dairy-based product or a soy-based product.
  • the invention therefore also includes a food or food supplement containing the present composition.
  • Typical food products that may be prepared in the framework of the present invention may be milk-powder based products; instant drinks; ready-to-drink formulations; nutritional powders; milk-based products, such as yogurt or ice cream; cereal products; beverages such as water, coffee, malt drinks;
  • the composition may further contain at least one prebiotic.
  • prebiotic means food substances intended to promote the growth of probiotic bacteria in the intestines.
  • the prebiotic may be selected from the group consisting of oligosaccharides and optionally contains fructose, galactose, mannose, soy and/or inulin; and/or dietary fibers.
  • the composition of the present invention may further contain prebiotics. Prebiotics may be dietary fibers.
  • Dietary fibers may be selected from the group consisting of fructo-oligosaccharides, galacto-oligosaccharides, xylo- oligosaccharides, isomalto-saccharides, soya oligosaccharides, pyrodextrins, transgalactosylated oligosaccharides, lactulose, beta-glucan, insulin, raffmose, stachyose. Dietary fibers also have the advantage of being resistant to a number of conditions including heating and long storage times. They furthermore may contribute to a treatment in the framework of the present invention by improving gastrointestinal health and by increasing satiety.
  • composition can be combined with other adjuvants such as antacids to dampen bacterial inactivation in the stomach.
  • adjuvants such as antacids to dampen bacterial inactivation in the stomach.
  • Acid secretion in the stomach could also be
  • H2-antagonists pharmacologically suppressed using H2-antagonists or proton pump inhibitors.
  • the H2-antagonist is ranitidine.
  • the proton pump inhibitor is omeprazole.
  • composition of the present invention may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co- compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents, gel forming agents, antioxidants and antimicrobials.
  • protective hydrocolloids such as gums, proteins, modified starches
  • binders film forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co- compounds, dispersing agents
  • composition according to the invention may comprise a source of protein.
  • Any suitable dietary protein may be used, for example animal proteins (such as milk proteins, meat proteins and egg proteins); vegetable proteins (such as soy protein, wheat protein, rice protein, and pea protein); mixtures of free amino acids; or combinations thereof.
  • the proteins may be intact, hydrolyzed, partially hydrolyzed or a mixture thereof.
  • the composition may also contain a source of carbohydrates and a source of fat.
  • a source of carbohydrate may be added to the composition. Any suitable carbohydrate may be used, for example sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrins, and mixtures thereof. Dietary fiber may also be added if desired.
  • the pharmaceutical compositions of the present invention can be, e.g., in a solid, semisolid, or liquid formulation. Compositions can also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, emulsions, suspensions, or any other appropriate compositions.
  • the present composition may be in the form of: an enema composition which can be reconstituted with an appropriate diluent; enteric-coated capsules or microcapsules; powder for reconstitution with an appropriate diluent for naso-enteric infusion, naso-duodenal infusion or colonoscopic infusion; powder for reconstitution with appropriate diluent, flavoring and gastric acid suppression agent for oral ingestion; or powder for reconstitution with food or drink.
  • an enema composition which can be reconstituted with an appropriate diluent; enteric-coated capsules or microcapsules; powder for reconstitution with an appropriate diluent for naso-enteric infusion, naso-duodenal infusion or colonoscopic infusion; powder for reconstitution with appropriate diluent, flavoring and gastric acid suppression agent for oral ingestion; or powder for reconstitution with food or drink.
  • composition may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, including, but not limited to, water, gelatine of any origin, vegetable gums, ligninsulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like. In all cases, such further components will be selected having regard to their suitability for the intended recipient.
  • conventional pharmaceutical additives and adjuvants, excipients and diluents including, but not limited to, water, gelatine of any origin, vegetable gums, ligninsulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fill
  • a sufficient dose of the recombinant bacteria is usually consumed per day in order to achieve successful colonization.
  • the daily dose of probiotics in the composition will depend on the particular person or animal to be treated. Important factors to be considered include age, body weight, sex and health condition.
  • Daily doses generally range from about 10 2 to about 10 14 cfu (colony forming units), from about 10 2 to about 1012 cfu, from about 104 to about 1012 cfu, from about 10 6 to about 10 10 cfu, from about 10 6 to about 10 14 cfu, about 10 7 to about 10 13 cfu, about 10 10 to about 10 14 cfu, about 10 11 to about 10 13 cfu, about l-4xl0 12 cfu, or from about 10 7 to about 10 9 cfu per day.
  • U.S. Patent No. 8,021,656 U.S. Patent No. 8,021,656.
  • the dosage of the present recombinant bacteria in the gut can be adjusted by those skilled in the art to the designated purpose. Any dose showing an effect may be suitable.
  • Appropriate frequency of administration can be determined by one of skill in the art and can be administered once or several times per day (e.g., twice, three, four or five times daily).
  • the compositions of the invention may also be administered once each day or once every other day.
  • the compositions may also be given twice weekly, weekly, monthly, or semi-annually.
  • the present compositions and methods may be used for the treatment and/or prophylaxis of a disorder associated with the presence in the gastrointestinal tract of a mammalian host of abnormal (or an abnormal distribution of) microbiota.
  • the method comprises administering an effective amount of the present composition.
  • the donor bacterium may be from a natural gut microbiota of a healthy mammal or a mammal with the following disorders.
  • Such disorders include but are not limited to those conditions in the following categories: gastro-intestinal disorders including irritable bowel syndrome (IBS, or spastic colon), and intestinal inflammation, functional bowel disease (FBD), including constipation predominant FBD, pain predominant FBD, upper abdominal FBD, non-ulcer dyspepsia (NUD), gastro- oesophageal reflux, inflammatory bowel disease including Crohn's disease, ulcerative colitis, indeterminate colitis, collagenous colitis, microscopic colitis, chronic Clostridium difficile infection, pseudomembranous colitis, mucous colitis, antibiotic associated colitis, idiopathic or simple constipation, diverticular disease, AIDS enteropathy, small bowel bacterial overgrowth, coeliac disease, polyposis coli, colonic polyps, chronic idiopathic pseudo obstructive syndrome; chronic gut infections with specific pathogens including bacteria, viruses, fungi and protozoa (e.g., Clostridium difficile infection (CDI));
  • viral gastrointestinal disorders including viral gastroenteritis, Norwalk viral
  • gastroenteritis gastroenteritis, rotavirus gastroenteritis, AIDS related gastroenteritis;
  • liver disorders such as primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver or cryptogenic cirrhosis;
  • rheumatic disorders such as rheumatoid arthritis, non-rheumatoid arthritidies, non rheumatoid factor positive arthritis, ankylosing spondylitis, Lyme disease, and Reiter's syndrome;
  • immune mediated disorders such as glomerulonephritis, haemolytic uraemic syndrome, type 1 or type 2 diabetes mellitus, mixed cryoglobulinaemia, polyarteritis, familial
  • autoimmune disorders including systemic lupus, idiopathic thrombocytopenic purpura, Sjogren's syndrome, haemolytic uremic syndrome or scleroderma;
  • neurological syndromes such as chronic fatigue syndrome, migraine, multiple sclerosis, amyotrophic lateral sclerosis, myasthenia gravis, Gillain-Barre syndrome, Parkinson's disease, Alzheimer's disease, Chronic Inflammatory Demyelinating Polyneuropathy, and other degenerative disorders;
  • psychiatric disorders including chronic depression, schizophrenia, psychotic disorders, manic depressive illness;
  • regressive disorders including Asperger's syndrome, Rett syndrome, autism, attention deficit hyperactivity disorder (ADHD), and attention deficit disorder (ADD);
  • SIDS sudden infant death syndrome
  • anorexia nervosa anorexia nervosa
  • metabolic disorders that can be treated or prevented by the above use include obesity, insulin resistance, hyperglycemia, hepatic steatosis, and small intestinal bacterial overgrowth (SIBO), U.S. Patent No. 8,110,177.
  • the present recombinant bacteria may additionally confer benefits to a subject. These additional benefits are generally known to those skilled in the art and may include managing lactose intolerance, prevention of colon cancer, lowering cholesterol, lowering blood pressure, improving immune function and preventing infections, reducing inflammation and/or improving mineral absorption.
  • Subjects which may be treated according to the present invention include all animals which may benefit from the present invention.
  • Such subjects include mammals, preferably humans (infants, children, adolescents and/or adults), but can also be an animal such as dogs and cats, farm animals such as cows, pigs, sheep, horses, goats and the like, and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
  • shotgun libraries for functional metagenomics of mammalian- associated microbiota has been demonstrated ex vivo, such as by growing the library in media with different substrates to characterize carbohydrate active enzymes (Tasse et al, 2010), prebiotic metabolism (Cecchini et al, 2013), glucuronidase activity (Gloux et al, 2011), salt tolerance (Culligan et al, 2012), and antibiotic resistance genes (Sommer et al, 2009), or by using filtered lysates of the library to screen for signal modulation in mammalian cell cultures (Lakhdari et al, 2010).
  • This metagenomic shotgun library approach has yet to be demonstrated on a large-scale in vivo.
  • Our approach marries two massively parallel strategies to identify genes that are enriched during functional selection.
  • Our strategy involves expression and selection of a functional genomic library followed by characterization of library composition and abundance over time through deep-sequencing. The method is culture -independent so "unculturable" microbes and metagenomic sources can be functionally interrogated without the need for cultivation in the lab.
  • the present approach tracks an important temporal component of gut colonization by measuring allele frequencies over time to monitor the dynamic changes during colonization.
  • a 2.2 kb E. coli expression vector, GMVlc was constructed to include the strong constitutive promoter pL and a ribosomal binding site upstream of the cloning site for input DNA fragments (Figure 1).
  • Figure 1 We cloned in 2-5 kb fragments of donor genomic DNA from Bt, and generated a library of -100,000 members, corresponding to >50X coverage of the donor genome.
  • Figure 2A and El We sequenced the library on the Illumina HiSeq instrument to confirm sufficient coverage of the Bt genome.
  • the distribution of member insert sizes in the input library was verified to be centered around 2-3 kb ( Figure 2B), a size range allowing for the full-length representation of almost all Bt genes.
  • Clones harboring the empty vector were the most fit library member: in both LB and MC conditions, these clones initially constituted 70%> of the library and increased to 90%> by the end of two weeks, albeit at a slower rate in anaerobic MC.
  • enriched genes included metabolic enzymes, such as chitobiase (BT 0865), which degrades chitin, and stress response proteins, such as glycine betaine/L-proline transport system permease (BT 1750), which is involved in the import of osmoprotectants glycine betaine or proline that mitigate effects of high osmolality (Haardt et al, 1995).
  • metabolic enzymes such as chitobiase (BT 0865), which degrades chitin
  • stress response proteins such as glycine betaine/L-proline transport system permease (BT 1750)
  • BT 1750 glycine betaine/L-proline transport system permease
  • BT 0369 endo-l,4-beta- xylanase
  • BT 0370 galactokinase
  • BT 0371 glucose/galactose transporter
  • BT 0372 aldose 1-epimerase
  • One cohort was colonized with our library; the other cohort with a control GMVlc vector carrying the 5.9 kb luciferase operon (luxCDABE from Photorhabdus luminescens,
  • Fecal pellets were collected on days 0.5, 0.75, 1.5, 1.75, 2.5, 3, 4, 7, 10, 14, 21, 25, and 28 after inoculation.
  • BT 0370 and BT 0371 transition from galactokinase and glucose/galactose transporter (BT 0370 and BT 0371) to glycoside hydrolase (BT 1759) occurred four days earlier in Mouse 5 than in Mouse 2, and the emergence of fructokinase (BT 1757) was detectable only in Mice 2, 4, and 5.
  • time-averaged relative abundance TA-RA
  • time-averaged normalized effective coverage TA-NEC
  • the TA-RA value is conceptually similar to a time-integrated pharmacological dose value (Byers & Sarver, 2009); in our analysis, it represents the average "dose" of a particular donor gene, relative to all other donor genes present in vivo over a period of time.
  • the TA-NEC value quantifies the fraction of the gene that is effectively covered by reads over a period of time.
  • coli GmhB is highly selective for ⁇ -anomers while Bt GmhB prefers a-anomers during hydrolysis of D-glycero-D-manno-heptose l ?,7-bisphosphate (Wang et al, 2010).
  • BT 1730 rfbD; rmlD
  • rmlD is involved in dTDP-rhamnose biosynthesis involved in production of O-antigen, a repetitive glycan polymer in LPS, and potentially other cell-membrane components. Deletion of rmlD in Vibrio cholera results in a severe defect in colonization of an infant mouse model (Chiang & Mekalanos, 1999), and uropathogenic E.
  • Bt rmlD could allow the recipient E. coli to alter its antigenicity or resistance to host factors that would impede its initial colonization of the gnotobiotic gut.
  • outer membrane lipoprotein SilC (BT 0297), cell surface protein (BT 1771), and outer membrane protein OmpA (BT 1511).
  • BT 0297 outer membrane lipoprotein SilC
  • BT 1771 cell surface protein
  • OmpA outer membrane protein
  • These genes could confer increased capabilities for E. coli to attach to the mucosal surface of the mammalian GI tract, or increased adaptations to the gut chemical environment.
  • Bacteroides fragilis lacking OmpA are more sensitive to SDS, high salt, and oxygen exposure (Wexler et al, 2009).
  • OmpA plays a role in intestinal adherence (Sato et al, 2010), and in Klebsiella pneumoniae, activates macrophages (Soulas et al, 2000).
  • Bt GMP synthase guaA Bt GMP synthase guaA
  • Inhibiting GMP synthase induces stationary phase genes in Bacillus subtilis (Ratnayake-Lecamwasam et al, 2001), and nucleotide concentrations drop when E. coli transition from growth to stationary phase (Buckstein et al, 2008).
  • extra GMP synthase may further protect E. coli from incorporating mutagenic deaminated nucleobases that would interfere with RNA function and gene expression (Pang et al, 2012).
  • sucrose utilization was enabled when we reconstituted the 4 nt truncation found in many of the Day 7 and Day 28 Sanger-sequenced clones into the starting E. coli strain.
  • the operon structure of the tRNA region may be lysT-valT-lysW-valZ-lysYZQ (Blattner et al, 1997), or valZ-lys Y could be a separate operon as predicted in EcoCyc (Keseler et al, 2011), in which case the SNV could affect transcription of the downstream tRNAs.
  • the traFpromoter variant the -35 hexamer has been documented to be TTTACC (Gaudin & Silverman, 1993).
  • the SNV T>C changes it to CTTACC, which could weaken the promoter to decrease expression of TraY, a DNA-binding protein involved in initiation of DNA transfer during conjugation.
  • E. coli GalR binds operator sequences upstream of the galETK operon (Weickert & Adhya, 1993), and the amino acid substitution of arginine for leucine could be disruptive to binding.
  • Galactose plays a substantial role in selection in our experiment, as all three of the observed E. coli genomic mutations (in galK, lacY, and galR) affected galactose utilization, and we observed selection for Bt galactokinase (BT 0370) and glucose/galactose transporter (BT 0371) in vivo.
  • BT 0370 Bt galactokinase
  • BT 0371 glucose/galactose transporter
  • Galactose is a component of the hemi-cellulose that makes up part of the 15.2% neutral detergent fiber in mouse chow, although galactose composition was not explicitly provided by the manufacturer.
  • Galactose is also a component of mammalian mucin in the GI tract (Juge, 2012).
  • sucrose-utilizing probiotic strains to occupy the sucrose niche could also be an effective strategy to resist pathogen colonization.
  • Bt has been investigated previously using transposon mutagenesis systems coupled to mouse gut colonization experiments (Goodman et al, 2009), facilitating comparison of our results to the prior study.
  • Goodman et al. found no difference in abundances of galactokinase (BT 0370) mutants in vitro but BT 0370 mutants were underrepresented in vivo.
  • the Bt galactokinase was selected for not only in vivo, but also in vitro.
  • Goodman et al. found dTDP-4-dehydrorhamnose reductase (BT 1730) and GMP synthase (BT 4265) mutants were underrepresented both in vitro and in vivo.
  • BT 1730 and BT 4265 seemed to confer fitness only in vivo.
  • the in vitro discrepancies may be a result of slightly different culturing and media conditions.
  • the in vivo results are in agreement for BT 0370, BT 1730, and BT 4265, though the other genes we identified in our experiments were not significantly altered in representation in the transposon mutagenesis experiments, highlighting the different capabilities of the two approaches.
  • the putative unfitness of the strain in vivo allows for stronger selection signals from clones harboring functional donor genes.
  • the recipient strain also plays a role in the co-evolution of the insert library and the strain's own genome.
  • Bt galactokinase Bt glycoside hydrolase
  • single nucleotide variations in E. coli galR and lac Y loci that boosted galactose utilization in individual clones harboring functional Bt genes. Given that co-evolution drives genomic changes in the recipient strain, using a well-characterized recipient strain may facilitate mechanistic interpretation of these changes.
  • mice germfree mice were mono-associated with the library. We expect selection results will differ in the setting of mice pre-colonized with a microbiota due to changes in nutrient availability and other ecological interactions, including competition or syntrophy. For instance, co-colonization experiments have demonstrated that probiotic strains and commensal bacteria adapt their substrate utilization. Bt shifts its metabolism from mucosal glycans to dietary plant polysaccharides when in the presence of Bifidobacterium animalis, Bifidobacterium longum, or Lactobacillus casei (Sonnenburg et al, 2006).
  • OMVs outer membrane vesicles
  • surface glycoside hydrolases or polysaccharide lyases e.g., surface glycoside hydrolases or polysaccharide lyases
  • Bacteroides thetaiotaomicron VPI-5482 was grown anaerobically in a rich medium based on Brain Heart Infusion with other supplements added.
  • the genomic library was maintained in an Escherichia coli K-12 strain, NEB Turbo (New England Biolabs, Ipswich, MA).
  • E. coli strains were grown in Luria broth (LB) and supplemented with carbenicillin (final concentration 100 ⁇ g/mL) as needed.
  • LB Luria broth
  • carbenicillin final concentration 100 ⁇ g/mL
  • Mouse chow (MC) filtrate was prepared by adding 150 mL deionized water to 8 g of crushed mouse chow (Mouse Breeding Diet 5021, LabDiet, St. Louis, MO). The mixture was heated at 95°C for 30 minutes with mixing, passed through a 0.22 ⁇ filter, and autoclaved. The sterility of the MC filtrate was confirmed by incubating at 37°C in aerobic and anaerobic conditions and observing no growth after several days.
  • Bacteroides thetaiotaomicron genomic DNA was isolated (DNeasy Blood & Tissue Kit, Qiagen, Venlo, Netherlands), fragmented by sonication to 3-5 kb (Covaris E210, Covaris, Woburn, MA), and size-selected and extracted by gel electrophoresis (Pippin Prep, Sage
  • the fragments were end-repaired (End-It DNA End-Repair Kit, Epicenter, Madison, WI) and cloned into a PCR-amp lifted GMVlc backbone vector via blunt-end ligation.
  • the reaction was transformed into NEB Turbo electrocompetent E. coli cells (New England Biolabs). The library size was quantified by counting colonies formed on selective media (LB
  • Plasmid retention Individual stool pellets from Days 0.75, 1.5, 1.75, 2.5, 4, 10, 14, 21, 25, and 28 were homogenized in 10% PBS and plated on LB agar with or without carbenicillin (carb). To obtain accurate counts, platings were performed in triplicate and repeated at 100X dilutions if the plates were overgrown. Plasmid retention was calculated as the number of colonies grown on LB-carb plates divided by the number of colonies grown on LB only plates.
  • LB cultures were grown in aerobic conditions with shaking and passaged every day for two weeks.
  • MC cultures were grown in anaerobic conditions without shaking and passaged every two days for two weeks, since the cultures took more time to reach saturation compared to the LB condition.
  • mice were orally gavaged with ⁇ 2 x 10 8 CFU of bacteria in a volume of 200 on Day 0. Mice inoculated with the library were separately housed. Fecal pellets were collected at 0.5, 0.75, 1.5, 1.75, 2.5, 3, 4, 7, 10, 14, 21, 25, and 28 days post-inoculation and stored at -80°C in 10% PBS buffer.
  • Bowtie (Langmead et al, 2009) was applied instead of Bowtie2 for higher sensitivity. Default parameters were used for building a Bowtie index with the B. thetaiotaomicron chromosome and plasmid sequences. Paired-end reads were aligned to the reference genome with parameter -X 300 using Bowtie. SAM files from the Bowtie alignment were converted to indexed and sorted BAM files using SAMtools (Li, et al. 2009).
  • Cuffdiff (Trapnell et al, 2013) was applied to test differential representation of genes (i.e., the library grown in rich medium at time 0 versus the library grown in rich medium at day 7, and the library grown in MC medium at time 0 versus the library grown in MC medium at day 7).
  • B. thetaiotaomicron genomic DNA inserts were amplified from isolated E. coli plasmids using our improved PCR protocol (see above). After Nextera sequencing library preparation, paired-end reads of 101 bp length were generated on the HiSeq 2500 (Illumina) instrument at the Baylor College of Medicine Alkek Center for Metagenomics and Microbiome Research. All reads passed quality control (base quality >30) using FastQC (Babraham Bioinformatics). To eliminate plasmid DNA sequences in reads, the reads were trimmed using custom Perl scripts that removed all flanking regions matching 15bp of the plasmid DNA on the 5' and 3' ends of B. thetaiotaomicron insert fragment. Reads less than 20bp after trimming were discarded, and the others were matched as pairs with the forward read and reverse reads.
  • Sequencing reads were mapped onto the reference genome of B. thetaiotaomicron using Bowtie2 (Langmead et al, 2009). Default parameters were used for building the Bowtie2 index using the B. thetaiotaomicron chromosome and plasmid sequences, and for aligning reads to the reference sequence. SAM files generated from Bowtie2 alignment were converted to indexed and sorted BAM files using SAMtools (Li et al, 2009). In SAMtools, 'mpileup' with parameter '-B' was used to obtain the depth of coverage of the reference genome. Across all samples, the mean of the mapped bases to the B. thetaiotaomicron genome was 1.17 x 10 9 , with a minimum of 4.31 x 10 8 and maximum of 2.49 x 10 9 bases per sample.
  • EPD effective positional diversity
  • r ti represents the fraction of reads at time t mapping to nucleotide i in a reference sequence totaling P nucelotides (e.g., the Bt genome).
  • TA-RA time-averaged relative abundance
  • ti and 3 ⁇ 4 denote the bounds of the time-interval of interest
  • f g represents a continuous-time function for gene g.
  • the function f g was estimated as follows. We fit a cubic smoothing spline, using the Matlab function csaps, applied to the log fold change in Fragments Per Kilobase per Million mapped reads (FPKM) for gene g at each time-point t (i.e., the FPKM value at time-point t divided by the FPKM value for the gene in the starting library). The smoothing spline was used to account for non-uniform temporal sampling and noise in the data.
  • FPKM Per Kilobase per Million mapped reads
  • the time-averaged normalized effective coverage (TA-NEC), a gene-level measure of coverage, was calculated using the formula:
  • l g denotes the length of gene g
  • h g represents a continuous-time function for gene g.
  • the function h g was estimated as follows. We fit a cubic smoothing spline, using the Matlab function csaps, applied to the effective coverage, EC(g,t) for the gene at each time -point:
  • s g denotes the start of the gene.
  • MacConkey base agar with a final concentration of 1% lactose or galactose was also used to characterize lactose or galactose utilization.
  • Example 2 Identifying genes that improve microbial fitness in the healthy and inflamed gut using in vivo temporal functional metagenomics
  • the temporal functional metagenomics of the present invention will be used to systematically dissect the genetic determinants underling microbial colonization of the healthy and inflamed gut and to understand the long-term adaptation of microbes to the mammalian gut. This can help understand how these genetic determinants may play a role in the maintenance of healthy and development of diseased states.
  • the approach involves a) construction of a DNA fragment library from a donor microbe or a metagenomic source, b) transformation and heterologous expression of the library in a recipient microbe, c) application of selective pressure on the population over time, and d) characterization of the changes in library composition and abundance by deep-sequencing.
  • Example 1 show a statistically significant enrichment for certain metabolic and transporter genes cloned into Escherichia coli from Bacteroides thetaiotaomicron that confer improved fitness during in vitro batch growth.
  • Bt Bacteroides thetaiotaomicron
  • the following research will be conducted: discover genetic determinants that promote microbial colonization of the healthy gut using genome-wide approaches; identify genetic factors in inflammation-associated microbiota that lead to retention in the chronically diseased gut and develop counteracting strategies; and characterize and understand the long-term adaptation and coevolution between microbial colonizers and the mammalian gut throughout neonatal development.
  • Aim 1 To systematically identify and characterize genes from commensal microbes that promote improved colonization in the healthy gut of gnotobiotic and conventional mice.
  • a temporal functional metagenomics approach will be used to gain insight into what types of genes from the microbiome gene pool can specifically improve microbial fitness of a gut colonizer.
  • E. coli a facultative anaerobe, as a suitable Gram-negative (GN) model because it is normally found in the gut at low levels, reflecting its lower fitness compared to other dominant commensals.
  • GN Gram-negative
  • Cb-Lp library or Cb-Bs library
  • L. plantarum or Bacillus subtilis
  • This aim includes construction and in vitro characterization of Cb-Bs genomic library, and in vivo mice selection of Cb-Bs library and deep-sequencing of fecal output.
  • GN/GP systems Since E. coli will serve as a Gram-negative (GN) recipient for Gram- negative DNA sources, we will also construct a Gram-positive (GP) expression system in parallel using Bacillus subtilis, which is not a native colonizer of the gut. Alternatively, the Gram(+) bacteria Lactobacillus plantarum (ATCC 14917) may be used as a recipient. This system can be developed for other probiotics including L. reuteri, L. rhamnosus, and B. longum.
  • Clostridum butyricum will be used as a representative Gram-positive donor as it is a natural gut colonizer and has been shown to interfere with the growth of gut pathogen C difficile. Woo et al. Inhibition of the cytotoxic effect of Clostridium difficile in vitro by Clostridium butyricum MIYAIRI 588 strain. J Med Microbiol 11 : 1617-25 (2011).
  • Metagenomic donor DNA sources Based on validation of the Cb-Lp library (or Cb-Bs library) generation protocol, we will further use metagenomic DNA from natural gut microflora as a source of donor DNA to build expression libraries in Lp or Bs to identify GCFs that can improve its gut retention.
  • Diet perturbations In addition to the standard low-fat, plant polysaccharide-rich diet fed to the mice, other modified diets will be used to assess selection for different genes by different dietary regimens. These additional diets includes: high- fat, high-sugar (Western diet) and combinations of defined macro-nutrients (casein for protein, corn oil for fat, cornstarch for polysaccharide, and sucrose for simple sugar) described previously.
  • High- fat, high-sugar Wood diet
  • macro-nutrients casein for protein, corn oil for fat, cornstarch for polysaccharide, and sucrose for simple sugar
  • GCFs gut colonization factors
  • Aim 2 To identify specific genetic factors in inflammation-associated microbiota that lead to colonization and retention of the chronically diseased gut and to develop strategies to reverse the diseased state by enhancing better re-colonization of the dysbiotic gut.
  • H/I-D/R health/inflamed - donor/recipient genomic libraries
  • in vivo mice selection of H/I-D/R library and sequencing of fecal output and in vivo selection using C. diff ' model to test pathogen exclusion.
  • HIDR Healthy/Inflamed Donor/Recipient study: Our donor library will be built using fecal samples from healthy human volunteers and from IBD patients. These libraries will be put into both our Gram-positive (B. subtilis) and Gram-negative (E. coli) expression systems. We will transplant these libraries into two C57BL/6 mice groups, a healthy gut group and an inflamed gut group. Prior to microbiota inoculation, well-established chemical routes will be used to induce chronic colitis by addition of dextran sodium sulfate (DSS) to the drinking water.
  • DSS dextran sodium sulfate
  • Clostridium difficile infection is a growing clinical challenge with very high rates of reoccurrence and is responsible for > 14,000 deaths per year in the US.
  • CDI Clostridium difficile infection
  • a similar strategy through fecal transplantation to recolonize the diseased gut with microbiota from healthy donors has shown clinical success in reversing CDI. Brandt et al., Fecal microbiota transplantation for recurrent Clostridium difficile infection. J Clin Gastroenterol. 45: 159-67 (2011).
  • HF high-fitness
  • dijf-associated murine model and apply the temporal functional metagenomics to identify commensal genes that are enriched in the presence of a persistent infection.
  • We will measure C. difficile titers to assess selection against the pathogen and enrichment of our desired higher fitness strains.
  • C. difficile titers In addition to assessing the microbiota throughout colonization, other murine immunological assays can also be done to measure serum IgG, IgA, and T-cell activation levels.
  • Aim 3 To characterize the long-term adaptation of microbial gut colonizers in gnotobiotic mice as a model for understanding neonatal colonization and host-microbe coevolution throughout development and maturation. Recent studies have highlighted the intimate link between microbial colonization and intestinal maturation during neonatal development. Koenig et al., Succession of microbial consortia in the developing infant gut microbiome. Proc. Natl. Acad. Sci. 108:4578-4585 (2011). The gut microbiome plays a key role in sensitizing the immune system to properly distinguish harmless bacteria from pathogens.
  • LTE long-term evolution
  • This Aim will include conducting long-term evolution experiments in vivo, isolate resulting clones and perform deep-sequencing and analysis of evolved strains, and phenotypic assays for clone fitness versus wild-type.
  • Diet & antibiotic resistance Host diet and nutritional availability is a key factor during LTE. We will maintain the same diet throughout this LTE experiment using a defined mouse chow (DMC) composed of defined macronutrients - casein for protein, corn oil for fat, cornstarch for polysaccharide, and sucrose for simple sugar, amino acids and vitamins. A defined rich diet is important for limiting batch-to-batch variation in mouse chow to control fluctuations in selective pressure and allow us to better measure nutritional utilization. Presently, an alarming level of antibiotics including fluoroquinolones (Ciprofloxacin) is being used during food processing leading to subinhibitory concentrations in our foods.
  • GCF gut colonization factors
  • Example 1 Based on the GCFs identified in Example 1 and Example 2, we will apply detailed characterization to quantify the level of fitness benefit that different GCFs impart on the recipient probiotic bacteria. These characterizations will initially be performed in vitro. Our results (Example 1) show that certain metabolic factors including those associated with polysaccharide utilization can improve gut-retention and provide colonization resistance against other bacteria (potentially pathogens). We will apply phenotypic assays including growth profiling of cells containing GCFs on various defined nutritional media (e.g. polysaccharides and glycans found in the diet). Co-culturing competition with fluorescently labeled wild-type L. plantarum will quantitatively determine the degree to which GCFs improve probiotic fitness.
  • phenotypic assays including growth profiling of cells containing GCFs on various defined nutritional media (e.g. polysaccharides and glycans found in the diet). Co-culturing competition with fluorescently labeled wild-type L. plantarum will quantitatively determine the degree
  • GCFs will not only improve colonization of probiotic bacteria, but can potentially impact the compositional structure of the gut microflora.
  • the goal is to enhance probiotic infiltration and establishment amongst the natural microflora to improve function and stability of the gut microbiome.
  • HF high-fitness
  • This work aims to assess how individual genes (or gene groups) can directly impact the rest of the gut microbiota to functionally dissecting these complex microbe-microbe interactions.
  • Genome Biol. 10 R25 Lee SM, Donaldson GP, Mikulski Z, Boyajian S, Ley K & Mazmanian SK (2013) Bacterial colonization factors control specificity and stability of the gut microbiota. Nature 501: 426- 9

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé puissant et systématique pour identifier des gènes qui améliorent la santé bactérienne dans le tractus gastro-intestinal (GI) de mammifères. Les gènes identifiés peuvent ensuite être utilisés pour générer des bactéries recombinantes qui peuvent améliorer la santé d'un mammifère, tel qu'un être humain, ou peuvent être utilisées pour lutter contre un trouble gastro-intestinal.
PCT/US2014/066173 2013-11-18 2014-11-18 Amélioration de la santé microbienne dans l'intestin de mammifères WO2015074054A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361905530P 2013-11-18 2013-11-18
US61/905,530 2013-11-18

Publications (1)

Publication Number Publication Date
WO2015074054A1 true WO2015074054A1 (fr) 2015-05-21

Family

ID=53058186

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/066173 WO2015074054A1 (fr) 2013-11-18 2014-11-18 Amélioration de la santé microbienne dans l'intestin de mammifères

Country Status (1)

Country Link
WO (1) WO2015074054A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017044885A1 (fr) * 2015-09-09 2017-03-16 uBiome, Inc. Procédé et système pour des diagnostics dérivés du microbiome et agents thérapeutiques pour des affections associées à la santé cérébro-carniofaciale
WO2017044871A1 (fr) * 2015-09-09 2017-03-16 uBiome, Inc. Procédé et système pour diagnostics dérivés du microbiome et agents thérapeutiques contre l'eczéma
WO2017044880A1 (fr) * 2015-09-09 2017-03-16 uBiome, Inc. Procédé et système pour diagnostics dérivés du microbiome et agents thérapeutiques pour une maladie infectieuse ou d'autres états de santé associés à l'utilisation d'antibiotiques
CN109852597A (zh) * 2019-03-21 2019-06-07 云南师范大学 一种β-半乳糖苷酶galRBM20_1及其制备方法和应用
WO2022013269A1 (fr) * 2020-07-15 2022-01-20 Danmarks Tekniske Universitet Bactéries probiotiques génétiquement modifiées pour la régulation de la colonisation sensible aux prébiotiques
CN117305189A (zh) * 2023-11-29 2023-12-29 杭州微致生物科技有限公司 一种德氏乳杆菌保加利亚亚种vb183及其培养装置和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100183559A1 (en) * 2008-11-11 2010-07-22 Alimentary Health Limited Bifidobacterium longum
US20110053273A1 (en) * 2007-10-08 2011-03-03 Synthetic Genomics, Inc. Methods for cloning and manipulating genomes
US20120164275A1 (en) * 2009-09-01 2012-06-28 Chr. Hansen A/S Lactic bacterium with modified galactokinase expression for texturizing food products by overexpression of exopolysaccharide
WO2012122522A2 (fr) * 2011-03-09 2012-09-13 Washington University Mise en culture d'un prélèvement d'une communauté microbienne intestinale

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110053273A1 (en) * 2007-10-08 2011-03-03 Synthetic Genomics, Inc. Methods for cloning and manipulating genomes
US20100183559A1 (en) * 2008-11-11 2010-07-22 Alimentary Health Limited Bifidobacterium longum
US20120164275A1 (en) * 2009-09-01 2012-06-28 Chr. Hansen A/S Lactic bacterium with modified galactokinase expression for texturizing food products by overexpression of exopolysaccharide
WO2012122522A2 (fr) * 2011-03-09 2012-09-13 Washington University Mise en culture d'un prélèvement d'une communauté microbienne intestinale

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FAITH ET AL.: "Identifying Gut Microbe-Host Phenotype Relationships Using Combinatorial Communities in Gnotobiotic Mice", SCIENCE TRANSLATIONAL MEDICINE, vol. 6, 22 January 2014 (2014-01-22), pages 1 - 24 *
RIESENFELD ET AL.: "METAGENOMICS: Genomic Analysis of Microbial Communities", ANNUAL REVIEW OF GENETICS, vol. 38, 14 July 2004 (2004-07-14), pages 525 - 552 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017044885A1 (fr) * 2015-09-09 2017-03-16 uBiome, Inc. Procédé et système pour des diagnostics dérivés du microbiome et agents thérapeutiques pour des affections associées à la santé cérébro-carniofaciale
WO2017044871A1 (fr) * 2015-09-09 2017-03-16 uBiome, Inc. Procédé et système pour diagnostics dérivés du microbiome et agents thérapeutiques contre l'eczéma
WO2017044880A1 (fr) * 2015-09-09 2017-03-16 uBiome, Inc. Procédé et système pour diagnostics dérivés du microbiome et agents thérapeutiques pour une maladie infectieuse ou d'autres états de santé associés à l'utilisation d'antibiotiques
US11773455B2 (en) 2015-09-09 2023-10-03 Psomagen, Inc. Method and system for microbiome-derived diagnostics and therapeutics infectious disease and other health conditions associated with antibiotic usage
CN109852597A (zh) * 2019-03-21 2019-06-07 云南师范大学 一种β-半乳糖苷酶galRBM20_1及其制备方法和应用
WO2022013269A1 (fr) * 2020-07-15 2022-01-20 Danmarks Tekniske Universitet Bactéries probiotiques génétiquement modifiées pour la régulation de la colonisation sensible aux prébiotiques
CN117305189A (zh) * 2023-11-29 2023-12-29 杭州微致生物科技有限公司 一种德氏乳杆菌保加利亚亚种vb183及其培养装置和应用
CN117305189B (zh) * 2023-11-29 2024-03-22 杭州微致生物科技有限公司 一种德氏乳杆菌保加利亚亚种vb183及其培养装置和应用

Similar Documents

Publication Publication Date Title
Crook et al. Adaptive strategies of the candidate probiotic E. coli Nissle in the mammalian gut
Buron-Moles et al. Uncovering carbohydrate metabolism through a genotype-phenotype association study of 56 lactic acid bacteria genomes
Poroyko et al. Gut microbial gene expression in mother-fed and formula-fed piglets
Broadbent et al. Physiological and transcriptional response of Lactobacillus casei ATCC 334 to acid stress
WO2015074054A1 (fr) Amélioration de la santé microbienne dans l'intestin de mammifères
Pamp et al. Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB)
Patrick et al. Twenty-eight divergent polysaccharide loci specifying within-and amongst-strain capsule diversity in three strains of Bacteroides fragilis
Mwangi et al. Regional and global changes in TCRαβ T cell repertoires in the gut are dependent upon the complexity of the enteric microflora
Bonacina et al. A genomic view of food-related and probiotic Enterococcus strains
Lengfelder et al. Complex bacterial consortia reprogram the colitogenic activity of Enterococcus faecalis in a gnotobiotic mouse model of chronic, immune-mediated colitis
Morelli et al. Genetics of lactic acid bacteria
Turroni et al. Ability of Bifidobacterium breve to grow on different types of milk: exploring the metabolism of milk through genome analysis
Yaung et al. Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics
Douillard et al. Comparative genome analysis of L actobacillus casei strains isolated from A ctimel and Y akult products reveals marked similarities and points to a common origin
Béchon et al. Autotransporters drive biofilm formation and autoaggregation in the diderm firmicute Veillonella parvula
Rice et al. Interspecies commensal interactions have nonlinear impacts on host immunity
Prajapati et al. Genomic analysis of dairy starter culture Streptococcus thermophilus MTCC 5461
Zakharevich et al. Complete genome sequence of Bifidobacterium longum GT15: identification and characterization of unique and global regulatory genes
Vannucci et al. Comparative transcriptional analysis of homologous pathogenic and non-pathogenic Lawsonia intracellularis isolates in infected porcine cells
Jones et al. Cyclopropane fatty acid synthase mutants of probiotic human-derived Lactobacillus reuteri are defective in TNF inhibition
Yaung et al. Recent progress in engineering human-associated microbiomes
Aziz et al. Molecular signatures of non-typeable Haemophilus influenzae lung adaptation in pediatric chronic lung disease
Mokszycki et al. A simple in vitro gut model for studying the interaction between Escherichia coli and the intestinal commensal microbiota in cecal mucus
Huang et al. The probiotic and immunomodulation effects of Limosilactobacillus reuteri RGW1 isolated from calf feces
Tsuchida et al. Genomic characteristics of Bifidobacterium thermacidophilum pig isolates and wild boar isolates reveal the unique presence of a putative mobile genetic element with tetW for pig farm isolates

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14862382

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14862382

Country of ref document: EP

Kind code of ref document: A1